猪繁殖与呼吸综合征病毒与非典型瘟病毒双重RT-PCR检测方法的建立与应用
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摘要
为建立同时快速简便地检测猪繁殖与呼吸综合征病毒(PRRSV)与非典型瘟病毒(APPV)两种病毒的方法,本研究根据GenBank中登录的PRRSV、APPV的基因序列,选取PRRSV的ORF2基因和APPV的NS2基因分别设计特异性引物,通过优化反应条件,建立了能够同时检测PRRSV与APPV的双重RT-PCR方法。利用该方法对PRRSV、APPV重组质粒、猪瘟病毒、乙型脑炎病毒、猪流行性腹泻病毒的cDNA和阴性对照进行检测,结果显示除PRRSV和APPV重组质粒扩增结果为阳性外,其余均为阴性,表明其特异性良好;该方法对APPV和PRRSV质粒标准品的最低检出量分别为2.17×10~4拷贝/μL、3.13×10~3拷贝/μL,敏感性较高;同时批间和批内重复性试验表明该方法重复性好。利用该方法对四川地区临床73份疑似仔猪颤抖病与猪繁殖与呼吸综合征病的病料样品进行检测,结果与单一RT-PCR的检测符合率为100%。本研究建立的双重RT-PCR方法具有良好的实用性,可用于临床样品的检测,对临床早期诊断具有重要意义。
In order to quickly and easily detect both porcine reproductive and respiratory syndrome virus(PRRSV) and atypical prion virus(APPV), a duplex RT-PCR assay was established for PRRSV and APPV detections with 2 pairs of specific primers targetting the ORF2 gene of PRRSV and NS2 gene of APPV. By optimizing the reaction conditions, the duplex RT-PCR method was capable of specifically detecting PRRSV and APPV simultaneously, and no amplification was found for swine fever virus, Japanese encephalitis virus and porcine epidemic diarrhea virus. The lowest copy number detected by this method was 2.17×10~4 copies/μL for APPV and 3.13×10~3 copies/μL for PRRSV, respectively. At the same time, the inter-batch and intra-batch tests showed that the method were reproducible. Moreover, 73 clinical samples collected from the piglets with suspected trembling disease and the pigs with reproductive and respiratory syndrome in Sichuan area were detected by the duplex assay, and the results were 100% coincident with single RT-PCR. The established RT-PCR method in this study can be used for the detection of clinical samples, which is of great significance for early clinical diagnosis.
In order to quickly and easily detect both porcine reproductive and respiratory syndrome virus(PRRSV) and atypical prion virus(APPV), a duplex RT-PCR assay was established for PRRSV and APPV detections with 2 pairs of specific primers targetting the ORF2 gene of PRRSV and NS2 gene of APPV. By optimizing the reaction conditions, the duplex RT-PCR method was capable of specifically detecting PRRSV and APPV simultaneously, and no amplification was found for swine fever virus, Japanese encephalitis virus and porcine epidemic diarrhea virus. The lowest copy number detected by this method was 2.17×10~4 copies/μL for APPV and 3.13×10~3 copies/μL for PRRSV, respectively. At the same time, the inter-batch and intra-batch tests showed that the method were reproducible. Moreover, 73 clinical samples collected from the piglets with suspected trembling disease and the pigs with reproductive and respiratory syndrome in Sichuan area were detected by the duplex assay, and the results were 100% coincident with single RT-PCR. The established RT-PCR method in this study can be used for the detection of clinical samples, which is of great significance for early clinical diagnosis.
引文
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[10]王新港,周峰,王傲杰,等.猪繁殖与呼吸综合征病毒和盖他病毒双重RT-PCR检测方法的建立与应用[J].病毒学报,2018,34(02):232-238.
[11]刘光清,仇华吉,蔡雪晖.猪繁殖与呼吸综合征病毒的持续感染[J].中国预防兽医学报,2002,24(4):316-318.
[12]李小璟,龚双燕,陈瑛琪,等.猪繁殖与呼吸综合征病毒与猪流行性腹泻病毒双重RT-PCR检测方法的建立与应用[J].中国兽医科学,2017,(5):544-550.
[13]龚双燕,李小璟,陈瑛琪,等.猪流行性腹泻病毒与圆环病毒2型复合PCR方法的建立[J].浙江农业学报,2017,29(7):1086-1092.
[14]王振华,曹晓涵,宋禾,等.猪繁殖与呼吸综合征病毒的危害及免疫策略[J].中国畜牧兽医,2014,41(02):239-244.
[2]代军,雷蕾,任志华.猪繁殖与呼吸综合征防控研究进展[J].动物医学进展,2014,35(4):97-101.
[3]Groof A D,Deijs M,Guelen L,et al.Atypical porcine pestivirus:A possible cause of congenital tremor type A-II in newborn piglets[J].Viruses,2016,8(10):E271.
[4]Blomstr?m A L,Ley C,Jacobson M.Astrovirus as a possible cause of congenital tremor type AII in piglets?[J].Acta Vet Scand,2014,56(1):1-6.
[5]张文波,吴松松,邓舜洲,等.一种新型仔猪先天性震颤及其病原的初步研究[J].中国畜牧兽医,2017,44(7):2147-2154.
[6]Arruda B L,Arruda P H,Magstadt D R,et al.Identification of a divergent lineage porcine pestivirus in nursing piglets with congenital tremors and reproduction of disease following experimental inoculation[J].PLo S One,2016,11(2):e0150104.
[7]杨本勇,曹东,顾贵波,等.天津株(TJM-F92)高致病性猪繁殖与呼吸综合征活疫苗免疫后病毒血症和抗体消长规律的试验报告[J].中国兽医杂志,2017,(7):56-58.
[8]Gatto I R H,Arruda P H,Visek C A,et al.Detection of atypical porcine pestivirus in semen from commercial boar studs in the United States[J].Transbound Emerg Dis,2017,65(2):e339-e343.
[9]王婕敏,张居作,薛立群.猪繁殖与呼吸综合征的公猪感染情况和检测研究进展[J].湖南畜牧兽医,2016,(2):52-55.
[10]王新港,周峰,王傲杰,等.猪繁殖与呼吸综合征病毒和盖他病毒双重RT-PCR检测方法的建立与应用[J].病毒学报,2018,34(02):232-238.
[11]刘光清,仇华吉,蔡雪晖.猪繁殖与呼吸综合征病毒的持续感染[J].中国预防兽医学报,2002,24(4):316-318.
[12]李小璟,龚双燕,陈瑛琪,等.猪繁殖与呼吸综合征病毒与猪流行性腹泻病毒双重RT-PCR检测方法的建立与应用[J].中国兽医科学,2017,(5):544-550.
[13]龚双燕,李小璟,陈瑛琪,等.猪流行性腹泻病毒与圆环病毒2型复合PCR方法的建立[J].浙江农业学报,2017,29(7):1086-1092.
[14]王振华,曹晓涵,宋禾,等.猪繁殖与呼吸综合征病毒的危害及免疫策略[J].中国畜牧兽医,2014,41(02):239-244.