National Public Health Laboratory Version No. 1
covlD-19 soP Copy no: 1
NPHL/ C19/SOP/04/EH Date of issue: 15/04/2020
Controlled copy Page no:1
Manual Extraction of Viral RNA using Ill-Media kit
Standard Operating procedure of National Public Health Laboratory Tripura Marg, Kathmandu Nepal
Prepared by: smriti Shrestna{t{' Authorised byi Dr. Runa Jha ned byl
Reviewed by: Rachana Mehta Director and Quality manager tha IN Quality and traininB unit \P WU I National Public Health Laboratory Version No. 1
covtD-19 soP Copy no: 1
I NPHL/ C19lSOP/04/EH Date of issue: 15104/)020
I Controlled copy PaSc nor2
SUMMARY
I'his SOP is part of a suit of SOPs that are set up to allow processing of COVID-19 samples in the National Public Health Laboratory OPHI-). Staffshould be aware of associated Risk Assessments and have received sufficient training to be able to demonstrate competency before perfonning this task alone.
This SOP details procedures for manual extraction ofviral RllA using the IIiPuraA viral RNA purification kit. Samples are loaded onto Hielute Miniprep spin columns where the released viral RNA is bound to the spin column membrane. Contaminants are removed by washing before high-quality RNA is eluted, yielding purified intact RNA ready tbr downstream RT-PCR.
SAFETY
All sentences written in red bold text and denoted with A, indicate a Safety Critical stcp or comment and as such extra attention must be given when undertaking them.
I All statTshould be familiar with Risk Assessments and have undertaken spccilic training
1.0 CROSS RF],FF],RENCES
NPHL/COVID- 19/RA/001 -General COVID-19 Laboratory RA NPHL/COVID- 19/FORM/001 - Sample tracking form
Manual : HiPurA viral RNA Purification Kit (Himedia)
2.0 TRAINING All staffshould have undertaken specific training before carrying out this procedure.
Prepared byr Smriti Shrestha W Althorised by: Dr. Runa Jha Mai ined by Reviewed byi Rachana Meht Director and Quality manager Mrs hrestha av^ V lnchar Quality and training unit National Public Health Laborato.y Version No. 1
(} covrD,19 soP Copy no: 1 *L,P NPHr/ C19lSOP/04/€H Date of issue: I5/M/2O2O '!.:Y'r Controlled copy Page no:3
3.0 RUQUISITES and REAGEN'IS
3.1 Reaqents HiPurA viral RNA Purification Kit M86I5
Ethanol (96- l 00%) Sigma #51976
3.2 E ul ment Collection tubes (2 ml)
+ or generic equivalent
Eppendrofftube 1.5 ml
RNase- free pipette tips (aerosol barrier)
Tabletop Microcentrifu ge Vortex Mixer
3.3 Clinical samples
3.3.1 'l'he first steps ofthis procedure include viral inactivation and must be carried out in a Class II Biological Safety Cabinet.
ABefore samples can be safely handled on the bench (i.e. out of containment), they must first be inactiyated by a validated method (refer to pathogen/task- specific guidance).
1.0 PROCIiDURE Peribrrn all pipetting steps using positivc displacement or aerosol resistant pipettc tips.
Staffcarrying out viral inactivation in an isolator should wear a disposable laboratory gown, a single layer ofgloves and suitablc footwear with dedicated lab tbotwear e.g. clogs. Iror eye protection, wear safbty glasses as a minirnum; face shield/goggles are pref'erable.
A,Proper attention to the use of required/specified PPE is critical to mitigatc thc risk of accidental exposure to pathogenic material and chemical hazards.
Prepared by: Smriti Shrestha Authorised by: Dr. Runa Jha Maint bvl neviewed bv: nachana MehtQp Director and Quality manager Mrs. Lil tha
lnchaage ality and training unit National Public Health Laboratory Version No. 1
covtD-19 soP Copy no: 1.
NPHr/ c19lSOP/04lEH Date of issue: 15/O4/2020 1rr,t..,-t.:-"
Controlled copy Page no:4
4.1 Advance preparation
4.t.1 Carrier RNA is supplied lyophilised with the HiPuraA Viral RNA Kit.
In advance, reconstitute carier RNA as following with elution buffer (RNase free water) and store at -20 oC in convenient sized aliquots to avoid repeated freeze and thaw.
Number of Preps Carrier RNA Elution Buller
250 3.5 mg 3.5 ml
4.1.2 Prior to use: Prepare AVL buffer containing carrier RNA and template internal control and water if required.
Select relevanl internal control (lC) depending on downstreom application (note, most PCR kits will contoin their own IC - this should not be substituted).
Calculate the volume of lysis buffer as 560p1 per sample.
Calculate the volume of carier RNA as 5.6 pl/sample
4.t.3 Dilute Wash solution concentrale (Ws) (DS0012) supplied with HiPuraA Viral RNA Kit. Dif ute 75 ml wash concentrate with 225 ml ethanol (96-100%). Record date reconstituted on bottle. Store closed at room temperalure.
4,2 Procedure
4.2.t For each batch ofpatient samples, a negative extraction control can be added that is treated identically. For different sample matrices use the following controls:
. For plasma/serumJ use pooled human serum/plasma or serum/plasma tiom known negative sample(s)
. For wet swabs, use VTM
. For dry swabs, use RNAse-free water
4.2.2 Lysis and virus inactivauon:
Prepared byr SmritiShrestha Authorised by: Dr. Runa Jha vaint\lner by: $tr ed Reviewed by: Rachana Mehta Director and Quality manager ,^,,,k stha w f,\p ,nanrrr"\ ality and training u nil National Public Health taboratory Version No. 1
covrD-19 soP Copy no: 1
NPHr/ C19lSOP/04/EH Date of issue: 15/04/2020
Controlled copy Page noiS
Pre- I Take aliquots of pre-prepared AVl/internal extractioIl control/carrier RNA mix. Choose the correct IC .for the downslreum RT-PCR asscty label tube with lab- assigned sample ID numbcr. Transfer the aliquoted patient- samples (from the refrigerator where aliquots are stored) for PCF. AND aliquots ofnegative extraction controls (plasmo, WM or water) - iJ to be included.
Within 2 Take 140 pl of sample (in l.5ml eppendorfftube) and add Biosafety 560trr1 of carrier RNA/Lysis solution (560p1 AVL and 5.6p1 Cabinet carier RNA/sample). Class II Mix thoroughly by inverting several times Or Pplse vortex for l5 seconds Briefly centrifuge tube to remove drops from inside of lid. Incubate I0 min at room temperature (15-25'C)
.A,cnrrrcal srAGE FoR sAFE TNACTIVArroN oF SAMPI-E
A,T,NSUNS BUFFER USED IS AVL AND TIMINGS ARE ADHERED TO 3 Add l0pl olExtraction control, voftex and pplse spin.
4 Carefplly add 560p1 ofchilled ethanol (Molecplar-grade if available, Absolute if required).
Mix by gentle pipetting
Briefly centrifuge tube to remove drops from inside of lid.
AeNsung SAMpLES ARE ALLowED lo REAC I' WITH ETOH 5 Remove samples from catlinet and transfer to RNA extraction .ar.,*. \ ,
Prepared by: SmritiShrestt a Authorised by: Dr. Runa.lha vJi\tained bv: lfr$J Reviewed by, Rachana MehtaqjP Director and Qualitv manager v.'$,1sr,,o,tn, n^0 lncha\e, Quality and traininB unrt r- Natronal Publrc llealth I aboratory Version No. 1 i I
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NPHr/ C19/SOP/04/EH Date of issue: 15/04/2020
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4.2.3 RNA purification using QIAamp Mini spin columns in RNA extraction area of rnain lab: ln I Label spin column caps with sample tracking number. RNA Apply 630 pl of the solution from step 4 of 4.2,2 to a HiElute extrac spin column (in a 2ml collection tube). Close cap tion This marks end o/ ethanol inuclir,otion.
a rea 1 Centrifuge at rprn (6000 x g) for I min I -8000 f Put HiElute spin column into clean 2ml collection tube. Discard filtrate. Add remaining 630 pl ofthe solution from step 4 of 4.2,2 to a HiElute spin column. Close cap. .t Centrifuge at -8000 rpm (6000 x g) tbr I rnin f 5 Put HiElute spin column into clcan 2ml collection tube; discard filtrate. Open the HiElute spin column. Add 500 pl diluted wash solution(WS) (DS00l2). Close cap. 6 Centrifuge at -8000 rpm (6000 x g ) lbr I min. f 7 Place HiElute spin column in clean 2 ml collection tube: discard fi ltrate. Open the HiElute spin column. Add another 500 pl diluted wash solution(WS) (DS00l2). Closc cap. ti Centrifuge at lpll speed 14.000 rpm (20,000 g) lbr 3 min. f 9 Put HiElute spin column into clcan 2rnl collection tube. Centrifuge at lpll speed 14.000 rpm (20,000 g) for I min. f l0 Put HiElute spin column in clean LABELLED 1.5 ml microfuge tube and open lid. Add 60 pl Buffer AVE (Elution Sol"). Close cap and incubate I min at room temperature. il Centrifuge at -8000 rpm (6000 x g ) tbr I min. Store eluate at 4 "C until PCR analysis (same day). i t2 Proceed to PCR t3 For longer periods ofstorage store eluted RNA for up to I year
at -20oC or -70oC . 4.2.4 Discard all the sample preparation waste (tubes, tips. filtrate) into a leak-proofbag with absorbent nraterial (e.g. tissue or absorbcnt spillage granples) and seal. Treat as dr) waste.
Prepared by: SmritiShresttra Authorised by: Dr. Runa Jha Main ed by: l(IPl Reviewed by: Rachana Meh,?s Directo. and quality manager Mrs. til estha
lncharge ality and training unit trv ,z I Netional public Health Laboratory Version No. 1 1
I
covtD-19 soP Copy no: 1
NPHr/ C19ISOP/04/EH Date of issue: 15/04/2020
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CAUTION .A$uffers AVL and AWI contain guanidine hydrochloride/thiocyanate. AOO NOf mix with sodium hypochlorite solutions. In event ofspillage wipe up with detergent, then rinse with waler. then rinse with sodium hypochlorite.
5.0 RESPONSIBILITIES All trained staff or new staffundergoing training must adhere Io this SOP.
Prepared by: SmritiShrestha Authorised by: Dr. Runa Jha ftl1(/' Rev,ewed bv: Ra.han, Mehta Director and Qualitv manager
au $t ffii:*::,,rannsun, I National Public Health taboratory Vcrsion No 1
covtD-19 soP Copy no: l
NPHL/ Cr9l5OP/05/EQ Oale of issue; 15/O4l7O).O
Controlled copy PaBe no r1
Manual extraction of viral RNA using the OIAamp Viral RNA kit
Standard Operating procedu rc of National Public Health Laboratory Tripura Marg, Kathmandu Nepal
Prepared by: Smriti Shrestha Authorised by: Dr.RunaJha \l frns Revicucd by: nachana NIe Director and Quality nranager \lls.l.i rcstha Qf2 f\4-- lrchargc. ualitv and tlaininr unit National Public Health laboratory
covtD-19 50P Copy noi 1
NPHr/ C19/SOP/os/EQ Date of issue: 15/04l2O2O
Controlled copy Page no:2
I
SI.JMMARY
This SOP is part of a suit of SOPs that are set up Io allow processing of COVID-19 samples in the National Public Health Laboratory (NPHL). Staffshould be aware of associated Risk Assessments and have received sulTicient training to be able to demonstrate competency before perlorming this task alone.
This SOP details procedures fbr manual extraction of viral RNA using the QIAamp Viral RNA kit. lt can be used instead ofautomated nucleic acid extraction (e.9. E7.ll.
Samples are loaded onto QIAamp Mini spin columns where the released viral RNA is bound to the QIAamp membrane. Contaminants are removed by washing before high-quality RNA is eluted, yielding purified intact RNA ready for downstream RT-PCR.
SAFEI'Y I
All sentences written in red bold text and denoted with A, indicate a Safety Critical step or comment and as such extra attention must be given when undertaking them.
