Merlin Biomedical (Xiamen) Co., Ltd. Validation of this test has not been reviewed by FDA. Review under the EUA program is pending.
Novel Coronavirus (COVID-19) RT-PCR Kit Instruction for Use
Product Novel Coronavirus (COVID-19) RT-PCR Kit Cat No. CP01020011
Specification 50T
Intended Use Novel Coronavirus (COVID-19) RT-PCR Kit is a real-time RT-PCR test intended for the SARS-CoV-2 ORF1ab (open reading frame, ORF1ab) and N (Nucleoprotein, N) gene qualitative detection of RNA from the SARS-CoV-2 in nasopharyngeal, and oropharyngeal swab specimens from individuals suspected of COVID-19 by their healthcare provider.
Testing is limited to laboratories certified under the Clinical Laboratory Improvement Amendments of 1988 (CLIA), 42 U.S.C. §263a, to perform high complexity tests, or by similarly qualified non-U.S. laboratories.
Results are for the identification of SARS-CoV-2 RNA. The SARS-CoV-2 RNA is generally detectable in upper respiratory specimens during the acute phase of infection. Positive results are indicative of the presence of SARS-CoV-2 RNA; clinical correlation with patient history and other diagnostic information is necessary to determine patient infection status. Positive results do not rule out bacterial infection or co-infection with other viruses. The agent detected may not be the definite cause of disease. Laboratories within the United States and its territories are required to report all test results to the appropriate public health authorities.
The Novel Coronavirus (COVID-19) RT-PCR Kit is intended for use by qualified and trained clinical laboratory personnel specifically instructed and trained in the techniques of real-time PCR and in vitro diagnostic procedures. Validation of Novel Coronavirus (COVID-19) RT-PCR Kit has not been reviewed by FDA. The test is distributed in accordance with Section IV.C. 2 of the FDA Policy for Coronavirus Disease-2019 Tests During the Public Health Emergency (Revised).
Negative results do not preclude SARS-CoV-2 infection and must not be used as the sole basis for patient management decisions. Negative results must be considered in the context of a patient’s recent exposures, history, presence of clinical signs and symptoms consistent with COVID-19.
The results of this kit can’t be used for clinical diagnosis.
Summary Coronaviruses are a large family of viruses that are enveloped single stranded positive sense RNA viruses which cause illnesses in both human and animals. The illness has ranged from common cold (e.g. respiratory syndrome, fever, cough, etc.) to more severe diseases such as pneumonia and severe acute respiratory syndrome.
Test Principle The kit is based on real-time PCR method and technology of fluorescent probes to detect specific nucleic acid sequences of SARS-CoV-2 ORF1ab (open reading frame, ORF1ab) and N (Nucleoprotein, N) gene from human respiratory tract secretion samples which indicates the infection of the above pathogens.
The PCR reagent of the kit contains UDG enzyme to selectively cleave the uracil glycosidic bond of uracil-containing PCR fragment for effectively reducing false positives caused by PCR product contamination.
Kit Contents The kit contains the following materials. Tube No. Contents Volume (μL/tube) Quantity 1300μL after ① Freeze-dried Buffer Mix 1 vial reconstituted ② Diluent 1400μL 1 vial 50μL after ③ Buffer PC 1 vial reconstituted ④ Buffer NC 100μL 1 vial
Storage and Stability Store at 2~8℃ in dark. Transport at 2 ~28℃ within 15 days. Shelf life: 6 months.
Note: Freeze-dried Buffer Mix should be stored at -20 ± 5℃ after reconstituted, valid for 48 hours. Buffer PC should be stored at -20 ± 5 ℃ after reconstituted, valid for 6 months. Repeated thawing and freezing reagents more than 3 times should be avoided as this may affect performance.
Materials Required (but not provided) 1) Compatible PCR instruments: Real time PCR System Gentier 96E(Xian Tianlong Science & Technology Co., Ltd.), LightCycler480II(Roche), ABI 7500 (Thermo Fisher) 2) DNA/RNA Extraction Kit (Magnetic Bead, Manual) (manufactured by Merlin Biomedical(Xiamen) Co., Ltd.). 3) Biosafety cabinet. 4) Vortex.
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5) Mini centrifuge with rotor for centrifuge tubes. 6) Personal Protective Equipment (PPE): Disposable powder-free gloves, goggles/face shield, respirators (NIOSH-certified N95 is recommended.) 7) Nuclease-free centrifuge tubes. 8) Nuclease-free PCR tubes and caps. 9) Adjustable pipettors and sterile, nuclease-free pipet tips. 10) Biohazard waste container. 11) Tube racks.
