AquaScreen® species qPCR Detection Kit

Instructions for Use

FOR USE IN RESEARCH AND QUALITY CONTROL Symbols

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Storage temperature

Number of reactions

Manufacturer INDICATION Legionella are ubiquitous in surface water and moist soil. Legionella are well known as opportunistic pathogens causing Legionnaires' disease and Pontiac fever. The incubation period is 20-10 days. An airborne transmission is unlikely; thus infections occur through inhalation of aer- osols bearing amoeba, the natural habitat of legionella.

The AquaScreen® Legionella species qPCR Detection kit is specifically designed for the quantita- tive detection of Legionella species in water samples prepared with the AquaScreen® FastExtract kit.

EXPLANATION OF THE TEST The AquaScreen® Legionella species kit utilizes qPCR for the quantitative detection of legionella in water samples. In contrast to time-consuming cell culture methods, the AquaScreen® ap- proach needs less than six hours including sample preparation and qPCR to reliably detect le- gionella bacteria. In addition, our qPCR assay is superior in terms of sensitivity and specificity as the assay only detects legionella species.

The AquaScreen qPCR assay is insensitive to contamination with other bacterial genera. Also, the assay’s robustness is unsurpassed with linear detection up to 1 x 106 particles per sample. Thus there is no need for diluting sample material.

The kit’s design complies with the requirements of AFNOR T90-471 and ISO/TS 12869:2012

TEST PRINCIPLE The PCR system targets a segment of the 16S rRNA region in the legionella genome. The target region is highly preserved within the genus Legionella. Clinically relevant species that are detect- ed are e.g. L. pneumophila (all serogroups), L. longbeachae, L. dumoffii, Fluoribacter bozemanii (L. bozemanii) and L. gormanii (see page 7 for the full list). Cross-reactivity to other waterborne microorganisms are not known.

The kit contains the nucleotide dUTP instead of dTTP. The heat-labile Uracil-DNA Glycosylase (UNG) is suitable to prevent carry-over contamination between PCRs. This technique relies on the incorporation of deoxyuridine triphosphate (dUTP) during all amplified reactions and the pre- treatment of all successive PCR mixtures with the heat-labile UNG. UNG is not included. The L. species mix contains all required primer, probes, dNTPs and Taq polymerase.

33-2025/2100/2250 Product Version 2 | Document Version 2 1 REAGENTS

Each kit contains reagents for 25, 100 or 250 reactions. The expiry date of the unopened pack- age is marked on the package label. The kit components must be stored at +2 to +8 °C until use. The rehydrated components must be stored at < -18 °C.

Quantity Kit component 25 reactions 100 reactions 250 reactions Cap Color Order No. 33-2025 Order No. 33-2100 Order No. 33-2250

1 vial 4 vials 10 vials L. species Mix red freeze-dried freeze-dried freeze-dried

1 vial 1 vial 3 vial Rehydration Buffer blue 1.8 ml 1.8 ml 1.8 ml each

1 vial 1 vial 1 vial Positive Control DNA green freeze-dried freeze-dried freeze-dried

1 vial 1 vial 2 vials Internal Control DNA yellow freeze-dried freeze-dried freeze-dried

1 vial 1 vial 1 vial PCR-grade Water white 2 ml 2 ml 2 ml

The lot specific Certificate of Analysis (CoA) can be downloaded from our website (www.minerva- biolabs.com).

USER-SUPPLIED CONSUMABLES AND EQUIPMENT The AquaScreen qPCR kit contains all necessary reagents for the PCR. Additional consumables and equipment is supplied by the user:  qPCR device with filter sets for detecting the fluorescence dyes FAMTM and ROXTM  PCR reaction tubes for the specific qPCR device  1.5 ml reaction tubes, DNA- and RNA-free  Microcentrifuge for 1.5 ml PCR reaction tubes  Pipettes with corresponding filter tips (10, 100, and 1000 µl)  For DNA standard curves, we recommend our PCR Quantification Standard (Cat-No.: 52-0101).

SPECIMEN

For sample preparation please see the AquaScreen® FastExtract instructions for users. Extracted samples must be stored at < -18 °C for up to one year. Repeated freeze/thaw must be avoided as it is detrimental to the DNA’s integrity.

