一种快速定量检测猴组织中SIV/SHIV的方法

Quantitative detection of SIV/SHIV in Chinese rhesus macaque tissues by TaqMan real-time FQ-PCR

目的以猴-人免疫缺陷病毒(simian-human immunodeficiency virus,SHIV)gag基因为特异性扩增靶标,建立一种快速、灵敏、特异的TaqMan实时荧光定量PCR法检测猴血浆、外周血单核细胞和淋巴结中的猴免疫缺陷病毒(simian immunodeficiency virus,SIV)和SHIV。方法以SHIVSF162p3n gag基因为模板,用普通PCR方法扩增出109 bp DNA片段用于克隆构建标准品质粒pGEM^®-T-SHIV gag。通过对SHIV标准品质粒定量分析,优化反应体系,检测TaqMan实时荧光定量PCR方法的灵敏度、特异度和重复性。结果TaqMan实时荧光定量PCR法能有效地检测出10^2~10^7 copies/μL SIV/SHIV gag基因mRNA,线性系数为R^2=0.998,slop=-3.304。方法重复性检测显示CV%在0.6%~1.1%之间。结论该TaqMan实时荧光定量PCR法具有快速、灵敏和特异性高的优点,适用于SIV/SHIV感染猴动物模型的研究工作。

Objective To develop a rapid,high-sensitivity,and high-specificity assay based on TaqMan realtime fluorescent quantitative PCR(TaqMan real-time FQ-PCR)targeting the gag gene of simian-human immunodeficiency virus(SHIV)for quantitative measurement of simian immunodeficiency virus(SIV)and SHIV in plasma,peripheral blood mononuclear cells,and lymph nodes from rhesus macaques.Methods A 109-bp fragment of the SHIVSF162p3n gag gene was amplified using reverse-transcription PCR and cloned into the pGEM^®-T vector to construct the pGEM^®-T-SHIV gag plasmid,which was used as a standard for the assay.Results TaqMan real-time FQ-PCR efficiently detected 10^2–10^7 copies/μL SIV/SHIV gag mRNA with a correlation coefficient(R^2)of 0.998 and slope of-3.304.The coefficient of variability was 0.6%–1.1%.Conclusions This rapid assay exhibited both high sensitivity and high specificity.Thus,this method is suitable for studies involving SIV/SHIV infection macaque models.