一种基于VP6基因的A组轮状病毒拷贝数实时荧光定量PCR检测方法的建立
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摘要
目的建立一种基于VP6基因的A组轮状病毒(rotavirus,RV)拷贝数的实时荧光定量PCR(RT-qPCR)检测方法。方法提取病毒液中RV基因组RNA,经RT-PCR扩增VP6基因片段,回收PCR产物克隆至pMD-19T载体,将鉴定正确的阳性菌株常规培养制备标准品质粒,建立RT-qPCR病毒拷贝数检测方法,以标准品质粒Ct值为横坐标,拷贝数的对数为纵坐标绘制标准曲线,依据标准曲线方程检测样品中RV拷贝数。结果标准品质粒经酶切及测序鉴定证明构建正确;获得RV标准品质粒的标准曲线方程为y=-0. 246 3 x+10. 957,R~2=0. 995 5;原倍及稀释度为1×10~(-3)的样品溶液的RV病毒拷贝数分别为265 319. 903 5、1 682. 228 735 copies/μL。结论建立的RT-qPCR拷贝数检测方法,能在早期快速准确的检测RV,该方法稳定性较好,可据此标准曲线方程对样品中的RV进行绝对定量,为RV研究及临床检查提供了检测工具。
Objective To establish a real-time fluorescent quantitative RT-PCR(RT-qPCR)assay for gene copy number of group A rotavirus(RV)based on VP6 gene. Methods Genomic RNA was extracted from culture supernatant of RV,with which VP6 gene was amplified by RT-PCR and cloned into vector pMD-19T. The constructed recombinant plasmid was transformed to competent E. coli DH5α. The positive bacterial strain identified by agarose gel electrophoresis was subjected to routine culture to obtain the standard plasmid,based on which a real-time fluorescent RT-qPCR assay was developed. A standard curve was plotted using the Ct value of standard plasmid as abscissa while the logarithm of copy number as ordinate. The RV copy number was calculated according to the equation of the standard curve. Results Restriction analysis and sequencing proved that the standard plasmid was constructed correctly. The equation of standard curve was as follows: y =-0. 246 3 x + 10. 957,R~2 = 0. 995 5. The gene copy numbers in primary samples and the samples at a dilution of 1 × 10~(-3) were 265 319. 903 5 and 1 682. 228 735 copies/μL respectively. Conclusion The developed RTqPCR assay may be used for the early and rapid test for RV,which showed high stability. According to the equation of standard curve,the RV in test samples may be quantified absolutely. It provided a tool for research and clinical examination of RV.
Objective To establish a real-time fluorescent quantitative RT-PCR(RT-qPCR)assay for gene copy number of group A rotavirus(RV)based on VP6 gene. Methods Genomic RNA was extracted from culture supernatant of RV,with which VP6 gene was amplified by RT-PCR and cloned into vector pMD-19T. The constructed recombinant plasmid was transformed to competent E. coli DH5α. The positive bacterial strain identified by agarose gel electrophoresis was subjected to routine culture to obtain the standard plasmid,based on which a real-time fluorescent RT-qPCR assay was developed. A standard curve was plotted using the Ct value of standard plasmid as abscissa while the logarithm of copy number as ordinate. The RV copy number was calculated according to the equation of the standard curve. Results Restriction analysis and sequencing proved that the standard plasmid was constructed correctly. The equation of standard curve was as follows: y =-0. 246 3 x + 10. 957,R~2 = 0. 995 5. The gene copy numbers in primary samples and the samples at a dilution of 1 × 10~(-3) were 265 319. 903 5 and 1 682. 228 735 copies/μL respectively. Conclusion The developed RTqPCR assay may be used for the early and rapid test for RV,which showed high stability. According to the equation of standard curve,the RV in test samples may be quantified absolutely. It provided a tool for research and clinical examination of RV.
引文
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[7]PAN X X,ZHANG S,CHEN Y D.Study advances on the VP6protein immunology of rotavirus[J].Chin J Vaccin Immun,2011(2):169-172.(in Chinese)潘小霞,张顺,陈元鼎.轮状病毒VP6蛋白免疫学研究进展[J].中国疫苗和免疫,2011(2):169-172.
