添加扩增内标的逆转录重组酶聚合酶扩增技术检测三种大豆病毒  

RT-RPA Technology Included with IAC for Detection of Three Soybean(Glycine max) Viruses

作　　者：袁俊杰 龙阳 渭婷玉 刘二龙 冯黎霞 刘思思 陈文 魏霜 YUAN Jun-Jie;LONG Yang;WEI Ting-Yu;LIU Er-Long;FENG Li-Xia;LIU Si-Si;CHEN Wen;WEI Shuang(Zhanjiang Customs,Zhanjiang 524022,China;Xining Customs,Xining 810000,China;Huangpu Customs,Guangzhou 510700,China;Guangzhou Customs,Guangzhou 510000,China;College of Science,Huazhong Agricultural University,Wuhan 430074)

机构地区：[1]湛江海关,湛江524022 [2]西宁海关,西宁810000 [3]黄埔海关,广州510700 [4]广州海关,广州510000 [5]华中农业大学理学院,武汉430074

出　　处：《农业生物技术学报》2020年第12期2261-2269,共9页Journal of Agricultural Biotechnology

基　　金：国家重点研发计划(2018YFC0809200);海关总署科技计划项目(2020HK147,2019HK052)。

摘　　要：假阴性结果是影响核酸检测结果的主要因素之一。扩增内标(internal amplification control,IAC)在监控PCR过程中假阴性结果发挥着重要作用。为探索扩增内标在逆转录重组酶聚合酶常温扩增(reverse transcription-recombinase polymerase amplification,RT-RPA)检测过程中的应用,解决RT-RPA检测过程中的假阴性问题,本研究通过复合引物法人工构建了扩增内标,并应用于大豆花叶病毒(Soybean mosaic virus,SMV)、菜豆荚斑驳病毒(Bean pod mottle virus,BPMV)及南方菜豆花叶病毒(Southern bean mo⁃saic virus,SBMV)三种大豆病毒RT-RPA检测过程。实验结果显示,所建立的方法能够实现目的基因特异性扩增的同时指示假阴性结果。灵敏度试验结果表明,当50μL反应体系中扩增内标添加量为1.83×104 copies时,该体系对SMV及BPMV的检测灵敏度为0.05 ng;扩增内标添加量为1.83×103 copies时,该体系对SBMV的检测灵敏度为0.05 ng。利用该方法对10批实际大豆(Glycine max)样品进行检测,与对应反转录PCR(reverse transcription-PCR,RT-PCR)结果一致,表明其具有良好的适用性。研究结果表明本方法可有效指示3种病毒RT-RPA检测过程中的假阴性,提高检测结果的可信度,为相关产品的监管提供技术支持和参考依据。False-negative result is a major factor affecting the results of nucleic acid testing. The internal amplification control(IAC) plays an important role in monitoring false negative results during PCR. In order to solve the false-negative result, the application of the IAC in reverse transcription-recombinase polymerase amplification(RT-RPA) was established. The IAC was constructed by the composite primer method and applied to the detection of three soybean viruses, Soybean mosaic virus(SMV), Bean pod mottle virus(BPMV)and Southern bean mosaic virus(SBMV), during RT-RPA. The results showed that the methods were able to achieve specific amplification of the target gene while indicating false negative results. Sensitivity experiments showed that the detection limit of this assay for SMV and BPMV was 0.05 ng, when the content of the IAC was 1.83×10~4 copies in 50 μL reaction system. And when the content of the IAC was 1.83×10~3 copies in 50 μL reaction system, the detection limit of this assay for SBMV was 0.05 ng. The methods were used to test 10 soybean(Glycine max) samples and the results were consistent with those of the corresponding reverse transcription-PCR(RT-PCR), indicating its good applicability. In conclusion, the IAC-RT-PRA methods for SMV,BPMV and SBMV were established in this study could indicate false negatives and improve credibility of test results.

关 键 词：大豆花叶病毒(SMV) 菜豆荚斑驳病毒(BPMV) 南方菜豆花叶病毒(SBMV) 逆转录重组酶聚合酶常温扩增(RT-RPA) 扩增内标(IAC) 

分 类 号：S41-30[农业科学—植物保护]