PATHfinder User Guide
Contents
1. Vibrio splendidus ATCC 33125 Vibrio vulnificus ATCC 27562 Yersinia enterocolitica subsp Enterocolitica ATCC 23715 Yersinia pseudotuberculosis ATCC 29833 According to scientific literature only HEX channel amplification was shown by 147 strains of Legionella pneumophila belonging to 2 15 serogroups and no amplification was shown by 50 Legionella spp not pneumophila strains whereas 257 strains of L pneumophila SG1 gave positive amplification in both FAM and HEX channel Reference strains used in QC operations are e L Pneumophila SG1 ATCC 33152 as positive control for FAM e L Pneumophila SG6 DSM 25182 as positive control for HEX User guide PATHfinder Legionella pneumophila Serogroup 1 ID Assay Rev 1 GSeneren Advanced Transfer Technologies 8 Troubleshooting Concomitant no target nor IAC amplification or amplification plots grossly abnormal Possible causes and corrective actions e An excess of DNA in the target might inhibit the reaction and IAC may be affected due to an excess of DNA and or PCR inhibitors Test samples diluted 1 10 and 1 100 Please use DNase RNase Free Water to prepare dilutions Inadequate sealing of optical caps film caused sample evaporation Redo the analysis using proper tools and proper optical caps film to secure perfect sealing Did not use the proper consumables Redo the analysis and use only optical grade 96 well plates and optical adhesive seal or optical 8 well strips and2. caps Samples were not properly prepared Remake the sample DNA preps Ensure that the DNA extraction method is properly performed Positive Control reactions failed to amplify but other reactions appear correct e g the IAC is amplified Positive Control DNA was not added to the reaction wells If other reactions look normal there may be no need to repeat the run Ill Negative Control reactions are positive e Contamination of the DNase RNase Free Water vial or the PATHfinder PCR mix with PATHfinder positive DNA Use more care to prevent contamination while handling assay reagents and setting up assays In case support is needed contact Generon at support generon it 9 Disclaimers The product is intended for research use only Generon makes no warranty of any kind either expressed or implied except that the materials from which its products are made of standard quality If any materials are defective Generon will provide a replacement product Generon shall not be liable for any damages including special or consequential damage or expense arising directly or indirectly from the use of this product Please do not interchange components between assays of different lot numbers This assay is designed to be used by laboratory personnel following the common molecular biology precautions User guide PATHfinder Legionella pneumophila Serogroup 1 ID Assay Rev 1 9 ENER N Advanced Transfer Technologies Quick Re
3. Gseneren Advanced Transfer Technologies PATHfinder Legionella pneumophila Dual ID assay Cat n PMB15D SG User Guide G Sennen Advanced Transfer Technologies 1 Introduction The genus Legionella is a pathogenic group of Gram negative bacteria that includes the species L pneumophila causing Legionellosis including a pneumonia type illness called Legionnaires disease and a mild flu like illness called Pontiac fever Legionella species typically exist in nature at low concentrations it has been found in groundwater lakes and streams In the right environmental conditions it may reproduce and reach high concentrations per liter especially in plumbing and cooling towers Legionella is traditionally detected by culture on buffered charcoal yeast extract BCYE agar Legionella requires the presence of cysteine and iron to grow and therefore does not grow on common blood agar media used for laboratory based total viable count After incubation for up to 10 days suspect colonies are confirmed as Legionella if they grow on BCYE containing cysteine but not on agar without cysteine added Legionella is common in many environments including soil and aquatic systems with at least 53 species and 70 serogroups identified L pneumophila is responsible for more than 90 of clinical cases and among the 15 serogroups SG characterized within the species L pneumophila SG1 is responsible for about 85 of all cases worldwide Given this epid
