Fan et al. BMC Infectious Diseases 2014, 14:541 http://www.biomedcentral.com/1471-2334/14/541 RESEARCH ARTICLE Open Access Detection of a novel avian influenza A (H7N9) virus in humans by multiplex one-step real-time RT-PCR assay Jian Fan1†, David Cui1†, Siuying Lau3, Guoliang Xie1, Xichao Guo1, Shufa Zheng1, Xiaofeng Huang3, Shigui Yang2, Xianzhi Yang1, Zhaoxia Huo1, Fei Yu1, Jianzhou Lou1, Li Tian1, Xuefen Li1, Yuejiao Dong1, Qiaoyun Zhu1 and Yu Chen1,2* Abstract Background: 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. Methods: Clinical 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. Results: The 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. Conclusions: These 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. Keywords: Avian influenza, H7N9, Detection, Rnase P, Multiplex real-time RT-PCR Background been characterized in avian species, while the H17N10 The influenza A virus (FluA) is a negative-sense, single- subtype is found in bats [3,4]. Most subtypes of FluA virus stranded RNA virus belonging to the family Orthomyx- such as H5 and H7 subtypes are not pathogenic in oviridae. FluA is further classified on the basis of two poultry, but outbreaks in poultry and wild birds are corre- surface glycoproteins, hemagglutinin (H) and neuramin- lated with the highly virulent FluA virus, which can cause idase (N) [1,2]. All subtypes of FluA comprise various severe economic losses owing to massive numbers of combinations of the H and N glycoproteins. Sixteen H deaths of domestic poultry [4-6]. subtypes (H1-H16) and nine N subtypes (N1-N9) have Human infections are generally associated with the H1, H2, and H3 subtypes, although sporadic cases or out- * Correspondence: [email protected] breaks of avian FluA subtypes H5N1 [3], H7N2 [5], H7N3 †Equal contributors [5], H7N7 [6], H10N8 [7], H10N7 [8], and H9N2 [9] have 1 Department of Clinical Laboratory, the First Affiliated Hospital, School of resulted from direct transmissions from domestic poultry Medicine, Zhejiang University, 79 Qingchun Road, Hangzhou 310003, China 2State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, the and wild birds to humans. Most patients infected with First Affiliated Hospital, School of Medicine, Zhejiang University, 79 Qingchun these subtypes exhibit mild symptoms, such as conjunctiv- Road, Hangzhou, China itis and acute upper respiratory tract infections associated Full list of author information is available at the end of the article © 2014 Fan et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Fan et al. BMC Infectious Diseases 2014, 14:541 Page 2 of 9 http://www.biomedcentral.com/1471-2334/14/541 with fever and sore throat, with or without gastrointestinal Moreover, clinical specimens were also collected from pa- symptoms. However, the H5N1 virus exhibits a high mor- tients with confirmed H7N9 viral infections. All specimens tality rate of over 50%, the H7N7 virus has caused one fa- were stored at −80°C. Specimens were tested for the pres- tality, and three deaths have resulted from the H10N8 ence of FluA and the novel H7N9 subtype using a real- virus [3,5,6]. time RT-PCR assay, and highly virulent avian influenza A The avian influenza A (H7N9) virus has been found in (H7N9) virus and other common respiratory pathogens poultry and birds, although no human infections have were cultured in the State Key Laboratory for Diagnosis been documented, and its pathogenicity was found to be and Treatment of Infectious Diseases. According to the low in previous studies [4]. Since February 2013, a novel, Declaration of Helsinki (1964), informed consents for par- reassortant avian influenza A (H7N9) virus associated ticipation were obtained from the participants or guardians with human deaths has emerged in eastern China [10,11]. of the patients, and the Medical Ethical Committee of our Up to February 28, 2014, this novel, highly virulent H7N9 hospital approved this study. virus for humans has infected over 381 patients who pre- sented with influenza-like illness (ILI) and severe acquired Viral RNA extraction pneumonia, resulting in 118 deaths. It will be a challenge RNA from viral