All staff should be familiar with Risk Assessments and have undertaken specific training
1.0 CROSS REFf,RENCES
NPHL/COVID-19/RA/001 - General COVID-19 Laboratory RA NPHI./COV I D- I 9/F'ORMi00 I - Sample tracking form
Manuals: QIAamp Viral RNA Mini Handbook (Qiagen)
2.0 TRAINING All staffshould have undertaken specific training befbre carrying out this procedure.
Prepared by: Smriti Shrestha [' Authorised by: Dr.RunaJha j\,tai ined by
Revie\Yed by: nacnana MehQ[, Director and Quality manager ivl rs. eShrestha W\P lnc QualiR and trainiDg unit National Public llealth Laboratory Version No. 1
covrD-19 soP Copy no: 1
NPHL/ C19lSOP/o5/EA Date of issuei 75/O4/202O
Controlled copy Pagc nor3
3.0 REQUISITES and REAGENTS
-l. I Reagents
QIAamp Viral RNA Kit Qiagen #52904 or #52906 Ethanol (96- 100%) Sigma#51976
Pooled human serum/plasma (Sigma #H6914) or pooled negative human serum/plasma or RNAse free H2O.
3.2 Equipment
Collection tubes (2 mls) Qiagen # 19201 * or generic equivalent
J.J Clinical samples
3.3.1 The first steps of this procedure include viral inactivation and must be carried out in a Class II Biological Safcty Cabinet.
.A,B"fu." samplcs can be safely handled on the bench (i.e. out of containment), they must first be inactivated by a validated method (refer to pathogen/task- specific guidance),
4.0 PROCEDURE Perform all pipetting steps using positive displacement or aerosol resistant pipeffe tips.
Staffcarrying out viral inactivation in an isolator should wear a disposable laboratory gown, a single layer ofgloves and suitable lootwear with dedicated lab footwear e.g. clogs. For eye protection, wear safety glasses as a minimum; face shield/goggles are preferable.
AProper attention to the use of required/speci{ied PPE is critical to mitigate the risk of accidental exposure to pathogenic material and chemical hazards.
Prepared by: Smriti Shrestha tr Authorised by: Dr.RunaJha Mai ined by Reviewed by: Rachana ven\p!7 Director and Quality manager Mrs hrcstha N_- lncha Quality and training unil National Public Health Laboratory Version No 1
covrD-19 soP Copy no 1
NPHr/ C19/SOP/0slEQ D.te of issue: 15104/2020
Controlled copV Page no:4
4.1 Advancepreparation
4.1 .l Carrier RNA (su{ficient fbr 48 sample preps) is supplied lyophilised with the QIAamp Viral RNA Kit. oC In advance, reconstitute carrier RNA with 3 l0 pl buffer AVE and store at -20 in convenient sized aliquots e.g. 40 pl
Note: 310 pl reconstituted carrier RNA is sufficient for 50-55 samples.
Thaw before use.
4.t.2 Prior to use: Prepare AV[- buffer containing carrier RNA and template internal control and water il requircd.
Select relevant internal conlrol (IC) depending on downstream application (nole, most PCR kils will conloin their ovn IL' - this should not be substilulad).
4.1.3 Dilute Buffer AWI supplied with QIAamp Viral RNA Mini Kit: Dilute l9 ml Awl concentrate with 25ml ethanol (96-100%). Record date reconstituted on the bottle with a permanent marker. Store closed at room temperature.
.1.l.+ Dilute BufferAW2 supplied with QlAamp Viral RNA Mini Kit: Dilute l3 ml AW2 concentrate with 30ml ethanol (96-100%). Record date reconstituted on Ihe bottle with a permanent marker. Store closed at room temperature.
4.2 QIAamp procedure
4.2.1 For each batch of patient samples. a negative extraction control can be added that is treated identically. For different sample matrices use the following controls:
. For plasma./serum, use pooled human serum/plasma or serum/plasma tiom known negative sample(s)
. Forwet swabs, use VTM
. For dry swabs, use RNAse-free water
Prepared by: Smriti Shrestha (O' Authorised by: Dr.Runajha Mai incd b1
Reviewed by: nu.nunu M"hrQh Director and Quality manager Mrs Shrcstha I^o v--- Inch c. Qualin and training Lrnit National Public Health taboratory Version No. 1
covrD-19 50P Copy no: 1
NPHL/ c19lSOP/oslrO Date of issue: 15/O4/)02O
Controlled copy Page no:5
4.2.2Lysis and virus inactivation:
Pre- I Take aliquots of pre-prepared AVL/internal control/carrier extraction RNA mix. Choose the correct IC for the downstream RT-PCR a.rsay label tube with lab-assigned sample ID number.
Transfer the aliquoted patient- samples (from the refrigeralor where afiquols are stored) for PCR AND aliquots of negetive extrqclion controls (plasma, VTM or waterl - ifto be included.
-) Within Take 140 pl of sample(in l.5ml eppendorfftube) and add 560p1 of Biosafety canier RNA/Lysis solution (560plAW and 5.6p1 canier Cabinet RNA/sample). Class II Mix thoroughly by inverting several times. OR Pulse vortex lor l5 seconds Briefly centrifuge lube to remove drops from inside of lid. Incubate l0 min at room temperature ( I 5-25oC)
Acntrtcel srAGE FoR sAFE INACTIvATIoN oF SAMPLE
AeNSunE BUFFER USED IS AYL AND TIMINCS ARE ADHERED TO
Add lOpl of Extraction control, vortex and pulse spin.
4 Carefullyadd 560p| of chilled ethanol (Molecplar-grade if available, Absolute if required).
Mix well by pulse vonexing for l5 seconds
Briefly centrifuge tube to remove drops from inside of lid.
AeNsuR.p sA,rutpLES ARE ALLowED To REACT wrrH ETHANOL
Prepared by: smriti shrestha\ir Authorised by: Dr.RunaJha N4a[ftarncd b1:
Reviewed by: Rachana uer,t1trfa Director and Quality manager r,,tr.[xsn,"rrtu t$tr.- lnch$sc, Qualirl and training unit Natio nal Public Health L;boratory Version No. 1
covtD-19 soP Copy no: 1
NPHr/ C19lSOP/OslEQ Dalc of issue: 15/O4/2O2O
Controlled.opy Page no:6
5 Rcmove samples from cabinet and transfer to RNA extraction area. I
4.2.3 RNA purification using QIAarnp Mini spin columns in RNA extraction area of main lab: ln I Label spin column caps with sample tracking number. RNA Apply 630 pl ofthe solution from step4 of4.2.2 to a QIAamp spin column (in a 2ml collection tube). Close cap extrac This marks end of ethanol inaclivalion. tio n area 2 Centrifuge at -8000 rpm (6000 x g) for I min. ..'l- + Put QlAamp spin column into clean 2ml collection tube. Discard filtrate. Add remaining 630 pl of the solution from step 4 of 4.2.2to a QIAamp spin column. Close cap. ,t Centrifug€ at -8000 rpm (6000 x g) for I min ? ) Pul QlAamp spin column into clean 2ml collection tube; discard filtrate. Open the QIAamp spin column. Add 500 pl buffer AWt Close cap. 6 Centrifug€ at rpm (6000 x g for I min. -8000 ) I 1 Place QIAamp spin column in clean 2 ml collection tube; discard filtrate. Open the QIAamp spin column. Add 500 td buffer AW2 Close cap. (20,000 g) 8 Centrifuge at full speed 14,000 rpm for 3 min. +t 9 Put QlAamp spin column into clean 2rnl collection tube. Ccntrifu at full s ed 14.000 20,000 for I min ? t0 Put QIAamp spin column in clean LABELLED 1.5 ml microcentrifuge tube and open lid. Arld 60 pl Buffer AVf, (Elution Solution). Close cap and incubate i min at room temperature Centri at -8000 rpm (6000 x g ) for 1 min. Store eluate at 4 'C until PC analysis sallte ? M ined b1: Preparcd by: Smriti Shre Authorised by: Dr.RunaJha
M l-ri hrestha Reviewed by: nachana MehtQ[ Director ard Quality^untS"t'- |( L/- lnc Quality and training unil Natio I nal Pubjic Hea,th t t I COVI D..19 SOp
Copy no: 1 NPHL/ c1s/soP/05/EQ Dal e ofissue 15/04/2020 ControJl ed copy Page no:7 l2 Proceed to PCR IJ For on pen ods o I storage store the 0.c or c uted R N fb 70.c I up to I vcar al 4.2.4 D rscar d all the sampI"preparation absorbent material waste (tubes. tips, filtrate) e.g. tissue or absorbent into a leak_p roof spillage granu bag with les) and seal CAUTION . Treat as dry waste. .ABuffers AVL and AWI contain guanidine ILDO hydrochloritle/thiocyanatc. NOT mix with sodium hypochlorite solulions. *"n detergent' then rinse ffiffiTti:;::#;Xi"lo with watcr, rhcn rinsc
5.0 RESPONSIBILITIES AII trained staffor new sraffundergoing training must adhere ro this SOp.
Prcpared by: Smriti Shreslha\.1,$f Authorised by: Dr.Runalha \1 aincd b1'
Rerierved h): nacfrana ulehta[p Director and Quality manager \Ir ceShresthil N,9_ I e. Quality and training unit National Public Health Laboratory Version No. 1
I covlD-19 soP Copy no:1
I NPHL/ C19/50P/06,/PM Date of lssue: 25103/2020
Controlled copy Pagc no:1
RT- PCR of covid- 19 using
Mal-slQllE:cetc atd BdBP) (es.-L?50Q)
Standard Operating procedure of National Public Health Laboratory Tripura Marg, Kathmandu
Nepal
Prepared by: Dr.lonathan Ashcroft Authorised by: Dr. Runa Jha by: Reviewed by: Smriti Shrestha I Director and Quality manager I'J (\\,L ,rh alitv and training unit National Public Health taboratory Version No. 1
covrD-19 soP Copy no:1
NPHr/ C19lSOP/06/PM Dale of issue: 25103/2020
Controlled copy Pagc no:2
SUMMARY
This SOP is part of a suit of SOPs that are set up to allow processing of COVID- l 9 samples in the National Public Health Laboratory (NPHL). Staff should be aware of associated Risk Assessments and have received sufficient training to be able to demonstrate competency before performing this task alone.
This SOP details procedures for RT-PCR detection ofthe 2019-nCoV (COVID-19) virus using the MOLBIOL LightMix CoV E- and RdRP-genes assay run on the AIll 7500 plattbrm.
SAFETY
All scntences n'rittcn in red bold text and denoted rvith 4,, intlicatc a Safeh'Critical step or commcnt and as such extra attention must bc given rvhen undertaking thcm.
Rtt staffsnouta be familiar with Risk Assessments and have undertakcn spccific training. I ]
BACKGROTIND Purpose
This SOP is adapted from the protocol of Corman et al,2020 and used at NPHL for detection ofE Gene and RdRP genes of COVID-I9.
Special Note
Coronaviruses (CoV) are positive-stranded RNA viruses from the Coronaviridae family. The four common human pathogen strains 229E and NL63 from the Alpha group, and OC43 and HKUI fromthe Beta group cause usually only a common cold, but the 2003 SARS-CoV pandemy with more than 800 fatal cases and the curent MERS-CoV pandemy originating from Arabia made this virus family well worldwide known.The Wuhan CoV 2019 was described end of December 2019 after some of visitors to the seafood market developed severe pneumonia. The genome sequence was published Jan I lth (Genbank acc. MN908947) and shows a high similarity to the SARS virus. Dcscription
A 76 bp long fragment from a conserved region in the E gene is detected with FAM labeled hydrolysis probes (530 channel). This assay will detect SARS and Wuhan 2019 CoV pneumonia virus as well as other bat-associated SARS-related viruses (Sarbecovirus).
Prepared by: 0r.lonathan Ashcroft Authorised by: Dr. Runa Jha vaintainLd b'
Reviewed by: SmritiShrestha Director and manager V,.s.Lifco\ \\t N Quality \f' NP tncharge, Q\ ity and training unit National Public Health Laboratory Version No- 1
(; covtD-19 soP Copy no:1 4*i" NpHL/ C19lSOP/06/PM Date of issue: 25103/2020 Controlled copy I Pagc no:3
A 100 bp long fragment from a conserved region ol the RNA-dependent RNA polymerase (RdRP) gene is detected with a Wuhan-specific FAM labeled hydrolysis probes (530 channel). No degenerate broad-range Sarbeco-virus probe contained (without probe Pl from the WHO protocol). This assay will detect Wuhan 2019 SARS-like CoV pneumonia virus but not all other SARS-like viruses. No cross reactivity with common human respiratory CoV NL63, 2298, LIKU, OC43 or MERS.
