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.
"Multiplex RT-PCR was designed to simultaneously amplify three genes from three different viruses which commonly infected in poultry in a single step reaction to detect M gene of AI virus, F gene of ND virus and VP2 gene of IBD virus using specific nucleotide primers for each gene (see Table). Conventional methods for detection and differential diagnosis of AI, ND and IBD require the inoculation and propagation into embryonated fowl eggs or tissue culture followed serological tests (Wei et al., 2006 "
[Show abstract][Hide abstract] 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
"Conventional laboratory methods for H5N1 virus detection include virus isolation in embryonated eggs or Madin-Darby canine kidney (MDCK) cells, followed by subsequent HA and NA subtype identification using serological methods [10,11]. Molecular detection methods such as reverse transcriptase PCR (RT-PCR) have been widely applied for the laboratory diagnosis of influenza infections and HA subtype identification [12,13]. However, these methods are technically demanding and time consuming, or requiring high level biosafety facility. "
[Show abstract][Hide abstract] ABSTRACT: Rapid diagnosis and surveillance for H5 subtype viruses are critical for the control of H5N1 infection.
In this study, H5 Dot ELISA, a rapid test for the detection of avian H5N1 influenza virus, was developed with two complementary H5 monoclonal antibodies. HA sequencing of escape mutants followed by epitope mapping revealed that the two Mabs target the epitope component (189th amino acid) on the HA protein but are specific for different amino acids (189Lys or 189Arg). Gene alignment indicated that these two amino acids are the most frequent types on this position among all of the H5 AIV reported in GeneBank. These two H5 Mabs were used together in a dot ELISA to detect H5 viral antigen. The detection limit of the developed test for multiple clades of H5N1 viruses, including clades 0, 1, 2.1, 2.2, 2.3, 4, 7, and 8, was less than 0.5 hemagglutinin units. The specificity of the optimized dot ELISA was examined by using 100 H5 strains, including H5N1 HPAI strains from multiple clades, 36 non-H5N1 viruses, and 4 influenza B viruses. No cross-reactivity was observed for any of the non-H5N1 viruses tested. Among 200 random poultry samples, the test gave 100% positive results for all of the twelve RT-PCR-positive samples.
Considering that the test is convenient for field use, this H5 Dot ELISA can be used for on-site detection of H5N1 infection in clinical or environmental specimens and facilitate the investigation of H5N1 influenza outbreaks and surveillance in poultry.
[Show abstract][Hide abstract] 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%.
Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.