乙型流感病毒TaqMan荧光定量PCR分型检测方法的建立及初步应用
Establishment and application of a TaqMan fluorescence quantitative PCR method for typing influenza B virus
赵文欣 1朱翔宇 2肖妍 2韩继成 3哈卓 2张赫 2谢宇飚 2鲁会军 2金宁一1
作者信息
- 1. 东北农业大学动物医学学院,黑龙江哈尔滨 150030;中国农业科学院长春兽医研究所,吉林长春 130122
- 2. 中国农业科学院长春兽医研究所,吉林长春 130122
- 3. 中国农业科学院长春兽医研究所,吉林长春 130122;长春中医药大学院士工作站,吉林长春 130117
- 折叠
摘要
通过比对GISAID登录的乙型流感病毒Victoria谱系、Yamagata谱系HA基因序列,选择保守区域设计并合成1对引物及2条荧光探针,以实验室保存的乙型流感毒株RNA为模板分别扩增Victoria谱系和Yamagata谱系HA基因片段,构建重组质粒标准品pUC57-Vic和pUC57-Yama,经PCR及测序鉴定正确后作为质粒标准品,初步建立乙型流感病毒的 TaqMan 荧光定量 PCR(real-time quantitative reverse transcription PCR,qRT-PCR)分型检测方法.结果显示,该方法可以特异的鉴别检测乙型流感Victoria谱系、Yamagata谱系病毒RNA,对甲型流感病毒、EB病毒、腺病毒等RNA/DNA的检测结果均为阴性;对重组质粒标准品的检测限分别为333、2 940拷贝/μL;批内与批间的重复性试验的变异系数均小于2％,表明所建立的检测方法特异性良好、灵敏度较高、重复性好;利用该方法与常规反转录PCR(RT-PCR)方法对10份流感样病例咽拭子进行检测,检测结果一致(3/10).结果表明,本试验建立的乙型流感病毒TaqMan荧光定量PCR分型检测方法能够检测乙型流感病毒,为乙型流感病毒Victoria谱系及Yamagata谱系的鉴别提供更加快速准确的定量检测手段.
Abstract
In order to establish a rapid and accurate method for genotyping influenza B virus,the HA gene sequences of Victoria lineage and Yamagata lineage of influenza B virus were downloaded from GISAID and used select the conserved regions for synthesis of a pair of primers and two fluo-rescent probes.Fragments of HA gene of Victoria lineage and Yamagata lineage were amplified u-sing the RNA of Influenza B virus strain preserved in the laboratory and used as a template.The recombinant plasmid standards pUC57-Vic and pUC57-Yama were constructed and identified by PCR and sequencing as plasmid standards.A real-time fluorescence quantitative PCR genotyping method for influenza B virus was preliminarily established.The specificity results showed that the method specifically identified and detected the RNA of Influenza B/Victoria and B/Yamagata Line-ages with no cross-reaction with the RNA/DNA of influenza A virus,EBV,ADV and others.The sensitivity of the detection method was 333 copies/μL and 2 940 copies/μL.The intra-lot CV and inter-lot CV repeatability tests was less than 2％.These data suggested that the established detec-tion method had good specificity,high sensitivity,and good repeatability.The method was used to detect 10 throat swabs of influenza-like cases with conventional reverse transcription PCR(RT-PCR),and the results were consistent(3/10).The fluorescence quantitative PCR method estab-lished in this study can detect and identify influenza B virus infection and provide a more rapid and accurate quantitative detection method for the identification of influenza B virus Victoria lineage and Yamagata lineage.
关键词
乙型流感病毒/TaqMan荧光定量PCR/血凝素Key words
influenza B virus/TaqMan fluorescent quantitative PCR/hemagglutinin(HA)引用本文复制引用
基金项目
国家重点研发计划(2021YFC2302503)
出版年
2024
NETL
NSTL
万方数据