Slomka M J, Coward V J, Banks J, Löndt B Z, Brown I H, Voermans J, Koch G, Handberg K J, Jørgensen P H, Cherbonnel-Pansart M, Jestin V, Cattoli G, Capua I, Ejdersund A, Thorén P, Czifra G
Virology Department, Veterinary Laboratories Agency, Woodham Lane, Addlestone, Surrey KT15 3NB, United Kingdom.
Avian Dis. 2007 Mar;51(1 Suppl):227-34. doi: 10.1637/7674-063006R1.1.
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.
许多不同的聚合酶链反应（PCR）方案已被用于禽流感（AI）病毒分离株的检测和特性分析，主要是在研究环境中。欧盟六个实验室进行了盲法环式试验，以确定最灵敏和特异的禽流感PCR方案。在环式试验的第1部分，各实验室使用自己的方法检测一组10份经过重构的匿名临床标本，最佳方法被选为第2部分的推荐方案，在第2部分中对16份RNA标本进行了检测。这两组标本均包含H5、H7、其他禽流感亚型以及非禽流感禽类病原体。结果包括：1）通过高度灵敏和特异的M基因实时PCR对禽流感进行通用鉴定；2）对H5和H7病毒检测和鉴定有效的常规PCR。后者包括通过扩增子测序进行病毒致病型鉴定。在第2部分中所有六个实验室使用推荐方案后结果都有所改善，这反映出灵敏度和特异性的提高。这包括在第2部分所有实验室中观察到的H5/H7鉴定和致病型鉴定的改善。本文提供了这些PCR方法的详细信息。总之，本研究有助于欧盟实验室禽流感PCR方案的统一，并对2005年秋季欧洲首次报告H5N1高致病性禽流感后的禽流感实验室应急计划产生了影响。
