Valle L, Amicizia D, Bacilieri S, Banfi F, Riente R, Durando P, Sticchi L, Gasparini R, Esposito C, Icardi G, Ansaldi F
CIRI-IV, Department of Health Sciences, University of Genoa, Italy.
J Prev Med Hyg. 2006 Dec;47(4):127-33.
Two real time one-step RT-PCR assays were developed for simultaneous detection and typing of influenza A and B viruses and detection of Respiratory Syncytial Virus (RSV). As regard influenza, primers were designed to amplify specific sequences of gene M of A/H1N1, A/H3N2, A/H5N1, A/H7N7 and A/H9N2 viruses and of gene NP of type B viruses belonging both Yamagata and Victoria lineage. Specificity, analytical and clinical sensitivity, dynamic range, linearity of the new assays were evaluated.
Dynamic ranges for Influenza A and B, and RSV were at least five logs and linearity was conserved. In order to evaluate the specificity, 80 nasopharyngeal swabs resulting Influenza and RSV negative by multiplex nested PCR and cell culture, were tested and 79 resulted negative. The detection limits for influenza A and B, calculated by 95% probit, was 0.008 and 0.09 PFU, respectively, resulting more sensible than nested PCR. A total of 75 specimens (10 A/H1N1, 3 A/H1N2, 8 A/H3N2 Johannesburg/94-like, 10 A/H3N2 Panama/2007/99-like, 10 A/H3N2 Fuijian/411/02-like, 2 A/H5N1, 2 A/H7N7 and 2 A/H9N2, 15 B/Yamagata-like and 13 B/Victoria-like) collected between 1994 and 2004 or received by WHO Influenza Centre, London, were chosen as representative of the circulating strains and tested. All samples resulted positive although one B/Victoria sample was not clear typed. Thirty swabs nested RT-PCR positive for RSV collected during the four seasons, were also analysed by realtime PCR, resulting positive. To evaluate the performance of the new assay on fresh material, 250 specimens, collected during the 2004/05 seasons, were tested by nested-PCR, cell culture and real-time PCR.
The new assays provide accurate and sensitive diagnosis of influenza and RSV infection and they represent a sensitive tool for virological surveillance and management of patient with ILI.
开发了两种实时一步法逆转录聚合酶链反应（RT-PCR）检测方法，用于同时检测甲型和乙型流感病毒并进行分型，以及检测呼吸道合胞病毒（RSV）。对于流感，设计引物以扩增甲型H1N1、H3N2、H5N1、H7N7和H9N2病毒基因M的特定序列，以及属于山形系和维多利亚系的乙型病毒基因NP的特定序列。对新检测方法的特异性、分析和临床敏感性、动态范围及线性进行了评估。
甲型和乙型流感病毒以及呼吸道合胞病毒的动态范围至少为5个对数，且线性良好。为评估特异性，对80份经多重巢式PCR和细胞培养检测为流感和呼吸道合胞病毒阴性的鼻咽拭子进行检测，其中79份结果为阴性。通过95%概率计算得出，甲型和乙型流感病毒的检测限分别为0.008和0.09 PFU，比巢式PCR更灵敏。选取了1994年至2004年间收集或由伦敦世界卫生组织流感中心提供的75份标本（10份甲型H1N1、3份甲型H1N2、8份约翰内斯堡/94样甲型H3N2、10份巴拿马/2007/99样甲型H3N2、10份福建/411/02样甲型H3N2、2份甲型H5N1、2份甲型H7N7和2份甲型H9N2、15份山形样乙型、13份维多利亚样乙型）作为流行毒株的代表进行检测。所有样本结果均为阳性，尽管1份维多利亚样乙型样本分型不明确。对四季期间收集的30份巢式RT-PCR检测为呼吸道合胞病毒阳性的拭子也进行了实时PCR分析，结果为阳性。为评估新检测方法对新鲜样本的性能，对2004/05季节收集的250份标本进行了巢式PCR、细胞培养和实时PCR检测。
新检测方法可对流感和呼吸道合胞病毒感染进行准确、灵敏的诊断，是病毒学监测及流感样疾病患者管理的灵敏工具。
