Applicability of a sensitive duplex real-time PCR assay for identifying B/Yamagata and B/Victoria lineages of influenza virus from clinical specimens
Applicability of a sensitive duplex real-time PCR assay for identifying B/Yamagata and B/Victoria lineages of influenza virus from clinical specimens
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DOI：
10.1007/s00253-011-3710-8
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发表时间：
2012-01-01
影响因子：
5
通讯作者：
Lu, Zuxun
中科院分区：
文献类型：
--
作者：
Zhang, Naixing;Fang, Shisong;Lu, Zuxun
B型流感病毒是主要的流行毒株之一，导致相当大的死亡率和发病率。在流感季节，需要快速和准确地识别不同的B型流感病毒谱系，即B/Yamagata(B/Y)和B/Victoria(B/V)。然而，现有的快速技术缺乏敏感性，通常识别流感病毒的方法需要在组织培养或胚胎母鸡卵中进行病毒扩增。因此，我们开发了几组能够在单个实时荧光PCR检测中检测和区分B/Y和B/V的引物对。与至少一条针对B/Y和B/V系HA基因的3‘端显示嘌呤的特异性引物结合使用，我们可以准确地识别每微升大约10(2)个拷贝的B/Y和B/V，批内和批间变异系数(CV)为4%。对2006-2010年流感监测季采集的17765份咽拭子标本进行检测时，该方法对流感病毒的检测、分型和分型与血凝抑制法相当，特异度和敏感度分别为100%和100%。综上所述，该方法为世卫组织疫苗成分的常规诊断和及时流行病学检查提供了灵敏和可靠的工具。
Type B influenza virus is one of the major epidemic strains and responsible for considerable mortality and morbidity. Rapidly and accurately identifying different influenza B virus lineages, i.e., B/Yamagata (B/Y) and B/Victoria (B/V), is desirable during the flu season. However, the available rapid techniques lack sensitivity, and the usual methods for identifying influenza viruses require expansion of virus in tissue culture or embryonated hen's eggs. Thus, we developed several sets of primer pairs that were able to detect and distinguish B/Y and B/V in a single real-time PCR assay. Used in conjunction with two sets of specific primers that exhibited purine at 3' end of at least one primer targeting on HA gene of B/Y and B/V lineages allows us to accurately identify approximately 10(2) copies per microliter for B/Y and B/V with intra- and inter-assay coefficient of variation (CV) < 4%. When it was used to test 17,765 throat swab specimens obtained in the 2006-2010 influenza surveillance season, this method was comparable to hemagglutination inhibition assay in detection, typing and subtyping of influenza viruses with 100% true-negative (specificity) and 100% true-positive (sensitivity). Taken together, this method provides sensitive and robust tool for routine diagnosis and on-time epidemiological examination for WHO decisions on vaccine composition.