Detection of a novel avian influenza A (H7N9) virus in humans by multiplex one-step real-time RT-PCR assay

Fan, Jian Gao; Cui, David; Lau, Siu-Ying; Xie, Guoliang; Guo, Xichao; Zheng, Shufa; Huang, Xiaofeng; Yang, Shigui; Yang, Xianzhi; Huo, Zhaoxia; Yu, Fei; Lou, Jianzhou; Tian, Lu; Li, Xuefen; Dong, Yuejiao; Zhu, Qiaoyun; Chen, Yu

doi:10.1186/1471-2334-14-541

Cited by 18 publications

(17 citation statements)

References 23 publications

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“…To verify the positive results amplified by the multiplex assay, 12 positive samples with the H3N2 virus, 8 positive samples with the pandemic (H1N1) 2009 virus and 5 positive samples with the reassortant avian H7N9 virus were randomly selected to analyse their HA genomic sequences that were confirmed by the Sangon Biotech Co., Ltd (Shanghai, China). These results strongly indicated that the multiplex rRT-PCR assay in the study was completely in accordance with the assay systems previously described (Chen et al 2011; Kang et al 2014; World Health Organization 2013; Fan et al 2014; Nie et al 2013; Kuo et al 2014). …”

Section: Discussionsupporting

confidence: 85%

“…Sixteen H subtypes (H1–H16) and nine N subtypes (N1–N9) have been found in avian species, while the H17N10 and H18N11 subtypes have emerged in bats (Bedford et al 2015; Wu et al 2014; Fan et al 2014). Human infections with FluA viruses are commonly associated with the human H1, H2, and H3 subtypes that can cause outbreaks of FluA virus infection, such as the seasonal H3N2 virus and the pandemic (H1N1) 2009 virus (Cox and Subbarao 2000; Chen et al 2011; Bedford et al 2015; Davlin et al 2016; Yang et al 2015).…”

Section: Discussionmentioning

confidence: 99%

“…The “gold standard” for the diagnosis of viral infection is considered to be tissue culture (Chen et al 2013; Fan et al 2014). To further evaluate the clinical sensitivity and specificity of the multiplex assay, each of the 523 clinical samples from the patients with ILIs was inoculated into MDCK cells for influenza A virus isolation, and the three viruses were detected by the multiplex assay and the referenced methods.…”

Section: Discussionmentioning

confidence: 99%

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Simultaneous detection of influenza A subtypes of H3N2 virus, pandemic (H1N1) 2009 virus and reassortant avian H7N9 virus in humans by multiplex one-step real-time RT-PCR assay

et al. 2016

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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.MethodsClinical 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.ResultsThe 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.ConclusionsThese 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.

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Section: Discussionsupporting

confidence: 85%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Simultaneous detection of influenza A subtypes of H3N2 virus, pandemic (H1N1) 2009 virus and reassortant avian H7N9 virus in humans by multiplex one-step real-time RT-PCR assay

et al. 2016

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“…A method was developed for the detection of avian influenza A/ H7N9 using real-time PCR with a detection limit of 3.2 Â 10 À4 and 6.4 Â 10 À4 haemagglutination units (HAUs) for the H7 and N9 genes, respectively [27,28]. A microfluidics chip (fabricated using polydimethylsiloxane) with real-time fluorescence detection for the rapid identification of AIV H5 was suggested by Zhu et al [29].…”

Section: Conventional Methods: Elisa and Pcrmentioning

confidence: 99%

Advanced nanotechnologies in avian influenza: Current status and future trends – A review

Moulick

Richtera

Milosavljević

et al. 2017

Analytica Chimica Acta

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In the last decade, the control of avian influenza virus has experienced many difficulties, which have caused major global agricultural problems that have also led to public health consequences. Conventional biochemical methods are not sufficient to detect and control agricultural pathogens in the field due to the growing demand for food and subsidiary products; thus, studies aiming to develop potent alternatives to conventional biochemical methods are urgently needed. In this review, emerging detection systems, their applicability to diagnostics, and their therapeutic possibilities in view of nanotechnology are discussed. Nanotechnology-based sensors are used for rapid, sensitive and cost-effective diagnostics of agricultural pathogens. The application of different nanomaterials promotes interactions between these materials and the virus, which enables researchers to construct portable electroanalytical biosensing analyser that should effectively detect the influenza virus. The present review will provide insights into the guidelines for future experiments to develop better techniques to detect and control influenza viruses.

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“…Internal standard primers and probes based on the sequences of Homo sapiens ribonuclease P were designed to monitor specimen extraction, RT-PCR amplification, and microarray hybridization. This marker has been described as a reliable internal positive control marker in several publications (Dare et al 2016; Fan et al 2014). All the primers and probes were confirmed using BLAST program of NCBI and then synthesized using Sangon Biotech Co., Ltd. (Shanghai).…”

Section: Methodsmentioning

confidence: 99%

Genotyping and detection of common avian and human origin-influenza viruses using a portable chemiluminescence imaging microarray

et al. 2016

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Background Influenza viruses are divided into three types, A, B, and C. Human influenza A and B viruses can cause seasonal epidemics, but influenza C causes only a mild respiratory illness. Influenza A virus can infect various host species. In 2013, human-infectious avian influenza A (H7N9) was first reported in China. By the second week of 2014, there were 210 laboratory-confirmed human cases in the country, and the mortality rate eventually reached 22 %. Rapid and accurate diagnosis of influenza viruses is important for clinical management and epidemiology. MethodsIn this assay, a cost-effective chemiluminescence (CL) detection oligonucleotide microarray was developed to genotype and detect avian influenza A (H7N9), avian influenza A (H5N1), 2009 influenza A (H1N1), seasonal influenza A (H1N1), and seasonal influenza A (H3N2). Influenza A viruses and influenza B viruses were also generally detected using this microarray.ResultsThe results of detection of 40 cultivated influenza virus strains showed that the microarray was able to distinguish the subtypes of these influenza viruses very well. The microarray possessed similar or 10 fold higher limit of detection than the real-time RT-PCR method. Sixty-six clinical swab samples were detected using this microarray and verified with real time RT-PCR to evaluate the efficiency of this microarray for clinical testing.ConclusionsA reliable CL detection oligonucleotide microarray had been developed to genotype and detected these influenza viruses.

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Detection of a novel avian influenza A (H7N9) virus in humans by multiplex one-step real-time RT-PCR assay

Cited by 18 publications

References 23 publications

Simultaneous detection of influenza A subtypes of H3N2 virus, pandemic (H1N1) 2009 virus and reassortant avian H7N9 virus in humans by multiplex one-step real-time RT-PCR assay

Simultaneous detection of influenza A subtypes of H3N2 virus, pandemic (H1N1) 2009 virus and reassortant avian H7N9 virus in humans by multiplex one-step real-time RT-PCR assay

Advanced nanotechnologies in avian influenza: Current status and future trends – A review

Genotyping and detection of common avian and human origin-influenza viruses using a portable chemiluminescence imaging microarray

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