33-2025/2100/2250 2 Product Version 2 | Document Version 2 PRECAUTIONS

The AquaScreen qPCR kit is for research use only. The kit should be used by trained laboratory staff only. All samples should be considered as potentially infectious and handled with all due care and attention. Always wear suitable lab coat and disposable gloves. This kit does not contain hazardous substances. Remnants can be discarded according to local regulations.

IMPORTANT NOTES

 These instructions must be understood to successfully use the AquaScreen® qPCR Detec- tion kit. The reagents supplied should not be mixed with reagents from different LOT and used as an integral unit. The reagents of the kit must not be used beyond their shelf life.  Follow the exact protocol. Any deviation may affect the test method and can affect the re- sults.  PCR inhibition is likely to be caused by the sample matrix, or, in case of extracted DNA, caused by the elution buffer. Thus we recommend our AquaScreen® FastExtract kit for sam- ple preparation. Any other DNA extraction kit needs to be qualified.  It is important to include control samples on a regular basis to monitor the reliability of your results. Positive and negative controls are essential in case of troubleshooting.  The control samples must be processed in the same manner as the test samples. You may want to include other laboratory specific control samples such as high, median and low DNA

level (e.g. 3x LOD95). Please note that Minerva Biolabs also offers to participate in external quality control programs.

APPENDIX

Limited Product Warranty This warranty limits our liability for replacement of this product. No warranties of any kind, express or implied, including, without limitation, implied warranties of merchantability or fitness for a particular purpose, are provided. Minerva Biolabs shall have no liability for any direct, indirect, consequential, or incidental damages arising from the use, the results of use, or the inability to use this product.

Trademarks LightCycler is a registered trademark of a member of the Roche Group. ABI Prism is a registered trademark of Applera Corporation or its subsidiaries in the US and certain other countries. FAM™ and ROX™ are trade- marks of Applera Corporation or it's subsidiaries in the US and certain other countries. Mx3005P is a trade- mark of Agilent Technologies. RotorGene is a registered trademark of Qiagen GmbH. AquaScreen is registered trademark of Minerva Biolabs GmbH.

33-2025/2100/2250 Product Version 2 | Document Version 2 3 PROCEDURE - OVERVIEW

AquaScreen® Legionella species

Included Duration Additionally required

L. species Rehydration · PCR reaction tubes Mix Buffer · 1.5 ml reaction tubes L R · Tools: Microcentrifuge Pipettes with corresponding filter tips Positive Internal PCR- qPCR device (+ filter sets for FAM™/ Control Control grade P I W < 45 min ROX™detection) DNA DNA Water Optional: PCR Quantification Standard

Procedure

1. Reagent Preparation a)

Full speed + 365 µl R L 5 sec RT b) L R P I W L P I 5min LPI Full speed P I + 300 µl W 5 sec

2. Reaction Mix Preparation Master Mix (x=∑samples) + x∙14 µl + x∙1 µl X 5 à 15 µl L I

3. Add samples 1. Negative Control +10 µl 4. W

2. Positive Control +10 µl P Full speed 5 sec 3. Test samples / Standard curve samples:+10 µl

4. Start PCR amplification 95°C 95°C 5 min 30 sec Start PCR 45 cycles 60°C program 55°C 45 sec 30 sec

Storage Legend

Store kit components at +2 to +8 °C. L. species Master Mix Vortex The rehydrated components must be stored Rehydration Buffer Complete PCR at < -18 °C. Incubate Positive Control Master Mix Extracted samples must be stored Test samples/ Centrifuge at < -18 °C. Internal Control Repeated freeze/ thaw must be avoided. PCR-grade Water Standard curve

33-2025/2100/2250 4 Product Version 2 | Document Version 2 PROCEDURE

1. Reagent preparation After reconstitution, the reagents can be stored at 2 to 8 °C for up to one day. Long time storage must be at < -18 °C. Repeated freeze/thaw of rehydrated components must be avoided as it might affect the assays’ sensitivity. We recommend to store these components in aliquots.