[8]ZHANG Z.Full genomic analysis of group A rotavirus isolated from an adult with acute gastroenteritis[D].Wuhan:Central China Normal University,2011.(in Chinese)张喆.成人腹泻粪便样品中A组轮状病毒全基因分析[D].湖北武汉:华中师范大学,2011.
[9]JERE K C,CHAGUZA C,BAR-ZEEV N,et al.Emergence of double-and triple-gene reassortant G1P[8]rotaviruses possessing a DS-1-like backbone after rotavirus vaccine introduction in Malawi[J].J Virol,2018,92(3):e1217-e1246.
[10]DE LA CRUZ HERNANDEZ S I,ANAYA MOLINA Y,GOMEZSANTIAGO F,et al.Real-time RT-PCR,a necessary tool to support the diagnosis and surveillance of rotavirus in Mexico[J].Diagn Microbiol Infect Dis,2017.
[11]ZENG J,TAN Y.Evaluation of different methods for the detection of group A rotavirus in infants[J].J Clin Exp Med,2013(7):543-544.(in Chinese)曾静,谭滢.三种婴幼儿A组轮状病毒检测方法的方法学评价[J].临床和实验医学杂志,2013(7):543-544.
[2]RENG J P.Progress in rotavirus epidemiology and vaccines[J].Zhejiang J Prevent Med,2015,27(8):801-804.(in Chinese)任江萍.轮状病毒流行病学与疫苗研究进展[J].浙江预防医学,2015,27(8):801-804.
[3]KOTLOFF K L,NATARO J P,BLACKWELDER W C,et al.Burden and aetiology of diarrhoeal disease in infants and young children in developing countries(the Global Enteric Multicenter Study,GEMS):a prospective,case-control study[J].Lancet,2013,382(9888):209-222.
[4]STAAT M A,PAYNE D C,DONAUER S,et al.Effectiveness of pentavalent rotavirus vaccine against severe disease[J].Pediatrics,2011,128(2):e267-e275.
[5]CUI P W.Study on disease burden of rotavirus diarrhea and epidemiological characteristics of intussusception among children in china[D].Beijing:Chinese Center for Disease Control and Prevention,2016.(in Chinese)崔朋伟.儿童轮状病毒腹泻疾病负担及肠套叠流行特征研究[D].北京:中国疾病预防控制中心,2016.
[6]LUO M,GONG C,SHI L L,et al.Evaluation on performance of five routine rotavirus detection assays and application strategies[J].Dis Surveillance,2014(3):223-227.(in Chinese)罗明,龚成,史玲莉,等.五种常用轮状病毒检测方法的评价及应用策略[J].疾病监测,2014(3):223-227.
[7]PAN X X,ZHANG S,CHEN Y D.Study advances on the VP6protein immunology of rotavirus[J].Chin J Vaccin Immun,2011(2):169-172.(in Chinese)潘小霞,张顺,陈元鼎.轮状病毒VP6蛋白免疫学研究进展[J].中国疫苗和免疫,2011(2):169-172.
[8]ZHANG Z.Full genomic analysis of group A rotavirus isolated from an adult with acute gastroenteritis[D].Wuhan:Central China Normal University,2011.(in Chinese)张喆.成人腹泻粪便样品中A组轮状病毒全基因分析[D].湖北武汉:华中师范大学,2011.
[9]JERE K C,CHAGUZA C,BAR-ZEEV N,et al.Emergence of double-and triple-gene reassortant G1P[8]rotaviruses possessing a DS-1-like backbone after rotavirus vaccine introduction in Malawi[J].J Virol,2018,92(3):e1217-e1246.
[10]DE LA CRUZ HERNANDEZ S I,ANAYA MOLINA Y,GOMEZSANTIAGO F,et al.Real-time RT-PCR,a necessary tool to support the diagnosis and surveillance of rotavirus in Mexico[J].Diagn Microbiol Infect Dis,2017.
[11]ZENG J,TAN Y.Evaluation of different methods for the detection of group A rotavirus in infants[J].J Clin Exp Med,2013(7):543-544.(in Chinese)曾静,谭滢.三种婴幼儿A组轮状病毒检测方法的方法学评价[J].临床和实验医学杂志,2013(7):543-544.