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

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

doi:10.1186/1471-2334-14-541

Jian Fan, Siuying Lau + Show 15 more

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https://doi.org/10.1186/1471-2334-14-541

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Abstract

BackgroundA novel avian influenza A (H7N9) virus emerged in eastern China in February 2013. 413 confirmed human cases, including 157 deaths, have been recorded as of July 31, 2014.MethodsClinical specimens, including throat swabs, sputum or tracheal aspirates, etc., were obtained from patients exhibiting influenza-like illness (ILIs), especially from those having pneumonia and a history of occupational exposure to poultry and wild birds. RNA was extracted from these samples and a multiplex one-step real-time RT-PCR assay was developed to specifically detect the influenza A virus (FluA). PCR primers targeted the conserved M and Rnase P (RP) genes, as well as the hemagglutinin and neuraminidase genes of the H7N9 virus.ResultsThe multiplex assay specifically detected the avian H7N9 virus, and no cross-reaction with other common respiratory pathogens was observed. The detection limit of the assay was approximately 0.05 50% tissue culture infective doses (TCID50), or 100 copies per reaction. Positive detection of the H7N9 virus in sputum/tracheal aspirates was higher than in throat swabs during the surveillance of patients with ILIs. Additionally, detection of the matrix (M) and Rnase P genes aided in the determination of the novel avian H7N9 virus and ensured the quality of the clinical samples.ConclusionsThese results demonstrate that the multiplex assay detected the novel avian H7N9 virus with high specificity and sensitivity, which is essential for the early diagnosis and treatment of infected patients.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2334-14-541) contains supplementary material, which is available to authorized users.

Highlights

A novel avian influenza A (H7N9) virus emerged in eastern China in February 2013. 413 confirmed human cases, including 157 deaths, have been recorded as of July 31, 2014
Human infections are generally associated with the H1, H2, and H3 subtypes, sporadic cases or outbreaks of avian FluA subtypes H5N1 [3], H7N2 [5], H7N3 [5], H7N7 [6], H10N8 [7], H10N7 [8], and H9N2 [9] have resulted from direct transmissions from domestic poultry and wild birds to humans
Using novel H7N9 sequences submitted to the National Center for Biotechnology Information (NCBI) database, we designed a new set of primers and probes to detect the novel H7N9 virus by a multiplex real-time RT-PCR assay in one reaction tube (Table 1)

Summary

Introduction

A novel avian influenza A (H7N9) virus emerged in eastern China in February 2013. 413 confirmed human cases, including 157 deaths, have been recorded as of July 31, 2014. All subtypes of FluA comprise various combinations of the H and N glycoproteins. Human infections are generally associated with the H1, H2, and H3 subtypes, sporadic cases or outbreaks of avian FluA subtypes H5N1 [3], H7N2 [5], H7N3 [5], H7N7 [6], H10N8 [7], H10N7 [8], and H9N2 [9] have resulted from direct transmissions from domestic poultry and wild birds to humans. Most patients infected with these subtypes exhibit mild symptoms, such as conjunctivitis and acute upper respiratory tract infections associated with fever and sore throat, with or without gastrointestinal symptoms. The H5N1 virus exhibits a high mortality rate of over 50%, the H7N7 virus has caused one fatality, and three deaths have resulted from the H10N8 virus [3,5,6]

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