2种猪冠状病毒双重RT-PCR检测方法的建立与应用
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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.
【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.
引文
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[11]刘邓,袁秀芳,徐丽华,牛登元,王朝文,王一成.猪流行性腹泻病毒和猪传染性胃肠炎病毒双重RT-PCR鉴别方法的建立[J].动物医学进展,2009,30(10):1-5
[12]任玉鹏,陈弟诗,苏生禹,聂艳如,张斌,岳华.三种猪腹泻病毒性病原多重RT-PCR检测方法的建立及应用[J].中国兽医科学,2013,43(10):1047-1051
[13]王元,杜允斌,范红燕,贺笋,刘宏杰,苏晓月,沈亚安,张会萍.新疆部分规模化猪场猪病毒性腹泻流行病学调查与防控[J].猪业科学,2013,30(8):89-90
[2]Gao Y,Kou Q,Ge X,Zhou L,Guo X,Yang H.Phylogenetic analysis of porcine epidemic diarrhea virus field strains prevailing recently in China[J].Archives of Virology,2013,158(3):711-715
[3]孟凡伟,张雪,王俊,单晶晶,王鹏,周双海,王志军.猪流行性腹泻病毒Real-time PCR检测方法的建立[J].中国农学通报,2013,33(10):24-29
[4]罗满林.动物传染病学[M].北京:中国林业出版社,2103:195-200
[5]Kim S H,Kim I J,Pyo H M,Tark D S,Song J Y,Hyun BH.Multiplex Real-time RT-PCR for the simultaneous detection and quantification of transmissible gastroenteritis virus and porcine epidemic diarrhea virus[J].Journal of Virological Methods,2007,146(1-2):172-177
[6]张雪,杨倩,于红欣,王俊,孟凡伟,周双海.猪传染性胃肠炎病毒实时荧光定量PCR检测方法的建立与应用[J].中国兽医科学,2015,45(6):644-648
[7]贾超伟,柳迦鹏,张喜喜,刘芊麟,周双海,宋勤叶.3株猪流行性腹泻病毒M基因克隆与序列分析[J].北京农学院学报,2018,33(3):53-56
[8]陈芳,刘惠莉,孙红立,曹祥荣,李震,赵立平.多重RT-PCR检测猪传染性胃肠炎病毒和猪流行性腹泻病毒[J].中国兽医学报,2005,25(1):9-12
[9]贾锐,叶佳欣,俞向前,叶承荣,朱建国.猪传染性胃肠炎及流行性腹泻病毒二重RT-PCR检测方法的建立[J].上海交通大学学报,2014,32(4):71-75
[10]郑世民,周首龙,赵良友,刘超男,高雪丽,吕晓萍.猪传染性胃肠炎和流行性腹泻病毒二联RT-PCR检测方法的建立[J].东北农业大学学报,2014,45(6):57-60
[11]刘邓,袁秀芳,徐丽华,牛登元,王朝文,王一成.猪流行性腹泻病毒和猪传染性胃肠炎病毒双重RT-PCR鉴别方法的建立[J].动物医学进展,2009,30(10):1-5
[12]任玉鹏,陈弟诗,苏生禹,聂艳如,张斌,岳华.三种猪腹泻病毒性病原多重RT-PCR检测方法的建立及应用[J].中国兽医科学,2013,43(10):1047-1051
[13]王元,杜允斌,范红燕,贺笋,刘宏杰,苏晓月,沈亚安,张会萍.新疆部分规模化猪场猪病毒性腹泻流行病学调查与防控[J].猪业科学,2013,30(8):89-90