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2种猪冠状病毒双重RT-PCR检测方法的建立与应用 预览

Development and application of a duplex RT-PCR assay for two kinds of swine coronaviruses
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摘要 【目的】建立一种可同时检测2种猪冠状病毒即猪传染性胃肠炎病毒(TGEV)和猪流行性腹泻病毒(PEDV)的双重RT-PCR检测方法并进行临床应用。【方法】对TGEV和PEDV的单项RT-PCR方法分别进行条件优化后对2种病毒的双重RT-PCR进行条件优化并进行特异性与敏感性试验,然后用单项RT-PCR方法和双重RT-PCR对临床样品进行比较检测。【结果】双重RT-PCR检测方法的PEDV与TGEV的检测敏感度分别为59、49copies/μL,该方法对当前常见猪源RNA病毒的检测结果都为阴性。该方法对2013-2016年18个猪场的235份仔猪腹泻样品的检测结果显示,PEDV和TGEV的个体阳性率分别为73.6%和2.1%,与单项RTPCR方法检测结果无显著性(P>0.05)差异,临床样品中PEDV的猪场阳性率与个体阳性率都极显著(P<0.01)高于TGEV。【结论】建立了一种有较高灵敏性与特异性的PEDV与TGEV的双重RT-PCR检测方法,近年来猪传染性腹泻样品中PEDV检出率极显著高于TGEV。 【Objective】The aim of this study is to establish a duplex RT-PCR method for simultaneous detection of two kinds of swine coronaviruses,namely porcine transmissible gastroenteritis virus(TGEV)and porcine epidemic diarrhea virus(PEDV)in clinical samples.【Methods】The conditions of the single RT-PCR assay of TGEV and PEDV were optimized respectively,and then the duplex RT-PCR assay method for these 2 viruses was optimized including its specificity and sensitivity.Thereafter,several clinical samples were comparatively detected with the single RT-PCR assay and the duplex RT-PCR assay.【Results】The detection sensitivity of the duplex RT-PCR assay for PEDV and TGEV was 59 and 49 copies/μL respectively,and the detection results of the current epidemic swine RNA viruses by this method were all negative.The detection results of 235 piglet diarrhea samples from 18 pig farms between 2013 and 2016 with this method showed that the positive rate of PEDV and TGEV was 73.6% and 2.1%,respectively,and showed no significant difference(P>0.05)from those with the single RTPCR method.The positive rate of PEDV for both pig farms and individuals in the clinical samples was all very significant(P<0.01)higher than that of TGEV.【Conclusion】A duplex RT-PCR assay with high sensitivity and specificity for PEDV and TGEV was developed,and the detection rate of PEDV was very significant higher than that of TGEV in porcine infectious diarrhea samples in recent years.
作者 温海京 贾超伟 刘芊麟 张喜喜 刘鑫宇 周双海 WEN Haijing;JIA Chaowei;LIU Qianlin;ZHANG Xixi;LIU Xinyu;ZHOU Shuanghai(Animal Science and Technology College,Beijing University of Agriculture,Beijing 102206,China;Mentougou Center for Animal Epidemic Disease Prevention and Control,Beijing 102300,China)
出处 《北京农学院学报》 2019年第1期51-55,共5页 Journal of Beijing Agricultural College
基金 北京农学院大学生科研训练项目(2017).
关键词 猪冠状病毒 猪传染性胃肠炎病毒 猪流行性腹泻病毒 双重RT-PCR swine coronavirus TGEV PEDV duplex RT-PCR
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