L species Mix Red cap Spin down all freeze-dried components at max 1. Internal Control Yellow cap speed for 5 sec. Positive Control Green cap

2. L. species Mix Red cap Add 365 µl rehydration buffer (blue cap)

3. Internal Control Yellow cap Add 300 µl PCR-grade Water (white cap)

4. Positive Control Green cap Add 300 µl PCR-grade Water (white cap)

L. species Mix Red cap 5. Internal Control Yellow cap Incubate at room temperature for 5 min Positive Control Green cap

L. species Mix Red cap 6. Internal Control Yellow cap Vortex briefly and spin for 5 sec Positive Control Green cap

2. Reaction mix preparation The following steps 2 to 4 (reaction mix preparation, add samples and start PCR amplification) should be done in less than 45 minutes to avoid a reduction in the fluorescent signal. Follow these schemes and sequences to set up the test:

Prepare the required volume of master mix at room temperature in a 1.5 ml reaction tube for all control and test reactions. 1. for 1 reaction for 25 reactions L. species Mix 14 µl 350 µl Internal Control 1 µl 25 µl

2. Homogenize the reaction mix by pipetting (5-times).

3. Add 15 µl to each PCR tube, discard remaining material.

33-2025/2100/2250 Product Version 2 | Document Version 2 5 3. Add samples  Please note that a DNA standard curve is required for quantification. We recommend our PCR quantification standards as templates for generating standard curves (e.g. Legionella pneumophila, Cat-No.: 52-0101)  Set up at positive and negative control samples (non template control) in duplicate in each PCR.

1. Negative Control: add 10 µl elution buffer from DNA extraction or PCR-grade Water (white cap).

2. Test sample/standard curve sample: add 10 µl of each sample.

3. Positive Control: add 10 µl Positive Control (green cap).

4. Spin PCR tubes briefly and ensure that all tubes are closed tightly.

4. Start PCR amplification

1. Place PCR tubes in the qPCR device and close the lid.

Program the PCR cycler: 1 cycle 95 °C for 5 min 45 cycles 95 °C for 30 sec (Denaturation) 2. 55 °C for 30 sec (Annealing) 60 °C for 45 sec (Elongation and data collection) Fluorescence dyes: FAM™ and ROX™

3. Start the program

This assay was tested on the following qPCR devices:

qPCR device Manufacturer

CFX-96 Bio-Rad

LightCycler® 1.2 Roche Diagnostics

ABI Prism® 7500 Applied Biosystems RotorGene® 6000 Corbett Research Mx3005P® Agilent Technologies

33-2025/2100/2250 6 Product Version 2 | Document Version 2 DATA INTERPRETATION The presence of legionella is indicated by an increasing fluorescence signal in the FAM channel. The quantification is based on threshold cycle (Ct) values and a DNA standard curve. The exact procedure for obtaining Ct-values including baseline calculation/normalization depends on the particular qPCR device and cycler control software. Please see the documentation of your device for further details. We recommend to assess the amplification curve progression of any sample including control samples.

A positive PCR is indicated by Ct < 40. PCR reactions with Ct ≥ 40 are considered negative. In addition, a positive PCR is displayed by an increasing fluorescence signal in either the FAM™ and/or the ROX™ channel (given the Internal Control was added to the master mix). The Legionel- la DNA and Internal Control function as competitors in the PCR. Thus, the more Legionella DNA is in the sample, the higher the signal is in the FAM™ channel and the lower the Internal Control signal is in the ROX™ channel. The following matrix will help to interpret the PCR result:

Detection of Legionella species Internal control Interpretation FAMTM channel ROXTM channel positive irrelevant Legionella positive negative negative PCR inhibition negative positive Legionella negative

The calculation of legionella particles per sample is illustrated by the following example:

Sample volume for DNA extraction 500 ml, eluted with 100 µl Sample volume used for qPCR 10 µl (with one-tenth of the sample) DNA copies determined by qPCR 60 (one-tenth of the sample)

=> Total DNA copies (10 x 60 =) 600 in 500 ml

In the 500 ml water sample, 600 intact legionella particles were determined. This figure may consist of viable and cultivable legionella and viable but non-cultivable legionella (VBNC-state) as well as dead yet still intact legionella.

33-2025/2100/2250 Product Version 2 | Document Version 2 7 ASSAY CHARACTERISTICS Sensitivity and linear range

Detection was demonstrated from 1 genome equivalent (GE) per PCR. Robust and linear detec- tion of legionella species is between 20 to 1 x 106 GE / PCR.

Fig. 1. Sensitivity and efficiency of the PCR. The figure on the right shows amplification curves for a serial dilution of L. pneumophila DNA (PCR Quantifi- cation standard). The lower figure displays a charac- teristic standard curve. The PCR was conducted on a Mx3005P qPCR System (Agilent Technologies, Inc.).

