Rapid Detection of Avian Influenza Virus A and Subtype H5N1 by Single Step Multiplex Reverse Transcription-polymerase Chain Reaction
ABSTRACT Outbreaks of H5N1 highly pathogenic avian influenza (HPAI) virus caused great economic losses to the poultry industry and resulted in human deaths in Thailand and Viet Nam in 2004. Rapid typing and subtyping of H5N1 viruses, especially from clinical specimens, are desirable for taking prompt control measures to prevent the spread of the disease. Here, we developed a set of oligonucleotide primers able to detect, type and subtype H5 and N1 influenza viruses in a single step multiplex reverse transcription-polymerase chain reaction (RT-PCR). RNA was extracted from allantoic fluid or from specimens with guanidinium isothiocyanate reagent. Reverse transcription and PCR were carried out with a mixture of primers specific for influenza viruses of type A, subtype H5 and N1 in a single reaction system under identical conditions. The amplified DNA fragments were analyzed by agarose gel electrophoresis. All the H5N1 viruses tested in the study and the experimental specimens presented three specific bands by the method established here. The results presented here suggest that the method described below is rapid and specific and, therefore, could be valuable in the rapid detection of H5N1 influenza viruses in clinics.
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ABSTRACT: Avian Influenza (AI), Newcastle Disease (ND) and Infectious Bursal Disease (IBD) are highly contagious diseases with high occurrence in poultry. These 3 viral diseases are a major cause of disease problems in the poultry industry in Indonesia. The classical methods for detection and characterization of the etiological agents are by clinical sign, serological test, immunodiffusion test, pathology, histopathology and virus isolation. Since these conventional laboratory method have low sensitivity and specificity, the rapid diagnostic tool based on molecular technique are needed. Rapid detection and differential diagnosis for viral diseases have an important implication in clinical, economical and epidemiological aspects. RT-PCR amplification for diagnosis of viral disease in poultry industry is common used. This method can detect virus as etiological agent in poultry disease. Multiplex RT-PCR involves simultaneous amplification of more than one infectious agent using more than primer pair. In the present study, we developed a single step multiplex RT-PCR method, which can help in rapid detection and differentiation viruses as an etiological agent of AI, ND and IBD diseases. The method is highly sensitivity, specificity, fast and less expensive. The results showed that the single step multiplex RT-PCR method has been developed to rapid detection and differential diagnose for AI, ND and IBD viruses simultaneously in one step amplification reaction. This method is simple and easy for laboratory diagnosis application as well as specific and efficient to diagnose of viral diseases in poultry
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ABSTRACT: The design and characterization of a low-density microarray for subtyping influenza A is presented. The microarray consisted of 15 distinct oligonucleotides designed to target only the matrix gene segment of influenza A. An artificial neural network was utilized to automate microarray image interpretation. The neural network was trained to recognize fluorescence image patterns for 68 known influenza viruses and subsequently used to identify 53 unknowns in a blind study that included 39 human patient samples and 14 negative control samples. The assay exhibited a clinical sensitivity of 95% and clinical specificity of 92%.Analytical Chemistry 12/2006; 78(22):7610-5. DOI:10.1021/ac061739f · 5.83 Impact Factor
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ABSTRACT: Highly pathogenic avian influenza (HPAI) caused by the H5N1 subtype has given rise to serious damage in poultry industries in Asia. The virus has expanded its geographical range to Europe and Africa, posing a great risk to human health as well. For the control of avian influenza, a rapid diagnosis by detecting the causative virus and identifying its subtype is essential. In the present study, a rapid diagnosis kit combining immunochromatography with enzyme immunoassay which detects the H5 HA antigen of influenza A virus was developed using newly established anti-H5 HA monoclonal antibodies. The present kit specifically detected all of the H5 influenza viruses tested, and did not react with the other HA subtypes. H5 HA antigens were detected from swabs and tissue homogenates of chickens infected with HPAI virus strain A/chicken/Yamaguchi/7/04 (H5N1) from 2 days post inoculation. The kit showed enough sensitivity and specificity for the rapid diagnosis of HPAI.Microbiology and Immunology 02/2007; 51(9):903-7. DOI:10.1111/j.1348-0421.2007.tb03973.x · 1.31 Impact Factor