WSSV和IHHNV二重实时荧光PCR检测方法的建立

DEVELOPMENT OF A MULTIPLEX REAL-TIME PCR ASSAY FOR DETECTION OF WSSV AND IHHNV

根据基因库中对虾白斑综合症病毒W SSV(AF369029)和传染性皮下及造血器官坏死病毒IHHNV(AF218226)基因序列,设计了W SSV和IHHNV的两对特异性引物和两条用不同荧光基团标记的TaqM an探针。对反应条件和试剂浓度进行优化,建立了能够同时检测W SSV和IHHNV的二重实时荧光PCR方法。该方法特异性好,对W SSV和IHHNV的检测敏感性分别达到2和20个模板拷贝数;此外抗干扰能力强,对W SSV和IHHNV不同模板浓度进行组合,仍可有效地同时检测这二个病毒。对保存的30份经常规PCR检测仅为W SSV或IHHNV阳性的样品进行二重实时荧光PCR检测,结果都为阳性,其中1份为W SSV和IHHNV混合感染。本研究建立的二重实时荧光PCR方法用于W SSV和IHHNV的检测具有特异、敏感、快速、定量等优点。

White spot syndrome virus （WSSV） and Infectious hypodermal and haematopoietic necrosis virus （IHHNV） are responsible for significant economic loss in the shrimp industry. In order to simultaneously and massively identify WSSV and IHHNV,two pairs of primers and two TaqMan probes were designed and synthesized according to the conserved gene sequences of WSSV （AF369029） and IHHNV （AF218226） in GenBank. The reaction parameters such as the concentration of two pair of primers, two TaqMan probes and the reaction buffer were optimized to develop a multiplex real-time PCR assay for the rapid detection of WSSV and IHHNV. The multiplex real-time PCR assay was found to be specific and be able to detect and differentiate WSSV and IHHNV,and no positive results were observed when nucleic acid from Vibrio,Taura Syndrome Virus and Streptococcus were used as multiplex real-time PCR templates. The developed multiplex real-time PCR assay was compared with that of routine PCR. The sensitivity of multiplex real-time PCR assay was 2 and 20 template copies for WSSV and IHHNV respectively,and its sensitivity was 103 and 102 times higher than that of the routine PCR. The samples were examined using the multiplex real-time PCR repeatedly and the results indicated that the multiplex real-time PCR was reproducible. Different concentrations of WSSV and IHHNV could be identified when mixed together, which im- plied the assay could be applied to clinical confirmation for simultaneous infection of WSSV and IHHNV. The multiplex real-time PCR results of 30 routine PCR positive samples showed that one specific amplified curve was displayed when shrimp was infected by only one of these two viral pathogens, whereas two specific amplified curves were displayed when shrimp was infected by two viral pathogens. The result indicated that multiplex real-time PCR was able to detect and differentiate the presence of each pathogen in infected clinical shrimp. This multiplex real-time PCR assay is a quick,sensitive, specific and quantitative tool for detection of WSSV and IHHNV,and it will be useful for the control of WSS and IHHN in shrimp.