3型哺乳动物正呼肠孤病毒SYBR Green I荧光定量PCR方法的建立及应用

Development and application of SYBR Green I fluorescent quantitative PCR method for mammalian orthoreovirus 3 detection

哺乳动物正呼肠孤病毒(MRV)是双链RNA病毒,可以感染自然宿主的哺乳动物和脊椎动物。为建立一种针对3型哺乳动物正呼肠孤病毒(MRV-3)的特异且快速的检测方法,本研究根据Genbank登录的MRV-3(OQ627746-OQ627755)S1基因保守区设计1对特异性引物,并从MRV-3中扩增S1基因,构建重组质粒p MD18-S1,经PCR和测序鉴定正确后作为质粒标准品,经反应体系及反应条件优化后首次初步建立了检测MRV-3的SYBR Green I荧光定量PCR(q PCR)方法。将质粒标准品10倍倍比稀释后作为模板经该q PCR扩增,建立标准曲线,结果显示,质粒标准品在1.3×10~8拷贝/μL~1.3×10~3拷贝/μL与各自的Ct值均呈良好的线性关系,斜率为-3.1706,R~2为0.9999,熔解曲线为单峰。以牛病毒性腹泻病毒(BVDV)、牛肠道病毒(BEV)、水牛匈爱病毒(Buf Hu V)、牛冠状病毒(BCo V)、牛细小病毒(BPV)和MRV-3的基因组DNA/c DNA为模板,利用本研究建立的q PCR方法检测,评估该方法的特异性;将质粒标准品10倍倍比稀释至1.3×10~2拷贝/μL~1.3×10~8拷贝/μL后作为模板,分别利用本研究建立的q PCR和常规PCR检测,比较两种方法的检测结果,评估本研究建立q PCR方法的敏感性;以1.3×10~3拷贝/μL~1.3×10~7拷贝/μL 5个不同浓度的质粒标准品为模板,利用该方法分别进行批内和批间的重复性试验,评估该方法的重复性。结果显示,该方法只能检测出MRV-3,其他相关病原的检测结果均为阴性;该q PCR对质粒标准品的检测限为1.3×10~3拷贝/μL,比常规PCR敏感性高10 000倍;批内和批间重复性试验的变异系数均小于或等于1.0%,表明该q PCR方法特异性强、敏感性高、重复性好。利用该方法检测220份牛粪便样品,结果显示MRV-3的检出率(3.64%,8/220)高于常规PCR的检出率(1.36%,3/220),两种检测方法的阳性符合率达100%,阴性符合率为97.75%,总符合率为97.78%。综上所述,本研究首次建立的检测MRV-3的SYBR Green I q PCR方法可以用于临床牛腹泻病原的检测,为MRV-3尤其是牛源MRV-3提供了一种快速灵敏的检测手段,也为MRV-3的后续研究奠定了基础。

Mammalian orthoreovirus(MRV)are double-stranded RNA viruses that can infect mammals and vertebrates as their natural hosts.In order to establish a specific and rapid clinical assay for mammalian positive echidna orphan virus type 3(MRV-3),we designed one pair of specific primers based on the conserved region of the S1 gene of MRV-3(OQ627746-OQ627755),which was obtained from Genbank.The S1 gene amplified from MRV-3 was cloned to construct a recombinant plasmid pMD18-T-S1,and after being identified correctly by PCR and sequencing,it will be used as a plasmid standard.After optimizing the reaction system and reaction conditions,the SYBR GreenⅠfluorescence quantitative PCR(qPCR)method for the detection of MRV-3 was established for the first time.Dilute the plasmid standard at a series of 10-fold dilutions and use it as a template for qPCR amplification to establish a standard curve.The results showed that the plasmid standards showed good linear relationships with their respective Ct values from 1.3×108 copies/μL to 1.3×103 copies/μL,with a slope of-3.1706,R2 of 0.9999,and the melting curve showed a single peak.Genomic DNA/cDNA of bovine viral diarrhea virus(BVDV),bovine enterovirus(BEV),buffalo hunlovirus(BufHuV),bovine coronavirus(BCoV),bovine pinto virus(BPV),and MRV-3 were used as templates and detected using the qPCR method established in the present study to assess the specificity of the method.The plasmid standard was diluted at a 10-fold ratio from 1.3×102 copies/μL to 1.3×108 copies/μL as the templates,which were used to evaluate the sensitivity of the established qPCR and normal PCR,respectively.The plasmid standard at five different concentrations of 1.3×103 copies/μL-1.3×107 copies/μL were used to assess the intra-batch and inter-batch reproducibility of the method.The results showed that the method could only detect MRV-3,and no other related pathogens were detected.The detection limit of the qPCR for plasmid standards was 1.3×103 copies/μL,which was 10000 times more sensitive than that of ordinary PCR.The coefficients of variation for intra-and inter-batch reproducibility tests were less than or equal to 1.0%,indicating that the qPCR method was highly specific,sensitive,and reproducible.Two hundred twenty cattle fecal samples were subjected to detect MRV-3 using this method.The results showed that the detection rate of MRV-3(3.64%,8/220)was higher than that of ordinary PCR(1.36%,3/220),and the positive compliance rate of the two assays reached 100%.In contrast,the negative compliance rate was 97.75%,with a total compliance rate of 97.78%.In conclusion,the SYBR GreenⅠfluorescent quantitative PCR method for detecting MRV-3 was established for the first time in this study can be used for detecting clinical bovine diarrhea pathogens,and provid a rapid and sensitive detection means for detecting MRV-3,especially the bovine-origin MRV-3.Also,it lays the detection foundation for the subsequent research of MRV-3.