Specification
In this assay, SARS-related coronaviruses. including 2019-nCoV are distinguished from other respiratory pathogens including human seasonal coronaviruses by using primers and probes specific for a conserved region ofthe RdRp gene. The location of the viral genome target region is shown below.
These assays detect l0 genome equivalenl copies or less per reuction.
f.{(9o89a7 W utan{ lu.1 t - -
RdPP
1.0 CROSS REFERENCES
NPHL/COVID-19/RAi00l - General COVID-19 Laboratory RA
NPHL/GEN/SOP/O1/lR- Incident Reporting
NPHL/COVID-19/IORM/001 - Sample tracking form
NPHL/COVID-19/FORM/002 - Incident Reporting Form
2.0 TRAINING All staff should have undertaken specific training before carrying out this procedure
Prepared by: Dr. Jonathan Ashcroft Authorised byi Dr. Runa lha Ma in dbv
Reviewed bv: Smriti Shrestha t\ 0irector and Quality manager Mrs. Lil restha
lncharge, lity and training unit \.N N, L National Public Health Laboratory Version No. 1 e covtD-19 soP Copy no:1 I tt*j,;, NPHL/ ClqlSOP/06/PM Date of issue: 25/03/2020 Controlled copy Pagc no:4
3.0 REQUISITES and REAGENTS
3.1 Reagents
2019-nCoV primers and probes (TlB MOLBIOL)
OligonuclootidelO S€quenc€(5'-3') Comment
Rd Rp_SARST-F2 GTGARATGGTCATGTGTGGCGG use 600 nM per reaclion
Rd Rp_SARST-R I CARATGTTAAASACACTATTAGCATA use 800 nM per reaclion
Speciric for Wuhan-Cov, will not dete.t SARS-CoV RdRp_SARST-P2 CAGGTGGAACCTCATCAGGAGATGC. 1 ps. BBO use 00 nM reaclion and mir wth P1
Pan Sa,beco-Probe, will d€tect FAM, wuhan virus. SAR$CoV and bat- RdRp_SARST-P1 CCAGGTGGWACRTCATCMGGTGATGo- sARs-relaled covs BBo use I 0o nM per .eaclion and mix wilh P2
o Water (DNase- and RNase-fiee)
o Positive control material o Extracted RNA from samples (inc Extraction control - EVA)
3.2 Equipment
o Microcentrifuge r PCR Hood o Vortex . Gilson Pipetteman (or equivalent) . Filtered pipette tips (sterile and disposable) o ABI TaqMan 7500 Fast Thermal Cycler o 96 well ABI TaqManMicroAmp FAST plate o ABI plate cover and sealing tool o Plate holder o Ice bucket and ice . Tubes for Master Mix
3.3Clinical samples(Advance preparation)
Prepared by: Dr. Jonathan Ashcroft Authorised by: Dr. Runa lha Ma in ed by: Reviewed by: Smriti Shrestha.\ \ Director and Quality manager Mrs. Lil stha I W Vz lncha ity and training unit National Public llcalth laboratory Version No. 1
covrD 19 soP Copy no:1 c.) I NPHv C19/SOP/06/PM Date of issue: 25103/2020 "it','#, Controlled copy Page no:5
I
3.3.1 Type of samples: Respiratory clinical samples e.g. upper respiratorl, samples such nosc/throat swabs, and nasopharyngeal aspirates, also bronchoalvcolar lavagc (BAL), and sputum. Additionally, tissue culture fluid from virus isolates may be tested. Other sample types may less commonly be received, such as serum, faeces and urine.
3.3.2 Virus inactivation must have been carried out in a Class II Biological Safety Cabinet.
A,B.fo." samples can be safely handled on the bench (i.e. out of containment), they must first be inactivated by a validated method (refer to pathogen/task- specific guidance).
3.3.3RNA extraction would then have been performed by either manual or automated means (refer to specific SOPs and manufacturers instructions for guidance).
3.4 PPE 3.4.1 l-aboratory staff should wear the iollowing PPE foltowing inactivation & extraction ofthe samples: Lab gown, gloves, face mask (due to genera[ environment) and dedicated laboratory shoes.
AProper attention to the use of required/specified PPE is critical to mitigate the risk of accidental exposure to pathogenic material and chemical hazards.
{.0 PROCEDURE Perform all pipetting steps using positive displacement or aerosol resistant pipette tips.
4.lAdvance preparation of Reagents
4.1.1 Primers & Probes:To the vial (YELLOW topped-vile)containing primers and probe add 50pl PCR-grade water, mix (vortex) and pulse spin down.
Notes: Use 0.5u1 reagenl for u 20ul RCR reaction.
4.1.2 Preparation of the Positive Control: Add 160 or pl RNase/DNase-free 10 mM'lris buffer pI{ 8 - 8.5 to the vial containing the positive control dtrEE topped-vile). if using l0 ptl sample volume add 320 pl. Mix by pipetting up and dou,n l0 times. [f vortexing spin down to collcct the solution. Store dissolved controls fiozcn. Usc ol 'fris increases the stability in solution.
Notes:
Prepared by: Dr. Jonathan Ashcroft I Authorised by: Dr. Runa.iha Main ed by
Reviewed by: smriti 5n*"1"\ Director and Quality manager Mrs. Li stha I \0 Na lncharg ality and training unit Nationalpubl ic Health taboratory Version No. 1 I covtD-19 50P Copy no:1 l I NPHL/ c1,9/SOP/06/PM Date of issue: 25/03/2020
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o Opening this vial may cause contamination of the workspace. pulse spin vial BEFORE opening. . Use 5pl positive control for a 20pl pCR reaction (5ul + 15ul MM). 4.2Advance preparation of the Master Mix
Note, to decrease turn-around-time, this con be prepared concurrently to the RNA Extraction procedure.
4.2.1 Preparation of the Master Mix MUSToccur in a dedicated room that is physically separated from the template addition room (and the room storing the positive control). This is to minimise poter.rtial contanrination.
.1.2.l Ilnsure sufficient Master Mix is prepared to account for all the samples and required controls (e.g. PC, NTC).
Prepare Master Mix using chilled tubes and reagents as follows:
,or 5 pl extract Component 10 pl extract
10.4 pl Water, PCR-grade (colorless cap. provided with the Roche Master kit) 54 Ul 0.5 ptl Reagent mlx (parameter specific reagents containing prjmers and probes) 0.5 pl Control Reaction and additional assays (Multiplex PCR) 4.0 pl Roche Master (see Roche manual) 4.0 pl 0.1 pl RT Enzyme (see Roche manual) 0.1 pl pl 15.0 t,l Volume of Reaction Mix 10.0
Note: Mix gently, spin down and trcrnslbr l5ul (l1ul) per well.
4.3 Addition of Templatc to the Master Mix
4.3.1 TheTemplate(andpositivecontrolmaterial)mustNEVERenter/beused/sortedinthe room must be used' Master Mix preparation room - a separate Template addition
4.3.1 Add the temPlate as follows: (NTC = water' PC = positive To l5pl of Master Mix' add 5pl RNAfrom the extraction control included in the kit).
Mai ned by Authorised bY: Dr. Runa l ha Prepared bYl Dr.lonathan Ashcroft Mrs. restha Director and Quality manager Reviewed bY: Smriti Shrestha lncha ality and training unit National Public Health Laboratory Version No. 1
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5.0 RUNNING SAMPLIIS ON ABI 75OO
5.1 See the Instrument operator's manual lor details. Ensure sofiware is most currcnt version'- 2.3 (as of l8-Mar-20) Start programming belbre preparing the solutions.
The protocol consists offour program steps: l: Reverse Transcription ofthe viral RNA (Stage l) 2: Denaturation: sample denaturation and enzyme activation (Stage 2) 3: Cycling: PCR-amplification (Stage 3) 4: Cooling: cooling the inslrument (Not-shown below)
5.2 On the ABI TaqMan 7500 real time thermocycler, set reference dye to "(none)" for each sample. Add detectors for FAM * RdRp and ROX - EVA (extraction control). Run in 'Standard 7500' mode using the following cycling conditions:
C"r.'cling Conditions (shorvn trvo diffcrcnt ways).
Program Stepr RT Step Denaturation Cyc!ing Cooling Parameler Analysis Mode None None Quantification mode None
Cycles I 1 45 1 Target ["C] 55 95 95 60 72 40 Hold h.mm:ss 00:05r00 00:05.00 00:00:05 00:00.15 00:00:15 00i00:30 Ramp Rate ['C/s] 96 4.4 4.4 4.4 2.2 4.4 Ramp Rate ['C/s] 38d 4.6 4.6 4.6 4.6 2.0 Acquisition Mode None None None Single None None
Cycling Conditions Stage Repetilions Temperature Time 1 1 55 "C 1O:00 2 I 94'C 03:00 00:15 45 94'C 58 "C 00:30 Standard 7500Mode Data Collection: Stage 3 Step 2 ROX reference set to (none) PCR Volume: 25 pL Acquire data on 58'C extension cycle.
Prepared by: Dr. Jonathan Ashcroft Authorised by: Dr. Runa Jha Ma in ed by Revlewcd by: Smriti Shresth "\ Director and Quality manager Mrs. Lil h tha \d lnch arge ality and training unit Nationa, Public llealth Laborato.y Version No. 1
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5.2 At the end of the run, check that the default "sct baseline cycle start and end" settings are correct and re-set if required. Set the tkeshold to 7.63E+04 for RdRp by dragging the threshold bar above fluorescence detected within no template controls. Select analyse to calculate C1 (cycle threshold) values for samples and positive controls.
6.0 TI.]CHNICAL/SCIENTI}-ICVALIDATIONANDRESULT INTERPRI,I'l'ATION
6.1 Technical and scientific validation of each RdRp gene RT-PCR assay run is performed according to the lollowing criteria:
6.2 An RdRp gene RT-PCR assay run is considered valid ifthe positive control produces a positive signal in the FAM charrnel, in duplicate samples with expected Ct values that are within 3.2 Ct between duplicates, and no amplification signal is present in any of the negative controls.
6.3 An RdRp gene RT-PCR assay run is considered invalid if; o An amplification signal (Ct< 45.00) is seen in any ofthe negatives. o The absence of an amplification signal (Ct> 45.00 - undctectable) in the positive control. . No amplification signal (Ct> 45.00 = undetectable) in one duplicate of the positive control. o A difference of> 3.2 Ct's between duplicate wells in the positive control. o The absence of amplification signal (Ct> 45.00 : undetectable) for the Internal Control (lC : EVA) only invalidates THAT sample (not the batch).
Note: If deemed invalid, the run should be repcated from extraclion and reported (NPHL/GEN/SOP/0 I /IR).
6.4 A sample is considered NEGATIVE for target when no test signal is detected in either of the duplicate reaclions but the intemal control signal is. o If a sample is negative for all targets but the intemal control reactions for the same sample are also negative, the sample must be re-extracted as the extraction has failed or the PCR has been inhibited. . If a sample has a weak amplification signal in one or both duplicates tbr a specific target, or no amplification signal for a target in one duplicate, the sample is re-extracted and may be eluted in 25pl following discussion with
Prepared by: 0r. Jonathan Ashcroft Authorised by: Dr. Runa Jha Maintai by
Reviewed by: Smritistrestla \ Director and quality manager Mrs. Lilee ha
lncharge, lity and training unit \d' I 69I!--- National Public Health Laboratory Version No. 1
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I NPrlr./ C19/SOP/06/PM Date of issue: 2l;/0312020
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Senior Microbiologist, in order to concentrate the sample to enable/confirm delection.
6.5 Technical and scientific validation of test results is performed by lbllowing the tables below showing examples ol typical results that may be generated by the use of this assay.