12) RNase-Free ddH2O.
Precautions and Warnings General Precautions • For in vitro test only. Please read the instruction carefully and become familiar with all components of the kit prior to use, and strictly follow the instruction during operation. • Please check the compatible real-time PCR instruments prior to use. • DO NOT use the kit or any kit component after expiry date. • DO NOT use any other reagents from different lots in the tests. • DO NOT use any other reagent in this kit. • Thaw reagents shall be stored at -20℃ for use within 2 days avoiding repeated freezing and thawing. Handling Precautions • Handle all specimens and components of the kit as potentially infectious material according to safe laboratory procedures. • Only trained professionals can use this kit. Wear appropriate personal protective equipment (PPE) when working with clinical specimens. • Specimen processing should be performed in a certified Class II biosafety cabinet following biosafety level 2 or higher guidelines. • Avoid skin, eyes and mucous membranes contact with the chemicals. In case of contact, flush with water immediately. • DO NOT pipet by mouth. • Avoid aerosols. Safety Precautions • The kit contains positive control; strictly distinguish the positive control from other reagents to avoid contamination which may cause false positive. • PCR amplification is extremely sensitive to cross-contamination. The flow of tubes, racks, pipettes and other materials used should be from pre- amplification to post-amplification, and never backwards. • Gloves should be worn and changed frequently when handling samples and reagents to prevent contamination. • Using separate, dedicated pipettes and nuclease-free filtered pipette tips when handling samples and reagents to prevent exogenous RNA contamination to the reagents. • Please pack the post-amplification tubes with two disposable gloves and discard properly. DO NOT open the post- amplification PCR tubes. • All disposable materials are for one time using. DO NOT reuse. • The unused reagents, used kit, and waste must be disposed of properly. • After the experiment, wipe down the work area, spray down the pipettes and equipment with 75% ethanol or 10% hypochlorous acid solution.
Specimen Collection and Preparation • Viral RNA extracted from human respiratory specimens should be collected by nasopharyngeal swabs and oropharyngeal swabs. • Oropharyngeal swab specimen collection: The patient should tilt their head slightly upward, open their mouth wide and make a sound of "ah" to expose both pharyngeal tonsils. The disposable swab should be used to cross the tongue base. Wipe both pharyngeal tonsils back and forth with slight force for at least three times, and then wipe the posterior pharyngeal wall up and down for at least three times. • Nasopharyngeal swab specimen collection: Gently hold the patient’s head with one hand, carefully insert the swab into the nostril and slowly go deep along the bottom of the lower nasal passage. When the top of the swab reaches the back wall of the nasopharyngeal cavity, gently rotate it for one round (pause for a moment once reflex cough), and then slowly remove the swab. • Swab specimens should be collected only on swabs with a synthetic tip with aluminum or plastic shafts. Swabs with calcium alginate or cotton tips with wooden shafts are not acceptable. • The samples should be extracted RNA immediately. For short-term storage of up to 24 hours, the sample should be stored at 2~8℃. For long-term storage of over 24 hours, the sample should be stored at -70℃ and avoid repeated freezing-thawing.
Assay Procedure RNA Extraction Following the RNA extraction steps and get purification RNA samples. Viral RNA extraction reagents are not included in this kit. Viral RNA Detection 1) Take the Freeze-dried Buffer Mix and Diluent out of the kit from the freezer. 2) Add 1300μL Diluent to dissolve Freeze-dried Buffer Mix and ensure it is evenly blended. 3) Prepare enough Master Mix in separate sterile centrifuge tube according to the ratio in below Table. The volume of Master Mix should be sufficient for tests of
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Reverse 1 48℃ 15min / Transcription
Initial denaturation 1 93℃ 2 min /
93℃ 15s /
Cycle Reaction 45 Fluorescence 58℃ 40s Collection
Device Cooling 1 30℃ 30s /
Target Channel Quencher ORF1ab FAM None
N HEX/VIC/JOE None
Internal Control Cal Red 610/ROX/TEXAS RED None
12) Start the PCR run immediately. 13) When the PCR run is finished, analyze the data according to the “Results Interpretation” procedures.
