欢迎访问《中国农学通报》，
中国农学通报

中国农学通报 ›› 2014, Vol. 30 ›› Issue (5): 41-45.doi: 10.11924/j.issn.1000-6850.2013-2012

所属专题： 畜牧兽医

• 畜牧 动物医学 蚕 蜂 • 上一篇    下一篇

猪流行性腹泻病毒Real-time PCR检测方法的建立

孟凡伟 张雪 王俊 单晶晶 王鹏 周双海 王志军   

  • 收稿日期:2013-07-24 修回日期:2013-12-11 出版日期:2014-02-15 发布日期:2014-02-15
  • 基金资助:
    天津市宁河原种猪场科技计划 “规模化猪场重要疫病防控关键技术研究” (2011)。

Development of Real-time PCR Assay for Porcine Epidemic Diarrhea Virus

  • Received:2013-07-24 Revised:2013-12-11 Online:2014-02-15 Published:2014-02-15

PDF (PC)

853

摘要： 为定量检测猪流行性腹泻病毒(PEDV)载量，建立PEDV的Real-time PCR方法。用RT-PCR方法扩增PEDV的M基因片段，构建含有M基因片段的重组质粒，用该重组质粒进行SYBR Green I Real-time PCR，来建立检测PEDV的荧光定量PCR方法。结果显示：在(5.56×102~5.56×107)拷贝/μL范围内，所建立方法具有优良的线性关系，其决定系数为0.9996，扩增效率为99.5%，扩增产物的熔解曲线只有一个特异性峰，无引物二聚体峰，熔解温度为(81.18±0.21)℃。该方法对猪传染性胃肠炎病毒、猪瘟病毒、猪繁殖与呼吸综合征病毒、伪狂犬病病毒、猪圆环病毒II型等猪源病毒均检测不到扩增产物，重复性试验的变异系数小于3%，对临床样品的检出率高于常规PCR方法。研究结果表明：建立的Real-time PCR检测方法特异性强、重复性好、灵敏性高，可用于PEDV的定量检测及其早期感染的快速诊断。

关键词: 乳管细胞, 乳管细胞

Abstract: The objective of this study was to develop a Real-time PCR assay of porcine epidemic diarrhea virus (PEDV) and therefore to detect the load of PEDV quantitatively. A fragment of M gene in PEDV was amplified by RT-PCR to construct the recombinant plasmid with the M gene fragment, then, the Real-time PCR based on SYBR Green I with the recombinant plasmid was conducted to develop the fluorescenct quantitative PCR assay for detection of PEDV. The results showed that, the assay generated had an excellent linear reltionship with a determination coefficient of 0.9996 and an amplification efficiency of 99.5% from (5.56×102 to 5.56×107) DNA copies/μL. The melting curve analysis showed only one specific peak with a melting temperature of (81.18±0.21)℃, and no primer-dimers peak represented. No amplification was determined by this mothod from unrelated swine virus samples, including porcine transmissible gastoenteritis virus, classical swine fever virus, porcine reproductive and respiratory syndrome virus, pseudorabies virus and porcine circovirus type 2. the coefficient of variations was less than 3% in the reproducible assays, and the detection results of several clinical samples with this method was improved as compared to conventional PCR. The results indicated that this Real-time PCR assay was of high specificity, producibility and sensitivity, and could be used for the quantitative detection of PEDV and quick diagnosis for early infection of PEDV.