猴免疫缺陷病毒实时荧光环介导等温扩增快速检测方法的建立

DEVELOPMENT OF A REAL-TIME LOOP-MEDIATED ISOTHERMAL AMPLIFICATION FOR RAPID DETECTION OF SIMIAN IMMUNODEFICIENCY VIRUS

以猴免疫缺陷病毒(Simian immunodeficiency virus,SIV)的核心蛋白Gag区域的部分保守基因序列为检测靶标,通过软件设计内、外引物和环引物,借助恒温荧光扩增仪建立SIV实时荧光环介导等温扩增快速检测方法(real-time loop-mediated isothermal amplification,Realamp),根据有无"S"型扩增曲线判断检测结果。Realamp方法对含有猴免疫缺陷病毒目标片段的质粒标准品的检测灵敏度为300 copies/μL,对猴类的其他常见病原如猴D型逆转录病毒、猴T淋巴细胞趋向性病毒、猴B病毒等无非特异性扩增。通过33份临床样本的检测,Realamp检出的阳性2例,qPCR检出的阳性率为1例,两种检测方法的符合率为97%。本研究建立的Realamp检测技术所需设备简单、反应效率快、检测灵敏度高和特异性强,可用于偏远地区猴养殖单位相关病原的检测。

In order to develop a real-time loop-mediated isothermal application method for detection of Simian immunodeficiency virus(SIV),the inner,outer and loop primers were designed based on the conserved DNA sequence of gag protein.With the help of isothermal amplification equipment,Realamp assay was conducted and test results were evaluated by the curve.The sensitivity of Realamp assay was300copies/|xL for plasmids containing partial sequence of SIV.No amplification was found for Simian type D retrovirus,Simian T lymphotropic virus and Monkey B virus.Among33clinical samples,two were detected positive with Realamp assay while one of these two samples positive with qPCR.The Realamp developed here may be applied to detect SIV for monkey breeding business.