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: Multiple-target (multiplex) reverse transcription-PCR (RT-PCR) for detection, typing, and subtyping of the hemagglutinin gene of influenza type A (H3N2 and H1N1) and type B viruses was developed and applied prospectively to virological surveillance of influenza in England in the 1995-1996 winter season. During this season both influenza A H3N2 and H1N1 viruses were circulating, although at different times. Six hundred nineteen combined nose and throat swabs taken by general practitioners in sentinel practices from individuals presenting with "influenzalike illness" were analyzed by culture, multiplex RT-PCR, and immunofluorescence. Of the 619 samples, 246 (39.7%) were positive by multiplex RT-PCR compared with 200 (32.3%) which yielded influenza viruses on culture. There was 100% correlation between multiplex RT-PCR typing and subtyping and the influenza types and subtypes obtained from culture. There was also excellent correlation between the temporal detection of influenza A H3N2 and H1N1 viruses by multiplex RT-PCR and by culture. During the peak weeks of influenza virus activity, a total of 259 specimens were received, of which 101 (38.9%) yielded influenza viruses on culture while 149 (57.5%) were positive in multiplex RT-PCR, providing an increase in detection of influenza viruses of approximately 20%. The increased detection of influenza virus occurred in all the age groups sampled. Samples which were positive by multiplex RT-PCR but negative by culture were not detected significantly earlier or later in the winter of 1995-1996 but were detected during the peak weeks of clinical influenza virus activity. Multiplex RT-PCR was successfully used in surveillance of influenza to provide accurate, sensitive diagnosis directly on clinical specimens sent through the post.Journal of Clinical Microbiology 09/1997; 35(8):2076-82. · 4.07 Impact Factor
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ABSTRACT: Avian influenza viruses have 15 different hemagglutinin (HA) subtypes (H1-H15). We report a procedure for the identification and HA-subtyping of avian influenza virus by reverse transcription-PCR (RT-PCR). The avian influenza virus is identified by RT-PCR using a set of primers specific to the nucleoprotein (NP) gene of avian influenza virus. The HA-subtypes of avian influenza virus were determined by running simultaneously 15 RT-PCR reactions, each using a set of primers specific to one HA-subtype. For a single virus strain or isolate, only one of the 15 RT-PCR reactions will give a product of expected size, and thus the HA-subtype of the virus is determined. The result of HA-subtyping was then confirmed by sequence analysis of the PCR product. A total of 80 strains or isolates of avian influenza viruses were subtyped by this RT-PCR procedure, and the result of RT-PCR gave an excellent (100%) correlation with the result of the conventional serological method. The RT-PCR procedure we developed is rapid and sensitive, and could be used for the identification and HA-subtyping of avian influenza virus in organ homogenates.Journal of Virological Methods 10/2001; 97(1-2):13-22. · 1.90 Impact Factor
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ABSTRACT: Two viruses with a novel hemagglutinin (HA), A/duck/Australia/341/83 and A/shearwater/West Australia/2576/79, have been isolated from a duck and a shorebird in Australia. Hemagglutination inhibition and double immunodiffusion assays failed to reveal cross-reactivity with any of the known subtypes (H1 to H14). We therefore propose that these viruses constitute a new HA subtype, H15. Sequence analysis of the HA genes confirmed the serologic findings. When compared at the amino acid level, the HA1 region of the H15 subtype differs from those of the other subtypes by 30% and more. This degree of heterogeneity is also found among HA genes of other subtypes. Thus we propose that amino acid sequence data should be evaluated when determining the HA subtypes of influenza A viruses. Sequence comparison and phylogenetic analysis suggested that the HA subtype H15 is most closely related to the H7. Compared to the H7 HA, the H15 acquired a 30-nucleotide insertion within HA1 at position 253 which is located in the globular head of the molecule. This finding suggests that RNA recombination, although a rare event in nature, may play an important role in the evolution of influenza viruses.Virology 04/1996; 217(2):508-16. · 3.37 Impact Factor