Bacillus Cereus Real Time PCR Kit User Manual For In
Contents
1. Liferiver Revision No ZJ0007 Issue Date Jul 1 2012 Bacillus Cereus Real Time PCR Kit User Manual For In Vitro Diagnostic Use Only DD 0042 01 For use with LightC ycler 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 C 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 The Bacillus Cereus real time PCR Kit is a test for the detection of Bacillus Cereus in stool or vomit samples in 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 after2. the amplification 3 Product Description Bacillus cereus is an endemic soil dwelling Gram positive rod shaped beta hemolytic bacteria that causes foodborne illness It is the cause of Fried Rice Syndrome B cereus bacteria are facultative anaerobes and like other members of the genus Bacillus can produce protective endospores B cereus is responsible for a minority of foodborne illnesses 2 5 It is known to create heavy nausea vomiting and abdominal periods Generally speaking Bacillus foodborne illnesses occur due to survival of the bacterial spores when food is improperly cooked This problem is compounded when food is then improperly refrigerated allowing the spores to germinate Bacterial growth results in production of enterotoxin and ingestion leads to two types of illness diarrheal and emetic syndrome The Bacillus Cereus real time PCR Kit contains a specific ready to use system for the detection of the Bacillus Cereus using PCR polymerase chain reaction in the real time PCR system The master contains reagents and enzymes for the specific amplification of the Bacillus Cereus DNA Fluorescence is emitted and measured by the real time systems optical unit during the PCR The detection of amplified Bacillus Cereus DNA fragment is performed by ROB reaction mix in fluorimeter channel 530nm with the fluorescent quencher BHQ1 Toxin types are identified by HBL amp NHE reaction mix In addition the kit contains a system to identif
3. ction 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 Internal 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 shown in the 560nm 9 3 PCR Protocol The Master Mix volume for each reaction should be pipetted as follows 17 yl 18yl 0 4ul 1ul Opl Reaction Mix Enzyme Mix Internal Control Enzyme Mix per reaction multiply with mene a the number of samples which includes the 18 4 ul number of the controls standards and 1 The volumes of Reaction Mix and Master Mix sample prepared Molecular Grade Water is used as the negative control For reasons 2ul 18 ul of unprecise pipetting always add an extra Extraction DNA Master Mix virtual sample n the number of Oa ae reaction Mix completely then spin down P briefly in a centrifuge Plate Tube PCR Instrument Reaction Volume ROB Master Mix Volume HBL amp NHE Master Mix Volume 17ul x n 1 18ul x n 1 Internal control AC 1x tD gt o O 2 Pipet 18ul Master Mix with micropipets of sterile filter tips to each Real time PCR reaction plate tube Then separately add 2ul DNA sample positive and negative controls to different reaction plate tubes Immed
4. ease thaw the buffer thoroughly and spin down briefly in the centrifuge before use It s better to use commercial kits for nucleic acid extraction 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 Vomit samples 1 Take 1 ml vomit 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 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 Attention 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 extra
5. iately 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 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 Quality control Negative control positive control and internal control must be performed correctly otherwise the sample results is invalid Crossing point value Control Reaction Mix 560nm Molecular Grade Water proses e QS quantitative detection In channel 530nm of ROB Reaction Mix Correlation coefficient of QS curve lt 0 98 12 Data Analysis and Interpretation The following results are possible Positive Control ROB HBL amp NHE Reaction Mix Reaction Mix Result analysis 1 lt 35 Blank Blank Bacillus Cereus Positive and it is atoxic 2 lt 35 lt 35 Blank Bacillus Cereus Positive and it contains HBL toxin PE UNDET Bacillus Cereus Positive and it contains NHE toxin PCR Inhibition No diagnosis can be concluded X The crossing point value shows 35 40 please repeat again If the result still shows 35 40 it can be considered negative For further questions or problems please contact our technical support at trade lifer
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7. posable gloves powderless e Biohazard waste container e Refrigerator and Freezer e Tube racks 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 Ws 1 al ee ee ee ee 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 Pl
8. y possible PCR inhibition by measuring the 560nm fluorescence of the internal control IC 4 Kit Contents DNA Extraction Buffer 2 vials 1 5ml ROB Reaction Mix 1 vial 480u1 HBL amp NHE Reaction Mix 1 vial 480ul PCR Enzyme Mix 1 vial 22u1 Molecular Grade Water 1 vial 400u1 Internal Control IC 1 vial 30u1 Positive Control 1 vial 60ul Analysis sensitivity 1 X 10 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 system 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 5u1 1000u1 e Sterile filter tips for micro pipets e Sterile microtubes e Dis
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