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猪瘟病毒荧光RT-PCR快速检测方法的建立
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单松华1，夏懿2，黄忠荣1，周煜2，李春阳1
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(1.上海出入境检验检疫局，上海200135；2.上海复星医学科技发展有限公司，上海201400)
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摘要：
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以猪瘟病毒5′端非编码区为靶核酸序列设计引物和探针，建立了一步法荧光RT-PCR检测猪瘟病毒。荧光RT-PCR仅检测出猪瘟C株、T株，未能检测出牛病毒性腹泻病毒（BVDV）、猪呼吸系统冠状病毒、猪传染性胃肠炎病毒、猪细小病毒、伪狂犬病病毒、猪生殖与呼吸综合征病毒、PK-15细胞和牛睾丸原代细胞；对猪瘟病毒T株的扩增反应产物进行了测序分析，与预期序列相符。荧光RT-PCR的检测极限可达到1 TCID50/mL，整个试验流程只需2h。采用荧光RT-PCR和抗原捕获ELISA同时检测临床病料、猪副产品共207份样本，两种方法的检出率分别为17.4%和13.5%，两者符合率为95.7%（198/207）；荧光RT-PCR的检出率高于ELISA，两者差异显著。结果表明，建立的荧光RT-PCR可用于猪产品、临床病料中猪瘟病毒的快速检测。 |
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关键词：
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猪瘟病毒；荧光RT-PCR；猪产品；检测 |
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中图分类号：S 852.651：Q 503
文献标识码：
A 文章编号：1673-4696(2007)11-0950-05 |
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Development of fluorescent real-time RT-PCR for rapid detection of
classical swine fever virus |
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SHAN Song-hua1,XIA Yi2,HUANG Zhong-rong
1,ZHOU Yu2,LI Chun-yang 1
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(1.Shanghai Entry-Exit Inspection and Quarantine Bureau,Shanghai 200135,China;
2.Shanghai Fuxing Med-Tech Development Co.,Ltd.,Shanghai 201400,China) |
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Abstract: A fluorescent real time RT-PCR for quick detection of classical swine fever virus (CSFV) was developed. Primers and FAM-labeled Taqman-probes specific for CSFV were selected. Optimal concentrations of transcriptase, Taq polymerase, Mg2+ and dNTPs; annealing temperature; and PCR cycles of the real-time RT-PCR were identified using the Thiveral strain (T strain) of CSFV. Specificity evaluation of the fluorescent RT-PCR showed that the assay detected T and the lapinized Chinese (C) strains of CSFV; no amplification signal was observed in any of the non-CSFV pestiviruses which included the following: bovine viral diarrhoea virus, porcine respiratory and reproductive syndrome virus(PRRSV), porcine parvovirus, swine transmissible gastroenteritis virus (TGEV), porcine respiratory coronavirus (PRCV), pseudorabies virus (PRV), and cell lines such as PK-15 cells and primary bovine testicular cells. Sequence of the PCR product of T strain was expected. The detection limit of the assay was 1 TCID50/mL. Of 75 visceral organ samples from suspected-CSFV infected pigs (e.g., liver, spleen, heart and kidney) and 132 other swine products tested, the RT-PCR and the commercial antigen-capture ELISA assay showed a concordance of 95.7% （198/207）. The RT-PCR assay could be performed within 2h or less. The above results demonstrated that the RT-PCR could be used as a method for quick diagnosis and detection of CSFV. |
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Key words: classical swine fever virus(CSFV);real-time RT-PCR;swine product;detection |
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