G01022 - gerbion
Contents
1. Instruction Manual diarellaSalmonella real time PCR Kit For the in vitro detection of the DNA of Salmonella enterica in clinical specimens or environmental samples al m LL G01022 32 G01022 96 a ZM 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 E diarellaSalmonella Instruction Manual Version 1 4 27 10 2014 Index 1 COMO ONEN irana ea a a a a aa e a aaas 3 2 PUB Fel OS enr e ero E 5 5 Mans DO ana SOTO erpinen a A AAA A 5 4 A a a a 5 5 same AS rs ne et ARO ES 6 A O 4 74 A E EEEE EE AEA 4 8 Talis ola Bc dle a a S 4 9 PECE OFEA O Deen Mer ae cer neat re rROT aT a a a ORV TSR OT ETT ren Ie tern 4 10 Equipment and Reagents to be Supplied BY Use iia liada 3 La OA NO E or ia 5 E a A A Aree atte e 5 ES ASCANIO od 6 14 contol DNATA naa a E diana 6 E RE Re A a A ea Andrenaa east ata staan teens 7 Es all important Points Before Start icon 7 15 2 A A 7 G e AA e a N I 10 IZ TOUDE noon E e oi III tent nets 12 TS Qer POUC ogis ea cas ERA tt 14 diarellaSalmonella Instruction Manual Version 1 4 27 10 2014 1 Components The reagents supplied are sufficient for 52 or 96 reactions respectively Table 1 Components of the diarellaSalmonella real time PCR Kit Lid Colour Content 32 96 Reaction Mix LS al 2 x 768 ul Positive Control 1x 50 ul 1x 100 ul Negative Control2. K4 used as Extraction Control diarellaSalmonella Control DNA K4 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 K4 per extraction 5 ul x N 1 Mix well Perform the DNA isolation according to the manufacturer s instructions If the extraction protocol includes an incubation step of the sample in the first buffer the Control DNA K4 is to be added to each sample individually after incubation The Control DNA K4 must not be added to the sample material directly diarellaSalmonella Instruction Manual Version 1 4 27 10 2014 Control DNA K4 used as Internal Control of the real time PCR If control of the DNA extraction is not desired the Control DNA K4 can be used as Internal Control of the real time PCR only To that end the Control DNA K4 is to be added directly to the real time PCR Master Mix Set up the real time PCR according to protocol B 15 Real time PCR 15 1 Important Points Before Starting Please pay attention to the Important Notes on page 5 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 Positive Co
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 K4 is added directly to the Master Mix 16 0 ul Reaktion Mix K1 16 0 ul x N 1 0 5 ul Control DNA K4 0 5 ul x N 1 The increase in volume caused by adding the Control DNA K4 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 and the Negative Control K3 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 diarellaSalmonella Instruction Manual Version 1 4 27 10 2014 Table 4 Preparation of the real time PCR Master Mix Sample Total Volume For the real time PCR use the thermal profile shown in Table 5 Table 5 real time PCR thermal profile Initial Denaturation Amplification of DNA Denaturation 10 sec 95 C Annealing and Extension AO sec 60 C Aquisition at the end of this
4. in real time via hybridization and subsequent hydrolysis of the pathogen specific fluorescent probes The fluorescence is measured in the FAM channel diarellaSalmonella Instruction Manual Version 1 4 27 10 2014 Furthermore the diarellaSalmonella real time PCR Kit contains a Control DNA K4 which is detected in a second amplification system Added during DNA extraction the Control DNA K4 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 K4 is meassured 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 GO5004 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 diarellaSalmonella real time PCR must be performed by qualified personnel only Good Laboratory Practice GLP has to be applied All samples must be regarded 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
5. step diarellaSalmonella Instruction Manual Version 1 4 27 10 2014 10 16 Data Analysis The Salmonella enterica specific amplification is measured in the FAM channel The amplification of the Control DNA K4 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 Salmonella enterica DNA In this case detection of a signal of the Control DNA K4 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 K4 No signal in the FAM channel but a signal in the VIC HEX JOE TET channel is detected The result is negative the sample does not contain Salmonella enterica DNA The signal of the Control DNA K4 excludes the possibilities of DNA isolation failure in case the Control DNA K4 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 12 Neither in the FAM 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 Control DNA K4 was added during DNA i
6. 0 2014 13 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 channel of the Negative Control K3 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 Control 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 diarellaSalmonella Instruction Manual Version 1 4 27 10 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 as the complete Product Catalogue is available on www gerbion com Product NukEx Pure RNA DNA NukEx Collection Tubes NukEx PLUS 2 0 NukEx Universal Dilution Buffer NukEx Pestle 1 5 ml NukEx TS Proteinase K g
7. 1x 50 ul 1x 100 ul Control DNA 1x 160 ul 2 x 240 ul 2 Abbreviations PCR Polymerase Chain Reaction DNA Deoxyribonucleic acid 3 Transport and Storage The diarellaSalmonella 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 and K4 4 Intended Use The diarellaSalmonella real time PCR Kit is an assay for the detection of the DNA of Salmonella enterica in clinical specimens or environmental samples using real time PCR microplate systems e g Applied Biosystems Stratagene Corbett Research 5 Sample Material Starting material for the assay is DNA isolated or released from clinical specimens or environmental samples diarellaSalmonella Instruction Manual Version 1 4 27 10 2014 6 Quality Control In accordance with gerbion s ISO certified Quality Management System each lot of the diarellaSalmonella real time PCR Kit is tested against predetermined specifications to ensure 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 pr
