Vibrio cholera O1 Real Time PCR Kit User Manual For In
Contents
1. Liferiver Revision No ZJO007 Issue Date Oct 1 2012 Vibrio cholera O1 Real Time PCR Kit User Manual For In Vitro Diagnostic Use Only DD 0123 01 For use with LightC ycler1 0 2 0 Instrument e Obelis S A Boulevard G n ral Wahis 53 1030 Brussels BELGIUM Tel 32 2 732 59 54 Fax 32 2 732 60 03 E Mail mail obelis net CE wal Shanghai ZJ Bio Tech Co Ltd www liferiver com cn Tel 86 21 34680596 trade liferiver com cn Fax 86 21 34680595 2 floor No 15 Building No 188 Xinjunhuan road PuJiang Hi tech Park Shanghai China 1 Intended Use Vibrio cholera O1 real time PCR kit is used for the detection of Ol type Cholera Vivrion in stool or water samples by using real time PCR systems 2 Principle of Real Time PCR The principle of the real time detection is based on the fluorogenic 5 nuclease assay During the PCR reaction the DNA polymerase cleaves the probe at the 5 end and separates the reporter dye from the quencher dye only when the probe hybridizes to the target DNA This cleavage results in the fluorescent signal generated by the cleaved reporter dye which is monitored real time by the PCR detection system The PCR cycle at which an increase in the fluorescence signal is detected initially Ct is proportional to the amount of the specific PCR product Monitoring the fluorescence intensities during Real Time allows the detection of the accumulating product without having to re open the reac2. Storage and transportation e Collect samples in sterile tubes e Specimens can be extracted immediately or frozen at 20 C to 80 C e Transportation of clinical specimens must comply with local regulations for the transport of etiologic agents 9 Procedure 9 1 DNA Extraction DNA extraction buffer is supplied in the kit Please thaw the buffer thoroughly and spin down briefly in the centrifuge before use 9 1 1 stool samples 1 Take about 50mg samples to a 1 5ml tube add 1 0ml normal saline then vortex vigorously Centrifuge the tube at 13000rpm for 2 minutes carefully remove and discard supernatant from the tube without disturbing the pellet 2 Add 100u1 DNA extraction buffer close the tube then resuspend the pellet with vortex vigorously Spin down briefly in a table centrifuge 3 Incubate the tube for 10 minutes at 100 C 4 Centrifuge the tube at 13000rpm for 5 minutes The supernatant contains the DNA extracted and can be used for PCR template 9 1 2 Water samples 1 Take 1 5 ml water to a tube Centrifuge the tube at 13000rpm for 2 minutes carefully remove and discard supernatant from the tube without disturbing the pellet 2 Add 50u DNA extraction buffer close the tube then vortex for 10 seconds Spin down briefly in a table centrifuge 3 Incubate the tube for 10 minutes at 100 C 4 Centrifuge the tube at 13000rpm for 5 minutes The supernatant contains the DNA extracted and can be used for PCR template Atte
3. DNA Fluorescence is emitted and measured by the real time systems optical unit during the PCR The detection of amplified Vibrio cholera O1 DNA fragment is performed in fluorimeter channel 530nm with the fluorescent quencher BHQ1 An external positive control defined as 1x10 copies ml is supplied which allow the determination of the gene load For further information please refer to section 9 2 Quantitation 4 Kit Contents DNA Extraction Buffer 2 vials 1 5ml Vibrio cholera O1 Reaction Mix 1 vial 480u1 1 vial 12ul 1 vial 400u1 1 vial 30ul Analysis sensitivity 1 X 10 copies ml LOQ 2X10 1X10 copies ml Note Analysis sensitivity depends on the sample volume elution volume nucleic acid extraction methods and other factors If you use the DNA extraction buffer in the kit the analysis sensitivity is the same as it declares However when the sample volume is dozens or even hundreds of times greater than elution volume by some concentrating method it can be much higher 5 Storage e All reagents should be stored at 20 C Storage at 4 C is not recommended e All reagents can be used until the expiration date indicated on the kit label e Repeated thawing and freezing gt 3x should be avoided as this may reduce the sensitivity of the assay e Cool all reagents during the working steps e Reaction mix should be stored in the dark 6 Additionally Required Materials and Devices e Biological cabinet e Real time PCR syste
4. m e Desktop microcentrifuge for eppendorf type tubes RCF max 16 000 x g e Vortex mixer e Real time PCR reaction tubes plates e Cryo container e Pipets 0 5ul 1000u1 e Sterile filter tips for micro pipets e Sterile microtubes e Disposable gloves powderless e Biohazard waste container e Refrigerator and Freezer e Tube racks PCR Enzyme Mix Molecular Grade Water Vibrio cholera O1 Positive Control 1x10 copies ml 7 Warnings and Precaution e Carefully read this instruction before starting the procedure e For in vitro diagnostic use only e This assay needs to be carried out by skilled personnel e Clinical samples should be regarded as potentially infectious materials and should be prepared in a laminar flow hood e This assay needs to be run according to Good Laboratory Practice e Do not use the kit after its expiration date e Avoid repeated thawing and freezing of the reagents this may reduce the sensitivity of the test e Once the reagents have been thawed vortex and centrifuge briefly the tubes before use e Quickly prepare the reaction mix on ice or in the cooling block e Set up two separate working areas 1 Isolation of the RNA DNA and 2 Amplification detection of amplification products e Pipets vials and other working materials should not circulate among working units e Use always sterile pipette tips with filters e Wear separate coats and gloves in each area Ws 1 8 Sample Collection
