Multiplex real-time reverse transcription PCR for detection of influenza viruses

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Multiplex real-time reverse transcription PCR for detection of influenza viruses


Author: Yuen, Suk-han
Title: Multiplex real-time reverse transcription PCR for detection of influenza viruses
Degree: M.Sc.
Year: 2010
Subject: Hong Kong Polytechnic University -- Dissertations
Influenza viruses -- China -- Hong Kong
Polymerase chain reaction -- Diagnostic use
Department: Faculty of Health and Social Sciences
Pages: xi, 78 leaves : ill. ; 30 cm.
InnoPac Record:
Abstract: Influenza viral infection is a global concern due to its high morbidity and mortality rate. Anti-viral drugs for influenza virus infections should be initiated within 48 hours of onset symptoms. Viral culture is time-consuming for diagnostic purposes and a method that will rapidly detect viruses is essential for management of patients with upper respiratory tract infections. Reverse transcription polymerase chain reaction (RT-PCR) is a useful tool for detection of influenza viruses. This study developed a two-step multiplex RT-PCR assay for detection of influenza A. Species of influenza A and its subtypes (H1, H3, H5, and H1 human swine) were identified using SYTO9 and Roche LightCycler 480. Five sets of primer pairs (M gene, H1, H3, H5, and H1 human swine) were designed for these strains and were optimized. Each primer pair generated a unique amplicon, evaluated by melting temperature due to different sequences of nucleotide bases. Due to non-specific signals found in melting curve analysis, two sets of primer pairs (M gene and H3) failed to be optimized. The duplex assay for seasonal H1 influenza A & human swine influenza (HSI) was evaluated using 86 nasopharyngeal aspirate samples, collected from patients with respiratory viruses in 2009. The seasonal H1 and HSI-specific primer sets had a detection limit approximately equivalent to 2.47 x 10⁹ and 2.18 x 10⁹ genome copies/ul, respectively. Sensitivity and specificity of duplex seasonal H1 and HSI specific real-time RT-PCR were 62% and 98%, respectively.

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