AquaScreen Escherichia coli. qpcr Detection Kit

Transcription

1 AquaScreen Escherichia coli qpcr Detection Kit Instructions for Use FOR USE IN RESEARCH AND QUALITY CONTROL

2 Symbols Lot No. Order No. Expiry date Storage temperature Number of reactions Manufacturer

3 INDICATION Escherichia coli are faecal coliform bacteria, commonly found in the intestines of animals and humans. The presence of Escherichia coli in water indicates recent sewage or animal waste contamination which might contain disease-causing microorganisms like Giardia spp. and Cryptosporidium. According to the WHO guideline values for the bacteriological quality of drinking water, treated water entering the distribution system and treated water in the distribution system, Escherichia coli must not be detectable in any 100 ml sample. The AquaScreen Escherichia coli qpcr Detection kit is specifically designed for the quantitative detection of Escherichia coli in water samples prepared with the AquaScreen FastExtract kit. EXPLANATION OF THE TEST The AquaScreen Escherichia coli kit utilizes qpcr for the quantitative detection of E.coli (all serotypes) as well as Shigella spp. In contrast to time-consuming cell culture methods, the AquaScreen approach needs less than six hours including sample preparation and qpcr to reliably detect E. coli bacteria. In addition, our qpcr assay is superior in terms of sensitivity and specificity as the assay only detects Escherichia coli 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 3 x 10 6 particles per sample. Thus there is no need for diluting sample material. TEST PRINCIPLE The PCR system targets the uidr gene in the E.coli genome. The target region is highly preserved within Escherichia coli species. All E. coli serotypes including enteroinvasive E. coli (EIEC) and Shigella spp. will be detected. Cross-reactivity to other waterborne microorganisms is 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 pretreatment of all successive PCR mixtures with the heat-labile UNG. UNG is not included. The E. coli mix contains all required primer, probes, dntps and Taq polymerase. 1

4 REAGENTS Each kit contains reagents for 25, 100 or 250 reactions. The expiry date of the unopened package 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 Order No reactions Order No reactions Order No Cap Color E. coli Mix 4 vials 10 vials red Rehydration Buffer 1.8 ml 1.8 ml 3 vial 1.8 ml each blue Positive Control DNA green Internal Control DNA 2 vials yellow PCR-grade Water 2 ml 2 ml 2 ml white The lot specific Certificate of Analysis (CoA) can be downloaded from our website ( 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 FAM TM and ROX TM 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 Escherichia coli PCR Quantification Standard (Cat-No.: ). 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. 2

5 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 Detection 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 results. 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 sample 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 LOD 95 ). 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 trademarks of Applera Corporation or it's subsidiaries in the US and certain other countries. Mx3005P is a trademark of Agilent Technologies. RotorGene is a registered trademark of Qiagen GmbH. AquaScreen is registered trademark of Minerva Biolabs GmbH. 3

6 PROCEDURE - OVERVIEW AquaScreen Escherichia coli Included Duration Additionally required E. coli Rehydration E Mix R Buffer Positive Internal PCR- P Control I Control W grade DNA DNA Water Procedure 1. Reagent Preparation < 45 min PCR reaction tubes 1.5 ml reaction tubes Tools: Microcentrifuge Pipettes with corresponding filter tips qpcr device (+ filter sets for FAM / ROX detection) Optional: PCR Quantification Standard E R P I W Full speed 5 sec 2. Reaction Mix Preparation E+ 365 µl R P I µl W E P I RT 5min E P I a) b) Full speed 5 sec Master Mix (x= samples) + x 14 µl + x 1 µl E I X 5 à 15 µl 3. Add samples 1. Negative Control W +10 µl 2. Positive Control P +10 µl 3. Test samples / Standard curve samples:+10 µl 4. Full speed 5 sec 4. Start PCR amplification Start PCR program 95 C 95 C 5 min 30 sec 45 cycles 60 C 45 sec 55 C 30 sec Storage Legend Store kit components at +2 to +8 C. The rehydrated components must be stored at < -18 C. Extracted samples must be stored at < -18 C. Repeated freeze/ thaw must be avoided. E. coli Rehydration Buffer Positive Control Internal Control PCR-grade Water Master Mix Complete PCR Master Mix Test samples/ Standard curve Vortex Incubate Centrifuge 4