cultures of three novel human H7N9 vi- to control the H7N9 outbreak in humans before it spreads ruses, three human novel H1N1 viruses, five seasonal hu- further. Currently, sporadic cases, but no evident out- man H1N1 viruses, five seasonal human H3N2 viruses, breaks, of this novel virus have been documented in wild three human influenza B viruses, 83 specimens from pa- birds and poultry, and there is some limited evidence of tients without ILI, and 1,011 specimens from patients human-to-human transmission during unprotected expos- with ILI was extracted using the RNeasy Mini Kit (Qiagen, ure [12], and there are no vaccines for humans. Valencia, CA) according to the manufacturer’sprotocol. Thus, early detection and isolation of suspected pa- tients are the most effective measures to control and prevent further transmission of the virus. Here, we re- Multiplex real-time RT-PCR assay port a multiplex real-time RT-PCR assay allowing the The multiplex one-step real-time RT-PCR assays were simultaneous detection and discrimination of universal carried out using the One Step PrimeScript® RT-PCR Kit μ FluA strains and the novel H7N9 subtype in a single test (Perfect Real Time) (Takara, China). In brief, 1.0 lof μ μ tube. The results showed that the sensitivity and specifi- 20 M FluA, H7, N9 and RP primers, 0.5 l of the corre- μ μ city of this assay was comparable to a protocol recom- sponding probes. 5 lofRNA,25 lof2×OneStep μ μ mended by the World Health Organization (WHO). RT-PCR buffer, 2 lofExTaqHSPolymerase,2 l of PrimeScript RT Enzyme Mix and an appropriate vol- Methods ume of Rnase-free distilled water (dH2O) were combined μ Primer and probe design in a 50 l reaction volume. The thermal cycles were per- Primers and probes were designed to correspond to con- formed on an ABI 7500 real-time PCR system (Applied served regions of the FluA matrix (M) gene segment. The Biosystems, Foster city, CA) under the following condi- primers/probes for the novel H7N9 strain were designed tions: 30 min at 50°C for reverse transcription; 5 min at to detect subtype-specific H and N gene segments, and the 95°C, then 40 cycles at 95°C for 15 s and 55°C for 45 s for Rnase P gene (RP) served as an internal control to monitor PCR amplification. The PCR results were determined if the quality of the clinical specimens. These primers/probes the quality controls work. First, the specimen is positive if ≤ were designed according to the Influenza Primer Design Ct value is 38.0 with appropriate results. Second, the spe- Resource and primers/probes recommended by the WHO cimen is negative if the Ct value is undetectable. Third, protocol were listed in Table 1 [13]. the specimen with a Ct > 38 is suggested to be repeated, the specimen is considered positive if the repeat result is Clinical specimens and patients the same as before, conversely the specimen is considered According to diagnostic criteria of China for the detec- negative if the repeat result is undetectable. tion of avian influenza A(H7N9) virus highly virulent in humans, patients with acute respiratory infections with Viral culture influenza-like illness (ILIs), especially those having pneu- Madin-Darby canine kidney (MDCK) cells were used to monia and a history of occupational exposure to poultry isolate the novel avian influenza A (H7N9) virus using and wild birds, were examined by the Infection Depart- standard methods in a biosafety level three (BSL-3) la- ment of the First Affiliated Hospital of the College of boratory. MDCK cells were inoculated with fresh clinical Medicine of Zhejiang University. Clinical specimens, in- specimens. When the cytopathogenic effect (CPE) of the cluding throat swabs, nasal aspirates and washes, and spu- virus was observed, cells were harvested and tested for tum specimens, were collected beginning in April 2013. the novel H7N9 virus by PCR assay of WHO protocol. Fan et al. BMC Infectious Diseases 2014, 14:541 Page 3 of 9 http://www.biomedcentral.com/1471-2334/14/541 Table 1 Primers and probes designed and used in this study Set Primers and probes Sequences (5′ → 3′) Targeted genes Location (bp) GenBank accession no.