鼠痘病毒荧光定量Taqman-PCR检测方法的建立与应用

Establishment and application of a fluorescence quantitative Taqman-PCR detection method of mouse poxvirus

目的建立鼠痘病毒的荧光定量Taqman-PCR检测方法,以期能快速、准确、灵敏、特异的检出鼠痘病毒。方法经过比对和筛选,本研究选取鼠痘病毒的ERPV_027基因480-800位序列段,作为引物设计位点设计引物和探针,并对该引物对和探针的特异性、灵敏性、稳定性和重复性进行检测。结果本研究建立的方法,对鼠痘病毒的检测极限是68 copies/μL;该方法特异性强,只对鼠痘病毒特定片段进行特异性扩增,而对小鼠肝炎病毒、仙台病毒、沙门氏菌等其它病毒、细菌无扩增;该方法稳定性和重复性均较好。结论本研究成功建立了检测鼠痘病毒的荧光定量Taqman-PCR方法,该方法特异性强、灵敏度高,且具有较好的稳定性和重复性,具有广阔的应用价值。

Objective To establish a fluorescence quantitative Taqman-PCR method for rapid and accurate detection of mouse poxvirus. Methods After sequence alignment and comparison,ERPV027 gene was selected as the primer and probe design gene. Furthermore,the specificity,sensitivity,stability and reproducibility of these primers and probes were detected. Results The detection limitation of this method was 68 copies / μL. Data showed that this method has high specificity,which specifically amplifies mouse poxvirus,with no amplification signal of mouse hepatitis virus,Sendai virus,Salmonella and some other viruses and bacteria. This method also showed good stability and reproducibility.Conclusions This study has successfully established a fluorescence quantitative Taqman-PCR method for detection of mouse poxvirus,with high specificity,sensitivity,good stability and reproducibility,and a broad application potential.