為同時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5UTR序列設計特異引物和TaqMan探針，建立了一種檢測CSFV和BVDV的雙重熒光RT-PCR檢測方法，并對該方法的特異性、最低檢出限和重復性等進行了評價。結果顯示，該方法只對CSFV和BVDV呈現特異性擴增，對豬偽狂犬病病毒、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、豬圓環病毒2型不發生交叉反應，對陽性標準對照CSFV-5UTR-RNA、BVDV-1-5UTR-RNA和BVDV-2-5UTR-RNA，最低可分別檢出27、36和32拷貝/μL。該方法的組內和組間試驗Ct值變異系數介于0.11%~1.20%，具有良好的重現性。對152份豬組織樣本用該方法進行CSFV和BVDV核酸檢測，結果檢出CSFV陽性樣本16份，BVDV陽性樣本3份，CSFV和BVDV雙陽性樣本1份，與國標和OIE《陸生動物診斷試驗和疫苗》相應的熒光RT-PCR方法陽性符合率為100%。結果表明，本研究建立的雙重熒光RT-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手段。
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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