5. ntion A During the incubation make sure the tube is not open as the vapor will volatilize into the air and may cause contamination if the sample is positive B The extraction sample should be used in 3 hours or store at 20 C for one month C Different DNA extraction kits are available You may use your own extraction systems or the commercial kit based on the yield For the DNA extraction please comply with the manufacturer s instructions 9 2 Quantitation The kit can be used for quantitative or qualitative real time PCR For performance of quantitative real time PCR Standard dilutions must prepare first as follows Molecular Grade Water is used for dilution The step of dilution is not needed for performance of qualitative real time PCR Take positive control 1x10 copies ml as the starting high standard in the first tube Respectively pipette 36ul of Molecular Grade Water into next three tubes Do three dilutions as the following figures Dilution of Standards 4ul 4ul 4ul Y WV V Y 1X107 1X10 1X10 1X 104 copiesimi To generate a standard curve on the real time system all four dilution standards should be used and defined as standard with specification of the corresponding concentrations Attention A Mix thoroughly before next transfer B The positive control contains high concentration of the target DNA Therefore be careful during the dilution in order to avoid contamination 9 3 PCR Protocol The Master Mix v
6. olume for each reaction should be pipetted as follows Re ae la Mix Ser 1 The volumes of Reaction Mix and Enzyme Mix per reaction multiply with the number of samples which includes the number of the Tea K controls standards and sample prepared aster Mix Molecular Grade Water is used as the negative control For reasons of unprecise 2 pl 18 yi pipetting always add an extra virtual sample Extraction DNA Master Mix Mix the master mix completely then spin i il down briefly in a centrifuge Reaction 2 Pipet 18ul Master Mix with micropipets of Plate Tube sterile filter tips to each Real time PCR i reaction plate tube Then separately add 2pl PCR Instrument DNA sample positive and negative controls to different reaction plate tubes Immediately close the plate tubes to avoid contamination 3 Spin down briefly in order to collect the Master Mix in the bottom of the reaction tubes 4 Perform the following protocol in the instrument 37 C for 2min Selection of fluorescence channels 93 C for 5sec 60 C for 30sec Fluorescence measured at 60 C 10 Threshold setting Choose Arithmetic as back ground and none as Noise Band method then adjust the Noise band just above the maximum level of molecular grade water and adjust the threshold just under the minimum of the positive control 11 Calibration for quantitative detection Input each concentration of standard controls at the end of run and a standard curve will be automatically fo
7. rmed 12 Quality control Negative control positive control and QS curve must be performed correctly otherwise the sample results is invalid oa A Crossing point value 530nm Molecular Grade Water Positive Control qualitative assay QS quantitative detection Correlation coefficient of QS curve lt 0 98 13 Data Analysis and Interpretation The following results are possible Cr ne pomi value Result Analysis Below the detection limit or negative Target Nucleic Acid 2 Positive and the software displays the quantitative value 3 35 40 Re test If it is still 35 40 report as 1 For further questions or problems please contact our technical support at trade liferiver com cn
8. tion tube after the amplification 3 Product Description Cholera is an acute intestinal infection caused by ingestion of food or water contaminated with the bacterium Vibrio cholerae It has a short incubation period and produces an enterotoxin that causes a copious painless watery diarrhoea that can quickly lead to severe dehydration and death if treatment is not promptly given Vomiting also occurs in most patients Most persons infected with V cholerae do not become ill although the bacterium is present in their faeces for 7 14 days When illness does occur about 80 90 of episodes are of mild or moderate severity and are difficult to distinguish clinically from other types of acute diarrhoea Less than 20 of ill persons develop typical cholera with signs of moderate or severe dehydration Cholera remains a global threat and is one of the key indicators of social development While the disease no longer poses a threat to countries with minimum standards of hygiene it remains a challenge to countries where access to safe drinking water and adequate sanitation cannot be guaranteed Almost every developing country faces cholera outbreaks or the threat of a cholera epidemic Vibrio cholera O1 real time PCR kit contains a specific ready to use system for the detection of the Vibrio cholera O1 using PCR polymerase chain reaction in the real time PCR system The master contains reagents and enzyme for the specific amplification of the Vibrio cholera O1
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