对虾IHHNV荧光定量PCR检测方法的建立

Development of a Real-time TaqMan-quantitative PCR Assay for IHHNV Detection

根据GenBank中对虾传染性皮下和造血器官坏死病毒(IHHNV)保守基因序列(AF218226),设计合成了1对引物和1条TaqMan探针,建立了检测IHHNV的荧光定量PCR技术。将建立的荧光定量PCR检测方法与常规PCR对比。结果显示,所建立的荧光定量PCR方法灵敏度可达2个拷贝,比常规PCR灵敏度高1000倍。对保存的15份经常规PCR检测为IHHNV阳性的DNA样品进行荧光定量PCR检测,结果都为阳性,检测的病毒含量为2.15×107～4.21×104拷贝/μL。用该方法对不同浓度的样品进行了重复检测,表明该方法具有良好的重复性,可满足IHHNV的临床诊断需要。

A pair of primers and a TaqMan probe were designed and synthesized according to the conserved gene sequences of Infectious Hypodermal and Haematopoietic Necrosis （IHHNV） in GenBank （AF218226）, and then reaction parameters were optimized to develop a real -time TaqMan -quantitative PCR assay. The developed quantitative PCR assay was compared with that of routine PCR. This quantitative PCR assay could detect 2 template copies of plasmid DNA, and its sensitivity was 1 000 times higher than that of the routine PCR. The real - time Taq- Man - quantitative PCR results of 15 routine PCR positive clinical samples showed that concentration of the clinical samples were 2.15 ×10^7 -4.21 × 10^4 copies/μL. The samples were examined using the quantitative PCR repeatedly and the results indicated that the quantitative PCR was reproducible and could be used successfully for the diagnosis of IHHNV infection.