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Development of Multiplex Reverse Transcription Polymerase Chain Reaction (rt-pcr) for Simultaneous Detection of Matrix, Haemagglutinin and Neuraminidase Genes of H5n1 Avian Influenza Virus

R. Majumder
Department of Pathology, Faculty of Veterinary Science, Bangladesh AgriculturalUniversity, Mymensingh-2202, Bangladesh

E. H. Chowdhury
Department of Pathology, Faculty of Veterinary Science, Bangladesh AgriculturalUniversity, Mymensingh-2202, Bangladesh

R. Parvin
Department of Pathology, Faculty of Veterinary Science, Bangladesh AgriculturalUniversity, Mymensingh-2202, Bangladesh

J. A. Begum
Department of Pathology, Faculty of Veterinary Science, Bangladesh AgriculturalUniversity, Mymensingh-2202, Bangladesh

M. Giasuddin
Bangladesh Livestock Research Institute, Savar, Dhaka, Bangladesh

M. R. Islam
Department of Pathology, Faculty of Veterinary Science, Bangladesh AgriculturalUniversity, Mymensingh-2202, Bangladesh

Abstract/ Executive Summary:

Influenza A virus, subtype H5N1 causes a fatal disease in domestic poultry and could spread directly from poultry to humans. The aim of this study was to develop a multiplex reverse transcription polymerase chain reaction (mRT-PCR) for simultaneous detection of Type A influenza virus-specific matrix protein (M) gene as well as H5 and N1 genes of highly pathogenic avian influenza (HPAI) viruses. Finnzymes Phusion-Flash High- Fidelity PCR Master Mix (Finnzymes Oy, Finland) and Qiagen one-step RT-PCR enzyme mix (Qiagen, Germany) were used in a one-step RT-PCR. RNA was extracted from two known positive samples using Qiagen RNA extraction kit. RT-PCR was carried out with a mixture of primers specific for the Type A influenza virus matrix protein (M), and H5 and N1 genes of H5N1 HPAI viruses in a single reaction system. The mRT-PCR cDNA products were visualized by gel electrophoresis. The mRT-PCR yielded fragments of 245 bp for M, 545 bp for H5 and 343 bp for N1 genes of HPAI virus, which were clearly distinguishable. The mRT-PCR using the Finnzymes Phusion-Flash High-Fidelity PCR Master Mix (Finnzymes Oy, Finland) with Qiagen one-step RT-PCR Enzyme Mix (Qiagen, Germany) required only one hour and 20 minutes.

Keywords: RT-PCR, Haemagglutinin, Neuraminidase genes, H5N1 avian influenza virus

Location: Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh

Start Date: 00-00-2011

End Date: 00-00-2011

Program Area: Animal Health and Management

Commodity: Poultry

Objectives:

1. In the present study, a multiplex RT-PCR method was developed for rapid simultaneous detection of MP, H5 and N1 genes of H5N1 viruses from positive clinical samples of chickens.

Methodology:

Two H5N1-positive isolates, A/Chicken/Bangladesh/BL84T/2007 and A/Chicken/ Bangladesh/BL-343T/2008, were used in this study. RNA was extracted from positive clinical samples using Qiagen RNA extraction kit (Qiagen, Germany) according to the manufacturer’s instructions. Appropriate primer sequences for RTPCR were selected to amplify Type A influenza virus-specific matrix (M) protein gene, H5 haemagglutinin gene and N1 neuraminidase gene (http://www.who.int/csr/disease/avian_influenza/guidelines/ labtests/en/index.html) of AI viruses. One-step RT-PCR for MP, H5 and N1 genes was performed separately using Finnzymes Phusion-Flash High-Fidelity PCR Master Mix (Finnzymes Oy, Finland) and QIAGEN RT-PCR Enzyme Mix (Qiagen, Germany) in 20 μl volumes, in which the reaction mixture contained 10 μl Phusion-Flash Master Mix, 1 μl Qiagen RT-PCR Enzyme Mix, 0.5 μl of each primer, 5 μl of RNA template and RNase-free water to make 20 μl. The thermal profile of the RT-PCR reaction for the amplification of all three genes was: 50ºC for 30 min, 98ºC for 30 sec, 35 cycles of 98ºC for 10 sec, 52ºC for 30 sec and 72ºC for 30 sec, followed by 72ºC for 10 min in a thermocycler. RT-PCR was performed individually for MP, H5 and N1 genes as well as simultaneously for all three genes in the same reaction tube. Then the RT-PCR products were analysed by agarose gel (1.5%) electrophoresis and visualized by UV trans-illumination.

Source of Information: The Bangladesh Veterinarian (2011) 28(2) : 55 – 59

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

A rapid single-step multiplex RT-PCR using Phusion-Flash High-Fidelity PCR Master Mix and Qiagen RT-PCR Enzyme mix has been developed. The test can detect simultaneously Type A influenza virus-specific matrix protein (M) gene and H5 and N1 genes of HPAI influenza viruses.

Knowledge Management: Journal