建立环介导间接PCR同时检测3种主要猪源性病毒

doi:10.3864/j.issn.0578-1752.2011.21.019

摘要/Abstract

摘要： 【目的】建立同时检测猪圆环病毒2型（PCV2）、猪瘟病毒（CSFV）和猪繁殖与呼吸综合征病毒（PRRSV）的环介导间接PCR方法，实现3种病毒快速检测。【方法】根据GenBank数据库中猪圆环病毒2型（PCV2）、猪瘟病毒（CSFV）和猪繁殖与呼吸综合征病毒（PRRSV）的核苷酸序列设计3对特异性探针，利用PCR技术分别标记于大豆Lectin基因片段的两端形成3种不同的报告基因，此标记的报告基因与3种待测靶标基因经杂交、缺口补平、环化后，采用1对引物反向PCR扩增报告基因，建立了PCV2、CSFV和PRRSV的环介导间接PCR检测方法，扩增片段大小分别为564 bp、434 bp和338 bp，该方法即可用于单病毒检测又可用于多病毒检测。【结果】灵敏度和特异性试验结果表明，无论是单病毒检测还是多病毒检测，该法都具有高度特异性，与其它病原检测无明显交叉反应；对PCV2、CSFV和PRRSV 3种病毒同时检测的底限分别为0.8、25和6.2 pg•μL-1，和单个病毒的检测灵敏度相同。利用环介导间接PCR和常规PCR对20份临床样本进行比较检测，结果表明，无论单病毒检测还是多病毒检测,其结果均与常规PCR检测结果完全一致。【结论】环介导间接PCR检测技术可用于PCV2、CSFV和PRRSV 3种病毒的同时检测，是一种简便快速、灵敏、特异的病原学诊断工具。

关键词: 猪圆环病毒2型, 猪瘟病毒, 猪繁殖与呼吸综合征病毒, 环介导间接PCR

Abstract: 【Objective】 The aim of this study is to establish a method of loop-mediated indirect PCR assay for simultaneous identification of porcine circovirus type 2 (PCV2), classical swine fever virus (CSFV) and porcine reproductive and respiratory syndrome virus (PRRSV) in pigs. 【Method】 According to the nucleotide sequences of PCV2, CSFV and PRRSV from GenBank, three pairs of specific probes were designed. By PCR, the Lectin gene fragment of soybean was labeled with different probes to be used as reporter genes aiming to the nucleotide sequences of PCV2, CSFV and PRRSV, respectively. After hybridizing with the target gene, gap filling and cyclizing, these probe-labeled reporter genes were amplified by reverse PCR with a pair of primers to detect the three species of viruses indirectly. The loop-mediated indirect PCR assay was developed, and the specific PCR products were 564 bp for PCV2, 434 bp for CSFV and 338 bp for PRRSV, respectively. The assay will be useful for the specific detection of the three species of viruses, not only for single virus but also for multiple viruses. 【Result】 The specificity and sensitivity of the loop-mediated indirect PCR assay were analyzed for single virus and multiple viruses. The results showed that the specificity was high, and no amplification was achieved to the other pathogeneses. The detection limits of the loop-mediated indirect PCR assay for multiple viruses was 0.8 pg?μL-1 for PCV2, 25 pg?μL-1 for CSFV and 6.2 pg?μL-1 for PRRSV, respectively, which was the same with the results of single virus detection. Twenty clinical samples were comparatively detected. The data showed that the results of the loop-mediated indirect PCR assay, not only for single virus but also for multiple viruses, were in accordance with the results of conventional PCR.【Conclusion】 The loop-mediated indirect PCR developed in this study is a simple, fast and specific method for detecting PCV2, CSFV and PRRSV in clinical samples, and can be used to detect all these three species of viruses at the same time.

Key words: porcine circovirus type 2, classical swine fever virus, porcine reproductive and respiratory syndrome virus, loop-mediated indirect PCR

郑鸣, 边传周, 王老七. 建立环介导间接PCR同时检测3种主要猪源性病毒[J]. 中国农业科学, 2011, 44(21): 4499-4507.

ZHENG Ming, BIAN Chuan-Zhou, WANG Lao-Qi. Development of Loop-Mediated Indirect PCR for Simultaneously Detecting Three Species of Viruses in Pigs[J]. Scientia Agricultura Sinica, 2011, 44(21): 4499-4507.

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https://www.chinaagrisci.com/CN/Y2011/V44/I21/4499

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