分子生物学方法在儿童流行性感冒监测中的应用

朱汝南; 钱渊; 王芳; 刘成贵

文章摘要

朱汝南,钱渊,王芳,刘成贵.分子生物学方法在儿童流行性感冒监测中的应用[J].中华流行病学杂志,2003,24(1):9-14

分子生物学方法在儿童流行性感冒监测中的应用

Application of molecular biological techniques in the surveillance of influenza viruses in infants and young

收稿日期:2002-08-27 出版日期:2014-09-15

DOI：

中文关键词: 流感病毒多重逆转录-聚合酶链反应巢式-聚合酶链反应序列分析

英文关键词: Influenza virus Multiplex RT-PCR Multiplex nested-PCR Sequence analysis

基金项目:北京市卫生局专项基金;北京市自然科学基金;北京市基因诊断基础性研究实验室项目 (JS96004)

作者	单位
朱汝南	首都儿科研究所北京市感染与免疫中心研究室北京人类疾病基因诊断实验室, 北京 100020
钱渊	首都儿科研究所北京市感染与免疫中心研究室北京人类疾病基因诊断实验室, 北京 100020
王芳	首都儿科研究所北京市感染与免疫中心研究室北京人类疾病基因诊断实验室, 北京 100020
刘成贵	首都儿科研究所北京市感染与免疫中心研究室北京人类疾病基因诊断实验室, 北京 100020

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中文摘要:

目的: 建立一种能够快速、特异、有效地直接从临床标本中检测流行性感冒(流感)病毒并进行病毒型和亚型鉴别的方法, 同时了解近年来北京地区A₃型流感病毒血凝素重链(HA1)区基因的变异特点。方法:根据编码A、B型流感病毒膜蛋白M基因的核苷酸序列设计两对引物, 用于同一逆转录(RT)PCR反应中(多重RT PCR), 根据A、B型毒株扩增产物的大小(分别为506bp和261bp)鉴别A、B型流感病毒。另根据编码A₁和A₃型流感病毒糖蛋白HA基因的核苷酸序列设计两对引物, 与B型M基因引物用于同一RT PCR反应, 可区分A₁、A₃或B型流感病毒(扩增产物分别为944bp、1072bp和261bp)。为提高检测临床标本的敏感性, 针对A₁和A₃型病毒HA基因和B型病毒M基因又设计了3对引物作为外侧引物, 进行巢式PCR反应。根据第二次PCR的扩增产物在1.2 %的琼脂糖凝胶电泳中的大小即可确定型别。经PCR扩增1996～2002年间分离的北京地区A₃型流感病毒株HA1区基因, 测序并进行序列分析。结果: 用上述多重RT PCR鉴定流感病毒分离株156株, 与血凝抑制试验阳性符合率为100 %。用多重巢式PCR检测92份已经病毒分离确定为流感的儿科临床呼吸道标本, 与病毒分离和血凝抑制试验阳性符合率分别为76.9%(A₁型)、57.1%(A₃型))、86.5 %(B型)。对5株1996～2002年间A₃3型分离株HA1区基因序列分析结果显示, 不同年份的分离株HA1区基因具有较高的核苷酸和氨基酸同源性, 而且年份越近的毒株之间同源性越高。结论：多重RT-PCR 和巢式-PCR 可同时检测不同型别的流感病毒, 并可直接对临床标本进行检测, 为流感监测提供更加快速的新方法。北京地区A₃型流感病毒分离株HA1区基因持续不断地发生点突变, 糖基化位点不断增多, 可能是导致其抗原漂移的原因。

英文摘要:

Objective： To establish a rapid, specific and effective technique for identifying subtyping A₁, A₃ and B of influenza virus isolates and clinical specimens as well as to analyze the sequences of nucleotides and deduced amino acids of HA1 regio ns from isolates of influenza virus A₃ isolated from 1996 to 2002. Methods：Six inner and outer sets of oligonucleo tide primers were desig ned to detect, type and subty pe human influenza A and B. The first two corresponding sets differatiate type A and B of matrix (M) gene while, the second two corresponding sets identify the H1 and H3 subty pes of type A virus HA gene. To type and subtype influenza viruses in clinical isolates, a mix ture of inner primer sets specific for H₁, H₃ and B were used in a single PCR reaction tube. To detect influenza viruses in clinical specimens, a mixture of the outer primer sets were used in a single primary PCR tube, and the inner ones in a single second PCR reaction tube. Amplified products were visualized in 1.2% agrose gel containing e thidium bromide. HA1 regions of hemagglutinin of 5 field strains (H3N2) isolated from 1996 to 2002 in Beijing were amplified by RT-PCR and sequenced directly. Results: There was 100% co rrelation between multiplex RT-PCR and culture to type and subtype influenza viruses from clinical isolates. For typing and subtyping76.9%, 57.1% and 86.5% were positive for A₁, A₃ and B by multiplex nested-PCR compared withn virus isolation on culture , respectively. The sequence data of HA1 of A3 strains showed that there was a high homology of nucleotide and amino acid, and the closer the date of isolating was, the hig her homology showed. Conclusions:　Multiplex RT-PCR and nested-PCR for influenza viruses could provide a useful alternative to existing methods of influenza detected and identified from clinical isolate and specimens. There were certain, continuous mutations and increasing gly cosy lated sites which might cause the antigen drift in the A3 strains during 1996-2002 in Beijing area.

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