為同時(shí)檢測豬源臨床樣本中的豬瘟病毒（CSFV）和牛病毒性腹瀉病毒（BVDV），基于這兩種病毒的5'UTR序列設(shè)計(jì)特異引物和TaqMan探針，建立了一種檢測CSFV和BVDV的雙重?zé)晒釸T-PCR檢測方法，并對(duì)該方法的特異性、最低檢出限和重復(fù)性等進(jìn)行了評(píng)價(jià)。結(jié)果顯示，該方法只對(duì)CSFV和BVDV呈現(xiàn)特異性擴(kuò)增，對(duì)豬偽狂犬病病毒、豬繁殖與呼吸綜合征病毒、豬傳染性胃腸炎病毒、豬流行性腹瀉病毒、豬圓環(huán)病毒2型不發(fā)生交叉反應(yīng)，對(duì)陽性標(biāo)準(zhǔn)對(duì)照CSFV-5'UTR-RNA、BVDV-1-5'UTR-RNA和BVDV-2-5'UTR-RNA，最低可分別檢出27、36和32拷貝/μL。該方法的組內(nèi)和組間試驗(yàn)Ct值變異系數(shù)介于0.11%~1.20%，具有良好的重現(xiàn)性。對(duì)152份豬組織樣本用該方法進(jìn)行CSFV和BVDV核酸檢測，結(jié)果檢出CSFV陽性樣本16份，BVDV陽性樣本3份，CSFV和BVDV雙陽性樣本1份，與國標(biāo)和OIE《陸生動(dòng)物診斷試驗(yàn)和疫苗》相應(yīng)的熒光RT-PCR方法陽性符合率為100%。結(jié)果表明，本研究建立的雙重?zé)晒釸T-PCR方法可用于臨床樣本中的CSFV和BVDV檢測，從而為豬瘟防制和凈化提供了一種有效的技術(shù)手段。
Establishment of a Duplex Fluorescent RT-PCR Assay for Detection of Classical Swine Fever Virus and Bovine Viral Diarrhea Virus
In order to simultaneously detect classical swine fever virus（CSFV）and bovine viral diarrhea virus（BVDV）in clinical samples from pigs，the specific primers and TaqMan probes were designed based on 5'UTR sequence of the two kinds of viruses，and a duplex fluorescent RT-PCR was established，the specificity，minimum detection limit and repeatability were evaluated. The results showed that the assay could react specifically with CSFV and BVDV only，but failed to crossly react with other viruses including pseudorabies virus（PRV），porcine reproductive and respiratory syndrome virus（PRRSV），transmissible gastroenteritis virus（TGEV），porcine epidemic diarrhea virus（PEDV）and porcine circovirus-2（PCV-2）. The minimum detection limits were 27，36 and 32 copies/μL for the positive standard plasmid control of CSFV-5'UTR-RNA，BVDV-1-5'UTR-RNA and BVDV-2-5'UTR-RNA respectively. The coefficients of variation（CV）of intra-and inter-group ranged 0.11% to 1.20%，showing good reproducibility. Atotal of 152 tissue samples were tested for CSFV and BVDV by the assay，with the results of 16 CSFV positive samples，3 BVDV positive samples and 1 CSFV-BVDV dual positive samples，which were completely consistent with the national standard and the results of corresponding fluorescent RT-PCR assay specified in OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. In conclusion，the assay established in this study could be used for the detection of CSFV and BVDV in clinical samples，which provided an effective technical method for control and purification of the two diseases.
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