| Influenza virus based on the diversity of nucleoprotein antigen, is divided into A type, B type, C type. Influenza A virus (IAV) infected a wide range of host, due to its antigenic drift and antigenic shift result in new IAV serotypes appear constantly, and even produce highly pathogenicity strains, and may cross the species barrier to infect humans, causing local region and even worldwide flu epidemic outbreaks. Therefore, the rapid detection of IAV is of great significance for the prevention and control of IAV. At present the main IAV detection methods are aetiology, serology and molecular biology diagnostic technology. Aetiology method is time consuming, not suitable for timely detection; serological method is low specificity, prone to cross reaction; Molecular biological diagnostic technology is stronger sensitivity and specificity, but more two steps, and cannot detect pan-IAV serotypes at the same time. Therefore, this study is to establish a rapid sensitive and can detect pan-IAV serotypes method, and successfully applied to poultry IAV testing of live-bird markets in China.Establishment of a pan-IAV, one-step reverse-transcription FRET-qPCR methodThis study is mainly based on a highly conserved region of 68 IAV serotypes matrix (M) gene, design specific primers and probes, to establish a pan-IAV, one-step reverse-transcription FRET-qPCR method (169 bp of the target fragment). The specific primers and probes further BLAST search, confirmed with IAV serotypes highly match (> 80%) was 122, contained 28,206 IAV sequences. Application of the method of standard H1N1 serotypes strain were prepared, and the results showed that the sensitivity of the method achieved 10 copies/reaction; the sensitivity of nine IAV serotypes strains detection, showed that this method was superior to the traditional EID50 assay; the specificity of NDV detection, showed that this method cannot amplify the NDV. For the PCR method to explore the feasibility of this experiment, collecting SPF chickens throat and cloacal swabs after the different dilutions of H9N2 serotypes virus was added to swab samples for the one-step reverse transcription FRET-qPCR method and EID50 assay. In addition, this study based on IAV PA gene one-step reverse-transcription PCR (575 bp), to verify this screening of positive samples, showed that can amplify 9 IAV serotypes strains and IAV positive swabs. Through agarose gel electrophoresis and sequencing of its have further validation. This study is to establish a rapid sensitive, can detect all IAV serotypes, for influenza virus epidemic monitoring and controling to provide important technical support.Application of a pan-IAV, one-step reverse-transcription FRET-qPCR methodCollect the throat and cloacal swab samples of 1,839 poultry (chicken 1,521, duck 127, goose 100, pigeon 91) from live-bird markets of 24 provinces in China, application of a pan-IAV, one-step reverse-transcription FRET-qPCR method. The results showed that IAV positive rate of the poultry was 9.2%(169/1,839), including the highest chicken IAV positive rate (10.4%; 158/1,521), followed by duck (5.5%; 7/127), pigeons (4.4%; 4/91), goose (0%; 0/100); IAV positive rate of throat swabs (7.5%; 138/1,839) was significantly higher than IAV positive rate of the cloacal swabs (5.2%; 95/1,839; P< 10"4). At the same time, the throat swabs contains IAV copy number was also significantly higher than the cloacal swabs (106.07 vs.105.54; P< 10-4); Throat and cloacal swabs were positive for poultry of total positive poultry 27.8% (47/169), throat swabs positive for poultry only 47.9% (81/169), cloacal swabs positive for poultry only 24.3%(41/169).Application based on PA, HA and NA gene of PCR, judged IAV serotypes of positive samples. Results showed that in positive IAV poultry samples, H9N2 serotypes accounted for 56.8% (96/169), H7N9 serotypes accounted for 41.4% (70/169), H5N1 serotypes accounted for 1.8% (3/169). H9N2 serotypes of poultry, including chickens (95.8%; 92/96) and duck (4.2%; 4/96), in 96 H9N2 serotypes poultry, throat and cloacal swabs were H9N2 serotypes poultry accounted for 9.3%(9/96), only throat swabs for H9N2 serotypes poultry accounted for 71.9% (69/96), only cloacal swabs for H9N2 serotypes poultry accounted for 18.8%(18/96); H7N9 serotypes of poultry, including chickens (94.3%; 66/70), and pigeons (5.7%; 4/70), in 70 H7N9 serotypes poultry, throat and cloacal swabs were H7N9 serotypes of poultry 51.4%(36/70), only throat swabs for H7N9 serotypes poultry accounted for 15.7%(11/70), only cloacal swabs for H7N9 serotypes poultry accounted for 32.9%(23/70); H5N1 serotypes poultry detection only in duck (n=3), two poultry throat and cloacal swabs were H5N1 serotypes, the other one only throat swab for H5N1 serotypes.In conclusion, this study based on M gene of a pan-IAV, one-step reverse-transcription FRET-qPCR method, and based on PA, HA and NA genes of reverse-transcription PCR method for verification. Application of this method in the throat and cloacal swabs of live-bird markets of 24 provinces in China testing and classification, results showed that the poultry IAV positive rate was 9.2%(169/1,839), including the highest chicken IAV positive rate (10.4%; 158/1,521); The hroat swabs IAV positive rate (7.5%; 138/1,839) was significantly higher than the cloacal swabs (5.2%; 95/1,839; P< 10-4), at the same time, the throat swabs contains IAV copy number was also significantly higher than the cloacal swabs (106.07 vs.105.54; P< 10-4); In IAV positive poultry, most of H9N2 serotypes (56.8%; 96/169) and H7N9 serotypes (41.4%; 70/169). This study established one-step reverse-transcription FRET-qPCR method is high sensitivity, and can detect all IAV serotypes quickly and easily, to influenza A virus monitoring and controling to provide important reference and technical support, has important economic value and social significance. |
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