2. Specificity Based on DNA sequence alignments the following legionella species will be detected:

Legionella adelaidensis Legionella lytica Legionella anisa Legionella maceachernii Legionella beliardensis Legionella(Tatlockia) micdadei Legionella birminghamensis Legionella moravica (Legionella) Fluoribacter bozemanae Legionella nagasakiensis Legionella brunensis Legionella nautarum Legionella busanensis Legionella oakridgensis Legionella cherrii Legionella parisiensis Legionella cincinnatiensis Legionella pneumophila Legionella donaldsonii Legionella pneumophila subsp. fraseri Legionella drancourtii Legionella pneumophila subsp. pascullei Legionella dresdenensis Legionella pneumophila subsp. pneumophila Legionella (Fluoribacter) dumoffii Legionella quateirensis Legionella erythra Legionella quinlivanii Legionella fairfieldensis Legionella rubrilucens Legionella feeleii Legionella sainthelensi Legionella (Fluoribacter) gormanii Legionella santicrucis Legionella gratiana Legionella shakespearei Legionella gresilensis Legionella spiritensis Legionella hackeliae Legionella steelei Legionella israelensis Legionella steigerwaltii Legionella jamestowniensis Legionella taurinensis Legionella jordanis Legionella tucsonensis Legionella lansingensis Legionella wadsworthii Legionella londiniensis Legionella waltersii Legionella worsleiensis

33-2025/2100/2250 8 Product Version 2 | Document Version 2 Related pRoducts AquaScreen® FastExtract DNA-based system for quantitative detection of water pathogens. AquaScreen® combines water filtration, lysis of the collected microorganisms, DNA extraction and elution of the DNA in minimal volumes ready for PCR analysis.

Features

Description Rapid DNA extraction from water samples

Recommended Use / AquaScreen® FastExtract can be used with your established suction device (47 mm Scope frit) for the extraction of legionella and other microbial contaminations. AquaScreen® FastExtract is optimized for high flow and throughput and provides high quality DNA for subsequent PCR analysis.

Kit Components Membrane filters Incubation dishes Incubation, collection and sample storage tubes Lysis, wash and elution buffers

Package Sizes Cat.-No. 32-1010 10 extractions Cat.-No. 32-1050 50 extractions

Required lab devices Vacuum pump & reagents Micro centrifuge Filtration system, 47 mm frit Pipetting equipment and filtered tips Incubator (37 °C for petri dishes, 56 °C for reaction tubes) Ethanol (96-100 %)

Shelf Life and Storage Components are maintainable at room temperature for at least 6 months.

Compliance AFNOR XP T90-471 and ISO/TS 12869:2012 in combination with AquaScreen® qPCR kits Related pRoducts Meat ID™ Identification of animal species in meat and other foods by qPCR

Background

The identification of different meats in especially minced meat products is a serious task in food safety and ethi- cal perspective, especially for muslims. Authentication of forbidden or none declared ingredients such as pork or substandard meat is essential to ensure confidence in the supply chain and regulatory compliance. Meat ID is available for rapid and reliable analysis from various matrices including raw, or even highly processed and cooked meat products where the DNA may be significantly degraded. It is possible to identify relevant species down to a threshold level of 0.5% with a semi-quantitative result.

Features

Principle The assay is based on the TaqMan® principle and worked with FAM and HEX labled probes.

Target The target sequence is a mitochondrial multi-copy gene (cytochrome b). Therefore, even very small amounts of DNA can lead to positive results.

Sensitivity 1 Genom Unit/PCR, ≙ 10 DNA copies/PCR

Content Master Mix, freeze-dried Primer Probe Mix, freeze-dried Rehydration Buffer PCR Grade Water Internal Control Positiv Control

Sample Requirements The DNA can be isolated from sample materials either by using an extraction kit designed to isolate gDNA e.g. ExtractNowTM DNA Mini Kit or by an in-house method.

Intended Use For research only! Not for use in diagnostic procedures.

Time to Result 90 minutes

Storage Components are maintainable at +2 to +8 °C. After rehydratisation the reagents must be stored at -18 °C.

