Detection and characterization of Newcastle disease virus in clinical samples using real time RT-PCR and melting curve analysis based on matrix and fusion genes amplification
Aim: Newcastle disease is still one of the major threats for poultry industry allover the world. Therefore, attempt was made in this study to use the SYBR Green I real-time PCR with melting curves analysis as for detection and differentiation of NDV strains in suspected infected birds. Materials and...
| Main Authors: | , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Veterinary World
2013-10-01
|
| Series: | Veterinary World |
| Subjects: | |
| Online Access: | http://www.scopemed.org/fulltextpdf.php?mno=20951 |
| _version_ | 1818649445802180608 |
|---|---|
| author | Saad Sharawi Malik A Al-Habeeb M. H. A. Mohamed |
| author_facet | Saad Sharawi Malik A Al-Habeeb M. H. A. Mohamed |
| author_sort | Saad Sharawi |
| collection | DOAJ |
| description | Aim: Newcastle disease is still one of the major threats for poultry industry allover the world. Therefore, attempt was made in this study to use the SYBR Green I real-time PCR with melting curves analysis as for detection and differentiation of NDV strains in suspected infected birds. Materials and Methods: Two sets of primers were used to amplify matrix and fusion genes in samples collected from suspectly infected birds (chickens and pigeons). Melting curve analysis in conjunction with real time PCR was conducted for identifying different pathotypes of the isolated NDVs. Clinical samples were propagated on specific pathogen free ECE and tested for MDT and ICIP. Results: The velogenic NDVs isolated from chickens and pigeons were distinguished with mean T 85.03±0.341 and m 83.78±0.237 respectively for M-gene amplification and for F-gene amplification the mean T were 84.04±0.037 and m 84.53±0.223. On the other hand the lentogenic NDV isolates including the vaccinal strains (HB1 and LaSota) have a higher mean T (86.99±0.021 for M-gene amplification and 86.50±0.063 for F-gene amplification). The test showed no reaction with m unrelated RNA samples. In addition, the results were in good agreement with both virus isolation and biological pathotyping (MDT and ICIP). The assay offers an attractive alternative method for the diagnosis of NDV that can be easily applied in laboratory diagnosis as a screening test for the detection and differentiation of NDV infections. Conclusion: As was shown by the successful rapid detection and pathotyping of 15 NDV strains in clinical samples representing velogenic and lentogenic NDV strains, and the agreement with the results of virus isolation , biological pathotyping and pathogenicity indices. The results of this report suggests that the described SybrGreen I real-time RT-PCR assay in conjunction with Melting curve analysis used as a rapid, specific and simple diagnostic tools for detection and pathotyping of different NDVs in clinically infected birds. [Vet World 2013; 6(5.000): 239-243] |
| first_indexed | 2024-12-17T01:34:26Z |
| format | Article |
| id | doaj.art-8a8d2ba618b245f79fbb72991d5cac40 |
| institution | Directory Open Access Journal |
| issn | 0972-8988 2231-0916 |
| language | English |
| last_indexed | 2024-12-17T01:34:26Z |
| publishDate | 2013-10-01 |
| publisher | Veterinary World |
| record_format | Article |
| series | Veterinary World |
| spelling | doaj.art-8a8d2ba618b245f79fbb72991d5cac402022-12-21T22:08:29ZengVeterinary WorldVeterinary World0972-89882231-09162013-10-0165.00023924310.5455/vetworld.2013.239-243Detection and characterization of Newcastle disease virus in clinical samples using real time RT-PCR and melting curve analysis based on matrix and fusion genes amplificationSaad SharawiMalik A Al-HabeebM. H. A. MohamedAim: Newcastle disease is still one of the major threats for poultry industry allover the world. Therefore, attempt was made in this study to use the SYBR Green I real-time PCR with melting curves analysis as for detection and differentiation of NDV strains in suspected infected birds. Materials and Methods: Two sets of primers were used to amplify matrix and fusion genes in samples collected from suspectly infected birds (chickens and pigeons). Melting curve analysis in conjunction with real time PCR was conducted for identifying different pathotypes of the isolated NDVs. Clinical samples were propagated on specific pathogen free ECE and tested for MDT and ICIP. Results: The velogenic NDVs isolated from chickens and pigeons were distinguished with mean T 85.03±0.341 and m 83.78±0.237 respectively for M-gene amplification and for F-gene amplification the mean T were 84.04±0.037 and m 84.53±0.223. On the other hand the lentogenic NDV isolates including the vaccinal strains (HB1 and LaSota) have a higher mean T (86.99±0.021 for M-gene amplification and 86.50±0.063 for F-gene amplification). The test showed no reaction with m unrelated RNA samples. In addition, the results were in good agreement with both virus isolation and biological pathotyping (MDT and ICIP). The assay offers an attractive alternative method for the diagnosis of NDV that can be easily applied in laboratory diagnosis as a screening test for the detection and differentiation of NDV infections. Conclusion: As was shown by the successful rapid detection and pathotyping of 15 NDV strains in clinical samples representing velogenic and lentogenic NDV strains, and the agreement with the results of virus isolation , biological pathotyping and pathogenicity indices. The results of this report suggests that the described SybrGreen I real-time RT-PCR assay in conjunction with Melting curve analysis used as a rapid, specific and simple diagnostic tools for detection and pathotyping of different NDVs in clinically infected birds. [Vet World 2013; 6(5.000): 239-243]http://www.scopemed.org/fulltextpdf.php?mno=20951lentogenicmelting tempratureNewcastle virussyer green Ivelogenic |
| spellingShingle | Saad Sharawi Malik A Al-Habeeb M. H. A. Mohamed Detection and characterization of Newcastle disease virus in clinical samples using real time RT-PCR and melting curve analysis based on matrix and fusion genes amplification Veterinary World lentogenic melting temprature Newcastle virus syer green I velogenic |
| title | Detection and characterization of Newcastle disease virus in clinical samples using real time RT-PCR and melting curve analysis based on matrix and fusion genes amplification |
| title_full | Detection and characterization of Newcastle disease virus in clinical samples using real time RT-PCR and melting curve analysis based on matrix and fusion genes amplification |
| title_fullStr | Detection and characterization of Newcastle disease virus in clinical samples using real time RT-PCR and melting curve analysis based on matrix and fusion genes amplification |
| title_full_unstemmed | Detection and characterization of Newcastle disease virus in clinical samples using real time RT-PCR and melting curve analysis based on matrix and fusion genes amplification |
| title_short | Detection and characterization of Newcastle disease virus in clinical samples using real time RT-PCR and melting curve analysis based on matrix and fusion genes amplification |
| title_sort | detection and characterization of newcastle disease virus in clinical samples using real time rt pcr and melting curve analysis based on matrix and fusion genes amplification |
| topic | lentogenic melting temprature Newcastle virus syer green I velogenic |
| url | http://www.scopemed.org/fulltextpdf.php?mno=20951 |
| work_keys_str_mv | AT saadsharawi detectionandcharacterizationofnewcastlediseasevirusinclinicalsamplesusingrealtimertpcrandmeltingcurveanalysisbasedonmatrixandfusiongenesamplification AT malikaalhabeeb detectionandcharacterizationofnewcastlediseasevirusinclinicalsamplesusingrealtimertpcrandmeltingcurveanalysisbasedonmatrixandfusiongenesamplification AT mhamohamed detectionandcharacterizationofnewcastlediseasevirusinclinicalsamplesusingrealtimertpcrandmeltingcurveanalysisbasedonmatrixandfusiongenesamplification |