Development of a universal multiplex RT-PCR for simultaneous detection and screening of bird flu H5NI from Newcastle disease viruses in a single-step-single-tube reaction

Morita Kouichi, . and Mohammed Alimul Islam, . (2011) Development of a universal multiplex RT-PCR for simultaneous detection and screening of bird flu H5NI from Newcastle disease viruses in a single-step-single-tube reaction. [Proceedings Paper]

The aim of this study was to develop a highly sensitive and cost effective molecular method for rapid and simultaneous detection and screening of highly pathogenic avian influenza HPAI virus H5N1/A and very virulent strain of Newcastle disease viruses vNDV from avian species by a single tube-single step UMRT-PCR assay. A universal multiplex RT-PCR UMRT-PCR method was developed and standardised by designing primer sets forward and reverse selective for the H5 and Ni genes of bird flu virus and F gene of Newcastle disease virus. The mRNA of each virus was extracted by QIAGENE RNA extraction kit. The LA-Taq DNA polymerase and AMV-RT Reverse transcriptase were used for the amplification of each gene of the two viruses using Bio-Red thermal cycler. Sensitivity and specificity of the newly designed UMRT-PCR was evaluated by amplifying the viral RNA extracted from fifty samples of chicken from a total of twenty five outbreaks 15 outbreaks of the year 2008 and 10 from 2009 respectively. Of the 55 samples 34 samples were positive for H5N1/A virus only. Out of 16 remaining samples 10 samples were positive only for NDV and 6 samples were positive both for H5N1/A and NDV respectively. All the five samples of crows were negative in UMRT-PCR for either virus. Though the result of UMRT-PCR indicated that the prevalence of H5N1/A virus was predominant in the samples of both the years of outbreak but all the outbreaks were not caused by H5N1/A virus only. The primers designed for UMRT-PCR for rapid genome detection of H5N1/A and NDV by the single tube reaction in this study are unique. Misinterpretation of the result of diagnosis of other methods can be challenged by using UMRT-PCR in detecting either one or more genomes of the two deadly viruses of poultry anywhere in the world where the diseases are endemic.

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Abstract

The aim of this study was to develop a highly sensitive and cost effective molecular method for rapid and simultaneous detection and screening of highly pathogenic avian influenza HPAI virus H5N1/A and very virulent strain of Newcastle disease viruses vNDV from avian species by a single tube-single step UMRT-PCR assay. A universal multiplex RT-PCR UMRT-PCR method was developed and standardised by designing primer sets forward and reverse selective for the H5 and Ni genes of bird flu virus and F gene of Newcastle disease virus. The mRNA of each virus was extracted by QIAGENE RNA extraction kit. The LA-Taq DNA polymerase and AMV-RT Reverse transcriptase were used for the amplification of each gene of the two viruses using Bio-Red thermal cycler. Sensitivity and specificity of the newly designed UMRT-PCR was evaluated by amplifying the viral RNA extracted from fifty samples of chicken from a total of twenty five outbreaks 15 outbreaks of the year 2008 and 10 from 2009 respectively. Of the 55 samples 34 samples were positive for H5N1/A virus only. Out of 16 remaining samples 10 samples were positive only for NDV and 6 samples were positive both for H5N1/A and NDV respectively. All the five samples of crows were negative in UMRT-PCR for either virus. Though the result of UMRT-PCR indicated that the prevalence of H5N1/A virus was predominant in the samples of both the years of outbreak but all the outbreaks were not caused by H5N1/A virus only. The primers designed for UMRT-PCR for rapid genome detection of H5N1/A and NDV by the single tube reaction in this study are unique. Misinterpretation of the result of diagnosis of other methods can be challenged by using UMRT-PCR in detecting either one or more genomes of the two deadly viruses of poultry anywhere in the world where the diseases are endemic.

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Item Type:	Proceedings Paper
Additional Information:	Available at Perpustakaan Sultan Abdul Samad Universiti Putra Malaysia 43400 UPM Serdang Selangor Malaysia. QR22 M3I61 2011 vol.2 Call Number.
AGROVOC Term:	Newcastle disease virus
AGROVOC Term:	Newcastle disease
AGROVOC Term:	Avian influenza virus
AGROVOC Term:	Animal diseases
AGROVOC Term:	Crows
AGROVOC Term:	Chickens
AGROVOC Term:	Diagnosis
AGROVOC Term:	Diagnostic techniques
AGROVOC Term:	Extraction
AGROVOC Term:	Genes
Geographical Term:	MALAYSIA
Depositing User:	Ms. Suzila Mohamad Kasim
Last Modified:	24 Apr 2025 05:15
URI:	http://webagris.upm.edu.my/id/eprint/12949

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