RdRp lc lnterpretation Positive Positive Novel coronavirus detected Positive Negative 'Strong' novel coronavirus detected Negative Positive Negative for novel coronaviruses Negative Negative lnvalid result - repeat
I Channel 530 Channel 660 Besult (sample) Control Reaction No amplification Detectable Negative Not detectable Amplilacalion Cp < 39' Negative WH-CoV Positive No amolitication I Not detectable PCR laiiure Bepeat Amplilication siqnal I Positive Conlaminatioo Repeat
7.0 REPORTING OF RESULTS All results are to be reported via previously established NPHL reporting lines
8.0 RESPONSIBILITIES All trained staff or new statIundergoing training must adhere to this SOP.
Prepared by: Dr.lonathan Ashcroft Authorised by: Dr. Runa Jha ed by
Reviewed by: Smritisnrestt," \ Director and Quality manager tha \d f\il-^ [:h lity and training unit National Public l{ealth Laboratory Version No. 1
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NPHL/ C1elSOP 107 /PW Dal e of issue:10 / 04/)-020
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RT- PCR of COVID-19 -SARS- CoY -2 usins Wondfo kit (BIORAD)
Standard Operating Procedure of National Public Health Laboratory Tripura Marg, Kathmandu Nepal
Prepared by: Smriti Shrestha Authorised by: Dr. Runa Jha by:
Reviewed by: Bimlesh Kr Jha Director and Quality manager Mrs. Lilee
lncharge, nd training unil National Public Health Laboratory Version No. 1
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NPHL/ C19/SOP/o7lPW Date of issue:10 /04/2-020
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STJMMARY
This SOP is part of a suit of SOPs that are set Lrp to allow processing of COVID-19 samples in the National Public Health Laboratory OPHL). Staff should be aware of associated Risk Assessments and have received sufficient training to be able to demonstrate competency befbre performing this task alone.
is SOP details procedures for nucleic acid extraction using Wondfo; Hipure Viral Nucleic Acid Isolation Kit followed by R'|-PCR detection of the 2019-nCoV (COVID-19) virus using the Wondfo RT-PCR assay run on the BIORAD platform
SAFETY
All sentcnces written in red boltl text and denoted with A, indi"ute a Safety Critical step or comment and as such extra attention must be given when undertaking them.
All staff should be familiar with Risk Assessments and have undertaken training.
Prepared by: Smriti Shrestha Authorised by: Dr. Runa.lha ned by:
Reviewed by: Bimlesh Kr Jha [1'7 Director and Quality manager H:N a fvLQ-/ tnctrare\ and training unit National Public Health Laboratory Version No. 1
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NPHL/ C|9ISOPl07/PW Date of issue:10 /O4/2.O?.0
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BACKGROUND Purpose ]'his SOP is intended to use at NPHL for nucleic acid extraction and RT-PCR assay using Wondfoviral nucleic acid isolation kit and wondfo SARS-Cov-2 RT-PCR kit lor detection of N Gene and ORFlab gene.
Special Note Coronaviruses (CoV) are positive-stranded RNA viruses from the Coronaviridae family. The six coronavirus human pathogen strains are known before this pandemic. Among, four strains are responsible for common cold such as229E. and Nl-63 (Alpha group), and OC43 and HKUI (Beta group). However, other two strains such as SARS-CoV and MERS-CoV. are highly pathogenic and cause the scverc respiratory illness. SARS-CoV is also originated from China in 2003 and causcd more than 800 fatal cases. Similarly, MERS-CoV started in Saudi Arabia. The seven coronaviruse human pathogen strain, is known as novel- coronavirus or SARS-CoV 2, first appeared in Wuhan, China in 2019, and causingthe pandemic. The genome sequence of SARS-CoV 2 showed a high similarity to SARS-CoV (Genbank acc. MN908947). Description
Nucleic acid Extraction: Respiratory sample (nasopharyngeal or oropharyngeal swab) is collected in VTM and placed for lysis in VLF buffer and digested with protease. Then, release RNA is transferred to an adsorption plate and filter column. RNA is adsorbed on the silica gel memebrane of the filter column. while protein is not adsorbed and removed with filtration. After washing proteins and other impurities, RNA is finally eluted with low salt buffer( l0 Mm TRIS, ph 8.0).
RT-PCR for detection of SARS-CoY-2: This assay is based on the principle of multiplex RT-PCR with specific primers and fluorescent probes. The isolated RNA target is transcribed generating cornplementary DNA (cDNA) by reverse transcriptase which is followed by the arlplification of a conserved region of ORFlab and N genes for SARS-CoV-2 using specific primers and a fluorescent-labeled probe. Internal control provide with the kit isprepared with inactivated phages and is used to control for adequate processing of the target virus and to monitor the presence of inhibitor(S) in the RT-PCR assay.
ORFlab gene is amplified and detected in VIC channel, N gene is amplified and detected in FAM chan the internal control in CY5 channel Prepared by: Smriti Shrestha Authorised by: Dr. Runa Jha M by, Reviewed by: Bimlesh Kr Jha hh Director and Quality manager Mrs. Li Nx/s lncharge and training unil National Public Health Laboratory Version No. 1
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,i:.:i,,, NPHL/ C19/SOPloT/Pw Date of issue:10 /O4/207.O
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1.0 CROSS REFERENCES
NPHL/COVID-19/RA/001 - General COVID-19 Laboratory RA
NPHL/G EN/SOP/0 I /lR-lnc ident Reporting
NPHL/COVID- I 9/FORM/00 I - Sample tracking form
NPHL/COVID- lglFORMl002 - Incident Reporting Form
2.0 TRAINING All staff should have undertaken specific training before carrying out this procedure.
3.0 REQIJISITIiS AND RBAGENTS
3.1 Reagents
For RNA extraction:
. PK/carrier RNA o Protease Dissolve buffer . Buffer VLF . Buf{br CW o Buffer AVE
For RT-PCR assay
. 3 individual sealed pouches, each pouch contains lx RT-PCR lyophilized reagent (8-tube strip) 2x Desiccant pouch
. 3 individual sealed pouches, the pouch contains lx internal control (lyophilized, 8test/tube) 2x dessicant pouch . posltlve (l tubex8O0pl)
Prepared by: Smriti Shrestha Authorised by: Dr. Runa .lha by: Reviewed by: Bimlesh Kr Jha Uy Director and Quality manager q\^^€ ::} and training unit National Public l'lealth Laboratory Version No. 1
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o Negative control (l tubex8OOpl) o Dissolve buffer (l tubex800pl)
Conrponent Ingredients Concentration Reverse Transcriptase <0.1o/o DNA polymerase <0.1o dNTPs <0.lYo
Mgcl2 <6mM RT-PCR Lyophilised
Reagent Primers <0.lYo
Fluorescent labelled <0.loh
probes
'l'rehalose <404
Sucrose <40h Inactivated Escherichia Internal control coli bacteriophage MS2 'lrelialose <40 Sucrose <4Yo Nucleic acid containing <0.1o gene N and ORF-I ab parlial sequence Positive control Nat-l2PO4 <30 Na2HPO4 <30 NaCl Prepared by: Smriti Shrestha Authorised by: Dr. Runa Jha ned by: Reviewed by: Bimlesh Kr Jha Director and Quality manager M rs. & ftl)-- lncha lity and training unit National Public Health Laboratory Version No. 1 covtD-19 soP Copy no:1 q, ..1'" " . ;. \., :"'.;.. NPHL/ C1glsoP l07lPw Date of issue:10 /04/20?.0 Controlled copy Page no:6 Natl2PO4 <30 Negative control Na2HPO4 <3Yo NaCl Dissolve buffer DNase and RNase Free 100,h Water 3.2Equiprnent For RNA extraction: . Collection tubes (2 ml) . or generic equivalent o Eppendroff tube 1.5 ml o RNase- free pipette tips (aerosol barrier) o TabletopMicrocentrifuge o Vortex Mixer o Biosafety cabinet class II For RT-PCR . Microcentrifuge . PCR Hood o Vofiex . Gilson Pipetternan (or equivalent) o Filtered pipette tips (sterile and disposable) . BIORAD CFX96 3.3 Clinical samples 3.3.1 1'ype of samples: Respiratory clinical samples e.g. oropharyngeal swab nasopharyngeal swab transpofied in viral transport medium(VTM) 3.3.2 The first steps of this procedure include viral inactivation and must be carried out in a Class II Cabinet. Prepared by: Smriti Shrestha Authorised by: Dr. Runa Jha Mai Reviewed by: Bimlesh Kr Jha Director and Quality manager Mrs. Lilee llro lncharge, and training unit National Public Health Laboratory Version No. 1 COVID.19 SOP Copy no:1 NPHL/ C19lsOP/o7/PW Date of issue: 10 / 04 / 2o2o Controlled copy Page no:7 ABefore samples can be safely handled on the bench (i.e. out of containment), they must first be inactivated by a validated method (refer to pathogen/task- specific guidance). J.J.J RNA extraction is perfbrmed by rnanual means (refer to specific SOPs and manufacturer instructions for guidance). 3.4 PPE 3.4.1 Laboratory staff should wear the following PPE following inactivation & extraction of the samples: Lab gown, gloves, face mask (due to general environment) and ded icated laboratory shoes. AP.op", attention to the use of required/specified PPE is critical to mitigate the risk of accidental exposure to pathogenic material and chemical hazards. 4.0 PROCEDURB For RNA Extraction Perform all pipetting steps using positive displacement or aerosol resistant pipette tips. 4.1 Advance preparation of Reagents 4.1 . I Disolve PK/carrier RNA: Add 2.5m1 protease dissolve Buffer to the pk/ carrier RNA bottle, mix gently and store at -20oC after dissolution. 4.1.2Preparation of internal control(lC): Dissolve T2pldissolve buffer into lyophilised IC (both reagents present in wondfb SARS-CoV-2 RT-PCR kit) and vofiex for l0 sec and brielly centrifuge. 4.1.3 Preparation of Lysis buffer+ carrier RNA(PK) mix : For each sample and positive control and negative control prepare lysis buffer+ carrier RNA mix as follows 400 plBuffer VLF/ sample + lOplcarrier RNA (PKy sample Mix thoroughly by inverting several times. 4.2 Wondfo RNA extraction procedure: 4.2.1 Lysis and virus inactivation: Authorised by: Dr. Runa Jha ed by: .:::::l ;:r-"N Director and Quality manager q stha f\l^e tncha( lity and training unit National Public Health Laboratory Version No. 1 COVID.19 SOP Copy no:1 NPHL/ c1elsoP /o7 lPw Date of issue:10 /04/2020 Controlled copy Page no:8 Pre- For every PCR extraction lot, extract positive control and extraction negative control in parallel with sample extraction. Required volume of each sample, positive control and negative control is 195 pl. Take aliquots of pre-prepared carrier RNA(PK) from refrigerator. Allow it to thaw at room temperature(RT). Transfer from aliquotting cabinet the aliquotted patient samples for PCR I witt i, 2 Add 5 pl Internal control (IC) to each patient sample, I niosatety positive and negative control. Make final volurne 200 pl. I Cabinet cu" rr J Add 410p1Of Buffer VLF and PK/carier RNA mixture to I 200 pl sample /PCAIC (l95sample+spl IC). Do vertex for l5 seconds Briefly perfonn a centrifugation to remove drops from inside of lid. Incubate 5 rnin at room temperature (15-25"C) ^AcnrrrceL srAGE FoR sAFE INACTTvATToN oF SAMPt,E "A ENsunE BUFFER USED IS AVL AND TIMINGS ARE ADHERED TO RNA purification using Hipure Viral Mini column in RNA extraction area of main lab: ln Label Hipure Viral Mini columnspin column caps with sample RNA tracking number. extrac Transfer 610 pl of the sample fiom step 3 of 4.2.1 to the tion column. area 2 Centrifuge at for I min -10,000xg ? Prepared by: Smriti Shresth Authorised by: Dr, Runa Jha M ed by: t!. Reviewed by: Bimlesh Kr Jha Director and Quality manager M r5. lnr and training unit ^)/a National Public Health Laboratory Version No. 1 covrD-19 soP Copy no:1" \."--.]. NPHL/ C19ISOP /O7 /PW Date of issue: 10 / 04/ 2o2o Controlled copy Page no:9 Jf Discard the filtrate and place the column back into the collection tube Add 500p1 Iluffer CW to the column. Close the cap. 4 Centrifuge at 10,000xg for I min. ? 