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羊痘病毒实时荧光定量TaqMan PCR
检测方法的建立
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康文玉1，徐自忠2，花群义2，杨云庆2，周晓黎2，董俊2，尹尚莲2，高洪1
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（1.云南农业大学 动物科学技术学院，云南 昆明650201；
2.云南出入境检验检疫局 技术中心，云南 昆明650228）
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摘要：
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参照羊痘病毒（CaPV）P32的基因序列，设计合成了2套引物和1条探针，建立了实时荧光定量PCR技术，对细胞培养物、皮肤丘疹、痂皮等组织病料中的GPV进行了特异性检测和敏感性试验。结果显示，用300nmol/L引物浓度和200nmol/L探针浓度，获得的CT值较小，而ΔRn最大；可检测到相当于0.1 TCID50的病毒DNA；制作的标准曲线中各浓度范围内有极好的线性关系且线性范围宽，相关系数为0.9995以上；组内和组间试验重复性的变异系数分别为2.3%和34%；与常规的PCR相比较，该方法具有快速、特异、敏感、可定量，可同时检测大量样品等优点。表明，荧光TaqMan PCR是一种检测CaPV的良好方法，可对组织病料中低含量的CaPV或持续带毒宿主进行准确检测。 |
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关键词：
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羊痘病毒；荧光定量TaqMan PCR；检测 |
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中图分类号：S 852.659.1：Q 503 文献标识码：
A 文章编号：1673-4696(2006)07-0529-05 |
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Development of a real-time fluorescent TaqMan-quantitative
PCR assay for detection of capripoxvirus |
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KANG Wen-yu1, XU Zi-zhong2, HUA
Qun-yi2, YANG Yun-qing2,
ZHOU Xiao-li2, DONG Jun2, YIN Shang-lian2，GAO
Hong1 |
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(1.College of Animal Science and Technology, Yunnan Agricultural University, Kunming 650201,China；
2.Center of Technology, Yunnan Entry-Exit Inspection and Quarantine Bureau, Kunming 650228, China) |
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Abstract: A real-time fluorescent TaqMan-quantitative PCR assay using 2 pairs of primers and a probe designed and synthesized according to the P32 gene of the virus genome was developed for the specific detection of capripox virus in epithelial suspensions and cell culture preparations. The real-time fluorescent PCR assay detected specifically CaPV virus in samples with greater sensitivity than the conventional PCR procedure. The fluorescent PCR assay was fast, and could quantitatively assess the virus amounts and could handle more samples and/or replicates of samples in a single assay than the conventional PCR procedure. The results showed that this assay is a valuable complementary tool to the routine diagnostic procedures for the detection of capripox virus infection. |
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Key words: capripoxvirus; fluorescent TaqMan-quantitative PCR assay; detection |
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