Detection and quantification of influenza C virus in pediatric respiratory specimens by real-time PCR and comparison with infectious viral counts
Detection and quantification of influenza C virus in pediatric respiratory specimens by real-time PCR and comparison with infectious viral counts
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DOI：
10.1016/j.jcv.2012.02.012
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发表时间：
2012-06-01
影响因子：
8.8
通讯作者：
Hongo, Seiji
中科院分区：
文献类型：
--
作者：
Matsuzaki, Yoko;Ikeda, Tatsuya;Hongo, Seiji
背景：仅通过传统病毒培养筛查可能低估了丙型流感病毒感染的流行病学和临床影响。目的：评估新开发的实时聚合酶链式反应（PCR）检测作为诊断丙型流感病毒感染的工具。研究设计：根据 1967 年至 2010 年 9 个分离株的比对序列，设计用于实时 PCR 的引物和探针，以扩增核蛋白基因的保守区域。通过细胞培养和实时 PCR 检查 2010 年 1 月至 2010 年 8 月期间收集的儿童呼吸道标本中是否存在丙型流感病毒。使用含胚鸡蛋的感染性测定对实时 PCR 病毒呈阳性的样本进行进一步检查。结果：在检查的 1203 份样本中，34 份 (2.8%) 通过细胞培养检测呈丙型流感病毒阳性，51 份 (4.2%) 通过实时 PCR 检测呈阳性。细胞培养阳性标本的平均病毒载量和感染滴度分别为3.97 x 10(8)拷贝/ml和5.43 x 10(5) EID50/ml，细胞培养阴性标本的平均病毒载量和感染滴度分别为2.18 x 10(6)拷贝/ml和3.67 x 10(2) EID50/ml。在病毒载量低于10(5)拷贝/ml的临床标本中，不可能使用含胚鸡蛋分离病毒。临床标本的拷贝数与EID50的比率（范围为32至278,000）远高于培养液的拷贝数/EID50比率（范围为2.3至13.5）。结论：本文所述的实时PCR检测可作为诊断丙型流感病毒感染的灵敏方法。 (C) 2012 Elsevier B.V. 保留所有权利。
Background: The epidemiological and clinical impacts of influenza C virus infection may have been underestimated by conventional viral culture screening alone.Objective: To evaluate a newly developed real-time polymerase chain reaction (PCR) assay as a tool for diagnosing influenza C virus infection.Study design: The primers and probe for real-time PCR were designed to amplify the conserved region of the nucleoprotein gene based on the aligned sequences of nine isolates from 1967 to 2010. Respiratory specimens from children collected between January 2010 and August 2010 were examined for the presence of influenza C virus by cell culture and real-time PCR. Specimens that were positive for the virus using real-time PCR were further examined using an infectivity assay with embryonated hen's eggs.Results: Of the 1203 specimens examined, 34 (2.8%) tested positive for the influenza C virus by cell culture and 51 (4.2%) tested positive by real-time PCR. The mean viral load and infectivity titer in specimens that tested positive using cell culture were 3.97 x 10(8) copies/ml and 5.43 x 10(5) EID50/ml, respectively, and those in specimens that were negative using cell culture were 2.18 x 10(6) copies/ml and 3.67 x 10(2) EID50/ml, respectively. In the clinical specimens with viral loads less than 10(5) copies/ml, it was not possible to isolate the virus using embryonated hen's eggs. The copy number-to-EID50 ratio of the clinical specimens was much higher, ranging from 32 to 278,000, than those of culture fluid, ranging from 2.3 to 13.5.Conclusion: The real-time PCR assay described here can be used as a sensitive method for diagnosing influenza C virus infection. (C) 2012 Elsevier B.V. All rights reserved.