Quality Control Before data analysis, the result of control items should be checked in following Table: Result of Control ROX (Inner Control, β-actin) FAM (ORF1ab Gene) HEX (N Gene) Negative Control No Ct or Ct=0 No Ct or Ct=0 No Ct or Ct=0 Positive Control Ct＜40, with good amplification curve Ct＜42 Ct＜42
Results Interpretation If the negative/positive control results meet the above "Quality Control ", the result of samples shall be determined according to below Table: Result of Sample ROX (Inner Control, β-actin) FAM (ORF1ab Gene) HEX (N Gene)
Negative Ct＜40, No Ct or Ct=0 No Ct or Ct=0 with good Positive amplification curve Ct＜42 Ct＜42 Suspicious results: for FAM or HEX, 42≤Ct<45, repeat experiment. If Ct <42 with good amplification curve, the sample is judged to be positive; otherwise, it is negative. Note: The sample can be defined as positive when ORF1ab and N of SARS-CoV-2 both are positive. If the specimen is suspected to be positive, retest is recommended with other detection methodology on re-extracted sample.
Explanation of Results Baseline setting: Adaptive baseline. Threshold setting: Threshold line should be set just above the peak of the normal negative control amplification curve (irregular noise line).
Performance Characteristics Appearance: Clean, clearly marked, no leakage. Precision: CV≤5%. See performance evaluations below for coincidence rates observed.
Performance Evaluation Summary 1. Analytical Sensitivity/ Limit of Detection (LoD): Dilute the pseudovirus specimen ORF1ab/N-P01(1.0x105 copies/mL, which was determined prior to the extraction step) into 104 copies/mL,103 copies/mL,5xl02 copies/mL,102 copies/mL, with the sample matrix of VTM, namely Single Use Specimen Container model G03, manufactured by Merlin Biomedical, main components: Hank’s buffer, BSA, amino acid and antibiotics. Use DNA/RNA Extraction Kit (Magnetic Bead, Manual), manufactured by Merlin Biomedical(Xiamen) Co., Ltd., to purify above diluted sample. Test 20 times each specimen using Novel Coronavirus (COVID-19) RT-PCR Kit on Real time PCR System Gentier 96E, LightCycler480II and ABI 7500 with same PCR protocol respectively, among which at least 19 times the results shall be positive (>95%). The LoD is 500 copies/mL.
Detection rate Concentration (104 copies/mL) Concentration (103 copies/mL) Concentration (5xl02 copies/mL) Concentration (102 copies/mL)
Real time PCR System 20/20 20/20 20/20 3/20 Gentier 96E 100% 100% 100% 15% 20/20 20/20 20/20 9/20 LightCycler480II 100% 100% 100% 45% 20/20 20/20 20/20 11/20 ABI 7500 100% 100% 100% 55%
2. Inclusivity An alignment was performed with the oligonucleotide primer and detection sequences designed for the Novel Coronavirus (COVID-19) RT-PCR Kit using all publicly available sequences for SARS-CoV-2(n=100) found in NCBI Virus Resource as of Nov 17, 2020 to predict the inclusivity of the target. All alignments show 100% identity to the primer sequences utilized by the Novel Coronavirus (COVID-19) RT-PCR Kit.
3. Cross-Reactivity 3.1 Homology comparison
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Strains Rate Human coronavirus 229E, complete genome 54% Human Coronavirus NL63 55% Human coronavirus OC43 strain ATCC VR-759 56% Human coronavirus HKU1 56% Middle East respiratory syndrome-related coronavirus isolate HCoV-EMC/2012 57% SARS coronavirus Tor2 80% Human adenovirus 55 isolate Hebei/BD6728/2013 48% Human parainfluenza virus 1 isolate AR001 38% Human parainfluenza virus 2 strain GREER 39% Human parainfluenza virus 3 strain HPIV3/MEX/2545/2006 38% Human parainfluenza virus 4a strain HPIV4a/Seattle/USA/SC9717/2018 41% Influenza A virus (A/New York/PV305/2017(H1N1)) 7% Respiratory syncytial virus strain B/WI/629-Q0190/10 38% Enterovirus A71 strain 16L 21% Human rhinovirus 14 21% 3.2 Cross reactivity testing
