鳗鲡疱疹病毒与鳗鲡圆环病毒双重PCR检测方法的建立

Establishment and application of duplex PCR detection system between Anguillid herpesvirus and Eel circovirus

【目的】建立能同时检测鳗鲡疱疹病毒(Anguillid herpesvirus, AHV)与鳗鲡圆环病毒(Eel circovirus,EeCV)的双重PCR法。【方法】优选PCR引物组合,建立鳗鲡疱疹病毒与鳗鲡圆环病毒的双重PCR检测方法,探究双重PCR扩增条件,并检验该方法的特异性、灵敏性和临床样品检测效果。【结果】双重PCR法同时扩增出鳗鲡疱疹病毒690 bp和鳗鲡圆环病毒338 bp特异性片段,与猪圆环病毒(Porcine circovirus)、鸭圆环病毒(Duck circovirus)、锦鲤疱疹病毒(Koi herpesvirus)和鲤疱疹病毒(Cyprinid herpesvirus)的核酸不发生扩增反应。鳗鲡疱疹病毒和鳗鲡圆环病毒重组质粒标准品的最低全检出量为5.0×10^(-4)μg·mL^(-1)。用所建立的双重PCR方法对50份临床病鳗进行检验,其中鳗鲡疱疹病毒的感染率为16%,鳗鲡圆环病毒的感染率为68%,共感染率为8%。该感染率与单一PCR检测结果完全一致。【结论】建立的鳗鲡疱疹病毒和鳗鲡圆环病毒双重PCR检测法不仅特异性强、灵敏度高、重复性好,而且临床应用效果佳,为鳗鲡疱疹病毒和鳗鲡圆环病毒的快速检测提供了有效的技术支持。

【 Objective】 The aim of the present study was to establish a duplex PCR method for detection of Anguillid herpesvirus and Eel circovirus. 【Method】 Routine PCR primers were preferred to optimize the amplification conditions and test the specificity and sensitivity of the method. At the same time, the sick eel of suspected cases collected from different farm were detected by the duplex PCR method, and compared with the routine PCR method. 【Result】 The results showed that the specific fragments of 690 bp and 388 bp were amplified by using the duplex PCR from DNA samples of Anguillid herpesvirus and Eel circovirus, respectively. But the nucleic acid amplification results of Porcine circovirus, Duck circovirus, Koi herpesvirus and Cyprinid herpesvirus were negative. When the positive DNA concentrations of both Anguillid herpesvirus and Eel circovirus were diluted to 5.0×10^(-4)μg·mL^(-1), the corresponding target fragments could still be detected. 50 samples collected from suspected cases of sick eels were diagnosed by PCR. The infection rate of Anguillid herpesvirus was 16%. The infection rate of Eel circovirus was 68%. The infection rate of Anguillid herpesvirus and Eel circovirus was 8%.【Conclusion】 The results indicate that the duplex PCR detection method has certain specificity and sensitivity, and can be used for the detection and differential diagnosis of recessive cases of clinical infection of Anguillid herpesvirus and Eel circovirus.