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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 5 0 6bp和2 61bp)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反应中 ,可区分A1 、A3或B型流感病毒 (扩增产物分别为 944bp、10 72bp和 2 61bp)。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～ 2 0 0 2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 15 6株 ,与血凝抑制试验阳性符合率为 10 0 %。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 )、5 7.1%(A3型 )、
Objective To establish a rapid, specific and effective detection of influenza (influenza) virus directly from clinical specimens and virus and subtypes identification methods, as well as in recent years, Beijing area influenza A virus hemagglutinin heavy chain (HA1) region of the gene mutation characteristics. Methods Two pairs of primers were designed according to the nucleotide sequence of the M gene encoding membrane proteins of influenza A and B viruses. They were used in the same reverse transcription (RT) PCR reaction (multiplex RT PCR). According to the amplification products of strains A and B, (50 6bp and 2161 bp, respectively) were identified for influenza A and B viruses. In addition, two pairs of primers were designed according to the nucleotide sequences of the HA genes encoding the A1 and A3 influenza viruses glycoproteins, and the B type M gene primers were used in the same RT PCR reaction to distinguish the influenza A, A3 or B viruses The products were 944 bp, 10 72 bp and 2161 bp, respectively). In order to improve the sensitivity of the detection of clinical specimens, three pairs of primers were designed as outer primers for the HA gene of type A1 and A3 viruses and the M gene of type B virus to perform nested PCR. The type can be determined based on the size of the amplification product of the second PCR in 1.2% agarose gel electrophoresis. The HA1 gene of influenza virus type A3 isolated in Beijing from 1996 to 2002 was amplified by PCR, sequenced and sequenced. Results Fifteen 6 influenza virus isolates were identified by multiplex RT-PCR and the positive coincidence rate was 10 0% with the hemagglutination inhibition test. A total of 92 pediatric clinical respiratory specimens that had been identified as influenza by multiplex nested PCR were detected with positive coincidence rates of 76.9% (type A1), 51.1% (type A3), respectively, with virus isolation and hemagglutination inhibition tests