Microorganisms were tested at 106 CFU/mL for bacteria and 105 TCID50/mL for viruses or higher when possible at Merlin Biomedical (Xiamen) Co., Ltd. Stock organisms were diluted and combined with simulated NPS in UTM matrix prior to extraction. Each organism was extracted in triplicate on the Virus Nucleic Acid Extraction Kit or Nucleic Acid Detection Kit manufactured by Merlin Biomedical according to the instructions for use. For each concentration tested, the number of replicates that gave valid results (per the Interpretation Algorithm) was determined. No cross-reactivity was observed for all 3 replicates (all 3 replicates should be negative for both ORF1ab and N gene sets) for all organisms assessed in this study (see tables below). Virus Analyzed for Analytical Specificity (Cross Reactivity) Organism Vendor Catalog # Titer tested (N X 10n units/mL) Cross-reactivity (Y/N) Detected (n/3) Human coronavirus 229E Guangdong Hecin Scientific VR-740 1.0 x 105 TCID50/mL N 0/3 Human coronavirus NL63 Guangdong Hecin Scientific KY074-1 1.0 x 105 TCID50/mL N 0/3 Human coronavirus OC43 Guangdong Hecin Scientific VR-1558 1.0 x 105 TCID50/mL N 0/3 Pseudovirus containing MERSgene Guangdong Hecin Scientific KY074-2 1.0 x 105 TCID50/mL N 0/3 fragment Pseudovirus containing SARS-CoV gene Guangdong Hecin Scientific KY074-3 1.0 x 105 TCID50/mL N 0/3 fragment Adenovirus Serotype 1 Guangdong Hecin Scientific KY074-10 1.0 x 105 TCID50/mL N 0/3 Adenovirus Serotype 2 Guangdong Hecin Scientific KY074-11 1.0 x 105 TCID50/mL N 0/3 Adenovirus Serotype 3 Guangdong Hecin Scientific KY074-12 1.0 x 105 TCID50/mL N 0/3 Adenovirus Serotype 4 Guangdong Hecin Scientific VR-1572 1.0 x 105 TCID50/mL N 0/3 Adenovirus Serotype 5 Guangdong Hecin Scientific KY074-13 1.0 x 105 TCID50/mL N 0/3 Adenovirus Serotype 7 Guangdong Hecin Scientific KY074-14 1.0 x 105 TCID50/mL N 0/3 Adenovirus Serotype 55 Guangdong Hecin Scientific KY074-15 1.0 x 105 TCID50/mL N 0/3 Human Metapneumovirus (Type A2) Guangdong Hecin Scientific KY074-20 1.0 x 105 TCID50/mL N 0/3 Human Rhinovirus Type A（HRV A2） Guangdong Hecin Scientific KY074-19 1.0 x 105 TCID50/mL N 0/3 Human Rhinovirus Type B (HRV 70 ) Guangdong Hecin Scientific VR-1170 1.0 x 105 TCID50/mL N 0/3 Influenza A H1N1 Guangdong Hecin Scientific KY074-5 1.0 x 105 TCID50/mL N 0/3 Influenza A H1N1/New Caledonia/20/99 Guangdong Hecin Scientific KY074-4 1.0 x 105 TCID50/mL N 0/3 Influenza A H3N2 Guangdong Hecin Scientific KY074-6 1.0 x 105 TCID50/mL N 0/3 Influenza B(Yamagata) Guangdong Hecin Scientific KY074-7 1.0 x 105 TCID50/mL N 0/3 Influenza B(Victoria) Guangdong Hecin Scientific KY074-8 1.0 x 105 TCID50/mL N 0/3 Parainfluenza Virus 1 Guangdong Hecin Scientific VR-94 1.0 x 105 TCID50/mL N 0/3 Parainfluenza Virus 2 Guangdong Hecin Scientific VR-92 1.0 x 105 TCID50/mL N 0/3 Parainfluenza Virus 3 Guangdong Hecin Scientific KY074-16 1.0 x 105 TCID50/mL N 0/3 Respiratory Syncytial Virus (Type A) Guangdong Hecin Scientific VR-26 1.0 x 105 TCID50/mL N 0/3 Respiratory Syncytial Virus (TypeB) Guangdong Hecin Scientific KY074-9 1.0 x 105 TCID50/mL N 0/3 Enterovirus Type A ( EV71 ) Guangdong Hecin Scientific KY074-17 1.0 x 105 TCID50/mL N 0/3 Enterovirus Type B ( CB3 ) Guangdong Hecin Scientific KY074-18 1.0 x 105 TCID50/mL N 0/3 Enterovirus Type C ( CA24 ) Guangdong Hecin Scientific VR-1662 1.0 x 105 TCID50/mL N 0/3 Enterovirus Type D ( EV70 ) Guangdong Hecin Scientific VR-836 1.0 x 105 TCID50/mL N 0/3 EB virus