Multiple Polymerase Chain Reaction Multiple Applications

Part ? A Multiplex PCR Assay Mediated by Universal Primers for the Detection of 13 Genetically Modified Soybean EventsObjective: Based on the multiplex PCR detection system established in our laboratory for 12 transgenic soybean lines,the MON87705 line was introduced.Established a multiplex PCR assay mediated by universal primers with low cost,high specificity and high sensitivity to detect 13 Genetically Modified Soybean Events approved by China so far(MON87701,GTS40-3-2,MON89788,CV127,A2704,A5547,DP356043,DP305423,MON87705,MON87769,MON87708,305423 × 40-3-2,MON87701×MON89788,305423×40-3-2 and MON87701×MON89788 are gene stacked).Methods: Testing the specificity of the screened public primers with the new strain MON87705 genome,and designing gene-specific primers respectively from 3' flanking sequences of MON87705 according to GMDD and patents,and other ten kinds of GM soybean events' gene-specific primers from literature(MON87701,GTS40-3-2,MON89788,CV127,A2704,A5547,DP356043,DP305423,MON87769,MON87708,305423×40-3-2,MON87701×MON89788 and two of them are gene stacked).Formation of specific chimeric primers by connecting common primers at the 5'end of specific primers.Established an UP-M-PCR detection method and the sensitivity and the specificity were detected.In order to verify the commercial value of our UP-M-PCR system,73 soybean products that may contain genetically modified soybean ingredients were examined by the mPCR method.Results: To introduce MON87705,the 11 pairs of primers do not interfere with each other,and the sensitivity of the UP-M-PCR system was over 0.1% exceeding EU standards(0.9%).Among 73 soybean products,42 positive specimen(57.5%).Conclusion: Established a mPCR method mediated by universal primers for detecting 13 types of genetically modified soybean events at same time with high specificity,sensitivity and stability.Part ? Development of a multiplex PCR method for detecting six types of viruses causing encephalitisObjective: To develop a new mPCR method for rapid diagnosis of six types of encephalitis causing viruses of HSVI,HSVII,VZV,EBV,EV71 and CMV.Methods: Six pairs of specific primers for CMV,EV71,HSV I,VZV,EBV and HSV II were designed.The mPCR detection method was established and the sensitivity was detected.In order to verify the clinical application value of our multiplex PCR system,fifteen cerebrospinal fluid specimens of clinically suspected VE from The First Affiliated Hospital of Soochow University between 2014 and 2015 were examined by the mPCR method.Results: The 6 pairs of primers do not interfere with each other,and the sensitivity of the mPCR system was over 103 copies.Among 15 cerebrospinal fluid specimens from patients with suspected viral encephalitis,six specimens(6/15,40%)were tested positive by the mPCR.Among them,HSV I was 5 and CMV was 1.Conclusion: The mPCR method for detecting six types of encephalitis-associate-d virus at same time was established with high specificity,sensitivity and stability.