G01087 - gerbion
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1. g erb on Instruction Manual gastroplexBac real time PCR Kit For the in vitro detection of the DNA of Campylobacter jejuni Salmonella enterica and Listeria monocytogenes in clinical specimens environmental and food samples REF G01087 32 GO1087 96 52 96 18 C gerbion gmbH amp Co KG Remsstr 1 70806 Kornwestheim Germany phone 49 7154 806 200 fax 49 7154 806 20 29 e mail info gerbion com www gerbion com gastroplexBac Instruction Manual Version 1 1 05 09 2014 Index 1 COMO ONEN rennen a a a a aa a aaas 3 2 A ee 3 5 Mans DO ana Sterage rt A seine 3 4 mended US escuienta ae lese 3 5 A E RENO 5 6 OU OTEO AN 5 74 PROGUCE WA AN ea E EEEE EAEN 5 8 Talis to Bch dle anne eaeelersese 5 9 PERIEID ESOT U O 6 10 Equipment and Reagents to be Supplied by USEF a ada 7 Fe OA NO E en nee nn non fans 7 12 General Pe AUNO een E E e AEE EE 7 ES Saa A O IN 8 14 Control DNA RE a dba 8 E A le ee 9 Tal important Points Before Start a 9 15 2 A A A IN 9 193 Vegi gl qe Gems spn eo rn E PR OPO OE 11 Ee oO A hts Sane Ce eT Teen EA nissen 14 I OUDE NGONG eriari energie 17 LS OM AOS ee 19 gastroplexBac Instruction Manual Version 1 1 03 09 2014 1 Components The reagents supplied are sufficient for 52 or 96 reactions respectively Table 1 Components of the gastroplexBac real time PCR kit Reaction Mix Positive Control 1 Campylobacter Positive Control 2 Salmonella Positive Control 3 List2. DNA loss during isolation In case the Control DNA K6 was added during process extraction the lack of an amplification signal can indicate that the DNA isolation was not successful Make sure that you use an appropriate isolation method commercial kits are recommended and stick to the manufacturer s protocol Incorrect storage conditions Check the storage conditions and the date of expiry for one or more printed on the kit label If necessary use a new kit and components or kit expired make sure kit components are stored as described in Transport and Storage page 3 Detection of a fluorescence signal in the FAM ROX or Cy 5 channel of the Negative Control K5 Contamination during Repeat the real time PCR in replicates If the result is preparation of the PCR negative in the repetition the contamination occurred when the samples were pipetted into the optical PCR reaction tubes Make sure to pipet the Positive Controls K2 K3 K4 last and close the optical PCR reaction tube immediately after adding the sample If the same result occurs one or more of the kit components might be contaminated Make sure that work space and instruments are decontaminated regularly Use a new kit and repeat the real time PCR gastroplexBac Instruction Manual Version 1 1 05 09 2014 18 Other Products A number of products related to real time PCR and nucleic acid isolation is available from gerbion GmbH amp Co KG More information as well
3. Important Notes on page 7 Before setting up the real time PCR familiarise yourself with the real time PCR instrument and read the user manual supplied with the instrument The programming of the thermal profile should take place before the PCR set up In every PCR run at least one of each Positive Control K2 K3 K4 and one Negative Control K5 should be included Before each use all reagents should be thawed completely at room temperature thoroughly mixed do NOT vortex the Reaction Mix K1 but mix by pipetting up and down repeatedly and centrifuged very briefly Then place all reagents on ice or on a cooling block 2 to 8 C 15 2 Procedure If the Control DNA K6 is Used to control both the real time PCR and the DNA isolation procedure please follow protocol A If the Control DNA K6 is solely used to detect possible inhibition failure of the real time PCR please follow protocol B Protocol A The Control DNA K6 was added during DNA extraction see Control DNA page 8 In this case prepare the Master Mix on ice or in a cooling block 2 to 8 C according to Table 2 The Master Mix contains all of the components needed for PCR except the sample Prepare a volume of Master Mix for at least one sample more than required in order to compensate for pipetting inaccuracy gastroplexBac Instruction Manual Version 1 1 05 09 2014 10 Table 2 Preparation of the Master Mix Control DNA K6 was added during DNA ex