Guangdong Hecin Scientific CRL-1612 1.0 x 105 TCID50/mL N 0/3 Human cytomegalovirus Guangdong Hecin Scientific VR-2356 1.0 x 105 TCID50/mL N 0/3 Measles virus Guangdong Hecin Scientific VR-24 1.0 x 105 TCID50/mL N 0/3 Mumps virus Guangdong Hecin Scientific VR1438 1.0 x 105 TCID50/mL N 0/3 Bacteria Analyzed for Analytical Specificity (Cross Reactivity) Organisma Vendor Catalog # Titer tested (N X 10n units/mL) Cross- reactivity (Y/N) Detected (n/3) Bordetella pertussis Guangdong Hecin Scientific 9340/BAA-1335 1.0 x 107CFU/mL N 0/3 Candida albicans Bena Culture Collection BNCC186382 1.0 x 107CFU/mL N 0/3 Chlamydia pneumoniae Guangdong Hecin Scientific VR-2282 1.0 x 107CFU/mL N 0/3 Haemophilus influenzae Bena Culture Collection BNCC337544 1.0 x 107CFU/mL N 0/3 Legionella pneumophila Guangdong Hecin Scientific 33152 1.0 x 107CFU/mL N 0/3 Mycobacterium Bena Culture Collection BNCC336701 1.0 x 107CFU/mL N 0/3 tuberculosis Mycoplasma pneumoniae Guangdong Hecin Scientific 15531 1.0 x 107CFU/mL N 0/3 4 / 5 Merlin Biomedical (Xiamen) Co., Ltd. Validation of this test has not been reviewed by FDA. Review under the EUA program is pending.
Streptococcus Bena Culture Collection 6314 1.0 x 107CFU/mL N 0/3 pneumoniae Staphylococcus aureus Bena Culture Collection BNCC186335 1.0 x 107CFU/mL N 0/3 Klebsiella Pneumoniae Bena Culture Collection BNCC102997 1.0 x 107CFU/mL N 0/3 Candida Glabrata Bena Culture Collection BNCC337348 1.0 x 107CFU/mL N 0/3 Aspergillus fumigatus Bena Culture Collection BNCC338385 1.0 x 107CFU/mL N 0/3 Filobasidiella Bacillispora Bena Culture Collection BNCC339771 1.0 x 107CFU/mL N 0/3 Streptococcus Pyogenes Bena Culture Collection BNCC185918 1.0 x 107CFU/mL N 0/3
4. Clinical Evaluation The nasopharyngeal swabs(30 positive and 30 negative, 60 in total) and oropharyngeal swabs(30 positive and 32 negative, 62 in total) specimens were sourced from COVID-19 infected patients, purified by DNA/RNA Extraction Kit (Magnetic Bead, Manual), manufactured by Merlin Biomedical(Xiamen) Co., Ltd., then tested by Novel Coronavirus (COVID-19) RT-PCR Kit on Real time PCR System Gentier 96E, and parallel tested by the reference kit Novel Coronavirus (2019-nCoV) Nucleic Acid Diagnostic Kit (PCR-Fluorescence Probing) manufactured by Sansure BioTech Inc. which has been EUA authorized by FDA, on ABI 7500 Real-Time PCR System. The results are summarized in below table and demonstrated a PPA of 100% and NPA of 100% for both nasopharyngeal swabs and oropharyngeal swabs. Nasopharyngeal Sansure test result Oropharyngeal Sansure test result swabs Positive Negative swabs Positive Negative Positive 30 True positive(a) 0 False positive(b) Positive 30 True positive(a) 0 False positive(b) Result Result Negative 0 False negative(c) 30 True negative(d) Negative 0 False negative(c) 32 True negative(d)
Limitations Laboratories should include a statement such as The test has been validated but FDA’s independent review of this validation is pending, in patient test reports to their healthcare providers.
Reference Heid, C. A., Stevens, J., Livak, K. J., et al. Real time quantitative PCR [J]. Genome Research, 1996, 6(10), 986–994.
Instruction Approval and Revision Date Approval Date: 05 Jun 2020 Revision Date: 09 Oct 2020 Date of Issue: 09 Oct 2020
Catalogue number Temperature limit
Consult instructions for use Batch code
In vitro diagnostic medical Use-by date device Manufacturer Biological risks
This way up Keep dry
Warning Avoid overexposure to the sun
Don't use the product when the Authorized representative in the
package is damaged European Community
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