应用qRT-PCR方法检测鼠痘病毒在小鼠不同组织中的差异分布

APPLICATION OF QRT-PCR ON DETECTION OF ECTROMELIA VIRUS LOADS IN MOUSE TISSUES

鼠痘(mouse pox)是由鼠痘病毒(Ectromelia virus,ECTV)感染实验小鼠引起的一种毁灭性、烈性传染病,为SPF小鼠必须排除的病原之一。本研究基于ECTV主要核蛋白P4b基因片段建立了实时定量PCR方法(real-time quantitative PCR,qRTPCR),用于检测C57BL/6小鼠感染ECTV后心脏、肝脏、脾脏、肺脏、肾脏和皮肤中的病毒载量,并用病理组织学观察验证了qRT-PCR方法的特异性和可靠性。结果表明,所建立的qRT-PCR方法能够准确检测小鼠感染后d5和d7不同组织的病毒载量,d5不同组织中病毒载量低于d7,与病理组织学检测结果一致。本研究建立的qRT-PCR检测方法且具有较高的灵敏性、特异性和可靠性,可用于ECTV感染细胞和组织器官中病毒载量、分布和消长动态的定性定量检测。

Ectromelia virus (ECTV) is the causative agent of mouse pox and has been extensively studied in mice as an excellent model for the pathogenesis and immunobiology of smallpox. In the present study, a SYBR Green I-based real-time PCR method was developed for the detection and quantitation of ECTV loads in different tissues. A pair of specific primers was designed to target a highly conserved region of major core protein P4b gene. The PCR method developed here was used to detect viral loads in hearts, livers, spleens, lungs, kidneys and skin tissues that were collected at days 5 and 7 from the C57BL/6 mice infected with ECTV. The result showed that this method was specific, sensitive and reproducible for detection of ECTV infection.