为快速、灵敏、准确地同时检测和鉴别猪圆环病毒2型(PCV2)、猪细小病毒(PPV)和伪狂犬病毒(PRV)的方法,本研究采用连接酶检测反应(LDR)-PCR和基因芯片技术建立一种新型检测方法.首先在3种病毒的保守区内分别设计一对LDR探针,两端各连接一段通用序列,依次进行LDR、通用引物荧光标记扩增和芯片杂交,同时比较引物标记和Cy5 -dCTP标记方法的灵敏度.结果表明该方法可以特异地检测PCV2、PPV和PRV3种病毒,而对牛病毒性腹泻病毒、猪传染性胃肠炎病毒、猪流行性腹泻病毒、猪繁殖与呼吸障碍综合征病毒、猪瘟病毒、乙型脑炎病毒、猪圆环病毒1型检测结果均为阴性;对3种病毒的最低检测限少于10个拷贝;Cy5-dCTP标记检测的灵敏度显著高于引物标记.利用建立的方法对41例临床样品进行检测,与普通PCR检测结果符合率为97.6％～100％.该方法的建立为基础研究和临床应用提供了技术平台.%To establish a sensitive detection method for porcine circovims type 2 (PCV2), porcine parvovirus (PPV) and pseudorabies virus (PRV) which cause swine severe reproductive and/or respiratory failure, a novel diagnostic oligonucleotide microarray based on ligase detection reaction PCR (LDR-PCR) was developed in this study. According to alignment of the viral sequences, LDR probes for each virus were designed, which were flanked by universal sequences on both sides of the probes. Through asymmetric PCR enrichment of the ligation products by universal primers, labeling and microarray hybridization, the target viruses were detected and differentiated. Compared to the labeling method with Cy5-primer, Cy5-dCTP labeling was more sensitive in this assay. The specific test result showed that the assay had no cross reaction with bovine viral diarrhea (BVDV), transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), Japanese encephalitis virus (JEV) and PCV1. The sensitivity of the assay was able to detect as low as 10 copies of target virus. Forty one clinical samples were tested by the assay and traditional PCR, the results of both methods showed a concordance rate of 97.6% to 100%. The established assay provided a new technical platform for the basic research and clinical application.
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