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猪瘟病毒和牛病毒性腹泻病毒双重荧光RT-PCR检测方法的建立

2020-10-19 21:02:41来(lai)源:中国动(dong)物检(jian)疫作者(zhe):张宏伟,王建华,陈(chen)本龙,等
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为同时检(jian)(jian)测猪(zhu)(zhu)源临床样(yang)(yang)本(ben)(ben)(ben)中的猪(zhu)(zhu)瘟病(bing)(bing)(bing)(bing)(bing)毒(du)(du)(CSFV)和(he)牛(niu)病(bing)(bing)(bing)(bing)(bing)毒(du)(du)性(xing)腹(fu)泻病(bing)(bing)(bing)(bing)(bing)毒(du)(du)(BVDV),基于(yu)这两种病(bing)(bing)(bing)(bing)(bing)毒(du)(du)的5UTR序(xu)列设计特异(yi)(yi)引物和(he)TaqMan探针,建(jian)立了一种检(jian)(jian)测CSFV和(he)BVDV的双(shuang)重荧(ying)光RT-PCR检(jian)(jian)测方法(fa),并(bing)对(dui)该方法(fa)的特异(yi)(yi)性(xing)、最(zui)低检(jian)(jian)出(chu)(chu)(chu)限(xian)和(he)重复(fu)性(xing)等进(jin)行了评价。结果(guo)显示,该方法(fa)只对(dui)CSFV和(he)BVDV呈现特异(yi)(yi)性(xing)扩增,对(dui)猪(zhu)(zhu)伪狂(kuang)犬病(bing)(bing)(bing)(bing)(bing)病(bing)(bing)(bing)(bing)(bing)毒(du)(du)、猪(zhu)(zhu)繁殖与呼吸综合征病(bing)(bing)(bing)(bing)(bing)毒(du)(du)、猪(zhu)(zhu)传染性(xing)胃肠炎病(bing)(bing)(bing)(bing)(bing)毒(du)(du)、猪(zhu)(zhu)流行性(xing)腹(fu)泻病(bing)(bing)(bing)(bing)(bing)毒(du)(du)、猪(zhu)(zhu)圆环(huan)病(bing)(bing)(bing)(bing)(bing)毒(du)(du)2型不发生交叉反应,对(dui)阳性(xing)标准(zhun)对(dui)照CSFV-5UTR-RNA、BVDV-1-5UTR-RNA和(he)BVDV-2-5UTR-RNA,最(zui)低可(ke)分别检(jian)(jian)出(chu)(chu)(chu)27、36和(he)32拷贝/μL。该方法(fa)的组内和(he)组间试(shi)(shi)验(yan)Ct值变异(yi)(yi)系数介于(yu)0.11%~1.20%,具有良好(hao)的重现性(xing)。对(dui)152份(fen)猪(zhu)(zhu)组织样(yang)(yang)本(ben)(ben)(ben)用该方法(fa)进(jin)行CSFV和(he)BVDV核(he)酸检(jian)(jian)测,结果(guo)检(jian)(jian)出(chu)(chu)(chu)CSFV阳性(xing)样(yang)(yang)本(ben)(ben)(ben)16份(fen),BVDV阳性(xing)样(yang)(yang)本(ben)(ben)(ben)3份(fen),CSFV和(he)BVDV双(shuang)阳性(xing)样(yang)(yang)本(ben)(ben)(ben)1份(fen),与国标和(he)OIE《陆(lu)生动物诊(zhen)断试(shi)(shi)验(yan)和(he)疫苗》相(xiang)应的荧(ying)光RT-PCR方法(fa)阳性(xing)符合率为100%。结果(guo)表(biao)明,本(ben)(ben)(ben)研(yan)究建(jian)立的双(shuang)重荧(ying)光RT-PCR方法(fa)可(ke)用于(yu)临床样(yang)(yang)本(ben)(ben)(ben)中的CSFV和(he)BVDV检(jian)(jian)测,从而为猪(zhu)(zhu)瘟防制和(he)净化提供(gong)了一种有效(xiao)的技术手(shou)段(duan)。

Establishment of a Duplex Fluorescent RT-PCR Assay for Detection of Classical Swine Fever Virus Bovine Viral Diarrhea Virus

In order to simultaneously detect classical swine fever virus(CSFV) bovine viral diarrhea virus(BVDV)in clinical samples from pigs,the specific primers TaqMan probes were designed based on 5UTR sequence of the two kinds of viruses, a duplex fluorescent RT-PCR was established,the specificity,minimum detection limit repeatability were evaluated. The results showed that the assay could react specifically with CSFV BVDV only,but failed to crossly react with other viruses including pseudorabies virus(PRV),porcine reproductive respiratory syndrome virus(PRRSV),transmissible gastroenteritis virus(TGEV),porcine epidemic diarrhea virus(PEDV) porcine circovirus-2(PCV-2). The minimum detection limits were 27,36 32 copies/μL for the positive standard plasmid control of CSFV-5UTR-RNA,BVDV-1-5UTR-RNA BVDV-2-5UTR-RNA respectively. The coefficients of variation(CV)of intra- inter-group ranged 0.11% to 1.20%,showing good reproducibility. Atotal of 152 tissue samples were tested for CSFV BVDV by the assay,with the results of 16 CSFV positive samples,3 BVDV positive samples 1 CSFV-BVDV dual positive samples,which were completely consistent with the national standard the results of corresponding fluorescent RT-PCR assay specified in OIE Manual of Diagnostic Tests Vaccines for Terrestrial Animals. In conclusion,the assay established in this study could be used for the detection of CSFV BVDV in clinical samples,which provided an effective technical method for control purification of the two diseases.

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猪瘟病毒和牛病毒性腹泻病毒双重荧光RT-PCR检测方法的建立
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