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A duplex real-time RT-PCR assay for detecting H5N1 avian influenza virus and pandemic H1N1 influenza virus.

ABSTRACT: A duplex real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay was improved for simultaneous detection of highly pathogenic H5N1 avian influenza virus and pandemic H1N1 (2009) influenza virus, which is suitable for early diagnosis of influenza-like patients and for epidemiological surveillance. The sensitivity of this duplex real-time RT-PCR assay was 0.02 TCID50 (50% tissue culture infective dose) for H5N1 and 0.2 TCID50 for the pandemic H1N1, which was the same as that of each single-target RT-PCR for pandemic H1N1 and even more sensitive for H5N1 with the same primers and probes. No cross reactivity of detecting other subtype influenza viruses or respiratory tract viruses was observed. Two hundred and thirty-six clinical specimens were tested by comparing with single real-time RT-PCR and result from the duplex assay was 100% consistent with the results of single real-time RT-PCR and sequence analysis.

SUBMITTER: Kang XP

PROVIDER: S-EPMC2892456 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature

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A duplex real-time RT-PCR assay for detecting H5N1 avian influenza virus and pandemic H1N1 influenza virus.

Kang Xiao-ping XP Jiang Tao T Li Yong-qiang YQ Lin Fang F Liu Hong H Chang Guo-hui GH Zhu Qing-yu QY Qin E-de ED Qin Cheng-feng CF Yang Yin-hui YH

Virology journal 20100602

A duplex real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay was improved for simultaneous detection of highly pathogenic H5N1 avian influenza virus and pandemic H1N1 (2009) influenza virus, which is suitable for early diagnosis of influenza-like patients and for epidemiological surveillance. The sensitivity of this duplex real-time RT-PCR assay was 0.02 TCID50 (50% tissue culture infective dose) for H5N1 and 0.2 TCID50 for the pandemic H1N1, which was the same as that of ea ...[more]

PMID: 20515509

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Project description:BACKGROUND:Influenza A virus is a leading causative pathogen of human acute respiratory infection. Recently, the co-circulation of pandemic (H1N1) 2009 and seasonal H3N2 viruses was reported, and sporadic cases with reassortant avian H7N9 virus are continually reported in China. We aimed to establish a multiplex one-step real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay to simultaneously detect and discriminate FluA subtypes, including human seasonal H3N2 virus, pandemic (H1N1) 2009 virus and reassortant avian H7N9 virus, in one reaction tube. METHODS:Clinical samples, including throat swabs and sputum, were collected from the patients with influenza-like illness (ILIs). Total viral RNA from each sample or viral culture was extracted, and the specific detection of FluA virus and its subtypes was performed using a multiplex rRT-PCR assay. RESULTS:The limitation of detection (LOD) of the multiplex assay was 5.4 × 10-2 50% tissue culture infective dose (TCID50) per reaction or 4.8 × 101 copies per reaction for each virus of the three viruses. For simultaneously detecting the three viruses, the LOD was 1.8 × 10-2 TCID50 per reaction or 1.6 × 10 copies per reaction for testing the total FluA virus RNA and 5.6 × 10-2 TCID50 per reaction or 5.1 × 10 copies per reaction for the H3, H1, and H7 genes in one reaction tube. The multiplex assay specifically detected these viruses, and no cross-reaction with other pathogens was found. Moreover, the assay had reliable clinical sensitivity (100%) and valuable clinical specificity (>95%). The detection of FluA with the matrix (M) gene contributed to the further determination of these subtypes, and the Rnase P gene (RP) was considered an internal control to favourably evaluate the quality of the clinical samples. CONCLUSIONS:These findings indicate that the multiplex assay can simultaneously detect and discriminate FluA subtypes with reliable sensitivity and specificity, which is required for the early clinical diagnosis and viral surveillance of patients with FluA infection.

Dataset Information

A duplex real-time RT-PCR assay for detecting H5N1 avian influenza virus and pandemic H1N1 influenza virus.

Publications

A duplex real-time RT-PCR assay for detecting H5N1 avian influenza virus and pandemic H1N1 influenza virus.

Similar Datasets

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