4. as the complete Product Catalogue is available on www gerbion com Product NukEx Pure RNA DNA NukEx PLUS 2 0 NukEx Collection Tubes NukEx Universal Dilution Buffer NukEx Pestle 1 5 ml NukEx TS Proteinase K Description Spin column based kit for the isolation of RNA and DNA from a variety of sample matrices For 50 or 200 extractions Reagent for the enzymatic release of nucleic acids from swabs and cell culture Suspensions Very fast and convinient protocol Including NukEx Stop for chemical inactivation 500 NukEx Collection Tubes for use with NukEx Spin Columns Diluent for samples for real time RT PCR 100 disposable PBTP pestles for use in 1 5 ml reaction tubes Individually packed DNase free RNase free non pyrogenic Shredding material aliquoted in 1 5 or 2 0 ml safe lock tubes or 2 0 ml screw cap tubes for the manual or automated preparation of Samples such as tissue or insects Proteinase K from Tritirachium album 100 mg 19 Cat No GO5004 50 605004 200 G05016 G06008 GO1014 G06006 606007 1 5 G06005 2 0 G06005 2 0 SC 607001 gastroplexBac Instruction Manual Version 1 1 03 09 2014
5. illness while symptoms caused by enteritis Salmonella such as Salmonella enteritidis and Salmonella thyphimurium remain mild only infants and immune suppressed patients are likely to develop severe illness Listeria are gram positive nonsporeforming catalase positive rods The major human pathogen in the Listeria genus is L monocytogenes which is commonly found in soil stream water sewage plants and food It is usually gastroplexBac Instruction Manual Version 1 1 05 09 2014 the causative agent of listeriosis a serious infection caused by eating food contaminated with the bacteria The overt form of the disease has a mortality rate of about 20 percent The two main clinical manifestations are sepsis and meningitis Meningitis is often complicated by encephalitis a pathology that is unusual for bacterial infections However recent studies found that the consumption of food contaminated with Listeria can lead to severe gastroenteritis even in healthy people In these cases listerioses is often not diagnosed and subsequentely not treated in a targeted way Secondary symptoms can occur weeks later and their connection to the Listeria infection are often not recognised Although Listeria monocytogenes has low infectivity it is hardy and can grow in temperatures from 4 C to 57 C The disease affects primarily pregnant women newborns adults with weakened immune systems and the elderly Prompt treatment of listeria infections in pregnan
6. information about DNA isolation is to be found in the extraction kit manual or from the extraction kit manufacturer s technical service 14 Control DNA K6 The gastroplexBac real time PCR Kit contains a Control DNA K6 which allows the user to control the DNA isolation procedure and to check for possible real time PCR inhibition Control DNA K6 used as Extraction Control gastroplexBac Control DNA K6 is added prior to the DNA extraction To this end multiply the buffer volume needed per extraction with the number of samples including at least one water control N plus 1 to compensate for inaccuracies in pipetting N 1 Add 5 ul Control DNA K6 per extraction 5 ul x N 1 Mix well Perform the DNA isolation according to the manufacturer s instructions gastroplexBac Instruction Manual Version 1 1 05 09 2014 If the extraction protocol includes an incubation step of the sample in the first buffer the Control DNA K6 is to be added to each sample individually after incubation The Control DNA K6 must not be added to the sample material directly Control DNA K6 used as Internal Control of the real time PCR If the control of the DNA extraction is not desired the Control DNA K6 can be used as Internal Control of the real time PCR only To that end the Control DNA K6 is to be added directly to the real time PCR Master Mix 15 Real time PCR 15 1 Important Points Before Starting Please pay attention to the
7. Campylobacter jejuni Salmonella enterica Control DNA Listeria monocytogenes Campylobacter jejuni Salmonella enterica Control DNA Listeria monocytogenes Campylobacter jejuni Salmonella enterica Control DNA Listeria monocytogenes Campylobacter jejuni Salmonella enterica Control DNA Listeria monocytogenes Detection channel 485 5535 558 610 525 568 615 670 Green Orange Yellow Red Notes Color Compensation Kit Multiplex 1 GO7OMP1 cc required Reference Dye None Option Reference Dye ROX NO gastroplexBac Instruction Manual Version 1 1 05 09 2014 14 16 Data Analysis The Listeria monocytogenes specific amplification is measured in the Cy 5 channel the Salmonella enterica specific amplification in the ROX channel and the Campylobacter jejuni specific amplification in the FAM channel The amplification of the Control DNA K6 is measured in the VIC HEX JOE TET channel Following results can occur A signal in the FAM channel is detected The result is positive the sample contains Campylobacter jejuni DNA In this case detection of a signal of the Control DNA K6 in the VIC HEX JOE TET channel is inessential as high concentrations of virus DNA may reduce or completely inhibit amplification of the Control DNA K6 A signal in the ROX channel is detected The result is positive the sample contains Salmonella enterica DNA In this case detection of a signal of the C
