Virology
Development of a multiplex real-time polymerase chain reaction for the detection of influenza virus type A including H5 and H9 subtypes

https://doi.org/10.1016/j.diagmicrobio.2008.01.007Get rights and content

Abstract

Avian influenza viruses (AIVs) are endemic in wild birds and, if transmitted to poultry, can cause serious economic losses. The aim of this study was to develop a multiplex real-time reverse transcriptase polymerase chain reaction (RT-PCR) for rapid detection of influenza virus type A, including H5 and H9 subtypes. The selected primers and various labeled TaqMan reporter probes corresponding to matrix, H5, and H9 genes were used in a multiplex real-time RT-PCR to simultaneously detect triple fluorescent signals. The results showed that the multiplex real-time RT-PCR assay can be applied to detect RNA of influenza virus type A including H5 and H9 subtypes with a high specificity and a sensitivity of 10 copies per reaction. As a result of its short turnaround times and a high specificity and sensitivity, the assay is very suitable for large-scale screening during AIV outbreaks.

Introduction

Influenza viruses type A are enveloped negative-sense, segmented, single-stranded RNA viruses of the family of Orthomyxoviridae. Based on major differences within the hemagglutinin (HA) and neuraminidase (NA) proteins, 16 HA and 9 NA subtypes have been recognized, all of which have been isolated from avian species (Fouchier et al., 2005, Rohm et al., 1996, Webster et al., 1992). Only the H1, H2, and H3 subtypes have established stable human lineages. However, the zoonotic events in Asia and in Europe caused by H5N1, H9N2, and H7N7 subtypes, respectively, indicate that avian influenza viruses (AIVs) can also infect humans (Perdue and Swayne, 2005). The link between human and avian influenza strains has raised concerns among public health authorities and in the scientific community about the prevalence and pandemic potential of AIVs (Trampuz et al., 2004).

Most virology laboratories utilize conventional diagnostic methods for influenza detection, including cell culture with immunofluorescence (IF) staining and antigen subtyping based on hemagglutination inhibition (HI) using antisera raised against various reference strains. These approaches are highly accurate and sensitive for viral detection, but they are laborious and time consuming. Many laboratories also utilize commercially available immunoassays for rapid detection of influenza virus. Although these commercial kits are fast and easy to use, they have been shown to be less sensitive and specific compared with traditional culture methods (Boivin et al., 2001, Herrmann et al., 2001, Kaiser et al., 1999).

An effective approach for the rapid identification and detection of AIVs is the reverse transcriptase polymerase chain reaction (RT-PCR) (Lee et al., 2001, Munch et al., 2001, Ng et al., 2006, Starick et al., 2000). Previous studies have employed multiplex polymerase chain reaction (PCR) for the detection of human influenza viruses and subtypes (Choi et al., 2002, Daum et al., 2002, Payungporn et al., 2004, Poddar, 2002, Poddar et al., 2002, Stockton et al., 1998). However, these studies utilized multistep approaches with random hexamers or multiple PCR amplifications with nested primer sets. Recently, different formats of quantitative, fluorescence-based, real-time PCR assays have been developed (Di Trani et al., 2006, Lee and Suarez, 2004, Payungporn et al., 2006, Schweiger et al., 2000, Spackman et al., 2002, Stone et al., 2004). These methods have a broad dynamic range, detect all AIVs subtypes, and are highly sensitive.

Here, we describe a rapid multiplex real-time RT-PCR assay for the simultaneous detection of generic influenza virus type A as well as the H5 and H9 subtypes.

Section snippets

Virus strains and specimens

The following reference virus strains were obtained from the American Type Culture Collection (ATCC): influenza virus type A (H1 subtype: [A1/FM/1/47], ATCC VR-97; H3 subtype: [A/Hong Kong/1/68], ATCC VR-544); influenza virus type B ([B/Taiwan/40], ATCC VR-295); parainfluenza virus type 1 (ATCC VR-94); parainfluenza virus type 3 (ATCC VR-93); respiratory syncytial virus subtype A ([Long strain], ATCC VR-26) and subtype B ([B-1 wild type], ATCC VR-1400); rhinovirus 12(ATCC VR-1122), and

Interpretation of influenza virus type A detection by multiplex real-time RT-PCR

The primers and probes labeled with FAM, HEX, and CY5 fluorescent dyes (Table 1) were selected for multiplex real-time PCR detection of M, H5, and H9 gene fragments, respectively (Fig. 1). Triple fluorescent signals correspond to an infection with influenza virus type A of both H5 and H9 subtypes. FAM and HEX signals correspond to an H5 infection and FAM and CY5 to an H9 infection. Other subtypes of influenza virus type A would only be positive for FAM fluorescence of the M gene.

Standard curves

Standard curves

Discussion

Endemicity of AIV in Asia and, more recently, epizootics in some European regions have caused considerable public concern and require an effective surveillance and rapid virus detection in poultry. Also, zoonotic events in Hong Kong and mainland China, caused by H5N1 and H9N2 avian viruses have highlighted the need for rapid tests to detect viruses originating from nonhuman hosts and for the implementation of control measures to contain outbreaks in poultry and reduce human exposure.

Viral

Acknowledgments

The Technology Research Program of Guangzhou Province, China, supported this work, grant no. 2005A11601010. The authors thank Dr. Zhiying Ou, Guangzhou Children's Hospital (Guangdong Province, China), to strictly revise the manuscript.

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    All of the authors have no actual and potential conflict of interest for any persons and any organizations.

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