Real time reverse transcription (RRT)-polymerase chain reaction (PCR) methods for detection of pandemic (H1N1) 2009 influenza virus and European swine influenza A virus infections in pigs.
Real time reverse transcription (RRT)-polymerase chain reaction (PCR) methods for detection of pandemic (H1N1) 2009 influenza virus and European swine influenza A virus infections in pigs.
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DOI：
10.1111/j.1750-2659.2010.00149.x
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发表时间：
2010-09
影响因子：
4.4
通讯作者：
Brown IH
中科院分区：
文献类型：
--
作者：
Slomka MJ;Densham AL;Coward VJ;Essen S;Brookes SM;Irvine RM;Spackman E;Ridgeon J;Gardner R;Hanna A;Suarez DL;Brown IH
请引用本文：Slomka 等人。 (2010) 用于检测猪中大流行 (H1N1) 2009 流感病毒和欧洲猪 A 型流感病毒感染的实时逆转录 (RRT)-聚合酶链式反应 (PCR) 方法。流感和其他呼吸道病毒 4(5), 277–293。 背景 需要通过实时逆转录 (RRT) PCR 方法检测和区分 2009 年 H1N1 流感大流行 (H1N1v) 和已确定的猪甲型流感病毒 (SIV)。 目标 首先，修改现有的基质 (M) 基因 RRT PCR，以实现 H1N1v 和其他欧洲 SIV 的灵敏通用检测。其次，设计 H1 RRT PCR 来特异性检测 H1N1v 感染。 方法 RRT PCR 检测用于检测 SIV（n = 51；37 个欧洲和 14 个北美）、H1N1v（n = 5）和禽流感病毒（AIV；n = 43）的实验室分离株。计算从现场病例和实验感染 SIV 和 H1N1v 的猪收集的拭子 (n = 133) 和组织 (n = 116) 的诊断敏感性和特异性。 结果 “完美匹配”M 基因 RRT PCR 是该测试中检测已建立的欧洲 SIV 和 H1N1v 的最敏感变体。 H1 RRT PCR 专门检测到 H1N1v，但未检测到欧洲 SIV。临床标本验证包括与作为“金标准”的病毒分离 (VI) 进行比较，而猪的 H1N1v 现场感染则通过常规 RT PCR 扩增的 H1N1v 测序进行独立确认。 “完美匹配”M基因RRT PCR对拭子的敏感性为100%，特异性为95·2%，对组织的特异性为93·6%和98·6%。 H1 RRT PCR 对拭子的敏感性和特异性分别为 100% 和 99·1%，对组织的敏感性和特异性分别为 100% 和 100%。 结论 两个 RRT PCR 已得到验证，其目的是 (i) 欧洲猪中 SIV 和 H1N1v 感染的一般检测，以及 (ii) H1N1v（大流行性流感）感染的特异性检测。
Please cite this paper as: Slomka et al. (2010) Real time reverse transcription (RRT)‐polymerase chain reaction (PCR) methods for detection of pandemic (H1N1) 2009 influenza virus and European swine influenza A virus infections in pigs. Influenza and Other Respiratory Viruses 4(5), 277–293. Background There is a requirement to detect and differentiate pandemic (H1N1) 2009 (H1N1v) and established swine influenza A viruses (SIVs) by real time reverse transcription (RRT) PCR methods. Objectives First, modify an existing matrix (M) gene RRT PCR for sensitive generic detection of H1N1v and other European SIVs. Second, design an H1 RRT PCR to specifically detect H1N1v infections. Methods RRT PCR assays were used to test laboratory isolates of SIV (n = 51; 37 European and 14 North American), H1N1v (n = 5) and avian influenza virus (AIV; n = 43). Diagnostic sensitivity and specificity were calculated for swabs (n = 133) and tissues (n = 116) collected from field cases and pigs infected experimentally with SIVs and H1N1v. Results The “perfect match” M gene RRT PCR was the most sensitive variant of this test for detection of established European SIVs and H1N1v. H1 RRT PCR specifically detected H1N1v but not European SIVs. Validation with clinical specimens included comparison with virus isolation (VI) as a “gold standard”, while field infection with H1N1v in swine was independently confirmed by sequencing H1N1v amplified by conventional RT PCR. “Perfect match” M gene RRT PCR had 100% sensitivity and 95·2% specificity for swabs, 93·6% and 98·6% for tissues. H1 RRT PCR demonstrated sensitivity and specificity of 100% and 99·1%, respectively, for the swabs, and 100% and 100% for the tissues. Conclusions Two RRT PCRs for the purposes of (i) generic detection of SIV and H1N1v infection in European pigs, and for (ii) specific detection of H1N1v (pandemic influenza) infection were validated.