EVALUATION OF A MULTIPLEX TAQMAN REAL-TIME REVERSE TRANSCRIPTION-PCR ASSAYS FOR THE DETECTION OF A( H1N1) PDM INFLUENZA VIRUSES IN CLINICAL SPECIMENS
DOI:
https://doi.org/10.31548/dopovidi2016.05.006Keywords:
pandemic influenza virus A (H1N1), polymerase chain reaction in real time, diagnostic test kitAbstract
The pandemic virus of Influenza A (H1N1)pdm09 (new Californian strains of 2009) became the absolutely new variant of flu virus, which was not previously circulating among humans and to which most people don't have immunity emerges and it could be transmited among humans. The 2009 pandemic virus had been spread globally quikly, and on 11 June 2009, the World Health Organization (WHO) declared the first influenza pandemic since 1968–1969[1]. Genetic analysis of new virus A(H1N1)pdm09 showed that it is 4-fold reassortant and already contains internal segments, that belong to the viruses of human influenza, birds and two separate lines of flu of pigs: the North-American and Eurasian lines[2]. April 2010, laboratory-confirmed infections of pH1N1 influenza virus was identified in 212 countries and overseas territories in April 2010, and fact of more than 18,000 laboratory-confirmed deaths was reported to the WHO worldwide. And in Ukraine have been reported the information of more than 1128 deaths in Ukraine in the same period was reported by Ministry of Health of Ukraine[4].
Research evaluation of virus of Influenza A (H1N1)pdm09 was carried out by Ukrainian scientists and it was detected[3], Ukrainian isolates of influenza virus had a high genetic identity (99%) to the pandemic strains A(H1N1)pdm09, this was observed in the period of 2009-2010 inother countries.
This pandemic A(H1N1)pdm09 virus has been widely circulating across the globe since 2009, and now it is established in human populations as a seasonal influenza virus. But during the epidemic season of 2015-2016 inUkraine pandemic influenza virus subtypes A(H1N1) pdm09 caused a considerable deaths among the human population of Ukraine. During 40 week it was registered 370 laboratory confirmed deaths caused by influenza, and 81,4% cases were caused by pandemic influenza A (H1N1)pdm09 virus [4].
Therefore modern diagnostics of the Californian strains of virus A(H1N1) pdm09 and monitoring of pandemic strains of virus subtypes A (H1N1) pdm09 across the south and east areas of Ukraine, where the birds influenza viruses A(H5N1) circulate naturally[5] remain actual problem for today.
The goal of our research work was to evaluate a Multiplex TaqMan Real-Time RT-PCR for the rapid and specific diagnostic of pandemic influenza virus A (H1N1) pdm09 in clinical samples of patients with influenza.
Materials and methods. We used Human Influenza virus reference strains А/FM1/47 (H1N1), А/Panama/2007/99 (H3N2), А/New Caledonia/20/99 (H1N1), B/Hong Kong/330/01 were kindly provided for our study by Svitlana L. Rybalko and WHO Collaborating Center for Influenza (CDC, USA) for evaluation the specificity levels. Human respiratory samples (nasopharyngeal swabs and aspirates, sputum) from Viral Influenza (VI) patients (n=10) were kindly provided by Irina G. Kostenko (the Main Military Clinical Hospital of Ukraine). The clinical samples were validated by Seeplex® Influenza A(H1N1pandemic) RT-PCR assay (Seegene Inc., South Korea) and TaqMan Influenza A (H1N1) Assay Sets [10]. The polymerase chain reaction in real time (Real-Time RT-PCR) was done for Applied Biosystems(ABI PRIZM 7000/7500).
Results: It was found that the Multiplex TaqMan Real-Time RT-PCR assay, designed primers and the TaqMan-probes(test kit «DIA Influenza H1N1») identifed the RNA of influenza pandemic California strains of A ( H1N1) pdm09 in clinical samples with 100% sensitivity and specificity.
Ten-fold dilutions of A (H1N1) pdm09 recombinant plasmids pIMC-13(M-gene), pIHC-21(H5-gene) и pINC-20(N1-gene) were used for the determination of detection limits and the amplification efficiency of the assay. Samples were tested in triplicate for each dilution. Therefore, analytical sensitivity of test is 10 copies per reaction.
Outlook: The proposed TaqMan Real-Time RT-PCR assay is an effective tool for fast and precise detection and diagnostic of the pandemic strains of influenza virus A (H1N1)pdm09.
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