多重荧光聚合酶链反应对转基因能力验证样品的检测和验证

目的利用多重实时荧光聚合酶链反应(polymerase chain reaction,PCR)对转基因能力验证样品进行检测和验证。方法通过筛选出合适的多重引物和探针、核酸提取及多重扩增和分析方法,分别对9个转基因标准品和11个来自于中国合格评定国家认可委员会(China National Accreditation Service for Conformity Assessment,CNAS)和食品分析水平测试计划(food analysis performance assessment scheme,FAPAS)的能力验证样品进行多重荧光PCR检测,通过SPSS统计软件比较二重、三重实时荧光PCR的循环阈值(cycle threshold,Ct)与单重实时荧光PCR结果的差异性。结果二重实时荧光PCR可同时检测Ca MV35s和NOS 2个基因,三重实时荧光PCR可同时检测Ca MV35s,NOS和NPTII 3个外源基因,各基因之间无信号的交叉干扰。经统计分析,二重和三重实时荧光PCR相对于单重实时荧光PCR的结果无显著性差异。结论建立的二重和三重实时荧光PCR技术可用于能力验证和实验室间比对实验,简单快速,节约试剂成本,且结果准确度可满足日常检测需求。

Detection and verification of proficiency testing samples of transgenic crops with multiplex fluorescence polymerase chain reaction

Objective To test and verify genetically modified capability validation samples by using multiplex real-time fluorescent polymerase chain reaction technique (PCR). Methods The appropriate multiple primers and probes were selected, nucleic acid extraction and multiple amplification and analysis methods were established. Totally 9 transgenic standards and 11 proficiency test samples from China National Accreditation Service for Conformity Assessment (CNAS) and the food analysis performance assessment scheme (FAPAS) were detected by multiplex real-time fluorescent PCR. The duplex, triplex real-time fluorescence PCR cycle threshold (cycle threshold, Ct) and single real-time fluorescent PCR results were compared by SPSS statistical software. Results The duplex real-time fluorescence PCR could simultaneously detected CaMV35s and NOS genes, and the triplex real-time fluorescence PCR could simultaneously detected CaMV35s, NOS and NPTII exogenous genes, and there was no signal intersecting interference between genes. According to statistical analysis, there were no significant differences in duplex and triplex real-time fluorescent PCR with single real-time fluorescence PCR. Conclusion The established duplex and triplex real-time fluorescent PCR can be used for the ability verification and inter-laboratory comparison experiment, and the methods are simple and fast, can save the cost of reagents, and the accuracy of results can meet the daily testing needs.