摘要
目的:建立一种能同时检测婴幼儿腹泻粪便中常见病毒的多重荧光定量PCR技术。方法:根据GenBank上几种病毒基因组保守序列设计引物序列,建立多重荧光定量PCR方法,对所建立的多重荧光定量PCR方法的特异性、灵敏性及重复性进行验证;并以所建立的方法对150例婴幼儿病毒性腹泻患者粪便标本进行检测。结果:所建立的多重荧光定量PCR检测方法具有很好的特异性,灵敏性检测可达102拷贝/mL,检测不同病毒核酸浓度各自的检测Ct值标准差均较小,变异系数均低于1.0%,具有较好的重复性;检测150份粪便标本,多重荧光定量PCR的检出率为36%,胶体金方法检出率为38.67%,两者比较差别无统计学意义(χ2=6.91,P>0.05)。多重荧光定量PCR方法中轮状病毒、腺病毒、诺如病毒、星状病毒的检出率分别为12.67%、6.00%、13.33%和4.00%,测序结果与已知病毒株基因都具有高度同源性。结论:所建立的多重荧光定量PCR方法快速、特异、灵敏,可以作为临床病原诊断的一个重要工具且适用于流行病学调查研究。
Objective:To develop and evaluate a multiplex fluorescence quantitative PCR assay for the simultaneous detection of common virus of faeces in infants with diarrhea.Methods:Genbank sequences of Rotavirus,Adenovirus,Norovirus and Astrovirus were included as reference sequences.A multiplex fluorescence quantitative PCR assay was developed and the primers and probes were designed based on the reference sequences,and the specificity,sensitivity and reproducibility of the assay were evaluated.Fecal samples from 150 patients with viral diarrhea were detected and verified by gene sequencing.Results:There were high specificity of the multiplex real-time PCR assay for detecting Rotavirus,Adenovirus,Norovirus and Astrovirus.The sensitivity of the method was 102 copies/mL.The standard deviations of CT values of different viral nucleic acid concentrations were small,and the coefficient of variation was less than 1.0%,which showed good repeatability.The detection rate of multiplex quantitative PCR was 36.00%in the 150 stool samples of infants with diarrhea,and that of colloidal gold method was 38.67%.There was no significant difference between the two methods.The detection rates of Rotavirus,Adenovirus,Norovirus and Astrovirus were 12.67%,6.00%,13.33%and 4.00%,respectively with multiplex fluorescent quantitative PCR.The sequencing results were highly homologous with the genes of known virus strains.Conclusion:Rotavirus,Adenovirus,Norovirus and Astrovirus can be detected and identified rapidly by the multiplex fluorescence quantitative PCR assay with high specificity and sensitivity.The assay developed in this study can be applied to the clinical diagnosis and epidemiological investigation.
作者
张蝶
卢晋英
唐雪峰
刘树业
李会强
ZHANG Die;LU Jin-ying;TANG Xue-feng;LIU Shu-ye;LI Hui-qiang(School of Medical Laboratory,Tianjin Medical University,Tianjin 300203,China;Clinical Laboratory,Tianjin Third Central Hospital,Tianjin 300170,China)
出处
《天津医科大学学报》
2021年第1期83-87,共5页
Journal of Tianjin Medical University