8. alysis of the analysis does not comply Campylobacter jejuni specific amplification the ROX with the protocol channel for the Salmonella enterica specific amplification the Cy 5 channel for the Listeria monocytogenes specific amplification and the VIC HEX JOE TET channel for the amplification of the Control DNA K6 Incorrect configuration of Check your work steps and compare with Procedure the real time PCR on page 9 The programming of the Compare the thermal profile with the protocol Table thermal profile is incorrect 5 page 11 Incorrect storage conditions Check the storage conditions and the date of expiry for one or more kit printed on the kit label If necessary use a new kit and components or kit expired make sure kit components are stored as described in Transport and Storage page 3 Weak or no signal of the Control DNA K6 and simultaneous absence of a signal in the FAM ROX or Cy 5 channel real time PCR conditions do Check the real time PCR conditions page 9 not comply with the protocol real time PCR inhibited Make sure that you use an appropriate isolation method see Isolation of DNA page 8 and follow the manufacturer s instructions Make sure that the ethanol containing wash buffer of the isolation kit has been completely removed An additional centrifugation step at high speed is recommended before elution of the DNA gastroplexBac Instruction Manual Version 1 1 03 09 2014 18
9. cy is critical to prevent the bacteria from infecting the fetus Higher doses of antibiotics are sometimes given to pregnant women to ensure penetration of the umbilical cord and placenta Listeria infection of the fetus can lead to abort or severe damages of the organs liver lung brain skin Bacteria of the genus Campylobacter are gram negative spirally bent rods which can be distinguished from Enterobacteria by their positive oxidase and catalase reactions Together with Salmonella Campylobacter is the most common bacterial cause for diarrhoea in Europe The Campylobacter species found most frequently is C jejuni It mainly causes diahrroea but secondary conditions such as reactive Arthritis or Guillain Barre Syndrome can also occur Pets soil and contaminated drinking water are a permanent reservoir for Campylobacter The transmission however mainly happens through contaminated food e g raw meat and poultry meat as well as unpasteurised milk After a 1 5 5 day long incubation period acute enteritis with watery later bloody diarrhoea and abdominal pains occurs Asymptomatic infections are common however in 10 20 of all patients protracted symptoms arise and 5 10 suffer from relapses Septic generalisation occurs when C jejuni reaches the blood stream 9 Principle of the Test The gastroplexBac real time PCR Kit contains specific primers and probes labelled with a fluorescent dye for the analysis of the DNA of Campylobacter je
10. ed as potentially infectious material and all equipment used has to be treated as potentially contaminated 12 General Precautions Stick to the protocol described in the Instruction Manual Set up different laboratory areas for the preparation of samples and for the set up of the PCR in order to avoid contaminations Pipettes tubes and other materials must not circulate between those different laboratory areas gastroplexBac Instruction Manual Version 1 1 05 09 2014 Always use filter tips Regulary decontaminate equipment and benches with ethanol free decontaminant Do not combine gastroplexBac real time PCR Kit components of different lot numbers 13 Isolation of DNA The gastroplexBac real time PCR is suitable for the detection of Campylobacter jejuni Salmonella enteric and Listeria monocytogenes DNA isolated or released from clinical specimens e g Stool samples blood pus with appropriate isolation methods Commercial kits for DNA isolation are recommended e g NukEx Pure RNA DNA gerbion Cat No GO5004 Important In addition to the samples always run a water control in your extraction possible contaminations during DNA extraction will be detectable Treat this water control analogous to a sample Please note the chapter Control DNA on page 8 If the real time PCR is not performed immediately store extracted DNA according to the instructions given by the DNA extraction kit s manufacturer Further