8. and the with the protocol VIC HEX JOE TET channel for the amplification of the Control DNA K4 Incorrect configuration of Check your work steps and compare with Procedure the real time PCR on page 7 The programming of the Compare the thermal profile with the protocol Table thermal profile is incorrect 5 page 9 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 K4 and simultaneous absence of a signal in the bacteria specific FAM channel real time PCR conditions do Check the real time PCR conditions page 7 not comply with the protocol real time PCR inhibited Make sure that you use an appropriate isolation method see Isolation of DNA page 6 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 DNA loss during isolation In case the Control DNA K4 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 diarellaSalmonella Instruction Manual Version 1 4 27 1
9. astroplexBac real time PCR Kit Description Spin column based kit for the isolation of RNA and DNA from a variety of sample matrices For 50 or 200 extractions 500 NukEx Collection Tubes for use with NukEx Spin Columns 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 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 For the in vitro detection of the DNA of Campylobacter jejuni Salmonella enterica and Listeria monocytogenes in clinical specimens environmental and food samples 14 Cat No GO5004 50 605004 200 06008 G05016 G01014 G06006 606007 1 5 G06005 2 0 G06005 2 0 sc 607001 GO1087 diarellaSalmonella Instruction Manual Version 1 4 27 10 2014
10. ntrol K2 and one Negative Control K3 should be included Before each use all reagents should be thawed completely at room temperature thouroughly 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 K4 is used to control both the real time PCR and the DNA isolation procedure please follow protocol A If the Control DNA K4 is solely used to detect possible inhibition failure of the real time PCR please follow protocol B Protocol A The Control DNA K4 was added during DNA extraction see Control DNA page 6 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 diarellaSalmonella Instruction Manual Version 1 4 27 10 2014 Table 2 Preparation of the Master Mix Control DNA K4 was added during DNA extraction 16 0 ul Reaktion Mix K1 16 0 ul x N 1 0 0 ul Control DNA K4 0 0 ul x N 1 Protocol B The Control DNA K4 is used for the control of the real time PCR only see Control DNA page 6 In this case prepare the Master Mix on ice or in a cooling block 2 to 8 C according to Table 3
11. oduct 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 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 It 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 illness while symptoms caused by enteritis Salmonella such as Salmonella enteritidis and Salmonella thyphimurium remain mild only infants and immuno suppressed patients are likely to develop severe illness 9 Principle of the Test The diarellaSalmonella real time PCR Kit contains specific primers and probes labelled with a fluorescent dye for the analysis of the DNA of Salmonella enterica isolated or released from clinical specimens or environmental samples The detection of the amplification is carried out
12. order to avoid contaminations Pipettes tubes and other materials must not circulate between those different laboratory areas Always use filter tips Regulary decontaminate equipment and benches with ethanol free decontaminant Do not combine diarellaSalmonella real time PCR Kit components of different lot numbers diarellaSalmonella Instruction Manual Version 1 4 27 10 2014 13 Isolation of DNA The diarellaSalmonella real time PCR is suitable for the detection of Salmonella enterica DNA isolated from clinical specimens or environmental samples with appropriate isolation methods Commercial kits for DNA isolation are recommended e g e 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 6 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 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 K4 The diarellaSalmonella real time PCR Kit contains a Control DNA K4 which allows the user to control the DNA isolation procedure and to check for possible real time PCR inhibition Control DNA
13. solation and not directly to the PCR Master Mix the Negative Control K3 is negative in both channels diarellaSalmonella Instruction Manual Version 1 4 27 10 2014 11 Figure 1 and Figure 2 show examples for positive and negative real time PCR results 2 4 6 8 10 12 14 16 18 20 22 24 26 26 30 32 N 3506 38 WO 2 4 Cycles Figure 1 The positive sample shows specific amplification in the FAM channel whereas no fluorescence signal is detected in the negative sample Fluorescence GR 2 4 6 8 0 2 4 06 18 20 22 24 26 28 320 32 N 356 BW Q y 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 K4 in the negative sample shows that the missing signal in the specific FAM channel is not due to PCR inhibition or failure of DNA isolation but that the sample is a true negative diarellaSalmonella Instruction Manual Version 1 4 27 10 2014 12 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 channel of the Positive Control K3 The selected channel for Select the FAM channel for analysis of the Salmonella analysis does not comply enterica specific amplification
Download Pdf Manuals
Related Search