An SYBR Green-based real-time RT-PCR assay for the detection of H5 hemagglutinin subtype avian influenza virus

详细信息    查看全文

• 作者：Lester J. Pé ; rez^a ; ¹ ; H. Dí ; az de Arce^a ; ¹ ; ^{heidydal@infomed.sld.cu} ; ^{heidydal@yahoo.es} ; F. Cilloni^b ; A. Salviato^b ; S. Marciano^b ; C.L. Perera^a ; A. Salomoni^b ; M.S. Beato^b ; A. Romero^b ; I. Capua^b ; G. Cattoli^b
• 关键词：SYBR Green ; Real-time RT-PCR ; Avian influenza ; Subtype H5
• 刊名：Molecular and Cellular Probes
• 出版年：2012
• 出版时间：June, 2012
• 年：2012
• 卷：26
• 期：3
• 页码：137-145
• 全文大小：1217 K

文摘

Increasing diversity among H5 hemagglutinin (HA) subtype avian influenza (AI) viruses has resulted in the need of novel sensitive and specific molecular assays. In this study, an SYBR Green-based real-time reverse transcription-PCR (RRT-PCR) assay was developed for the detection of H5 subtype AI virus. Sequence analysis of the Mexican lineage H5N2 isolates (subgroup B) revealed several mismatches in the primer/hydrolysis probe set reported in the commonly used RRT-PCR assay for the detection of H5 North American lineage. The present assay was designed to circumvent the challenge that these viruses represent for the specific detection of H5 subtype AI viruses. This RRT-PCR assay successfully detected a range of different H5 subtype AI strains from both Eurasian and North American lineages representing different avian H5 HA clades from diverse geographical locations. The sensitivity of the present method was determined by using in?vitro-transcribed RNA and 10-fold serial dilutions of titrated AI viruses. High sensitivity levels were obtained, with limits of detection of 10⁰ 50 % egg infectious dose (EID₅₀)/mL and 4.2?gene?copies/¦Ìl. The linear ranges of the assay span within 10⁶-10⁰?EID₅₀/mL and 10⁶-10⁰?gene copies/¦Ìl. The results obtained from this method were directly compared with those of the H5 RRT-PCR assay recommended by the OIE. The comparison was performed with 110 tracheal and cloacal swabs from various bird species collected during field and laboratory investigations in Eurasia and Africa in 2006 and 2008 and showed 100 % agreement. This assay is recommended as an alternative method, also allowing a ¡®double check?approach detection, to be use mainly in outbreak scenarios with higher risk of poultry infections by Central American/Caribbean H5 AI viruses.