DATA SHEET Vibrio parahaemolyticus Real Time PCR Kit
Contents
1. l DATA SHEET GENTAUR Vibrio parahaemolyticus Real Time PCR Kit Cat No DD 0038 02 For use with ABI Prism 7000 7300 7500 7900 Smart Cyclerll iCycler iQ 4 iQ 5 Rotor Gene 6000 Mx3000P 3005P MJ Option2 Chromo4 LightCycler 480real time PCR systems User Manual E For in vitro Diagnostic use only European Authorized Representative E A R Obelis S A 34 Av De Tervuren bte 44 B 1040 Brussels Belgium Phone 32 2 732 59 54 Fax 32 2 732 60 03 E mail mail obelis net 1 Intended Use Vibrio parahaemolyticus real time PCR kit is used for the detection of Vibrio parahaemolyticus 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 reaction tube after the amplification2. 3 Product Description Vibrio parahaemolyticus is a curved rod shaped Gram negative bacterium found in brackish saltwater which when ingested causes gastroeintestinal illness in humans V parahaemolyticus is oxidase positive facultatively aerobic and does not form spores Like other members of the genus Vibrio this species is motile with a single polar flagellum Outbreaks tend to be concentrated along coastal regions during the summer and early fall when higher water temperatures favor higher levels of bacteria Seafood most often implicated includes squid mackerel tuna sardines crab shrimp and bivalves like oysters and clams The incubation period of 24 hours is followed by explosive watery diarrhea accompanied by nausea vomiting abdominal cramps and sometimes fever Vibrio parahaemolyticus symptoms typically resolve with in 72 hours but can persist for up to 10 days in immunocompromised individuals As the vast majority of cases of V parahaemolyticus food infection are self limiting treatment is not typically necessary In severe cases fluid and electrolyte replacement is indicated Vibrio parahaemolyticus real time PCR Kit contains a specific ready to use system for the detection of the Vibrio parahaemolyticus by polymerase chain reaction PCR in the real time PCR system The master contains reagents and enzymes for the specific amplification of Vibrio parahaemolyticus DNA Fluorescence is emitted and me
3. asured by the real time systems optical unit during the PCR The detection of amplified Vibrio parahaemolyticus DNA fragment is performed in fluorimeter channel FAM with the fluorescent quencher BHQ1 DNA extraction buffer is available in the kit and excreta samples are used for DNA extraction In addition the kit contains a system to identify possible PCR inhibition by measuring the HEX VIC JOE fluorescence of the internal control IC An external positive control 1x107copies ml allows the determination of the gene load For further information please refer to section 9 3 Quantitation Gentaur Molecular Products Voortstraat 49 1910 Kampenhout Belgium 4 Kit Contents DNA Extraction Buffer 2 vials 1 S5ml VP Reaction Mix 1 vial 9501 PCR Enzyme Mix 1 vial 12 Molecular Grade Water 1 vial 400 1 Internal Control IC 1 vial 30 VP Positive Control 1x10 copies ml 1 vial 30ul 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 system e Vortex mixer e Real time PCR reaction tubes plates e Cryo container
4. 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 0m 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 100ul 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 3 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 100ul 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 Attention A During the incubation make sure the tube is not open as the vapor will volatilize into the air and may cause co
5. e Ct value of internal control in HEX VIC JOE channel shows 25 33 Correlation coefficient of standard curve should be lt 0 98 otherwise the result is invalid 13 Data Analysis and Interpretation The following results are possible 1 The Ct value in channel FAM shows lt 35 The result is positive The sample contains vibrio parahaemolyticus DNA 2 The Ct value in channel FAM shows 35 40 please repeat again If the result still shows 35 40 it can be considered negative 3 In channel FAM no signal is detected at the same time a HEX VIC JOE signal from the Internal Control appears The sample does not contain any vibrio parahaemolyticus DNA It can be considered negative 4 Neither in channel FAM nor in channel HEX VIC JOE signal is detected A diagnostic statement can not be made Inhibition of the PCR reaction Gentaur Molecular Products Voortstraat 49 1910 Kampenhout Belgium
6. e Pipets 0 5ul 1000ul 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 e Desktop microcentrifuge for eppendorf type tubes RCF max 16 000 x g 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 8 Sample Collection 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
7. les which includes the number of the controls standards and sample prepared Molecular Grade Water is used as the negative control For reasons of unprecise pipetting always add an extra virtual sample Mix the master mix completely then spin down briefly in a centrifuge 2 Pipet 36ul 22 5ul for SmartCycer II Master Mix with micropipets of sterile filter tips to each Real time PCR reaction plate tube Then separately add 4ul 2 5ul for SmartCycer Il DNA Gentaur Molecular Products Voortstraat 49 1910 Kampenhout Belgium 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 2 min 1 cycle 94 C for 2 min 1 cycle 93 C for 15 sec 60 C for 60 sec 40 cycles Fluorescence is measured at 60 C FAM and HEX VIC JOE channels should be chosen 5 If you use ABI Prism system please choose none as passive reference and quencher 10 Baseline setting just above the maximum level of molecular grade water 11 Calabration for quantitative detection Input each concentration of standard controls at the end of run and a standard curve will be automatically formed 12 Quality control The Ct value of molecular grade water and positive control in FAM channel shows UNDET and lt 35 respectively Th
8. ntamination 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 Gentaur Molecular Products Voortstraat 49 1910 Kampenhout Belgium commercial kit based on the yield For the DNA extraction please comply with the manufacturer s instructions 9 2 Internal Control and positive control It is necessary to add internal control IC in the reaction mix Internal Control IC allows the user to determine and control the possibility of PCR inhibition Add the internal control IC 1ul rxn and the result will be got in the HEX VIC JOE channel Attention It is necessary to dilute the internal control supplied in the kit by 10 times with molecular grade water before detection and close the tube immediately then vortex for 10 seconds Because of transportation with carbon dioxide ice there may be white precipitate in tubes of internal control and positive control but it will disappear in a few minutes when it is incubated at room temperature Besides the white precipitate have no effection on the detection result 9 3 Quantitation The kit can be used for quantitative or qualitative real time PCR A positive control defined as 1x107copies ml is supplied in the kit For performance of quantitative real time PCR Standard dilutions must prepare first as follows Molecular Grade Wa
9. ter is used for dilution The step of dilution is not needed for performance of qualitative real time PCR Take positive control 1x107copies 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 Aul Aul l Y VY Y Y 1X10 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 1x107copies ml contains high concentration of the target DNA Therefore be careful during the dilution in order to avoid contamination 9 4 PCR Protocol The Master Mix volume for each reaction should be pipetted as follows 35pl 0 4ul ipl Reaction Mix Enzyme Mix internal Control 36 4 Master Mix dul 36y Extraction DNA Master Mix Reaction Plate Tube l PCR Instrument OR 21 5 ul 0 4 ul Tul Reaction Mix Enzyme Mix Intemal Control ne ain 22 9 Master Mix 2 5ul 22 Syl Extraction DNA Master Mix ete Reaction Plate Tube This system is only for PCR inelument Smart Cycler Il PCR system without HEX VIC JOE channel may be treated with 1ul Molecular Grade Water instead of Iyl IC 1 The volumes of Reaction Mix and Enzyme Mix per reaction multiply with the number of samp
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