非洲马瘟病毒RT-RAA快速检测方法的建立

Establishment of a Novel RT-RAA Fluorescence Assay for African Horse Sickness Virus

根据GenBank中非洲马瘟病毒结构蛋白VP7的编码基因序列,针对不同血清型间VP7中的保守序列设计引物和探针,建立非洲马瘟病毒核酸的RT-RAA检测方法,并对引物和探针浓度进行优化,确定最佳反应浓度。然后对该方法的特异性、敏感性和重复性进行评估。结果显示:本研究建立的非洲马瘟病毒RT-RAA检测方法在42℃恒定温度下20min能够有效检出目标基因片段,与其他马属动物疫病病原及重要经济动物疫病病原核酸无交叉反应,对非洲马瘟病毒阳性质粒的检测灵敏度达到102 copies/μL,重复性好。利用建立的非洲马瘟病毒RT-RAA检测方法对收集的88份马血清、10份马全血和4份非洲马瘟病毒模拟阳性样本进行检测,检测结果与OIE推荐的荧光RT-PCR检测结果一致。本研究建立的非洲马瘟病毒RT-RAA方法特异性强、敏感性高、稳定性好,在20 min内就能够检出样本中非洲马瘟病毒核酸,适用于非洲马瘟病毒核酸的快速检测。

Based on the coding gene sequence of African horse sickness virus(AHSV)structural protein VP7 in GenBank,primers and probes were designed for the conserved sequence of VP7 among different serotypes,and the RT-RAA assay of AHSV nucleic acid was established.The primer and probe concentrations were optimized to determine the best responses.The specificity,sensitivity and repeatability of this method were then investigated.The results showed that the RT-RAA detection method for AHSV established in this study could effectively detect the target gene fragment at a constant temperature of 42℃for 20 minutes,and had no cross reaction with the nucleic acids of other equine animal epidemics and important economic animal epidemics.The detection sensitivity of AHSV positive plasmid reached 102 copies/μL,with good repeatability.The established RT-RAA detection method for AHSV was used to detect 88 serum samples,10 unclotted whole blood samples and 4 mock positive samples collected in the laboratory,and the detection results were consistent with the fluorescent RT-PCR detection results recommended by OIE.Therefore,the established RT-RAA for AHSV,which had a high level of specificity,repeatability and sensitivity,can be employed to the rapid detection of AHSV.