摘要
本研究旨在建立多重Real-time PCR方法检测猪繁殖与呼吸综合征病毒(PRRSV)、猪圆环病毒2型(PCV2)、猪伪狂犬病毒(PRV)和猪细小病毒(PPV)。根据GenBank数据库中PRRSV、PCV2、PRV、和PPV的核苷酸序列设计特异性引物和探针,通过优化反应条件,检测系列稀释的纯化阳性质粒,建立检测PRRSV、PCV2、PRV和PPV的单项和多重Real-time PCR方法。灵敏度和特异性试验结果表明,无论是单项和多重Real-timePCR检测,该方法都具有高度特异性,与其它病原检测无明显交叉反应;对4种病毒的最低检测量为<101拷贝或1TCID50,检测灵敏度比常规PCR高100倍。对40份临床样本进行检测,多重Real-time PCR检测结果和单项Real-time PCR检测结果完全一致。建立的同时检测PRRSV、PCV2、PRV和PPV的多重Real-time PCR方法具有快速、灵敏、特异、重复性好和能对样品进行定量检测等优点。
The aim of this study is to establish a multiplex real-time PCR assay to simultaneously detect and discriminate porcine reproductive and respiratory syndrome virus （PRRSV）, porcine circovirus type 2 （PCV2）,porcine pseudorabies virus（PRV）and porcine parvovirus （PPV） in a single test tube. According to the nucleotide sequences of PRRSV,PCV2, PRV and PPV from GenBank, four pairs of specific probes were designed. By optimizing the probe＇s concentration, primers concentration and sample DNA extraction, a multiplex real-time quantitative PCR assay for detection of these viruses was established. The specificity and sensitivity of the multiplex real- time quantitative 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 pathogene- ses. Both multiplex and singleplex assays were consistently able to detect 〈10^1 copies of plasmid templates or 1 TCID50 virus per reaction. 40 clinical samples were comparatively detected using the multiplex and singleplex assay. The data showed that the results of the singleplex and multi- plex assay were in accordance with the results of conventional PCR. A multiplex real-time PCR assay was developed to simultaneously detect and discriminate PRRSV, PCV2, PRV and PPV in a single test tube, the method is rapid, sensitive, specific and accurate.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2012年第12期1998-2002,共5页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
河南省教育厅自然科学研究计划项目(2011A230016)
河南省重点科技攻关项目(092102110073)
