Human Influenza A Virus (H5N1) Detection by a Novel Multiplex PCR Typing Method

Abstract

We report the use of ResPlex III for genotyping influenza A viruses. The performance characteristics of the assay with regard to H5N1 are further evaluated. The ResPlex system incorporates a novel multiplex PCR technology, target-enriched multiplex PCR, to simultaneously amplify multiple molecular targets in one reaction. The ResPlex III assay targets the H1, H2, H3, H5, H7, H9, N1, and N2 genes from the influenza A virus as well as the NS genes from influenza A (NSA) and B (NSB) viruses, providing detection and genotyping of influenza A and B viruses. The analytical sensitivities for detecting the H5, N1, and NSA genes were 1, 10 ؊1 , and 10 50% tissue culture infectious doses/200 l/reaction, respectively. A total of 217 sequential clinical samples including 14 samples with human H5N1 infections were tested by the ResPlex III assay, and the results were compared to a reference standard combined with results of viral culture and conventional reverse transcriptase and real-time PCR. The clinical sensitivity and specificity for detecting H5N1 were 93.3% and 100%, respectively, indicating that different subtypes of influenza A virus can be quickly and correctly identified using the ResPlex III genotyping approach.

Indications that Live Poultry Markets Are a Major Source of Human H5N1 Influenza Virus Infection in China

“…Live poultry markets, backyard poultry breeding, and domestic poultry farms are all potential sources of human H5N1 infection, and visiting live poultry markets has been identified as an important factor in such infections in China (15,36).…”

“…A confirmed case of H5N1 infection was defined as a case of pneumonia-or influenza-like illness with laboratory evidence of H5N1 infection based on multiple methods, including viral culture, reverse transcriptase PCR (RT-PCR), real-time PCR, and/or a serology test (30,36). Briefly, each sample was tested by real-time PCR using matrix gene-specific primers, as well as H5 hemagglutinin (HA) and N1 neuraminidase (NA) gene-specific primers.…”

“…In fact, no reported avian outbreaks were temporally or geographically related to any human case identified after 11 February 2006 (see Table S1 in the supplemental material). However, many patients who presented after February 2006 had visited live poultry markets shortly before the onset of clinical disease (15,36).…”

Design of Multiplexed Detection Assays for Identification of Avian Influenza A Virus Subtypes Pathogenic to Humans by SmartCycler Real-Time Reverse Transcription-PCR

“…Recent techniques have developed a one-step TaqMan rRT-PCR specific for the detection of less than 1 PFU of H5 AIV from clades 0, 1, 2, and 3 (10, 27). The limit of detection in reaction 2 of 10 copies of RNA molecule and 0.01 50% lethal dose of virus was similar to limits obtained using one of the WHOrecommended protocols and to those previously published (13,22,55,56). Two H1 and 37 H3 virus subtypes positive for the M gene in rRT-PCR or RT-PCR (Table 6) were subtyped using reaction 3.…”

Microarray Multiplex Assay for the Simultaneous Detection and Discrimination of Influenza A and Influenza B Viruses

“…The rapid and precise laboratory diagnosis of influenza during pandemic periods is closely related to the control of influenza prevalence and the treatment of critically ill g foot and mouth disease virus; h infectious bronchitis virus; i infectious bursal disease virus; and j control Fig. 3 Results of the assessment of chip sensitivity [9][10][11][12]. The present study explored the possibility of testing for the influenza virus using a microarray.…”