Development of multiplex RT-PCR assays for rapid detection and subtyping of influenza type A viruses from clinical specimens

College of Medicine and Medical Research Institute, Chungbuk National University, Cheongju, Korea.
Journal of Microbiology and Biotechnology (Impact Factor: 1.53). 07/2008; 18(6):1164-9.
Source: PubMed


We developed multiplex RT-PCR assays that can detect and identify 12 hemagglutinin (H1-H12) and 9 neuraminidase (N1-N9) subtypes that are commonly isolated from avian, swine, and human influenza A viruses. RT-PCR products with unique sizes characteristic of each subtype were amplified by multiplex RT-PCRs, and sequence analysis of each amplicon was demonstrated to be specific for each subtype with 24 reference viruses. The specificity was demonstrated further with DNA or cDNA templates from 7 viruses, 5 bacteria, and 50 influenza A virus negative specimens. Furthermore, the assays could detect and subtype up to 105 dilution of each of the reference viruses that had an original infectivity titer of 106 EID50/ml. Of 188 virus isolates, the multiplex RT-PCR results agreed completely with individual RT-PCR subtyping results and with results obtained from virus isolations. Furthermore, the multiplex RT-PCR methods efficiently detected mixed infections with at least two different subtypes of influenza viruses in one host. Therefore, these methods could facilitate rapid and accurate subtyping of influenza A viruses directly from field specimens.

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    • "Based on this results, it indicates that detection and differential diagnosis of 3 viruses, as etiological agent of AI, ND and IBD diseases by using single step multiplex RT-PCR holds potential and reliable as a rapid diagnostic method for the simultaneous detection of 3 viruses with material genetic of RNA in clinical and field samples from poultry (Malik et al., 2004). This single step multiplex RT- PCR method could be used to investigate the presence of each AI, ND and IBD for rapidly detect and differential diagnosis of each virus from clinical specimens (Chang et al., 2008). A multiplex RT-PCR that can rapidly differentiate between these infectious agents will be very important for the control of disease transmission not only in poultries but also in humans, along with the identification of three most common pathogen viruses which often found as mixed infections in poultry disease, and hence economic losses will be reduced in poultry (Rashid et al., 2009). "
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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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    • "Molecular methods such as reverse transcription-polymerase chain reaction (RT-PCR) (Hoffmann et al., 2001; Lee et al., 2001; Phipps et al., 2004; Chan et al., 2006; Kyoung et al., 2008), real time RT-PCR (Stone et al., 2004; Ong et al., 2007), microsphere-based duplexed immunoassay (Yan et al., 2004), DNA microarray (Kessler et al., 2004), GreeneChipResp oligonucleotide microarray (Quan et al., 2007), and padlock probes combined with DNA microarray (Gyarmati et al., 2008) have been developed and used for the detection and/or typing of influenza viruses. One problem of the currently available RT-PCR based methods is the need to utilize multiple, subtype-specific primer sets for nucleic acid amplification (Hoffmann et al., 2001; Chan et al., 2006). "
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    ABSTRACT: We designed a degenerate primer set that yielded full-length amplification of hemagglutinin (HA), neuraminidase (NA), matrix (M), and non-structural protein (NSP) genes of influenza A viruses in a single reaction mixture. These four genes were amplified from 15 HA (1-15) and 9 NA (1-9) subtypes of influenza A viruses of avian (n=16) origin. In addition, 272 field isolates of avian origin were tested by this method. Full-length amplification of HA, NA, M, and NSP genes was obtained in 242 (88.9%), 254 (93.4%), 268 (98.5%), and 268 (98.5%) isolates, respectively. No gene was amplified in four isolates. Of these four isolates, two were subtyped as H4N6, one as H7N7, and one as H10N7. Amplification was successful for all 4 genes of H1N1, H2N3, and H3N2 isolates of swine influenza. Also, all four genes were amplified in one equine influenza (H3N8) isolate and seven isolates of human origin (H1N1 and H3N2). This appears to be the first study using degenerate primer set for full-length amplification of four genes of influenza A viruses in a single reaction. Further studies are needed to determine if this primer set can be used for subtyping of influenza virus isolates.
    Journal of virological methods 06/2009; 160(1-2):163-6. DOI:10.1016/j.jviromet.2009.05.003 · 1.78 Impact Factor
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