5 Discard the filtrate and place the column back into the collection tube. Add another 500p1 Buffer CW to the column. Close cap. 6 Centrifuge at 10,000x g for I min. ? 7 Discard the filtrate and place the column back into the collection tube. 8 Centrifuge at 13000 x g for 3 minutes to dry the column .+r { l0 Put the spin column in clean labelled 1.5 ml micro centrifuge tube. Add 50pl Buffer AVE to the center of the membrane of the column. ll Centrifuge at 13000 x g for I min. Store eluate at 4 oC until PCR analysis (same *h day). I t2 Proceed to PCR l3 Eluted RNA can be stored for long periods or up to I year at - 70oC or lower. Prepared by: Smriti Shresth\ .1) Authorised by: Dr. Runa Jha by: Reviewed by: Bimlesh Xr.Jf'r$$ Director and Quality manager n^a- ffi and training unit National Public Health Laboratory Version No. 1 COVID.19 SOP Copy no:1 NPHL/ C19ISOP/Ot/PW Date of issue:10 /O4/2.O20 Controlled copy Page no:10 4.2Advance preparation of the Master Mix The kit is provided with lyophilised RT-PCR reagent. Take out the kit from the refrigerator, quick spin to ensure that the lyophilised assay is at the bottom ofthe tube. 4.3 Addition of I'emplate to the Master Mix . Label each tube in the strip with patient tracking number. . Uncap the 8- tube strip (lyophilised RT-PCR reagent), . Add 25 plextracted nucleic acid of specimens, positive controland negative control into the lyophilised RT-PCR reagent. . Cap the tubes, then briefly centrifuge to collect' 5.0 RUNNING SAMPLES ON BIORAD 5.1 See the Instrument operator's manual for details. Ensure software is most current version = Staft programming before preparing the solutions 5.2 Steps: . Switch "ON" button present in the right lower corner of the Biorad thennocycler. o Switch on the laptop connected with the thermocycler. e Double click Bio-Rad CFX Maestro file present on the desktop o Click on File icon . Open protocol on the file- go to validation- go to wondfo PCR and click open . Check the cycling condition ( refer to step 5.4 of this SOP) o Click OK . Click Plate setup icon. Select the required plate tbr the sample o Go to create new o Click Select fluorophores and click FAM, VIC, CY5 (refer to step 5.3 of this SOP) . Click sample type as unknown and PC for positive control and NC for negative control o Click OK . Save change menu appears on the screen, click Yes-enter date and filename . open the lid of the Bio-Rad by pressing front bottom of the lid. . load sample on the smaller hole of the sample tray according to the plate setup . close the lid o Start the run Prepared by: Smriti Sh Authorised by: Dr. Runa Jha Ma by: I stha Reviewed by: Bimlesh Kr J Director and Qual Mrs. i\Utr lncha ality and training unit National Public Health Laboratory Version No. 1 covrD-19 SoP Copy no:1 b-"d NPHL/ C1glsoP /07lPW Date of issue:10 /O4/2o2.0 Controlled copy Page no:11 The protocol consists offour program steps: l: Reverse Transcription of the viral RNA (Stage l) 2: Denaturation: sample denaturation and enzyme activation (Stage 2) 3: Cycling: PCR-amplification (Stage 3) 4: Cooling: cooling the instrument(Not-shown below) 5.3 On the biorad realtime thermocycler, set Reporter dye: FAM-N gene , VIC- ORF I ab and CY5-IC Quencher dye: None Passive ref-erence dye: None Sample volume (25 pl) 5.4 Cycling Conditions Step Temp Time Cycles RT incubation (Hold 500c l0 min I l) Enzyme activation g50c 2 min I (Hold 2) Denaturation g50c 5s 40 cycle Annealing and 600c 32s Extensiott 6.0 TECHNICAL/SCIENTIFTCVALIDATIONANDRESULT INTERPRETATION 6.1 Technical and scientific validation of each RT-PCR assay run is performed according to thc fbll pr"par"a Uv' smriti Shrest]r)' Authorised by: Dr, Runa Jha MaintAinr by: - :. Reviewed by: Bimlesh *r rn.\$ Director and Quality manager ,r^ U\\. N,{ fnaf'arS\ lity and training unit National Public Health Laboratory Version No. 1- COVID-19 SOP Copy no:1 \-7 NPHL/ C1slSOPl0TlPw Date of issue:10 lO4l2O20 !, _;:- ," . . Controlled copy Page no:72 Result Detector Ct Result interpretation FAM VIC CY5 Positive <38 <_38 Not required SARS-CoV2 nucleic acid detected Negative >38 >38 >33 SARS-CoV2 nucleic acid not detected Result need to >38 <- 38 Not required Invalid result >38 >38 >33 Need to be re tested 6.2 RT-PCR assay is considered valid if the positive control produces a positive signal in the FAM channel for N gene and VIC channel for ORF lab gene in duplicate samples with expected Ct values within Cf38 between duplicates. and no amplification signal is present in any of the negative controls. 6.3 RT-PCR assay run is considered invalid if; o An amplification signal (Ct>38.00) is seen in any of the negatives. . The absence of an amplification signal (Ct> 38.00 : undetectable) in the positive control. o No amplification signal (CtZ 38: undetectable) in one duplicate o1'the positive control. Prepared by: Smriti Authorised by: Dr. Runa Jha Mafrrined bv: Reviewed by: Bimlesh Kr Jh Director and Quality manager MrsILltee Shrestha ('ul- r".r',r$g/rrity and trainins unit National Public l'lealth Laboratory Version No. 1 COVID.19 SOP Copy no:1 NPHL/ c1slSOP loT lPw Date of issue: 10 /04/2020 Controlled copy Page no:13 o The absence of amplification signal (Ct> 33.00 : undetectable) fbr the Internal Control (lC ) invalidates only THAT sample (not the batch)' Note: If deemed invalid, the run should be repeated from extraction and reported (NPHL/COVID-1 9/SOP/002). 6.4 A sarnple is considered NEGATIVE for target when no test signal is detected in either of the duplicate reactions but the internal control should have an amplification signal. . lf a sample is negative for all targets but the internal control reactions for the same sample are also negative, the sarnpte must be re-extracted as the extraction has failed or the PCR has inhibited. o lf a sample has a weak amplification signal in one or both duplicates for a specific target, or no amplification signal for a target in one dtrplicate, the sample is re-extracled and may be eluted in25p,l following discussion with Senior Microbiologist, in order to concentrate the sample to enable/confirm detection. 6.5 Technical and scientific validation of test results is performed by following the tables below showing examples of typical results that may be generated by the use of this assay. Result Detector Ct Result interpretation FAM VIC CY5 Positive Negative >38 >38 >33 SARS-CoV2 nucleic acid not detected Result need to 38 Not required Need to be re- be re tested tested, if the result is still positive in re- test. it indicates SARS-CoV-2 nucleic acid Prepared by: Smriti Shresth Authorised by: Dr. Runa Jha MaintaihAd by: Reviewed by: Bimlesh Kr Jha Director and Oualitv manaRer ,rr. r,,"S f)Al- fnat,rrC",{ and training unil National Public Health Laboratory Vcrsion No. 1 COVID-19 SOP Copy no:.1. NPrlL/ c1elsoP/o7/Pw tlal e of issue::t0 /OAD.O7.O Controlled copy Page no:14 detected >38 Invalid result >38 >38 >55 Need to be re tested Quality control 'fhe criteria for the valid test are as follows CT value control Quality FAM vtc CY5 Positive control Negative control >38 >38 7.0 REPORTING OF RESULTS All results are to be reported via previously established NPHL reporting lines. 8.0 RESPONSIBILITIES All trained staff or new staff undergoing training must adhere to this SOP Prepared by: Smriti Shrestha |,$1u Authorised by: Dr. Runa Jha ed by: Reviewed by: Bimlesh *r rnr|$ Director and Quality manager n^o h,h alitv and training unil. National Public llealth Laboratory Version No. 1 COVID-19 SOP Copy no:1 \--':. NPHL/ c1elsoP/o7/Pw Date of issue: 10 / 04 / 2020 Controlled copy Page no:15 Annex l: PCR run Start programming befbre preparing the solutions flowchart Switch on biorad thermocycter and also laptop connected rvith it' Make sure tltat your thermocycler is under remote control i e' your laptop Double click Bio-Rad CFX \'Iaestro file present on the laptop Click on File icon Open protocol on the lile- go to validation- go to wondtb PCR and click open Check the cycling condition( ref'er to step 5.4 ofthis SOP) and then click OK Click Plale setup icon. Select the required number olwells lbr the sarrple Click Selecr tluorophores and click FAM. vlc, cY5 (ref'er to step 5.3 of this sol'j) Click sample type as unknown and PC for positive control and NC tbr negative control Click OK Click on File icon Save change tnenu appears on the screen, click Yes-enter datc and tllenanre Prenare samnle after this nroctss Open the lid ofthe Bio-Rad bv pressing front botton oithe lid or fiom the laptop Load PCR tubes containing sample on the selected u'ells according to thc platc setup Close the lid and start the run Prepared by: Smriti Shrestha Authorised by: Dr. Runa Jha Maintained Reviewed by: Bimlesh Kr Director and Quality manager Mrs. Lilee Alg lncharge, training unit National Public Health Laboratory rsion No. 1 covtD-1950P NpHL/ C19lSOP/08/Ps ate of issue:15/04/2020 Controlled copy ) RT-PCR detection of COVID-19 -SARS- CoV-2 Bv See gene Korea kit Standard Operating procedure of National Public Health Laboratory Tripura Marg, Kathmandu NcPal al by: uthorised bY: Dr. Runa J ha parcd bY: Krishna Pd Ja ishi rs. Lil irector and Quality manager ewed bY: lYoti AcharYa and training unit N,a Nationat ion No. I CO vrD 19SOP NPtlL/ C19|SOP/OA/PS le of iss?e:75/04/202a Controiled copt aEe oo:2 I,O SUMMAR' I l'his SoP part is of a suit of Sops that are set up to arow processing of C,VID-r9 sampres in National Public Health the Laboratory (NPul-). Staffshould aware te ofassociated Risk Assessments and have received sufficient training to be able to,i"rnon.t.ut. belbre pertbrrning "orpetency this task alone Ih s So J) dcta s proc ed u lc s fbr R 'I C R d e1ect oIt of th 2 0 9- l1 ( C0 Co I) 9 ) fu S LI s ll h c PC R 1l LI 0resc ctlce prob l1 b Secgell q U v lbr al tat e detecti ()l'l o th C E ctl c, R f d R P n e & N C n es oI t1 ovc C oronav ru s by LI s l1 th C Biorad, ABI, R0 org t) c p la fonl') 2.0 SAFE,TY All sentences written in red bold text and denoted with rft, indicate a Safety Critical step or comment and as such extra attention must be given when undertaking them. All staff should be t'amiliar with Risk Assessmenrs and have undertaken specific training 3.0 BACKGROUND 3.1 Purposes/ Intended use This SOP is adapted from the protocol of AllplexrM Seegene 2019-nCoV Assay and used at NpHL for detection ofthe E gene, RdRP gene & N genes for novel coronavirus (SARS-cov-2). The Allplex 2019-nCoV Assay is an in vitro diagnostic (lVD) real+ime reverse transcriptase polymerase chain reaction (RT-PCR) test intended for the qualitative detection of nucleic acid from severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) in human nasopharyngeal swab, oropharyngeal swab, anterior nasal swab, mid-turbinate and sputum specimens from individuals with signs and symptoms of infection who are suspected ofCOVID-19 by their health care provider. 