Real Time Cycler • qTOWER (Analytik Jena) • TOptical (Analytik Jena) • Rotor-Gene® Q (Qiagen GmbH) • LightCycler® (Roche Diagnostic GmbH) • Mastercycler® ep realplex (Eppendorf) • CFX ConnectTM (Bio-Rad) • Amplifa (Illumina ECO) • StepOnePlus™ (Applied Biosystems) Related pRoducts Food Control™ qPCR Detect foodborne pathogens with easy interpretable lateral flow evaluation.

Features

Target • – invasion protein (invA) gene • – heat-stable enterotoxin A gene • Shigella spp. – invasion plasmid antigen (ipaH6) gene • Campylobacter spp. – acyl-[acyl-carrier-protein]--UDP-N-acetylglucosamine O-acyltransferase (lpxA) gene • Clostridium perfringens – phospholipase C alpha toxin (plc) gene • Shiga Toxin 1 – stx1 gene • Shiga Toxin 2 – stx2 gene • O157 – wbdR gene • Escherichia coli O104 – wckD gene • Listeria spp. – invasion associated protein p60 (iap) gene • Listeria monocytogenes – listeriolysin O (hly) gene • Salmonella spp. – spacer-region between 16S and 23S RNA genes

Sensitivity Down to 10 DNA copies/assay.

Principle TaqMan® assay based on FAM and HEX labeled probes.

Content qPCR Mix Species Mix Rehydration Buffer PCR Grade Water Internal Control Positive Control

Sample Requirements Isolated total DNA from potentially contaminated food serves here as starting material, typically after pre-cultivation of the sample growth medium.

Intended Use For research use only! Time to Result 150 minutes

Cycler • qTOWER (Analytik Jena) • TOptical (Analytik Jena) • Rotor-Gene® (Qiagen) • Rotor-Gene®6000 (Qiagen) • LightCycler® (Roche Diagnostics) • Mastercycler® ep reaplex (Eppendorf) • CFX Connect™ (Bio-Rad) • StepOnePlus™, ABI 7500 (Applied Biosystem®) • Mx3005P (Agilent Technologies) Related Products

AquaScreen® Detection kits for qPCR 33-2025/-2100/-2250 AquaScreen® Legionella species 25/100/250 reactions 34-2025/-2100/-2250 AquaScreen® Legionella pneumophila 25/100/250 reactions 34-6025/-6100/-6250 AquaScreen® 25/100/250 reactions 34-7025/-7100/-7250 AquaScreen® Escherichia coli 25/100/250 reactions

PCR Quantification Standards, 1x108 genomes / vial 52-0101 Legionella pneumophila 52-0071 Pseudomonas aeruginosa 52-0083 Escherichia coli See Minerva homepage for further available species

Genomic DNA Extracts - Specificity Standards, 10 ng ± 2 ng / vial 51-1370 Legionella dumoffii 51-1533 Legionella jordanis 51-0101 Legionella pneumophila 51-1514 Legionella pneumophila subs. fraseri 51-1515 Legionella pneumophila subs. pascullei 51-0071 Pseudomonas aeruginosa 51-0083 Escherichia coli See Minerva homepage for further available species

Contamination Control Kits 11-1025/-1050/-1100/-1250 Venor®GeM Classic Mycoplasma Detection Kit 25/50/100/250 tests 11-7024/-7048/-7096/-7240 Venor®GeM Advance Mycoplasma Detection Kit 24/48/96/240 tests 11-8025/-8050/-8100/-8250 Venor®GeM OneStep Mycoplasma Detection Kit 25/50/100/250 tests 12-1025/-1050/-1100/-1250 Onar® Bacteria Detection Kit 25/50/100/250 tests 11-9025/-9100/-9250 Venor®GeM qEP Mycoplasma Detection Kit 25/100/250 tests

PCR CleanTM (DNA Remover) 15-2025 DNA Decontamination Reagent, spray bottle 250 ml 15-2200 DNA Decontamination Reagent, refill bottles 4 x 500 ml

PCR CleanTM Wipes (DNA Remover Wipes) 15-2001 DNA Decontamination Reagent, in spender box 120 wipes 15-2002 DNA Decontamination Reagent, refill sachets 5 x 120 wipes

ZellShieldTM 13-0050/-0150 Contamination Prevention Reagent 1000 ml/ 5 x 1000 ml 100x concentrate

WaterShieldTM 15-3025/-3075 Water Disinfection Additive for incubators 30 x 5 ml / 500 ml and water baths (200x concentrate)

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