7 PROCEDURE - STEP BY STEP 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 the components in aliquots. 1. E. coli Mix Red cap Internal Control Yellow cap Positive Control Green cap Spin down all components at max speed for 5 sec E. coli Mix Red cap Add 365 µl rehydration buffer (blue cap) Internal Control Yellow cap Add 300 µl PCR-grade Water (white cap) Positive Control Green cap Add 300 µl PCR-grade Water (white cap) E. coli Mix Red cap Internal Control Yellow cap Positive Control Green cap E. coli Mix Red cap Internal Control Yellow cap Positive Control Green cap Incubate at room temperature for 5 min Vortex briefly and spin for 5 sec 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: 1. Prepare the required volume of master mix at room temperature in a 1.5 ml reaction tube for all control and test reactions. for 1 reaction for 25 reactions E. coli Mix 14 µl 350 µl Internal Control DNA 1 µl 25 µl 2. Homogenize the reaction mix by pipetting (5-times). 3. Add 15 µl to each PCR tube, discard remaining material. 5

8 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. E. coli, Cat- No.: ). 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. 2. Program the PCR cycler: 1 cycle 95 C for 5 min 45 cycles 95 C for 30 sec (Denaturation) 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 CFX-96 LightCycler 1.2 ABI Prism 7500 RotorGene 6000 Mx3005P Manufacturer Bio-Rad Roche Diagnostics Applied Biosystems Corbett Research Agilent Technologies 6

9 DATA INTERPRETATION The presence of E. coli 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 both the FAM and the ROX channel (given the Internal Control was added to the master mix). The E. coli DNA and Internal Control do not show significant target competition. Thus, the Internal Control signal in the ROX channel is expected to show an increasing fluorescence signal irrespectively of the E. coli DNA input. The following matrix will help to interpret the PCR result: Detection of E. coli. FAM TM channel Internal control ROX TM channel Interpretation positive irrelevant E. coli positive negative negative PCR inhibition negative positive E. coli negative The calculation of E. coli 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 E. coli particles were determined. This figure may consist of viable and cultivable E. coli and viable but non-cultivable E. coli (VBNC-state) as well as dead yet still intact E. coli. 7

10 ASSAY CHARACTERISTICS 1. Sensitivity and linear range Robust detection was demonstrated from 3 genome equivalent (GE) per PCR. Linear detection of E. coli is from 30 to 3 x 10 6 GE / PCR. Fig. 1. Sensitivity and efficiency of the PCR. The figure on the right shows amplification curves for a serial dilution of E. coli DNA (PCR Quantification standard). The lower figure displays the corresponding standard curve. The qpcr was conducted on a Mx3005P qpcr System (Agilent Technologies, inc.). 2. Specificity Escherichia coli (all serotypes) as well as Shigella spp. will be detected. Cross-reactivity with the following bacteria was tested negative: Bacillus cereus Bacillus subtilis Clostridium acetobutylicum Enterococcus feacalis Enterobacter aerogenis Legionella pneumophila Micrococcus luteus Proteus mirabilis Salmonella enterica Staphylococcus aureaus Lactobacillus acidophilus 8

11 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 Recommended Use / Scope Kit Components Rapid DNA extraction from water samples AquaScreen FastExtract can be used with your established suction device (47 mm 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. Membrane filters Incubation dishes Incubation, collection and sample storage tubes Lysis, wash and elution buffers Package Sizes Cat.-No extractions Cat.-No extractions Required lab devices & reagents Shelf Life and Storage Compliance Vacuum pump Micro centrifuge Filtration system, 47 mm frit Pipetting equipment and filtered tips Incubator (37 C for petri dishes, 56 C for reaction tubes) Ethanol ( %) Components are maintainable at room temperature for at least 6 months. AFNOR XP T and ISO/TS 12869:2012 in combination with AquaScreen qpcr kits

12 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 ethical 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 Target Sensitivity Content Sample Requirements Intended Use Time to Result Storage Real Time Cycler The assay is based on the TaqMan principle and worked with FAM and HEX labled probes. The target sequence is a mitochondrial multi-copy gene (cytochrome b). Therefore, even very small amounts of DNA can lead to positive results. 1 Genom Unit/PCR, 10 DNA copies/pcr Master Mix, Primer Probe Mix, Rehydration Buffer PCR Grade Water Internal Control Positiv Control 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. For research only! Not for use in diagnostic procedures. 90 minutes Components are maintainable at +2 to +8 C. After rehydratisation the reagents must be stored at -18 C. 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)

13 Related products Food Control qpcr Detect foodborne pathogens with easy interpretable lateral flow evaluation. Features Target Sensitivity Principle Content Sample Requirements Intended Use Time to Result Cycler Salmonella enterica invasion protein (inva) gene Yersinia enterocolitica 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 Escherichia coli 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 Down to 10 DNA copies/assay. TaqMan assay based on FAM and HEX labeled probes. qpcr Mix Species Mix Rehydration Buffer PCR Grade Water Internal Control Positive Control Isolated total DNA from potentially contaminated food serves here as starting material, typically after pre-cultivation of the sample growth medium. For research use only! 150 minutes 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)

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