4. der WORKING Mastermix with the aim of homogenizing the mix and excluding MgCl gradient that could impair the results Spin to collect contents at the bottom of the vial Note label GENERase PLUS vials with target name after OLIGO Mix addition Vortex briefly Positive Control and samples before proceeding further spin to collect contents at the bottom of the vial Transfer PATHfinder WORKING Mastermix and samples into the plate as follows Reagents per well Volume Unknown Sample extracted DNA or colony Positive Control Sul Negative Control PATHfinder WORKING Mastermix 15 pl Final Volume 20 pl Detector Setup Target Reporter Dye Quencher Dye Legionella pneumophila Serogroup 1 FAM BHQ1 NFQ Legionella pneumophila Serogroup 2 15 HEX BHQ1 NFQ IAC Internal Positive Control Cy5 BHQ2 NFQ According to your thermocycler you can replace HEX detector in the plate setting with VIC or JOE in case your own Real Time Platform does not possess the HEX reading channel User guide PATHfinder Legionella pneumophila Serogroup 1 ID Assay Rev 1 10 ENER N Advanced Transfer Technologies Quick Reference Guide Thermal cycling Duration UNG 50 10 min Taq Activation 95 10 min Denaturation 95 15 sec Annealing Extension Plate Reading 60 60 sec The thermal profile presented above was optimized for GENERase Mastermix Results analysis If the following conditions are met TEST L pneumophila SG1 FAM L pneumophila SG2 15 HEX Int
5. e damages possibly resulting from the disclosure or transmission of the data themselves to third parties under any form or circumstance This clause is automatically accepted by the user when purchasing the products The patent for performing PCR is held by Hoffmann La Roche Authorization to use PCR can be obtained on licence from Hoffmann LaRoche The product equipment and information included in the assay consists of assembled and reagents The licence and licence and authorisation for PCR use are not included in the assay The user is responsible for setting prefixed goals choosing whether or not to perform the PCR reaction and to apply for register his own licence The use assay is designed for the services supply quality control or any other application that is not exclusively an internal company s research and requires a specific licence for PCR use This PCR use licence to supply a service on food analysis field has to be requested directly from Applied Biosystems This assay requires the use of Taq Polymerise enzyme The product was internally tested by our quality control Any responsibility is waivered if the warranty of quality control does not refer to the specific product The user is personally responsible for data that he will obtained and or he will supply to third parties using this assay Once the sealed package is open the user accepts all the conditions without fail if the package is still sealed the product can be returned and the us
6. emiological background it is necessary to identify rapidly not only the presence of Legionella in water samples but simultaneously also the species and serogroups in order to estimate the risk of legionellosis New techniques for the rapid detection of Legionella in water samples are emerging including the use of polymerase chain reaction PCR This technology can typically provide much faster results This assay utilizes the polymerase chain reaction PCR to amplify a genetic target typical of the pathogen species Per Generon in house validation the LOD for PATHfinder Legionella pneumophila Serogroup 1 ID Assay was experimentally determined to be between 1 and 10 copies DNA was extracted using Bio Rad AquadienTM and Generon ION Force DNA Extractor FAST Cat HEXD001 User guide PATHfinder Legionella pneumophila Serogroup 1 ID Assay Rev 1 2 GSeneren Advanced Transfer Technologies 2 PATHfinder Legionella pneumophila Dual ID Assay When used along with GENERase Mastermix Colony ID Cat ENG001 ID this real time PCR assay detects Legionella pneumophila Serogroup 1 and Legionella pneumophila Serogroup 2 15 DNA The amplification of the target sequence is measured by the use of a specific fluorescence labeled probe FAM and HEX in less than 1 5 hours 2 1 Assay Content Box 50 reactions Box 100 reactions N vials Volume pl N vials Volume pl PATHfinder OLIGO Mix OLIGOS and Probe pre blended mix 1 250 2 250 Positive Co
7. er can be refunded User guide PATHfinder Legionella pneumophila Serogroup 1 ID Assay Rev 1 11
8. ernal Amplification Control Cy5 Positive Control Not significative Negative Control Then the possible results for any sample are TEST L pneumophila SG1 FAM L pneumophila SG2 15 HEX Internal Amplification Control Cy5 Positive SG1 Not significative Positive SG2 15 Not L Pneumophila In case of inhibition DNA isolation and purification for the sample need to be improved or you may need to dilute your sample before performing a new test Refer to the Troubleshooting paragraph section 8 in user manual for further suggestions Warning and Precaution Please do not interchange components of assays with different lot numbers This assay is designed to be used by laboratory personnel following the common molecular biology precautions GLP Disclaimer Generon s r l guarantees the buyer exclusively concerning the quality of reagents and of the components used to produce the Assay Generon S r l is not responsible and cannot anyway be considered responsible or jointly responsible for possible damages resulting from the utilization of the product by the user The user consciously and under his own responsibilities decides for the utilization purposes of the product and uses it the way he considers most suitable in order to reach his goals and or objectives Generon S r l is not responsible for the data resulting from the use of the products for the utilization that the user independently decides to make of them or for th
