Detection of influenza A viruses from different species by PCR amplification of conserved sequences in the matrix gene
Detection of influenza A viruses from different species by PCR amplification of conserved sequences in the matrix gene
复制标题
DOI：
10.1128/jcm.38.11.4096-4101.2000
复制
发表时间：
2000-11-01
影响因子：
9.4
通讯作者：
Osterhaus, ADME
中科院分区：
文献类型：
--
作者：
Fouchier, RAM;Bestebroer, TM;Osterhaus, ADME
最近提高了人们对新流感大流行的威胁的认识，激发了人们对人类和动物分泌物中流感病毒的检测的兴趣。仅病毒隔离是为此目的不满意的，因为其固有的有限敏感性以及缺乏普遍允许所有流感病毒的宿主细胞。先前描述的PCR方法更敏感，但主要针对人类循环的病毒菌株，因为引物集的序列对动物流感病毒的序列显示出相当数量的不匹配。因此，基于基质基因的高度保守区域的一组新底漆设计用于单管逆转录PCR，用于从多种物种中检测流感A的病毒。该PCR被证明是通过从鸟类，人类，猪，马和密封剂获得的25种遗传学病毒分离株的面板完全反应的，其中包括所有已知的流感亚型A A A A A型病毒。它与已测试的其他11种RNA病毒没有反应。与来自人类的喉咙拭子样品以及来自鸟类的粪便和泄殖腔拭子样品的比较测试证实，新的PCR比经典病毒隔离程序更快，高达100倍。
The recently raised awareness of the threat of a new influenza pandemic has stimulated interest in the detection of influenza A viruses in human as well as animal secretions. Virus isolation alone is unsatisfactory for this purpose because of its inherent limited sensitivity and the lack of host cells that are universally permissive to all influenza A viruses. Previously described PCR methods are more sensitive but are targeted predominantly at virus strains currently circulating in humans, since the sequences of the primer sets display considerable numbers of mismatches to the sequences of animal influenza A viruses. Therefore, a new set of primers, based on highly conserved regions of the matrix gene, was designed for single-tube reverse transcription-PCR for the detection of influenza A viruses from multiple species. This PCR proved to be fully reactive with a panel of 25 genetically diverse virus isolates that were obtained from birds, humans, pigs, horses, and seals and that included all known subtypes of influenza A virus. It was not reactive with the 11 other RNA viruses tested. Comparative tests with throat swab samples from humans and fecal and cloacal swab samples from birds confirmed that the new PCR is faster and up to 100-fold more sensitive than classical virus isolation procedures.