猪肠道α冠状病毒荧光定量PCR检测方法的建立
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摘要
为建立猪肠道α冠状病毒(SeACoV)荧光定量检测方法,本研究采用RT-PCR方法扩增SeACoV N基因保守区序列,将其克隆至p EASY-Blunt载体,构建重组质粒p EASY-Blunt-PA-N作为标准阳性质粒,以其为模板建立SeACoV的SYBR Green I荧光定量PCR检测方法,并进行特异性、敏感性和重复性试验验证。结果显示,建立的SYBR Green I荧光定量PCR检测方法 Ct值与标准品模板在9.47×101拷贝/μL～9.47×107拷贝/μL范围内呈良好的线性关系,相关系数为0.996,斜率为-3.91。本实验建立的方法对PEDV、TGEV、PDCo V、PRRSV等猪常见腹泻病样品检测无扩增,表明特异性良好;敏感性试验结果显示,该方法检测下限可达到9.47×101拷贝/μL;重复性试验结果显示,组内变异系数在0.82%～1.01%,组间变异系数在1.20%～1.69%,重复性较好。采用本研究建立的方法对广西和云南等地70份临床样品进行检测,结果显示除阳性对照外,临床样品均为阴性,表明该病毒尚未传播至广东以外的省份。本实验首次建立了SeACoV N基因SYBR Green I荧光定量PCR检测方法,为SeACoV快速检测和病毒感染预防提供技术手段。
This study was to establish a real-time fluorescence quantitative PCR method which can quickly and accuratelydetect SeACoV. The conserved region of SeACoV N gene was amplified by PCR and cloned into pEASY-Blunt vector. Theresulted pEASY-Blunt-N gene plasmid DNA was used as template to optimize assay condition of developing a SYBT Green Ireal-time PCR for detection of SeACoV. The standard curve produced a linear relationship between Ct value and initial amounts oftotal DNA at a range of 9.47×101-9.47×107 copies/μL, the correlation coefficient and slope were 0.996 and-3.91, respectively.The specificity assay showed no amplification of PEDV, TGEV, PDCoV and PRRSV which displayed a good specificity; Thesensitivity of this method was 9.47×101 copies/μL; Repeatability analysis showed that the coefficient of variation within the groupranged from 0.82% to 1.01%; The coefficient of variation between groups ranged from 1.20% to 1.69%. A total of 70 clinical samples from Guangxi and Yunnan were tested by this method and no positive samples were detected. The results showed thatSYBR Green I real-time PCR was a rapidly specific and sensitive method for the detection of SeACoV.
This study was to establish a real-time fluorescence quantitative PCR method which can quickly and accuratelydetect SeACoV. The conserved region of SeACoV N gene was amplified by PCR and cloned into pEASY-Blunt vector. Theresulted pEASY-Blunt-N gene plasmid DNA was used as template to optimize assay condition of developing a SYBT Green Ireal-time PCR for detection of SeACoV. The standard curve produced a linear relationship between Ct value and initial amounts oftotal DNA at a range of 9.47×101-9.47×107 copies/μL, the correlation coefficient and slope were 0.996 and-3.91, respectively.The specificity assay showed no amplification of PEDV, TGEV, PDCoV and PRRSV which displayed a good specificity; Thesensitivity of this method was 9.47×101 copies/μL; Repeatability analysis showed that the coefficient of variation within the groupranged from 0.82% to 1.01%; The coefficient of variation between groups ranged from 1.20% to 1.69%. A total of 70 clinical samples from Guangxi and Yunnan were tested by this method and no positive samples were detected. The results showed thatSYBR Green I real-time PCR was a rapidly specific and sensitive method for the detection of SeACoV.
引文
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[3]Wilde A,Zevenhoven-Dobbe J C,Beugeling C,et al.Coronaviruses and arteriviruses display striking differences in their cyclophilin A-dependence during replication in cell culture[J].JVirol,2018,517:148-156.
[4]Ummul H,Vasan S S,Ravindran T,et al.Development and evaluation of a one-step SYBR-Green I based real-time RT-PCRassay for the detection and quantification of Chikungunya virus in human,monkey and mosquito samples[J].Trop Biomed,2010,27(3):611-623.
[5]Bevins S N,Lutman M,Pedersen K,et al.Spillover of swine coronaviruses,United States[J].Emerg Infect Dis,2018,24(7):1390-1392.
[6]Santhosh S R,Parida M M,Dash P K,et al.Development and evaluation of SYBR Green I-based one-step real-time RT-PCRassay for detection and quantification of Chikungunya virus[J].J Clin Virol,2007,39(3):188-193.
[7]Akimkin V,Beer M,Blome S,et al.New Chimeric porcine coronavirus in swine feces,germany,2012[J].Emerg Infect Dis,2016,22(7):1314-1315.
[8]Gong Lang,Li Jie,Zhou Qing-feng,et al.A new bat-HKU2-like coronavirus in swine,China,2017[J].Emerg Infect Dis,2017,23(9):1607-1609.
[9]Peng Zhou,Hang Fan,Tian Lan,et al.Fatal swine acute diarrhoea syndrome caused by an HKU2-related coronavirus of bat origin[J].Nature,2018,556(7700):255-258.
[10]Pan Yong-fei,Tian Xiao-yan,Qin Pan,et al.Discovery of a novel swine enteric alphacoronavirus(SeACoV)in southern China[J].Vet Microbiol,2017,211:15-21.
[11]Calvo E,Escors D,Lopez J A,et al.Phosphorylation and subcellular localization of transmissible gastroenteritis virus nucleocapsid protein in infected cells[J].J Gen Virol,2005,86(8):2255-2267.
[12]Lin Ying,Shen Xu,Yang Rui-fu,et al.Identification of an epitope of SARS-coronavirus nucleocapsid protein[J].Cell Res,2003,13(3):141-145.
[13]Zhou Ling,Sun Yuan,Wu Jiao-ling,et al.Development of a Taq Man-based real-time RT-PCR assay for the detection of SADS-CoV associated with severe diarrhea disease in pigs[J].JVirol Methods,2018,255:66-70.
[14]Hu Hui,Kwonil J,Wang Qiu-hong,et al.Development of a one-step RT-PCR assay for detection of pancoronaviruses(alpha-,beta-,gamma-,and delta-coronaviruses)using newly designed degenerate primers for porcine and avian fecal samples[J].J Virol Methods,2018,256:116-122.