9. ference Guide Page 1 Product Line PATHfinder Part Number PMB15D SG Type Qualitative Storage Frozen Execution time about 120 minutes Expiry date see date on the packaging product validity refers to the product kept intact in its original packaging and constantly under suitable temperature conditions as mentioned above Assay Box Content Box 50 reactions Box 100 reactions N vials Volume pl N vials Volume pl PATHfinder OLIGO Mix OLIGOS and Probe pre blended mix 1 250 2 250 Positive Control 1 85 2 85 Negative Control 1 1 All reagents are supplied with a 5 of extra volume Not Provided Article GENERase PLUS Mastermix Colony ID ENG002 ID or GENERase Mastermix Colony ID ENG001 ID or equivalent The IAC Internal Amplification Control is available only with GENERase Mastermix Colony ID ENG002 ID Reaction Set Up Protect reagents from light exposure as far as OLIGO Mix reagents are photosensitive Before setting the analysis we strongly advise to leave the reagents to warm up at room temperature Vortex briefly OLIGO mix afterwards spin to collect contents at the bottom of the vials Spin GENERase PLUS Mastermix Colony ID ENG002 ID before opening it Prepare PATHfinder WORKING Mastermix by adding 250 ul of PATHfinder OLIGO Mix into each tube prefilled with 500 ul of GENERase PLUS Mastermix Colony ID ENG002 ID in order to obtain a single volume of 750 pl of PATHfinder WORKING Mastermix Vortex briefly PATHfin
10. ighing etc must be done according to GLP so that chance of cross contamination between samples is minimized It is recommended to use disposable equipment whenever possible This PATHfinder assay can be used for colony PCR experiments and allows the direct identification as L pneumophila either SG1 or SG2 15 of any colony isolated on GVPC BCYE plate to perform this technique is sufficient to pick one colony with a sterilized needle and to suspend it in 1 X Reaction Mix before running the PCR No DNA extraction is needed Colony PCR Diagram Pick a colony in ae in 1x working Mix SIE ariaa th Run PCR User guide PATHfinder Legionella pneumophila Serogroup 1 ID Assay Rev 1 5 GSeneren 5 Real Time PCR detection 5 1 Reaction setup Allow the reagents to thaw GENERase PLUS Mastermix PATHfinder OLIGO MIX Positive Control and Negative Control Vortex tubes when thawed and spin to collect contents at the bottom of the vial Mix 250 pl of PATHfinder OLIGO Mix with 500 ul of GENERase PLUS Mastermix to prepare PATHfinder Working Mastermix WMX Vortex briefly and spin down in order to homogenize the mix Transfer 15 ul of WMX into each well Add 5 ul of Negative Control into wells acting as negative controls Add 5 ul of each sample into wells testing the unknown samples When performing a Colony PCR experiment add 5 uL of DNAse RNAse Free Water for each well intended to be used for this technique and then pick one c
11. ldarius ATCC 27009 Alicyclobacillus acidoterrestris ATCC 49025 Alicyclobacillus herbarius DSM 13609 Bacillus cereus cereulide DSM 4312 Bacillus cereus ATCC 14579 Bacillus subtilis subsp Spizizenii ATCC 6633 Campylobacter coli ATCC 33559 Campylobacter fetus ATCC 27374 Campylobacter jejuni ATCC 33560 Campylobacter laridis lari ATCC 35221 Citrobacter freundi ATCC 8090 Clostridium perfringens ATCC 25768 Clostridium sporogenes ATCC 3584 Cronobacter sakazakii ATCC 29544 Edwardsiella tarda ATCC 15947 Enterobacter cloacae ATCC 13047 Escherichia coli ATCC 11775 Janthinobacterium lividum ATCC 12473 Klebsiella pneumoniae ATCC 13883 Lactococcus lactis subsp Lactis ATCC 19435 Listeria innocua ATCC 33152 Listeria ivanovi ATCC 33090 Listeria monocytogenes ATCC 19119 Morganella morgani subsp Morganii ATCC 15313 Proteus vulgaris ATCC 25830 Providencia stuartii ATCC 29905 Pseudomonas aeruginosa ATCC 29914 Pseudomonas fragi ATCC 10145 Pseudomonas syringae ATCC 4973 Pseudomonas fluorescens ATCC 19310 Salmonella enterica subsp Enterica ATCC 13525 Serratia marcescens ATCC 13076 Shewanella putrefacens ATCC 13880 Shigella boydii ATCC 8071 Shigella flexneri ATCC 8700 Shigella sonnei ATCC 29903 Staphilococcus aureus subsp Aureus ATCC 12600 Vibrio aerogenes ATCC 700797 Vibrio algynoliticus ATCC 17749 Vibrio mytili ATCC 51288 Vibrio parahaemolyticus ATCC 17802
