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豬瘟病毒和牛病毒性腹瀉病毒雙重熒光RT-PCR檢測方法的建立

2020-10-19 21:02:41來源:中國動物檢疫作者:張宏偉,王建華,陳本龍,等
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WEITONGSHIJIANCEZHUYUANLINCHUANGYANGBENZHONGDEZHUWENBINGDU(CSFV)HENIUBINGDUXINGFUXIEBINGDU(BVDV),JIYUZHELIANGZHONGBINGDUDE5UTRXULIESHEJITEYIYINWUHETaqManTANZHEN,JIANLILEYIZHONGJIANCECSFVHEBVDVDESHUANGZHONGYINGGUANGRT-PCRJIANCEFANGFA,BINGDUIGAIFANGFADETEYIXING、ZUIDIJIANCHUXIANHEZHONGFUXINGDENGJINXINGLEPINGJIA。JIEGUOXIANSHI,GAIFANGFAZHIDUICSFVHEBVDVCHENGXIANTEYIXINGKUOZENG,DUIZHUWEIKUANGQUANBINGBINGDU、ZHUFANZHIYUHUXIZONGHEZHENGBINGDU、ZHUCHUANRANXINGWEICHANGYANBINGDU、ZHULIUXINGXINGFUXIEBINGDU、ZHUYUANHUANBINGDU2XINGBUFASHENGJIAOCHAFANYING,DUIYANGXINGBIAOZHUNDUIZHAOCSFV-5UTR-RNA、BVDV-1-5UTR-RNAHEBVDV-2-5UTR-RNA,ZUIDIKEFENBIEJIANCHU27、36HE32KAOBEI/μL。GAIFANGFADEZUNEIHEZUJIANSHIYANCtZHIBIANYIXISHUJIEYU0.11%~1.20%,JUYOULIANGHAODEZHONGXIANXING。DUI152FENZHUZUZHIYANGBENYONGGAIFANGFAJINXINGCSFVHEBVDVHESUANJIANCE,JIEGUOJIANCHUCSFVYANGXINGYANGBEN16FEN,BVDVYANGXINGYANGBEN3FEN,CSFVHEBVDVSHUANGYANGXINGYANGBEN1FEN,YUGUOBIAOHEOIE《LUSHENGDONGWUZHENDUANSHIYANHEYIMIAO》XIANGYINGDEYINGGUANGRT-PCRFANGFAYANGXINGFUHELVWEI100%。JIEGUOBIAOMING,BENYANJIUJIANLIDESHUANGZHONGYINGGUANGRT-PCRFANGFAKEYONGYULINCHUANGYANGBENZHONGDECSFVHEBVDVJIANCE,CONGERWEIZHUWENFANGZHIHEJINGHUATIGONGLEYIZHONGYOUXIAODEJISHUSHOUDUAN。

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.

QUANWENXIAZAILIANJIE:http://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202009022&v=Bh8a8ZT44%25mmd2BXfswhduqZcZQVHMHhHXi9wSi2xg6rwQX6OsZDtjLz8LFamaifdRAHg


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豬瘟病毒和牛病毒性腹瀉病毒雙重熒光RT-PCR檢測方法的建立
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