11. eria Negative Control Control DNA Lid Colour 32 1x5121l 1x 50 ul 1x50 ul 1x50 ul 1x50ul 1x 160 ul 96 2 x 768 ul 1x 100 ul 1 x 100 ul 1 x 100 ul 1x 100 ul 2 x 240 ul 2 Abbreviations PCR Polymerase Chain Reaction DNA Deoxyribonucleic acid 3 Transport and Storage The gastroplexBac real time PCR Kit is shipped on dry ice All components must be stored at 18 C in the dark immediately after receipt Do not use reagents after the date of expiry printed on the package After initial usage reagents are stable for up to six months To avoid a loss of sensitivity the reagents should not be thawed and frozen more than two times If necessary aliquot kit components K1 K2 K3 K4 and K6 4 Intended Use The gastroplexBac real time PCR Kit is a screening assay for the detection of the Campylobacter jejuni Salmonella enterica and Listeria monocytogenes in clinical specimens e g stool samples blood pus environmental and food samples gastroplexBac Instruction Manual Version 1 1 05 09 2014 gastroplexBac Instruction Manual Version 1 1 03 09 2014 5 Sample Material Starting material for the assay is DNA isolated or released from clinical specimens e g stool samples blood pus environmental and food samples 6 Quality Control In accordance with gerbion s ISO certified Quality Management System each lot of the gastroplexBac real time PCR kit is tested against predetermined specifications to ens
12. juni Salmonella enterica and Listeria monocytogenes isolated or released gastroplexBac Instruction Manual Version 1 1 05 09 2014 from clinical specimens e g stool samples blood pus environmental or food samples The detection of the amplification is carried out in real time via hybridization and subsequent hydrolysis of the pathogen specific fluorescent probes The fluorescences are measured in the FAM Campylobacter jejuni ROX Salmonella enterica and Cy 5 channels Listeria monocytogenes Furthermore the gastroplexBac real time PCR Kit contains a Control DNA K6 which is detected in a heterologous amplification system Added during DNA extraction the Control DNA K6 allows not only for the detection of PCR inhibition but also detects possible mistakes during DNA extraction This greatly reduces the risk of false negative results The amplification of the Control DNA K6 is measured in the VIC HEX JOE TET channel 10 Equipment and Reagents to be Supplied by User DNA isolation kit e g NukEx Pure RNA DNA gerbion Cat No GOSO04 Sterile microtubes Pipets adjustable volume Sterile pipet tips with filter Table centrifuge Vortexer real time PCR instrument Optical PCR reaction tubes with lid Optional Liquid handling system for automation 11 Important Notes The gastroplexBac real time PCR must be performed by qualified personnel only Good Laboratory Practice GLP has to be applied All samples must be regard
13. ntrol DNA K6 was added during DNA isolation and not directly to the PCR Master Mix the Negative Control K5 is negative in both channels gastroplexBac Instruction Manual Version 1 1 05 09 2014 16 Figure 1 and Figure 2 show examples for positive and negative real time PCR results Fluorescence GR 2 4 6 8 10 12 14 6 18 20 22 24 26 26 30 2 N 3506 38 HO 2 4 Cycles Figure 1 The positive sample shows bacteria specific amplification in the FAM channel whereas no fluorescence signal is detected in the negative sample Fluorescence GR 2 4 6 8 10 12 14 10 18 2 2 2 2 28 30 32 4 BH RR A 2 4 Cycles Figure 2 The positive sample as well as the negative sample show a signal in the Control DNA specific VIC HEX JOE TET channel The amplification signal of the Control DNA K6 in the negative sample shows that the missing signal in the bacteria specific FAM channel is not due to PCR inhibition or failure of DNA isolation but that the sample is a true negative gastroplexBac Instruction Manual Version 1 1 05 09 2014 17 17 Troubleshooting The following troubleshooting guide is included to help you with possible problems that may arise when performing a real time PCR If you have further questions please do not hesitate to contact our scientists on info gerbion com No fluorescence signal in the FAM ROX Cy 5 channel of the Positive Controls K2 K3 K4 The selected channel for Select the FAM channel for an
14. ontrol DNA K6 in the VICO HEX JOE M TET channel is inessential as high concentrations of bacterial DNA may reduce or completely inhibit amplification of the Control DNA K6 A signal in the CY 5 channel is detected The result is positive the sample contains Listeria monocytogenes DNA In this case detection of a signal of the Control DNA K6 in the VICO HEX JOE M TET channel is inessential as high concentrations of bacterial DNA may reduce or completely inhibit amplification of the Control DNA K6 No signal in the FAM ROX and Cy 5 channel but a signal in the VIC HEX JOE TET channel is detected The result is negative the sample does neither contain Campylobacter jejuni DNA nor Salmonella enterica DNA nor Listeria monocytogenes DNA The signal of the Control DNA K6 excludes the possibilities of DNA isolation failure in case the Control DNA K6 is being used as an Extraction Control and or real time PCR inhibition If the C value of a sample differs significantly from the C value of the water control a partial inhibition occured which can lead to negative results in weak positive samples see Troubleshooting page 17 gastroplexBac Instruction Manual Version 1 1 03 09 2014 15 Neither in the FAM ROX Cy 5 nor in the VIC HEX JOE TET channel a Signal is detected A diagnostic statement cannot be made The DNA isolation was not successful or an inhibition of the PCR has occurred In case the Co