12. ntrol 1 85 2 85 Negative Control 1 200 1 200 reagents are supplied with a 5 of extra volume We suggest to use PATHfinder Legionella pneumophila Serogroup 1 ID Assay along with the following Polymerase Enzyme Ready to use mastermix GENERase Mastermix Colony ID Cat ENG001 ID or GENERase PLUS Mastermix Colony ID Cat ENG002 ID When using this GENERase PLUS an additional detection channel HEX becomes available to detect the Internal Amplification Control IAC to excluding false negative results due to a PCR inhibition 2 2 Storage amp Expiry information Expiry date see date on the packaging product validity refers to the product kept intact in its original packaging Protect reagents from light exposure as far as OLIGO Mix reagents are photosensitive Store frozen User guide PATHfinder Legionella pneumophila Serogroup 1 ID Assay Rev 1 3 ENER N Advanced Transfer Technologies 3 Materials and equipments needed 3 1 Isolation and colony picking Material Equipment BCYE agar Sterilized Needles Toothpicks 3 2 Extraction Material Equipment Extraction Kit Vortexer Thermal Water Bath or Heat block for 1 5 ml tubes Pipette sets Pipette tips Barrier Tube rack for 1 5 ml tubes 2 0 and 1 5 ml micro tubes Micro centrifuge for 1 5 2 0 ml micro tubes 3 3 Detection via real time PCR Material Equipment Real Time PCR System 2 PATHfinder Legionella pneumophila Dual ID Assay GENERase Mas
13. olony of interest with a sterilized needle from the agar plate and suspend it inside the selected well Add 5 pl of Positive Control into wells acting as positive control Close wells and ensure no bubbles are present at the bottom of the wells Spin briefly optical PCR tubes or plates 5 2 Instrument setup With GENERase Mastermix or GENERase PLUS Mastermix set the following parameters on your thermocycler Total Reaction volume 20 ul Fluorophores Quenchers Target Legionella pneumophila Serogroup 1 FAM BHQ1 NFQ Legionella pneumophila Serogroup 2 15 HEX BHQ1 NFQ Internal Amplification Control Cy5 BHQ2 NFQ Internal Amplification Control is present only in GENERase PLUS Mastermix Depending on your thermocycler you can replace HEX detector in the plate setting with VIC or JOE in case your own Real Time Platform does not possess the HEX reading channel Thermal profile Duration UNG 10 min Taq Activation 10 min Denaturation 15 sec Annealing Extension Plate Reading 60 sec User guide PATHfinder Legionella pneumophila Serogroup 1 ID Assay Rev 1 6 ENER N Advanced Transfer Technologies 5 Data Interpretation Results evaluation must be done according to the analysis software recommended by the Real Time PCR instrument manufacturer After performing PCR each individual sample is analyzed through the instrument software to produce a Cq value quantification cycle for each reporter dye These values are used
14. termix or GENERase PLUS Mastermix 3 Optical Adhesive Seal or Optical Caps Optical reaction plate or Optical Tube Strips Micropipette sets Source Generon or other Lab suppliers Generon or other Lab suppliers Source Bio Rad Aquadien Generon or other Lab suppliers Generon or other Lab suppliers Generon or other Lab suppliers Generon or other Lab suppliers Generon or other Lab suppliers Generon or other Lab suppliers Generon or other Lab suppliers Source Generon or other Lab suppliers Generon PMB15D SG Generon ENG001 ID or ENGOO2 ID Generon or other Lab suppliers Generon or other Lab suppliers Generon or other Lab suppliers 1 Equipment necessary only when ION FORCE Extractor FAST EXD001 is used 2 The assay can be used with Biorad CFX and MiniOpticon Stratagene MxSeries ABI 7300 7500 7900 StepONE StepONE Plus Light Cycler 480 Eppendorff realplex Rotor Gene Q etc The assay is not compatible with Roche Light Cycler and II 3 GENERase PLUS Mastermix includes the IAC Internal Amplification Control a system to assess the presence of false negatives due to substances inhibiting PCR in your extracted DNA In addition the IAC is a system to check on assay performance thus confirming whether PCR reagents are working and amplifying properly User guide PATHfinder Legionella pneumophila Serogroup 1 ID Assay Rev 1 GSeneren 4 DNA extraction Each step of sample preparation grinding transferring we
15. to determine the identity Qualitative Test of pathogen into the sample See below an example of the graphics obtained for a positive Legionella pneumophila Serogroup 1 Fig A a positive Legionella pneumophila Serogroup 2 15 Fig B and for the IAC amplification Fig C Amplification T SG1 FAM HEX 7T_ f SG2 15 FAM HEX After setting the baseline the analysis outcome should be evaluated following the indications below If the following conditions are met TEST L pneumophila SG1 FAM L pneumophila SG2 15 HEX Internal Amplification Control Cy5 Positive Control Not significative Negative Control Then the possible results for any sample are TEST L pneumophila SG1 FAM L pneumophila SG2 15 HEX Internal Amplification Control Cy5 Positive SG1 Not significative Positive SG2 15 Not L Pheumophila E In case of inhibition DNA isolation and purification for the sample need to be improved or you may need to dilute your sample before performing a new test Refer to the Troubleshooting paragraph section 8 for further suggestions User guide PATHfinder Legionella pneumophila Serogroup 1 ID Assay Rev 1 GSeneren Advanced Transfer Technologies 7 Inclusivity and Excusivity Panel The following DNA extracts showed no amplification curve in a 15 pl total reaction volume Alicyclobacillus acidiphilus DSM 14558 Alicyclobacillus acidocaldarius subsp Ca
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