Journal of Virological Methods 165 (2010) 294–296 Contents lists available at ScienceDirect Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet Short communication Detection of pandemic influenza A/H1N1/2009 virus by real-time reverse transcription polymerase chain reaction Weili Wu a,1 , Xiaoping Kang b,1 , Zhijun Bai c,1 , Licheng Liu a,1 , Jingjing Li d , Xinwei Wu c , Honghe Sun a , Tao Hu a , Min Yang a , Pengzhi Wang a , Li Liu d , Yinhui Yang b,∗ , Biao Di c,∗∗ , Weijun Chen a,d,∗ ∗ ∗ a Beijing Institute of Genomics, Chinese Academy of Sciences, Airport Industry B6, Beijing 101300, China State Key Laboratory of Pathogen and Biosecurity, Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Fengtai Dongdajie Road 20, Beijing 100071, China c Center for Disease Control and Prevention of Guangzhou, Guangzhou 510080, China d Beijing BGI-GBI Biotech Co., Ltd, Beijing 101300, China b a b s t r a c t Article history: Received 4 August 2009 Received in revised form 25 November 2009 Accepted 30 November 2009 Available online 4 December 2009 Keywords: Pandemic influenza A/H1N1/2009 virus Real-time reverse transcription polymerase chain reaction (RT-PCR) Detection The first case of pandemic influenza A/H1N1/2009 infection was reported in Mexico in mid-April, 2009, and to date the new H1N1 virus has spread to over 160 countries. Therefore, it is important to obtain reliable epidemiological data on the spread of this virus by novel molecular methods can be developed in detection of this new infectious agent. A new method was developed for detection of the pandemic influenza A/H1N1/2009 using real-time reverse transcription polymerase chain reaction (RT-PCR). Direct comparison with the specific primers and probes recommended by the World Health Organization (WHO) demonstrates that the new method has a much greater sensitivity, and may thus be employed as a rapid, alternative method in detecting of the pandemic influenza A/H1N1/2009 virus. © 2009 Elsevier B.V. All rights reserved. An influenza pandemic has emerged recently and has spread to over 160 countries worldwide within a short period of time, and some 134,500 people were infected with 816 deaths by July 27, 2009 (WHO, 2009a). This new influenza A, subtype H1N1 virus characterized by a unique combination of gene segments which had not been identified previously among human or swine influenza A viruses (New Swine-Origin Influenza A (H1N1) Virus Investigation Team, 2009; Fraser et al., 2009). Each year, vaccines for the seasonal influenza A and B viruses are used to induce immunity to circulating strains. However, genetic variation can introduce a new influenza virus which can infect the population (WHO, 2009b). Symptoms caused by the pandemic influenza A/H1N1/2009 virus and the seasonal influenza viruses, such as fever, cough and sneeze, appear very similar that the clinical diagnosis often fails to distinguish (New Swine-Origin Influenza A (H1N1) Virus Investigation Team, 2009). Therefore, a rapid diagnostic method in detecting of the pan- ∗ Corresponding author. Tel.: +86 10 66948610. ∗∗ Corresponding author. ∗ ∗ ∗Corresponding author at: Beijing Institute of Genomics, Beijing Airport Industry Zone B-6, Beijing 101300, China. Tel.: +86 10 80481554; fax: +86 10 80485404. E-mail addresses: yyh428@sina.com (Y. Yang), biao65di@yahoo.com (B. Di), chenwj@genomics.org.cn (W. Chen). 1 These authors contributed equally to this article. 0166-0934/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2009.11.033 demic influenza A/H1N1/2009 virus is urgent for controlling the spread of the virus. In this study, a sensitive and specific RT-PCR assay was developed for the detection of the pandemic influenza A/H1N1/2009 virus. The results showed that the new method is much more sensitive than that recommended by World Health Organization (WHO), and it allows rapid and accurate detection of the RNA of pandemic influenza A/H1N1/2009 virus in clinical sample. The alignment of pandemic influenza A/H1N1/2009 hemagglutinin (HA) gene sequences (published in April 26, 2009 on GISAID, including California/04, 05, 06, 07, 09 and Texas/04, 05) was performed with all HA genes of seasonal influenza A (H1N1) virus published formerly (data not shown). The specific primers and probe set were designed using the Primer Express Software (PE Applied Biosystems, Foster City, CA). The sequences of primers and probe set were listed here. Forward primer (HA389F): 5′ -GGTTYGAGATATTCCCCAAGACA-3′ , reverse primer (HA464R): 5′ -TGAGGACATGCTGCCGTTAC-3′ and probe (HA414): 5′ -FAM-TTCATGGCCCAATCATGACTCGAACA-TAMRA-3′ . The probe was labeled with the reporter FAM (6-carboxyfluorescein) and the quencher TAMRA (6-carboxytetra-methylrhodamine). The primers and probes for the H1 gene (swH1) and M gene (InfA) recommended by WHO (WHO, 2009c) were also synthesized in this study for direct comparison. All primers W. Wu et al. / Journal of Virological Methods 165 (2010) 294–296 and probes were synthesized by the Shanghai Sangon Company. To evaluate the amplification efficiency and to determine the detection limits of the assay, RNA standards of pandemic influenza A/H1N1/2009 H1 gene were generated and tested. The H1 gene was synthesized according to the aligned sequence of HA gene of pandemic influenza A/H1N1/2009 virus (Beijing Genomics Institute, Beijing, China). The resulting product was cloned into a pGEMTeasy vector (Promega Shanghai, Shanghai, China) and linearized by specific DNA restriction enzyme. RNA was generated by in vitro transcription of the linearized plasmid DNA using the RiboMax Express Large Scale RNA production system according to the manufacturer’s instruction (Promega, Madison, WI, USA). After digestion of the template DNA with RNase-free DNase I, the transcribed RNA was purified with an RNeasy kit (Qiagen GmbH, Hilden, Germany). The purified RNA was quantified spectrophotometrically at 260 nm, and divided into aliquots which were stored at −80 ◦ C for future use. Serial dilutions of the transcribed H1 RNA (5 × 107 to 5 copies/␮l at 10-fold; 5–0.625 copies/␮l at 2-fold) were subjected to RT-PCR analysis. RT-PCR was performed in a 20 ␮l reaction volume that contained 4 ␮l of the RNA dilution, 10 ␮l 2× Taqman one-step RTPCR Master Mix Reagents (ABI, 4309169), 0.5 ␮l 40× MultiScribe and RNase inhibitor mixture, 0.25 ␮M forward primer, 0.25 ␮M of reverse primer and 0.125 ␮M of probe in a fluorometric PCR instrument (ABI 7300). Thermal cycling conditions were 30 min at 42 ◦ C followed by 10 min at 95 ◦ C and a subsequent 40 cycles amplification (95 ◦ C for 15 s; 55 ◦ C for 30 s, fluorescence was collected at 55 ◦ C). WHO swH1 assay was also performed using the identical reaction conditions according to WHO protocol (WHO, 2009c). Detection of live virus was assessed by using allantoic fluid from chicken embryos infected with the virus. The virus was diluted from approximately 5 × 104 50% egg infective dose (EID50 /ml) to 0.5 EID50 /ml at 10-fold and from 0.5 EID50 /ml to 0.03125 EID50 /ml at 2-fold in PBS buffer. RNA from 140-␮l aliquots of each dilution was extracted by TRIzol (Invitrogen), and resuspended in 60 ␮l of DEPC-treated water. RT-PCR was performed in 20 ␮l reaction volume according to the method described above. Detection of eight other influenza viruses was assessed by using allantoic fluid from infected chicken embryos (all viruses concentrations were diluted to approximate 104 50% egg infective dose (EID50)/ml). These eight viruses were kindly provided by the Tianjin Institute of Animal Husbandry and Veterinary Science, the Academy of Military Medical Science and the National Institute for the Control of Pharmaceutical and Biological Products: influenza virus A/PR/8/34 (H1N1); influenza virus A/Beijing/30/95 (H3N2); influenza virus A/Beijing/01/2003 (H5N1); influenza virus A/duck/Taiwan/4201/99 (H7N7); influenza virus A/Swine/Guangdong/2/2001(H1N1); influenza virus A/Iowa/CEID23/2005(H1N1); influenza virus A/Swine/Shandong/nb/2003 (H9N2); influenza B virus (Hongkong/5/72). RNA from 140-␮l aliquots of each dilution was extracted and resuspended in 60 ␮l of DEPC-treated water. RT-PCR was performed in 20 ␮l reaction volume according to the 295 method described above. WHO swH1 and Infa assays were also performed according to recommended protocols (WHO, 2009c). There were 627 clinical throat swab samples that were collected from 192 patients with possible pandemic influenza A/H1N1/2009 virus by the Guangzhou CDC and Academy of Military Medical Sciences in different time after onset of symptoms based on World Health Organization criteria. The age of patients with pandemic influenza ranged from 6 months to 71 years old. Sixty-five percent of the patients were between 10 and 18 years old, and only 10% of the patients were 55 years of age or older. Among these patients, for whom clinical information was available, the most common symptoms included fever (100%), cough (99%), and sore throat (80%). The other 120 throat swab samples from patients with seasonal influenza and 80 throat swab samples from healthy participants without symptoms were collected in a previous study from the years of 2005 to 2008. The patients with seasonal influenza comprised 50 patients with influenza A virus H1N1 infection, 40 with influenza A virus H3N2 infection and 30 with influenza B virus infection. These samples were