3.2 Special Note Coronaviruses (CoV) are positive-stranded RNA viruses f-rom the Coronaviridae family. The four common human pathogen strains 2298 and NL63 from the Alpha group, and OC43 and HKUI fromthe Beta group cause usually only a common cold, but the 2003 SARS-CoV pandemic with more than 800 fatal cases and the current MERS-CoV pandemic originating from Arabia made this virus family well worldwide known. The Wuhan CoV 2019 was described at the end of December 2019 atter some of visitors to a seafood market in Wuhan developed severe pneumonia. The genome sequence was published Jan llth (Genbank acc. MN908947) and shows a high sirnilarity to the SARS v irus. 3.3 Description A 76 bp long fragment from a conserved region in the E gene is detected with FAM labeled hydrolysis probes (530 channel). This assay will detect SARS and Wuhan 2019 CoV pneumonia virus as well as other bat-associated SARS-related viruses (Sarbecovirus). A 100 bp long fiagmenl tiom a conserved region of the RNA-dependent RNA polymerase (RdRP) gene is detected with a Wuhan-specitic probcs' No degencratc broad-range Sarbeco-virus probe contained (without probe Pl tiom thc WllO protocol). This assay will detect Wuhan 2019 SARS-like CoV pneumonia virus b not all other SARS-like viruses. No cross reactivity with common human respiratory CoV NL63,229 I IKU. OC43 or MERS pared by: Krishna Pd Jaishi thorised by: Dr. Runa lha aintained and Qualt\ managel edblr l1o\r Acharla ncharge, Qu dD-Y \'J, Nat,onal Public Health taboratory No. 1 Ienion covrD'19 50P no:1 NPrlL/ C19/SOP/08/PS e of issuo:15/04/2020 no,r face C{1rb I a I 15,16r-15.46i Ral, 4.0 CROSSREFERENCES NPHL/COVID- l 9/RA/001 - General COVID- l 9 Laboratory RA NPHL/GEN/SOP/0 I /lR-lncident Reporting NPHI-/COVID- I g/FORM/001 - Sample tracking form N Pl lLlCOVI D- I 9/l-ORM/002- lncident Reporting Form 5.0 TRAINING All staffshould have undertaken specific training before carrying out this procedure. 6.0 REQUIR-EMENTS: 6.1 Reagents supplied in the kit include: . 2019-nCoV MOM o RNase free water . 5X Real time One step buffer r Real Time one step enzyme o Positive control & Negative Control o lnternal control All are rcady to use 6.2 Notc: Kit Stability: Expiry date is 8 months from the date of manufacture at < -20'C. Please refer to product label for final expiry date. T'his product can be used for 5 days after opening the vials (ifstored at 2-8'C. 'lhis product can be used tbr a maximum of seven repeats of ireezing and thawing. 6.3 Equipment & Materials required but not provided: Water (DNase- and RNase-fiee) pared by: Krashna Pd Jaishi ,w uthorised by: Dr. Runa Jha a ewed by: JyotiAcharya rector and Quality manager rs. Lilee frrv, ncharge, Qu and training !nit National Public Health Laboratory rsion No. 1 n-a covtD 19 soP no:1 i:- - 1.i NPHr/ C19lSOp/08/PS ate of is'ue:1s /o4 / 2o2o . Nucleic acid extraction or purilication reagents. . Extracted RNA from samples (inc Extraction control - EVA) . Disposable, powderless nitrile gloves and N95 mask . 1.5 ml centrifuge tube & 0.5 ml centrifuge tube o Voftex mixer o Adjustabie pipettes & pipette tips with filters (sterile and disposable) o 'Iable mode high speed centrifuge with rotor fbr 1.5 ml reaction tube . Real time PCR amplification apparatus . Specific reaction tube for PCR amplification apparatus (0.2m1 light reaction tube & glass capillary (0.2 ml 8 tube strip with out caps & Optical flatScapstrips) . PPE set o Microcentrifuge . PCR Hood . Vortex . Thermal Cycler (ABI TaqMan 7500 Fast, Biorad, Rotorgene) o Plate holder . lce bucket and ice/ice cooler . Tubes for Master Mix r Viral transport media with specimen 6.4 Spccimens: . Nasopharyngeal swab, . Oropharyngeal swab, o Anterior nasal swab- o Mid-turbinate and sputum specimens . Bronchoalveolar lavage (BAL) 6.5 Specimen handling & storage: . Specimens can be stored at 4 oC for up to 72 hours after collection. Ifany delay in extraction is expected, store specimens at -70oC or lower. . Extracted nucleic acids should be stored at -20'C or lower. 7.0 PROCEDURE 7.1 For Aliquoting & Nucleic Acid Extraction: Refbr to aliquot & Extraction SOP No ...... (Annex ll). NOTE: 10 pL of RP-V IC must be addcd to each specimen before nucleic acid extraction. 7.2 Preparation for Rcal timc PCR: Prepared by: Krishna Pd Jaishi Fo'YM thorised by: Dr. Runa Jha Iwaintai\ by Reviewed by: lyoti Acharya It Director and Quality mana8er lMrs Lrl4 h ha lity and training unit W*t wrt. Itncharee, Natio6al Public Health taboratory covrD 19 soP NPHr/ C19lSOP/08/PS ate of is5ue.15/04/2020 Controll€d copy age no:5 Note: Centrifuge all reagents stored al -20 oC after thawing them completely. Positive control (Green cap: PC 20l9nCoV) is ready for use and should be processed as fbr sample. Prepare following reagents in a labelled sterile 1.5 ml eppendorf tube. Set up all the reagenls on ice/ice cooler. (Refers to Annex I & Il) S.N Reagent For one reaction For n reaction (in pL) (in pL) I 201 9-nCoV MOM 5 5*n e.g. for 2 reactions 5+2=10 ttl 1 RNase free water 5 5tn ., 5x Real timc One step 5 5*n Bufler I -l Real time one step 2 2*n enzyme i To the 1.5 ml eppendorf tube, add the components according to the total number of tests. Mix by inverting the tube 5-6 times or quickly vortex and centrifuge briefly. ! Aliquot lTpL ofthe prepared RT PCR mastermix into PCR reaction tubes placed on the ice cooler. > Add 8 pL of NC (RNase free water) in A0l well. then each sample nucleic acid and PC. into the last reaction tube containing the prepared master mix. i Close the cap and briefly centrifuge the tube. Verify that the liquid containing all PCR components is at the bottom of each reaction tube, if not centrifuge again at a higher rpm and for a longer time. i Check for any air bubbles! Ifseen, eliminate by flicking and re-spin. ), Immediately initiate the PCR (within 45 minutes). 7.3 PCR RUN 7.3.1 Protocol set up i Set up the protocol on the Real time PCR instrument used (refer to the rnanual of respective PCR instrument) with the 45 cyclcs and following temperature and time. Stcp Number of cycles ''l-emperature Duration I I 50'c 20 min 2 I 95"C l5 min ) 45 94.C 15 sec 4 58'C 30 scc 5 GO TO Step 3, 44 more times > Check the sample volunre and set as 25 microliter. 7.3.2 Plate setup: l Set the plate by clicking create new from open plate editor window and select the following fluorophores: l-luorophore an alyte FAM E gene (Sarbecovirus E gene) Prepared by: Krishna Pd Jai5hi rised by: Dr. Runa jha ainta ed by eviewed by:.lyoti Acharya n,0) irector and Quality manager rs. Lil 5h ncha ity and training unit I Nataonal Public Health Laboratory [""' covrD-19 50P no:1 NPHL/ C19lSOP/08/PS ol issu.:ls1O4/2O2O controlled copy no:5 face tix Internal Control (lC) Cal Red 6l 0 COVID-19 RdRp gcne Quasar 670 COVID-19 N gene Click on the appropriate checkboxes (FAM, HEX, Cal Red 610 and Quasar 670) to specifu the fluorophores to be detected in the selected wells. Type in Sample Name and press enter key. Select the analyes in this ordcr: NC. samples. PC and click next to start run Click OK to save the new plate. Save with the unique name for that run with date, lot number and initials ofthe user. You will be returned to the Experiment Setup window. 7.3.1 Start Run o From Start Run tab in Experiment Setup, click Open Lid to add the reaction tubes according lo palte setup. Then click "Close Lid" to close the instrument lid. o Click "Start Run". . Store the run tlle in a designated folder. Input the file name, click SAVI:]. and the run will start. 7.3.4 Analysis ofdata and interpretation: (see annex [) A. Pre-setting for Data Analysis . Create one folder to save amplification curve detection results. Folder name may be as desired by the user. . After the test. Click one Fluorophore only, then select "Settings" tab, select Baseline threshold; change the threshold from auto-calculated to user defined (in the log phase of the amplification curve, usually 100). Click OK. . Now Click on one fluorophore and check the values whether valid or not according to QC criteria e.g. for IIEX it has to be <40 to be valid. Repeat the same for all fluorophores selected. Ct value Result Remark <.10 Detected IfCt value of IC is >40. conduct a > 40 or NiA Not detected re-tesl for that particular specimen afier re-extraction. i lnvalid lC only invalidates the particular specimen and not the whole run. i For data analysis please refer to manual ofPCR instrument and annex Il 8.0 Quality Assurance 8.1 Specificity: Cross reactivity of Allplex 2019 nCoV assay was tested using 49 standard materials and organism shown as not detected (in annex ll) 8.2 Sensitivity: It has a detection limit of 100 RNA copies/reaction. 8.3 Special Note: e For in vitro diagnostic use only. o Reliability ofthe results depends on adequate specimen collection, storage, transport, and processing proccdule pared by: Krishna Pd laishi thorised by: Dr. Runa Jha aintaine eviewed by:.lyoti Acharya 'w irector and Quality manager rs. Lilee S ncharge, (lu and training unit National Publl. Health Laboratory No.l l"""" covrD 19soP NPHr/ C19lSOP/08/PS ol ls:]')e'.751A4/? AZA Contro ied.opy t\o--7 r' . This test has been validated for the lbllowing specimen types: sputum, nasopharyngeal aspirate. throat & nasopharyngeal swab, and bronchoalveolar lavage. This test has not been validated for any olher types of spec imens. o Store RNA samples at 5 -20'C until use and keep on ice during use. o Sensitivity ofthe assay may decrease if samples are repeatedly frozen/thawed or stored for a longer period of time. . Workflow in the laboratory should proceed in a unidirectional manner. Use separated and segregated r.lorking areas lor each cxperimenl. . Wear disposable gloves and change them before entering different areas. Change gloves immediately if contaminated or treat them with DNA decontaminating reagent. .Supplies and equipments must be dedicated to working areas and should not be moved from one area to another. rAvoid contamination ofreagents when removing aliquots from reagent tubes. Use ofsterilized aerosol resistant disposable pipette tips is recommended. o Do not pool reagents from different lots or from different tubes ofthe same lot. . Do not use the product after its expiry date. . Do not reuse all disposable items. . Use screw-capped tubes and prevent any potential splashing or cross-contamination of specimens during preparation. . Please be careful not to contaminate reagents with extracted nucleic acids, PCR products, and positive control. To prevent contamination ofreagents, use of filter-tips is recommended. .To avoid contamination of working areas with amplified products, open PCR reaction tubes or strips only at designaled working areas after amplification. . Store positive materials separated tiom the kit's reagents. o Laboratory safety procedures (refer to Biosafety in Microbiological and Biomedical Laboratories & CLSI Documents) must be taken when handling specimens. Thoroughly clean and disinfect all work surfaces with 0.5% sodium hypochlorite (in de-ionized or distilled water). Product components (product residuals, packaging) can be considered as laboratory waste. Dispose of unused reagents and waste in accordance with applicable federal, state, and local regulations. . Expiry date is 8 months from the date of manufacture at < -20"C. Please refer to label for final expiry date. . This kit is a qualitative in vitro test for the single or multiple detection of3 types ofgene (E gene, RdRP gene, and N gene). repared by: Krishna Pd Jaishi Authorised by: Dr. Runa Jha aintain eviewed by: Jyoti Acharya Director and Quality manager rs. Lilee ftJroz ncharge, and training unit National Publc Health Laboratory covtD-19 soP NPHL/ C19lsOP/08/Ps te of issue:15/04l7020 Conlrolled copy ge no:8 9.0 Troubleshootin OBS[RVATION PROBABLE CAUSES SOLUTION The fluorophores lor data Select the correct fluorophores for data analysis and analysis do not colnply expoft the data again. There is no necd to rcpcat the test with the protocol in this case. Incorrect setting of real- PIease check the themal cycling corrditions and repeat time thermal cycler the test under the colTect settings. Please check the storage conditions and the expiry date Incorrect storage or past (refer to label) ofthe test kit and use a No signal expiry date ofthe test kit new kit if necessary. No signal including IC may