实时荧光定量PCR快速检测对虾IHHNV载量方法的建立和应用

Development and application of a real-time PCR for IHHNV

目的：建立一种快速、定量、特异、灵敏的PCR方法用于对虾传染性皮下及造血组织坏死病毒（IHHNV）的早期诊断和疫情监测。方法：根据GenBank登录的IHHNV毒株序列，使用生物学软件进行序列比对，在IHHNV保守区序列设计特异性引物与Taqman探针。对实时荧光定量PCR反应条件进行优化以缩短检测时间，提高特异性、灵敏度、重复性，并与普通PCR进行盲样测试的比对。结果：本法线性范围5×10^2～5×10^7拷贝／ml，检出限5×10^1拷贝／ml，灵敏度超过普通PCRl00倍，特异性高于普通PCR，重复性极好（S=0．035～0．39，CV=0．13％-1．05％），检测时间为普通PCR的1／5，现场盲样IHHNV检出阳性率高于普通PCR26．00％。结论：本研究建立Taqman实时荧光定量PCR用于对虾IH—HNV检测具有特异、灵敏、快速、定量、重复性好等优点。

Objective ：To establish a Taqman - based real - time PCR assay for detecting infectious hypodermal and hematpoietic necroses virus （IHHNV）. Methods：The IHHNV sequences obtained from genbank were aligned to find the conserved regions, and the specific primers and probes were designed according to these regions. PCR assay were optimized to reduce the detection time, improve the sensitivity, specificity and reproducibility, then compared to the general PCR method in the blind test. Results：The linear range and detection limit of our assay were 5 × 10^2-5 × 107 copies/ml, 5 × 10^1 copies/ml, while the sensitivity was 100 times and the specificity was higher than that of the general PCR method. Furthermore. It also had a excellent reproducibility （s =0. 035 -0. 29, CV =0. 13% - 1.05% ） and the time of detection was 1/5 of general PCR method. Positive rate in blind tests using our method was higher than the general PCR with the rate of 25.64%. Conclusion：This Taqman - based real - time PCR assay is a quick, sensitive, specific, quantitative and reproducible tool for the detection of IHHNV.