单管双重巢式聚合酶链反应及基因测序技术鉴别呼吸道合胞病毒A,B亚型

Identification of Respiratory Syncytial Virus A and B Subtypesby Tube Double Nested Polymerase Chain Reaction and Gene Sequencing Technology

目的建立呼吸道合胞病毒(RSV)A,B亚型的鉴别方法,以供临床科研应用。方法运用荧光定量聚合酶链反应技术(FQ-PCR)筛选出2015~2017年50例住院儿童咽拭子RSV阳性标本;根据RSV的G蛋白编码基因核苷酸序列设计单管双重巢式PCR引物进行基因分型;其A,B亚型产物经测序与GeneBank库中核苷酸序列进行比对分析鉴定;采用卡方检验对结果进行统计学分析。结果50例咽拭子RSV阳性患儿呼吸道合胞病毒A,B亚型及混合感染率分别为82.00%,14.00%和4.00%,差异有统计学显著性意义(χ2=81.06,P<0.01);RSV分型结果与基因测序结果完全符合。结论深圳东部地区以A亚型流行为主,混合感染并存;单管双重巢式聚合酶链反应(PCR)及基因测序技术适用于RSV的A,B亚型鉴别,具有灵敏度高、特异度强和准确率高的特点。

Objective Objective to establish a method for identification of respiratory syncytial virus（RSV）A and B subtypes for clinical research and application.Methods Using fluorescent quantitative polymerase chain reaction（FQ-PCR）screened50 cases of throat swab that RSV were positive in hospitalized children from 2015 to 2017.The genotyping was performed according to the nucleotide sequence of G protein coding gene,and a single tube double nested PCR primers was designed for it.A and B subgroup by sequencing to conduct comparative analysis with nucleotide sequence in the Genebank.The results were analyzed by chi-square test.Results In the 50 cases of throat swab RSV positive children,respiratory syncytial virus A and B subtype and mixed infection rates were 82.00%,14.00% and 4.00%,respectively.The difference was statistically significant（χ2=81.06,P〈0.01）.The RSV fractal results were consistent with the gene sequencing results.ConclusionThe eastern part of shenzhen was dominated by respiratory syncytial virus A subtype epidemic and mixed infection.Single tube double nested polymerase chain reaction（PCR）and gene sequencing technique is suitable for the identification of A and B subtypes of RSV.It is characterized by high sensitivity,specificity and high accuracy.