indicate loss of nucleic acid during extraction. Make sure that you Lrsc N ucleic acid extr action recommended extraction method. lfdue to irrhibitors. re- f'ailure cxtract the original specimen or dilute the spccirrcn (l/3-ll10) in sterile saline solution and repeat the test from extraction step. No lntemal lftarget pathogen signal is observed but not [C, then IC Control signal amplification may have been inhibited by high titcr of High load of pathogen's largel. pathogen. In order to confinn nucleic acid IC signal, dilute the specimen ( l/3-l /10) in sterile saline solution and repeat the test from cxtraction step. Presence of RT-PCR Please dilute the specimen ( I /3- l/ l0) in saline solution inhibitor and repeat the test from extraction step. Putative false Decontaminate all surfaces and instruments with sodium positive or hypochlorite and ethanol. Only use filter tips throughout target signals Contamination the procedure and change tips between tubes. Repeat the observed in entire procedure from nucleic acid extraction with a new Negativc Control set of reagents. Please check the specimen colleclion method. and re- Error in specimen collect specimen Please re-collect the specimcn and collection repeat the entire procedure. Incorrect storage of the Ensure that the specimen is stored as recornmended. specimen Erroaln spaai'nen Please check the specimen collection method. collection Putative false Error in nucleic acid Please check the nucleic acid extraction procedure as well extraction negative or no as nucleic acid concentration, and re-extract nucleic acid. signal observed in Error in addins nuc leic Check the sample numbers oftubes containing nucleic Positive Control acid to corresn"ondinc acid and make sure to add nucleic acid in to the correct PCR tubes PCR tubes and carefully repeat the test if'necessary Please dilute the specimen (l/3-l/10) in saline solution Presence of inhibitor and repcat the test from extractio! steP. Confirm that all components are added Io the RI'-PCR mixture (Sensitivity is comprorrised rl,ith pre-cornposed lncorrcct PCR rr ixture premix). All reagents must be homogenized and spun down before use. N Prepared by: Krishna Pd Jaishi 'g,'v* uthorised by: Dr. Runa Jha aintain by: eviewed by: lyoti Acharya irector and Quality manager rs. Lile a N-e-/ ncharge, and training unit National Public liealth Laboratory r"- No.1 covlD-19 50P topy no:1 N PH L/ C19lSOP/08/PS Ft";ffiCh4ror-o [+"*s Spikes in any Centrilirgc thc PCR tube bctbre run. cycles o{' Bubble in the PCR tube amplification curve 10.0 Reference: I . Allplex"r'' 2019-nCoV Assay kit instrucrion 2. World Health Organization (Wt'lO). Coronavirus. Geneva: WHO; 2020 [Accessed 2l Jan 2020]. Available from: https://www. who.int/health-topics/coronavirus. 3. Zhang Y-2. Novel 2019 coronavirus genome. Virological.[Accessed 2l Jan 2020). Available from: http://virolosical. org/t/novel-20 I 9-coronavirus-genome/3 I 9 pared by: Krishna Pd.,aishi c.r{P by: Dr. Runa lha aintained ewed by:.lyoti Acharya rector and Qualitv man(ilrta l.ilee Sh , arge, Qua nd training Lrnit I N-tional P!b ic l'lea th Laboratory t" covrD-19 sop NPHL/ C19l5OP/08/PS ate of issue:15/04/2020 Contro led copy age no:10 ANNEX I PCR LOG SHEET: Government ofNepal Ministry of Health and Populalion Deparlment of Health Sen ice: National Public Health Laboratory, Teku BIORAD CFX96 Real-Time PCR rvork shcet In ilial Date [.xperimcnl No._ Signature_ Master Mix Protocol r2 l9-nCoV Se ene - Korean Components Per rxn Total clin Conditions: Positive: FAM, HEX, Cal pl amount ld I 50'C for 20 mins 10, Quasar 670: <40 2019-nCov MoM 5 old I 95'C for 15 mins ,15 RNAsc Free 5 Cvcle - egative: FAM, Cal Red 610, Water 94'C for 15 sec uasar 670: <40 5x Real time one 5 58"C for 30 sec > ,10 step buffcr 1cs1: HllX (lC) or N/A Real time one 2 porter l)yes: step enzyme AM: E gene nconclusive resull: (IC) 610, Total mater mix 17 EX: Internal Control f FAM, Cal Red Quasar al Red 610: RdRp gene 70 all three are not <.10 at a Template 8 uasar 670: N c me the result is considered otal volume : 5 UN PC value(For dll: <40 nconclusive and must be UN NC Value(For all: >40 rcpeated. nvalid if >,10 rir N/A RUN TIME: F'rom: 'l'o: I 2 J 4 5 6 7 8 9 t0 1l l2 A B C D E, F G II REMARKS: repared by: Krishna Pd Jaishi uthorised byi Dr. Runa lha ar ed by Reviewed by: lyoti Acharya rrector and Quality managet rs. [i stha ^ / l\/v/ ncha lity and training u nit National Public Health Laboratory No. I fersion covto 19 soP NPHr/ C19lSOP/08/PS of $sue:15l04l)0O Controlled copy rr "o faee NIC Cal FAM I{EX Quas NIC Cal FA rIEX Quas NIC Cal FAM II IX Quas No Red ar No Red M ar No Red tr 610 670 610 670 610 670 -T-l by: Krishna Pd laishi by: Or. Runa Jha by:lyotiAcharya and Quality manager Lilee f\u, training unit National Public Health Laboratory covtD-19 soP Copy no:1 NPHr/ C19lSoP/09/pSY Dal,e ol issue: 25/O4/2020 Controlled copy Page no:1 RT-PCR detection of COVID-19 -SARS- COV2 (PCR- F'luorescence obin Ily Da An Gene Co,. Ltd. of Sun Yat-sen UniversitvRT- PCR O]IORAD) Standard Operating procedure of National Public Health Laboratory Tripura Marg, Kathmandu Nepal Prepared by: Krishna Pd Jaishi g".q Authorised by: Dr. Runa Jha Mai ned by: Reviewed by:.Jyoti Acharya Director and Quality manager Mrs. ee Shrestha .0,|'Y ft.!.)Q lnc Quality and training unit National Public Health taboratory Version No. 1 covrD-19 soP Copy no:1 :.i NPIL/ C19lSOP/O9lPsY Date ol issue: 25/04/2O2O Controlled copy Page no:2 SUMMARY This SOP is part of a suit of SOP5 that are set up to allow processing of COVID-1g samples in the National Public Health Laboratory (NPHL). Staff should be aware of associated Risk Assessments and have received sufficient training to be able to demonstrate competency before performing this task alone. This SOP details procedures for RT-PCR detection of the 2019-nCoV (COVID-1g) virus using the PCR fluorescence probing developed by Sun Yat-sen University for qualitative detection of the oRFlab &N genes of novel coronavirus by using the Biorad platform. SAF ETY All sentences written in red bold text and denoted with A, indicate a safety critical step or comment and as such extra attention must be given when undertaking them. All staff should be familiar with Risk Assessments and have undertaken specific training BACKGROUND Purposc This SOP is adapted from the protocol of Da An Gene Co., Ltd of SunYat-Sen university for Covidl9 and used at NPHL for detection of the @!.1f,\g9 for novel coronavirus (2019- nCoV). Special Note Coronaviruses (CoV) are positive-stranded RNA viruses from the Coronaviridae famill. 'l'hc four common human pathogen strains 229E and NL63 from the Alpha group. and OC43 and llKL.ll lromthe Bcta group usually cause only a common cold but thc 2003 SA ItS- Prepared by: krishna Pd.Jaish '$."', Authorised by: Dr. RLrna Jha "*t' Reviewed by: JyotiAcharya Oirector and Quality manager ,^. { e Shrestha r\rx rncna) ality and training unit National Public Health [aboratory Version No. 1 I covlD-19 soP Copy no:1 NPHL/ C19l5OP/09/PSY Date af issue: 25/o4l2o2o Controlled copy Page no:3 CoVpandemicwith more than 800 fatal cases and the recent MERS-CoV pandernicoriginating lrom Arabia rnade this virus family well known worldwide.'Ihe Wuhan CoV 2019 was described at the end of December 2019 after some visitors of a seafood market in Wuhan developed severe pneumonia. The genorne sequence was published on the ll'h of January (Genbank acc. MN908947) and shows a high similarity to the SARS virus. Description This assay will detect Wuhan 2019 SARSlike CoV pneumonia virus but not all other SARS-Iike viruses. No cross reaclivity with common human respiratory CoV Iike NL63, 229E. HKU, OC43 or MERS. Specification In this assay, SARS-related coronaviruses, including 2019-nCoV are distinguished from other respiratory pathogens including human seasonal coronaviruses by using primers and probes specific for a conserved region of the RdRp gcne. The location ofthe viral genome target region is shown below. - l\ I Prepared by: Krishna Pd Jai'n['-or Authorised by: Dr. Runa Jha Mai by: Reviewed byr Jyoti Acharya Director and quality manager Mrs, hrestha lnch lity and training unit l_ N)P/ National Public Health Laboratory Version No. 1 t-} covtD-19 soP Copy no:1 .':,:. I NPHr/ C19/5OP/09/PSY Date of issue: 25/04/2020 Controlled copy Pa8e no:4 1.0 CROSS REFERf,NCES NPHL/COVID-19/RA,/001 - General COVID-I9 Laboratory RA NPHL/GEN/SOP/O I fl R-lncident Reporting NPIlt./COVtD- l9/FORM/001 - Sample tracking form NPIIL/COVID- l9lFORM/002 - [ncident Reporting Form 2.It TRAINING All staffshould have undertaken specific training before carrying out this procedure 2.0 Requisites and Reagenls 3.1 Reagents Name ofreagent Contenl I Rernark PCR Derection PCR reaction Specific Primers, probes, reagent Package: solution A - NC tris-HCL, KCL, ORF lab.t,l (NHr):SOr, MgCl:. dNTPs (24 tesr/kit, 48 tests/kit and 96 PCR reaction Hot start Taq DNA testsikit ) solution B NC Polymerase, c-MMI-V ORF lab.4,,l enzyme. Rnasin etc. Quality control NC (ORFlab/Irl) Pseudovirus with internal package Negative control control segments NC ( ORFrab/l'{) Pseudovirus with target Positive control segments & internal control segment Prepared by: Krishna Pd Jai 'U"''q' Authorised by: Dr. Runa lha ,",\\"ned by Reviewed by: lyoti Acharya Director and Quality manaSer ,^.,H ha dA..A"T fN ,' fnaf,"r{ ality and training unit National Public Health taboratory covrD-19 soP Copy no:1 .l NPrlL/ C19lSOP/09/pSY Oare of issue: 2S/O412C,.O Controlled copy Pagc no:5 3,2 Equipment & Materials required but not provided: Water (DNase- and RNase-free) Nucleic acid extraction or purification reagents. Extracted RNA from samples (including Extraction control - EVA) Sterile Physiological saline solution Disposable powderless nitrile gloves and mask 1.5 ml centrifuge tube & 0.5 ml centrifuge tube Vortex mixer Table mode high speed centrifuge with rotor for 1.5 ml reaction tube Thermostat-controlled water bath or other thermostatic equipment Real time PCR amplification machine (Biorad/ABI 7500 Fast Thermal Cycler/Roche Lightcycler) Specific reaction tube for PCR amplification (0.2m1 light reaction tube/strips) PPE set Microcentrifuge PCR Hood Gilson Pipetteman (or equivalentAdjustable micropipettes) Filtered pipette tips (sterile and disposable) 96 well ABI TaqManMicroAmp FAST plate ABI plate cover and sealing tool Plate holder Ice bucket and ice Tubes for Master Mix Viral transport media with or without added trizol Prepared by: Krishna Pd laisha Authorised by: Dr. Runa Jha lV alnta in by: Pe'rw Reviewed by: .Jyoti Acha rya oirector and Quality mana8er Mrs.l-ilee H lncharge, and training unit National Public Health Laboratory Ver5ion No. 1 covtD 19 50P Copy no 1 NPHL/ C19lSoP/09/PSY Date of issue: 25/04/2020 Controlled copy PaBe noi6 3.3Spccimen type, collection, pre-treatment, transpoft & storage: 3.3.1 Tvoe of samples & collection: Respiratory clinical samples e.g. upper respiratory samples such nose/throat swabs, and nasopharyngeal aspirates, also bronchoalveolar lavage (BAL), and sputum. Additionally, tissue culture fluid from virus isolates may be tested. Other sample types may less commonly be received, such as serum, faeces and urine. 3.3.2 Specimen transport: In order to ensure high quality,specimens should be transported at controlled temperalure to the laboratory as soon as possible. Ice, dry ice or liquid nitrogen is neededfor transportation to the Iaboratory. Note: Transportation of specimens must comply with country and local regulations for the transport of etiologic agent. 