resuspended in 1 ml of PBS buffer and centrifuged for 10 min at 1000 × g at 4 ◦ C, and RNA was extracted from the supernatant for RT-PCR analysis as described above. WHO swH1 and Infa assays were also performed according to recommended protocols (WHO, 2009c). Specimens generating equivocal results were confirmed by nucleotide sequencing. Standard curves of serial diluted RNA versus threshold cycle were both generated to determine the efficiency of the RT-PCR and the limit of detection. Both WHO assay and new H1 assay have a wide linear range (beginning at 20 copies/reaction and extending through 2 × 107 copies/reaction of target RNA, R2 = 0.997, 0.996, respectively). Analysis of the Ct values for each concentration of pandemic influenza A/H1N1/2009 H1 RNA serial dilutions proved that the new method is more sensitive compared to the WHO method and new method has lower Ct values than the WHO assay: the detected Ct values of dilutions from the analysis above were 16.2, 19.9, 24.1, 27.7, 31.1, 34.2, 37.2 for WHO assay and 14.9, 18.3, 22.2, 25.3, 28.9, 31.7, 34.9 for the H1 assay in this study, respectively. The detection limit for pandemic (H1N1) 2009 RNA was <5 copies per reaction (Ct = 37.5) in new H1 assay, while it is <10 copies per reaction (Ct = 38.1) in WHO swH1 assay. Allantoic fluid from infected chicken embryo with pandemic (H1N1) 2009 virus was also diluted into serial aliquots to determine the detection limit for live virus. Analysis of RNA extracted from serial dilutions showed a detection threshold of 0.25 EID50 /ml (data not shown). To evaluate the specificity of the new H1 assay, eight cultured influenza viruses, 120 seasonal influenza samples and 80 samples from healthy people were tested. No cross-reaction with eight cultured influenza viruses to the new H1 assay was observed. Both the new H1 assay and the WHO swH1 assay were negative to the seasonal influenza and healthy people. The confirmed influenza A samples were positive by the WHO Infa assay. Of 627 samples from 192 patients infected with possible pandemic (H1N1) 2009 virus, the detection rates of the H1N1 virus varied as time increased from the primary infection. The results of the WHO swH1 assay and new H1 assay both indicated that the Table 1 Summary of the clinical samples. Assays Time after onset of symptom (day) 1 P WHO swH1 New H1 CT (WHO) CT (new H1) 2 N 53 7 56 4 28.6 ± 1.1 27.9 ± 2.1 P 3 N 84 19 84 19 27.7 ± 2.6 27.1 ± 3.3 P 4 N 68 24 68 24 28.9 ± 3.3 28.7 ± 3.6 P 5 N 52 36 58 30 30.1 ± 4.2 30.1 ± 3.3 P 6 N 48 29 53 24 29.9 ± 3.2 29.5 ± 3.4 P: positive; N: negative; CT : average cycle threshold value of positive clinical specimens. P 7 N 36 39 42 33 34.2 ± 3.3 33.4 ± 2.5 P 8 N 21 38 26 33 36.1 ± 1.1 35.7 ± 0.9 P 9 N 13 24 14 23 37.5 ± 2.0 36.9 ± 2.4 P ≥10 N 3 19 3 19 38.5 ± 0.7 38.3 ± 0.8 P N 2 12 2 12 39.1 ± 0.4 38.7 ± 0.8 296 W. Wu et al. / Journal of Virological Methods 165 (2010) 294–296 The results of this study indicated that the new H1 assay is more sensitive than the WHO swH1 assay for detection of the virus. Whether the infectious virus is still present in the samples collected at later time points remains to be determined. Acknowledgments The work was supported by the research funding from Infectious Diseases Special Project, Minister of Health of China (2008ZX10004-001). Thank Drs. Siqi Liu and Saijun Tang for their critical reading of the manuscript. Appendix A. Supplementary data Fig. 1. The detection rate of pandemic (H1N1) 2009 virus by the WHO swH1 assay and new H1 assay. Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jviromet.2009.11.033. References optimal time for detecting pandemic influenza A/H1N1/2009 virus may lie between the first 3 days after the onset development of the symptoms caused by the virus. After the initial 3 days, the detection rate declined significantly, it may be due to the titer of the pandemic influenza A/H1N1/2009 virus which was decreased following the host immunological response and/or the antiviral treatment. Using the new H1 assay, the detection rate was 93.3% on the first day after the onset of symptoms and decreases gradually in the following days (Table 1, Fig. 1). On the 9th day, the detection rate declined to 13.6% in the current study, however, on the 17th day, the virus was still present in some samples. Compared with the WHO swH1 assay, the new H1 assay was more sensitive, and there were 26 samples which positive only by new H1 assay but negative in WHO swH1 assay (P = 0.001 < 0.05) (Table 1, Fig. 1). These samples were confirmed to be the pandemic influenza A/H1N1/2009 virus by nucleotide sequencing (Supplementary 1). 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