Identification of sensitive and specific avian influenza polymerase chain reaction methods through blind ring trials organized in the European Union
Identification of sensitive and specific avian influenza polymerase chain reaction methods through blind ring trials organized in the European Union
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DOI：
10.1637/7674-063006r1.1
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发表时间：
2007-01-01
期刊：
影响因子：
1.4
通讯作者：
Czifra, G.
中科院分区：
文献类型：
--
作者：
Slomka, M. J.;Coward, V. J.;Czifra, G.
许多不同的聚合酶链反应（PCR）方案已用于检测和表征禽流感（AI）病毒分离株，主要是在研究环境中。进行了盲环试验，以确定来自六个欧盟（EU）实验室的最敏感和最特异的AI PCR方案。在环试验的第1部分中，实验室使用自己的方法检测了一组10份重构的匿名临床标本，并选择最佳方法作为第2部分的推荐方案，其中检测了16份RNA标本。两组均含有H5、H7、其他AI亚型和非AI禽类病原体。结果包括验证1）通过高灵敏度和特异性M基因实时PCR鉴定通用AI，以及2）有效检测和鉴定H5和H7病毒的常规PCR。后者包括通过扩增子测序进行病毒病理分型。使用推荐的协议导致所有六个实验室在第2部分的结果有所改善，反映了增加的灵敏度和特异性。这包括在第2部分所有实验室中观察到的H5/H7鉴定和病理分型的改进。提供了这些PCR方法的细节。总之，这项研究有助于协调AI PCR协议在欧盟实验室和影响AI实验室应急计划后，在2005年秋季的第一个欧洲报告的H5 N1高致病性AI。
Many different polymerase chain reaction (PCR) protocols have been used for detection and characterization of avian influenza (AI) virus isolates, mainly in research settings. Blind ring trials were conducted to determine the most sensitive and specific AI PCR protocols from a group of six European Union (EU) laboratories. In part 1 of the ring trial the laboratories used their own methods to test a panel of 10 reconstituted anonymized clinical specimens, and the best methods were selected as recommended protocols for part 2, in which 16 RNA specimens were tested. Both panels contained H5, H7, other AI subtypes, and non-AI avian pathogens. Outcomes included verification of 1) generic AI identification by highly sensitive and specific M-gene real-time PCR, and 2) conventional PCRs that were effective for detection and identification of H5 and H7 viruses. The latter included virus pathotyping by amplicon sequencing. The use of recommended protocols resulted in improved results among all six laboratories in part 2, reflecting increased sensitivity and specificity. This included improved H5/H7 identification and pathotyping observed among all laboratories in part 2. Details of these PCR methods are provided. In summary, this study has contributed to the harmonization of AI PCR protocols in EU laboratories and influenced AI laboratory contingency planning following the first European reports of H5N1 highly pathogenic AI during autumn 2005.