AquaScreen® Legionella species qPCR Detection Kit INSTRUCTIONS FOR USE FOR USE IN RESEARCH AND QUALITY CONTROL Symbols Lot No. Order No. Expiry date Storage temperature Number of reactions Manufacturer INDICATION Legionella bacteria 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 Legionella pneumophila 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.