斑点叉尾鮰疱疹病毒PCR-ELISA检测方法的建立

Development of a PCR-ELISA Assay for the Detection of the Channel Catfish Virus

针对斑点叉尾鮰疱疹病毒(CCV)的ORF6基因设计引物和探针,PCR产物在扩增过程中标记地高辛,探针5′端用生物素标记,建立了CCV的敏感、快速、环保的PCR-ELISA检测方法。对反应条件进行了优化,并评估了该方法的特异性和敏感性。结果表明:该方法可特异性检测CCV DNA,与各对照均无交叉反应,具有高度特异性;该方法对CCV DNA的检测阈值为5 fg,敏感性是常规PCR检测方法的10倍。用此方法对人工感染样品进行检测,能够检测到感染组织中的CCV DNA。该方法能够定性和半定量检测CCV DNA,可用于斑点叉尾鮰疱疹病毒的检测、斑点叉尾鮰暴发性流行病的诊断。

A rapid and sensitive PCR-ELISA assay was developed for detection of channel catfish virus （CCV）. The specific 135 bp fragment labeled with digoxigenin was amplified from the ORF6 gene of CCV. Approximate 5 fg of purified DNA could be detected by the PCR-ELISA system, indicating this method had high sensitivity,10 times as much as which detected by conventional PCR. No cross-reaction was observed against the controls. The result in this study showed that this sensitive and specific method was useful for early detection of CCV infection.