Development of multiplex real-time RT-PCR assays for influenza virus types and subtypes detection in respiratory samples

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Development of multiplex real-time RT-PCR assays for influenza virus types and subtypes detection in respiratory samples


Author: Poon, Ka Yan Inly
Title: Development of multiplex real-time RT-PCR assays for influenza virus types and subtypes detection in respiratory samples
Degree: M.Sc.
Year: 2015
Subject: Influenza -- Diagnosis
Hong Kong Polytechnic University -- Dissertations
Department: Dept. of Health Technology and Informatics
Pages: xi, 62 leaves : color illustraions ; 30 cm
Language: English
InnoPac Record:
Abstract: Influenza virus can cause zoonotic infections that can cause high morbidity and mortality in the extreme of age group and the immunocompromised patients. The virus causes a major global concern when it can emerge into new variant species and cross infect different species with serious illness. In particular, the avian flu’ tend to be more lethal and usually involves in pandemic outbreaks. Hence, early diagnosis is important for detection of influenza types and subtypes, particularly aid in better clinical management in treating patients, more profound surveillances in infection control and monitoring for any new emerging virus. This project was to develop a rapid, accurate and high throughput diagnostic test for early diagnosis and surveillance for detection of influenza types and subtypes. A multiplex RT-PCR assay was developed and optimized for detection of the influenza A, B and C, with primers for targeting the matrix protein of conserved region of the viruses. TaqMan hydrolysis probes principal was applied in this assay. The multiplex assay was performed and analyzed by LightCycler-96 (Roche) without the need of using any reference dye. Instead, four channels exhibiting different wavelength including FAM, Texad Red, VIC and Cy5 were used to detect the influenza A, B, C and RNase P, respectively. RNase P was served as an internal control in this assay. Amplification of RNase P was successful in all tested samples with threshold cycle from 20 to 35. This shows that no inhibitory substance present in the samples. The detection limits of multiplex PCR for the influenza typing are influenza A (1.25 TCID50/ml or 102 copies per reaction), influenza B (0.05 TCID50/ml or 101 copies per reaction) and influenza C (101 copies/ml). The sensitivity and specificity of the multiplex assay was found to be influenza A (80%, 100%), influenza B (84.2%, 100%) and influenza C (100%, 100%). The multiplex assay has also shown to be a higher detection rate of influenza virus, of approximately 50 % (21 vs 14) better in sensitivity than direct immunofluorescence assay. The time restraint causes subtyping assays that cannot be developed in the study period. However, using similar techniques and approach to develop multiplex RT-PCR assays for subtyping of seasonal and avian influenza are possible in the future study. This would further improve clinical management by rapid appropriate use of antivirals and antibiotics and facilitate implementation of infection control measures.

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