Journal of Microbial & Biochemical Technology

Simultaneous detection of human parainfluenza viruses 1, 2, 3, and 4 in patients with influenza-like illness using a multiplex real-time PCR

World Congress and Expo on Applied Microbiology

August 18-20, 2015 Frankfurt, Germany

Mahboobeh Ramezannia1,2, Fatemeh Fotouhi2, Seyed Masoud Hosseini1, Behzad Khansarinejad3, Vahideh Mazaheri2 and Peyvand Biglari2

1Shahid Beheshti University, Iran 2Pasteur Institute of Iran, Iran 3Arak University of Medical Sciences, Iran

Posters-Accepted Abstracts: J Microb Biochem Technol

Abstract:

Introduction: The human ParainfluenzaViruses (PIVs) are enveloped non-segmented, negative, single-stranded RNA viruses which belong to Paramyxoviridae Family. They are an important cause of upper and lower respiratory tract infections causing pneumonia, croup, and bronchiolitis in infants, children, and immune-compromised individuals. Four types of HPIV are circulating worldwide. The availability of HPIV-specific detection assays is important because there are other respiratory pathogens cause similar illnesses. Objective of the present study was to develop a one-step multiplex quantitative RT-PCR to detect clinical specimens from 69 hospitalized patients with influenza-like illness in a single test. Materials & Methods: Nasopharyngeal swabs were collected from pediatric and adult patients with influenza-like illness. Nucleic acid extraction was performed using High Pure RNA Kit (Roche, Germany). The gene sequences of parainfluenza viruses were obtained from NCBI and alignment was carried out using AlleleID software to identify conserved sequences for designing primers and probes. The parainfluenza viruses-specific hydrolysis probes were labeled at the 5â?² end with four different reporter dyes FAM (530 nm), TET (555 nm), ROX (610 nm) and Cy5 (660 nm), respectively. The assay was first optimized in a monoplex qRT-PCR for each type; subsequently, a one-tube multiplex real-time RT-PCR was performed to discriminate all known human parainfluenza viruses using a Rotor-Gene 6000 apparatus (Corbett Life Science). Results & Conclusions: Of the 69 specimens, 35 (50.72%) were positive by multiplex qRT-PCR. HPIV2 (26.08%) was the most frequent detected virus followed by HPIV1 (15.94%), HPIV3 (2.89%), and HPIV4 (5.79%). This assay is mostly recognized as a sensitive and specific method for detection of respiratory RNA viruses whose result can be achieved within 3 hours, which raise its clinical relevance. Thus, use of this qRT â??PCR assay would develop patient management and infection control.

Biography :

Mahboobeh Ramezannia has completed her BS from Azad University of Iran and is now an MSc student from Shahid Beheshti University of Iran.