Taqman实时荧光定量PCR快速检测白斑综合症病毒的方法研究

目的：建立一种快速、特异、灵敏的实时荧光定量PCR方法用于检测白斑综合病毒DNA，为控制对虾白斑综合症提供科学依据。方法：根据GenBank登录的白斑综合病毒株序列，使用生物学软件进行序列对比，在白斑综合症病毒保守区序列设计引物和Tagman探针并进行筛选。对实时荧光定量PCR反应条件进行优化，提高了该方法的特异性、灵敏度和重现性等，并与标准方法(核酸探针点杂交)进行比对，对现场32份虾样进行了检测。结果：Tagman实时荧光定量PCR与标准方法的特异性符合率为100％，灵敏度超过标准方法的10000-100000倍，检测时间为标准方法的1／24，重现性良好(s=0．1-0．49，CV=0．78％-3．72％)，并能准确定量。结论：本研究建立的Taqman实时荧光定量PCR方法用于白斑综合症病毒检测具有特异、灵敏、快速、定量、重现性好等优点。

The methodology study on rapid detection of WSSV by taqman-based real-time PCR

Objective:To estabilish a Tagman-based real-time PCR assay for detection of WSSV.Methods:The WSSV sequence downloaded from Genbank was aligned by biologic software and the specific primers and probes were designed in the conserved region of WSSV.The PCR conditions was optimized to improve the sensitivity, specificity and reproducibility. The assay was compared to the standard method (dot hybridization of nucleic acid probe), and 32 samples of Shrimp were detected.Results:The specificity of the assay was accorded with the standard method with a rate of 100%, the sensitivity of the assay was 10 000~ 100 000 times higher than that of the standard method, while the time consumed of the former was 1/24 of the latter. Furthermore, it also had a good reproducibility (s=0.1~0.49,CV=0.78%~3.72%)and could be used for the purpose of quantification.Conclusion:This Taqman-based real-time PCR assay was a quick, sensitive, specific, quantitative,reproducible tool for the detection of WSSV.