15. ration of the real time PCR Component Volume Master Mix Sample Total Volume 15 3 Instrument Settings For the real time PCR use the thermal profile shown in Table 5 Table 5 real time PCR thermal profile Discription Temperature Number of Cycles Initial Denaturation 95 C 1 Amplification of DNA Denaturation 10 sec 95 C Annealing and Extension AO sec BOT Aquisition at the end of this Step Samples can be tested for pathogens with a RNA genome in the same PCR run e g with the gastroplexVirus real time RT PCR Kit when a reverse transcription step is run prior to the amplification cycles The thermal profile has to be programmed according to Table 6 gastroplexBac Instruction Manual Version 1 1 05 09 2014 12 Table 6 real time RT PCR thermal profile Discription Temperature Number of Cycles Reverse Transcription 45 C 1 Initial Denaturation IC 1 Amplification of DNA Denaturation 10 sec J gt E Annealing and Extension 40 sec 60 C Aquisition at the end of this step Dependent on the real time instrument used further instrument settings have to be adjusted according to Table 7 gastroplexBac Instruction Manual Version 1 1 05 09 2014 13 Table 7 Overview of the instrument settings required for the gastroplexBac real time PCR Real time PCR Instrument LightCycler 480I Stratagene Mx3000P Mx3005P ABI 7500 Rotor Gene Q Rotor Gene 3000 Rotor Gene 6000 Parameter
16. traction 16 0 ul Reaction Mix K1 16 0 ul x N 1 0 0 ul Control DNA K6 0 0 ul x N 1 Protocol B The Control DNA K6 is used for the control of the real time PCR only see Control DNA page 8 In this case prepare the Master Mix on ice or ina cooling block 2 to 8 C according to Table 3 The Master Mix contains all of the components needed for PCR except the sample Prepare a volume of Master Mix for at least one sample more than required in order to compensate for pipetting inaccuracy Table 3 Preparation of the Master Mix Control DNA K6 is added directly to the Master Mix 16 0 ul Reaction Mix K1 16 0 ul x N 1 0 5 ul Control DNA K6 OS Mc Ni The increase in volume caused by adding the Control DNA K6 is not taken into account when preparing the PCR assay The sensitivity of the detection system is not impaired Protocol A and B real time PCR set up Put the number of optical PCR reaction tubes needed into the cooling block e Pipet 16 ul of the Master Mix into each optical PCR reaction tube Add 4 ul of the eluates from the DNA isolation including the eluate of the water control the Positive Controls K2 K3 K4 and the Negative Control K5 to the corresponding optical PCR reaction tube Table 4 Close the optical PCR reaction tubes immediately after filling in order to reduce the risk of contamination gastroplexBac Instruction Manual Version 1 1 03 09 2014 11 Table 4 Prepa
17. ure consistent product quality 7 Product Warranty gerbion guarantees the performance of all products when used according to the instructions given in the Instruction Manual The purchaser must determine the suitability of the product for its particular use Should any product fail to perform satisfactorily due to any reason other than misuse gerbion will replace it free of charge or refund the price We reserve the right to change alter or modify any product to enhance its performance and design 8 Introduction Gastroenteritis or infectious diarrhea is an inflammation of the gastrointestinal tract Both the stomach and the small intestine are involved Typical symptoms are diarrhoea vomiting abdominal pain and cramps often followed by dehydration The causative agent can be viral or bacterial Salmonella are gram negative bacteria found worldwide in cold and warm blooded animals including humans as well as in the environment Infections with Salmonella called salmonellosis are zoonotic i e they can be transmitted from animals to humans and the other way round Typical symptoms of salmonellosis are diarrhoea fever vomiting and abdominal cramps 12 to 72 hours after infection Most infections are due to ingestion of contaminated food lt can be differentiated between enteritis Salmonella and typhoid paratyphoid Salmonella the latter because of a special virulence factor and a capsule protein virulence antigen can cause serious
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