3.3.3 Pre{reatment of specimens: Virus inactivation must tre carried out in a Class II Biological Safety Cabinet{by adding trizol (guanidinium thioc)'anate and acid phenol) or using heat inactivation ) Pre-treatment of sputum: Take the sputum specimen and add sterile physiological saline four times the volume of the specinen. Shake gently and l&e ovemight at 4"C for complete liquefaction. Mix properly with a pipette and process as for throat swab received in VTM. ABefore samples can be safely handled on the bench (i.e. out of conlainment), they must first be inactivatcd by a validated method (refer to pathogcn/task-specific guidance). 3.3.4 RNA extraction can be performed by either manual or automated means (refer to specific SOPs and manufacturer's instructions for guidance). It is recommended to take 200 pl of liquid specimen for nucleic acid extraction. Both negative and positive controls provided in the kit need to be extracted as for specimens. Prepared by: Krishna PdJtjshi g) Authorised by: Dr. Runa Jha d bv: Reviewed by: lyot; Acharya Director and Quality manager :","il[ ha tur-2 lncharge, lity and training u nit National Public llealth Laboratory covtD,19 soP Copy no:1 NPHU C19lSOP/09/PSY Date of issLre: 25104/2020 Controlled copy 3.4 I't> t,l 3.4.1 Laboratory staffshould wear the follorving PPE following inactivation & exlraction of the samples: l,ab gown, gloves. face mask (due to general environment) and dedicated laboratory shoes. A.Proper attention to the use of required/specified PPE is critical to mitigate the risk of accidental €xposure to pathogenic material and chemical hazards. 3.0 TESTPROCEDURE: Perform all pipetting steps using positive displacement or aerosol resistant pipette 1ips. For extraclion please refer to exlraclion SOP ..,.., 4.1 Advance preparation of Reagents 4.1.1 PCR reaction solution A & B are ready to use (Primers & Probes: Solution A (havinq white cap)& Enzvme mixture: Solution B (havinq Blue cap) 4.1.2 Use 5pl of extracted positive control & Negative control for a 20pl PCR reaction (Solution A lTpl+ Solution B 3pl)to prepare the Master mix. 4.2Advance preparation of the Master Mix Note: lo decreose lurn-aroundlime, thi.s can be prepared concurrently k; lhe RNA Exlrdction procedue. 4.2.1 Preparation of lhe Master Mix MUSToccur in a dedicated room that is physically separated fiom the template addition room (and the room storing the positive control) This is to minimize potential contamination. When calculating the total amount of Master mix reaction mixture, negative conlrol. positive control and onc extra per 2 strips should be calculatcd. prepared by: xrishna pd Authorised by: Dr. Runa Jha Maintained by: ",r@*d Reviewed by: lyoti Acharya Director and Quality manager Mrs. Lrlec Shrestha .!u,.$'")' l),P- lncharge, Quality and traini National Public Health Laboratory I I covtD,19 50P Copy no:1 I NPHr/ C19lSOP/09/PSY Date of issue: 25/04/2020 Controlled copy Page no:8 4.2.t Ensure suf'flcient Master Mix is prepared to account for all the samples and required controls (e.g. PC, NTC). 4.2.2 Prepare Master Mix using chilled tubes and reagents as follows'. Solation A l7 ul anl Solution B 3 ul per rcaction giving the total volume ol20 ul, 4.3 Addilion of Template to the Master Mix 4.3.1 The Template (and positive control material) must NEVER enter/be used/sorted in the Master Mix preparation room -- a separate Template addition room rnust be used. 4.3.1 Add the lemplate as follows: To 20pl of Master Mix, add 5pl RNAfrom the extraction (NTC = water, PC = positive control included in the kit). 5.0 RUNNING SAMPLES ON ABI 7500/ BioradCfx96/Rotorgene 5.I See the Instrument operator's manual for delails. Ensure software is the most current version 2.3 (as of l8-Mar-20). Start programming before preparing the solutions. The protocol consists of four program steps: l: Reverse Transcription ofthe viral RNA (Stage l) 2: Denaluration: sample denaturation and enzyme activation (Stage 2) 3: Cycling: PCR-amplification (Stage 3) 4: Coofing: cooling the instrument (Not-shown below) 5.2 On real time thermocycler, set reference dye to -(none)" for each samplc. Add detectors for FAM - N gene and VIC- ORFlab, Cy5 for Internal control. Run as pcr thc protocol & refer to SOP/manual ofthermocycler. (rishna Prepared by: Pd Jaishi &r$ Authorised by: Dr. Runa lha Maintained by: Reviewed by:.lyoti Acharya Director and Quality manager Mrs. Lilee Shrestha nlo lncharge, \VV Quality and trainin National Public Health taboratory Version No. 1 covtD-19 soP Copy noi 1 NPHr/ C19lsoP/o9/P5Y Oate of issuei 25/o4/2O2O Controlled copy Page no:9 Cycling Conditions Stage Repeats 'l'arget Running Time Analytical mode/ acquisition mode ec) I I 50 l5 Mins Nonc I 95 I 5 mins None 3 45 91 l5 secs None 55 45 secs Quantification/ single Note: For programming & procedure setup,refer to detailed information in operation manual of each instrument 5.2 At the end ofthe run, check that the default "set baseline cycle start and end" settings are correct and re-set if required. Set the threshold for ORFlab & N gene by dragging the threshold bar above fluorescence detected with 'NTC" the'No template controls". Select analyse to calculate Cr (cycle threshold) values for samples and positive controls. 6.0 1'I]CIINICAL/SCIENTII-ICVALIDATIONANDRESUI-TINTERPRETATION 6.1 Technical and scientific validation of each ORFlab & N gene RT-PCR assay run is perlbrrned according to the follorving criteria: Prepared by: Krishna Pd Jaish'&,r7 Authorised by: Dr. Runa Jha Maintained by: Reviewed by: lyoti A.harya Oirector and Quality manaSer Mrs. Lilee Shrestha lncharge, Quality and --UuM*/" {uf National Public Health Laboratory Version No. 1 covrD-19 soP Copv no:1 NPHL/ C19/50P/09/PSY oate of issue: 25104/2020 controlled copy Pa8c no:10 6.2 An N gene & ORFlab gene RT-PCR assay run is considered valid if the positive control produces positive signal in the FAM channel. & VIC channel in duplicate samples with expected Ct values that are within 3.2 Ct betrveen duplicates, and no amplification signal is present in any ofthe negative/no template controls. 6.3 An N gene & ORFlab R'I'-PCR assay run is considered valid if; Negative control (NC): No amplification curve in FAM & VIC, amplification curve seen in Cy5. Positive control (PC): Amplification curve must be seen in FAM & VIC with CT value less than or equal to 32.0, amplification curve may be seen or absent in Cy5. An amplification signal (FAM or VIC channel): Ct> 40.0 is seen in any of the Negative for nCoV-2019. Cy5 channel amplification curve has to be scen. An amplification signal (FAM & VIC channel): Not More Than (NM'l') 40.0 is Positive for nCoV-20 19. An amplification of test sample with CT value NMT 40 only in one channel among FAM & VIC, and no amplification in other channel, it is recommended to repeat the test. lf the result of re test is same with original, test is determined as positive for nCoV - 2019. If the result of retest is negative. test is determined as negative flor nCoV-20 I 9. A difference of> 3.2 Ct's befween duplicate wells in the positive control. The absence of arnplification signal (Ct> 40.00 = undetectablc) for the Intcrnal Control (lC - EVA) only invalidates TIIA'f sample (not the batch). Note: If deemed invalid, the run should be repeated from extraction and reported (NPrrL/COVTD- r 9/SOP/002). 6.4 A sample is considered NEGATIVE for target when no test signal is detected in either of the duplicate reactions but the intemal control signal is present. . Ifa sample is negative for all targets but the intemal control reactions for the same sample are also negative, the sample must be re-extracted as the extraction has tailed or the PCR has been inhibited. . lfa sample has a weak amplification signal in one or both duplicates for a specific target, or no amplification signal for a target in one duplicate, the sample is re-extracted Prepared by: Krishna Pd Jaishi 0.''o Authorised by: 0r. Runa lha Maintained byl Reviewed by: Jyoti Acharya Director and Quality manager Mrs. Lilee Shrestha illt)Q .-- lncharge, Quality and lrai nit National Publir Health [aboratory Version No. 1 I covlD,19 soP Copy no:1 NPHL/ C19/SOP/09/PsY Date of issuer 25/04/2020 Controlled copy PaBe noi1I and may be eluted in 25pl following discussion with Senior Microbiologist. in order to concentrate the sample to enable/confirm detection. 7.0 Rf,PORTING OF Rf,SULTS All results are to be reported via previously established NPHL reporting lincs 8.0 RESPONSIBILITIES Alltrained staffor neu staffundergoing training rnust adhere to this SOP 9.0 Technological spccifications: g.lsensitivity: analytical sensitivity ofall instruments is 5.0 X 102 copies/rn I by laboratory evaluation. The diagnostic sensitivity is l00o/o for use with ABI Prism 7500, Light cycler 480 and Biorad CFX96. 9.2 Specificity: the analytical specificity ofall instruments is 100% by laboratory evaluation diagnostic specificity is 100%o for use with ABI Prism 7500, Light cycler 480 and Biorad CFX96. 9.3 Precision: Not more than 5olo 9.4 Stability & storage: PCR fluorescence probes can be stored for 3 days 4'C. Perform acceleration testing at 37'C for 3 days which does not affect the performance ofthe kit. Long term stability under storage conditions of-20 +5'C is still in progress. ,\trlc: PCR omnlilittttion should be ocrlbrmcd x'ilhin 45 ntinules ol oreouritty lhc Mtslermi-r. 10.0 Product use limitations & Troubleshooting: . Polymerase repression may result in false negative result. . Proper specimen collection and transport gives reliable result. . The kit can not be uscd to assess a treatment. . 'fhe product is to be used by- personnel specially trained on PCR technique only . No fluorescent increase signal in reaction tube of Positive control erenared bv: Krishna pd la$o Authorised by: Dr. Runa lha Maintained by Reviewed by: lyoti Acharya Director and Quality manager Mrs. Lilee Shrestha lncharge, Quality and t nt National Public Health taboratory Version No. 1 covrD-19 soP Copy no:1 I NPHt/ C19lSOP/09/PSY Date of issuer 25/04/2020 Controlled copy PaBe nor12 Procedurc setup crror: check according to log sheet in annex I Note: For detailed instructions. Dlease refer to instruction use sheet $'hich is Drovided in kit box. prepared by: Krishna pd laisu+.e Authorised by: Dr. Runa Jha Maintained byl Reviewed by:.Jyoti Acharya Dire.tor and Quality manager Mrs. Lilee Shrestha M/ lncharge, Quality and nB unit National Public Heaith Laboratory Version No. I covtD-19 50P Copy no:1 NPHL/ C1915OP/09/PSY Date of issue: 25/04/2020 controlled copy PaBc no:13 ANNEX-I Govemment ofNepal Ministry of Health and Population Depanment oI Health Services Natinnal Public Health Laboratory, Teku lllORAD ( 1,'\96l,\lll/Rotorgen Real-Time P(lR work shcet lnilirl Datr Elpefiment \o. Sigllature_ r Mix Protocol for 2019-nCoV Sun atsen Unive (lomponrnls volume Totlll Cvclinq Condition: Inlcrprt}lation of tr\t resull: (Fl Amount Hold I 50"C for l5 mins Posilircr IrA\1. \ I( . (')5: I I 7 J ,l ll llr t2 I tl I B I ( D I L tr G I II I I Prepared by: Krishna Pd laisli Authorised by: Dr. Runa lha Maintained by: C.n$/ Reviewed byr Jyoti Acharya Director and Quality manager Mrs. [ilee Shrert lncharge, Quality rain ng unit National Public Health taboratory Version No.1 covlD-19 soP Copy no i1 NPHL/ C19/sOP/09/PSY Dale of issuet 25/04/2020 Controlled copy Page no:14 REMARKS: NIC cv5 1,'Alt vIC Rrmark NIC cv5 Ir.\ ]l vtc Rcmrrli No prepared by: Krishna pd t"tt\ Authorised by: Dr. Runa lha tr,tal"tainea UV, ,., N \\ Reviewed byi Jyoti Acharya Director and Quality manaFer Mrs. filee Shrestha\\ .-!!.s"f rurP'f lncharge, Ouality an\ ng unit