» Volume 5, Issue 12 pp. 709-780 » Volume 5, Issue 11 pp. 645-704 » Volume 5, Issue 10 pp. 581-638 » Volume 5, Issue 9 pp. 517-576 » Volume 5, Issue 8 pp. 453-511 » Volume 5, Issue 7 pp. 389-448 » Volume 5, Issue 6 pp. 325-384 » Volume 5, Issue 5 pp. 261-319 » Volume 5, Issue 4 pp. 197-254 » Volume 5, Issue 3 pp. 133-192 » Volume 5, Issue 2 pp. 69-127 » Volume 5, Issue 1 pp. 5-63 » Volume 4, Issue 12 pp. 533-574 » Volume 4, Issue 11 pp. 485-528 » Volume 4, Issue 10 pp. 437-479 » Volume 4, Issue 9 pp. 389-432 » Volume 4, Issue 8 pp. 341-382 » Volume 4, Issue 7 pp. 293-336 » Volume 4, Issue 6 pp. 245-286 » Volume 4, Issue 5 pp. 197-240 » Volume 4, Issue 4 pp. 149-192 » Volume 4, Issue 3 pp. 101-144 » Volume 4, Issue 2 pp. 53-96 » Volume 4, Issue 1 pp. 5-47 » Volume 3, Issue 12 pp. 533-569 » Volume 3, Issue 11 pp. 489-527 » Volume 3, Issue 10 pp. 445-484 » Volume 3, Issue 9 pp. 397-439 » Volume 3, Issue 8 pp. 353-392 » Volume 3, Issue 7 pp. 305-348 » Volume 3, Issue 6 pp. 257-300 » Volume 3, Issue 5 pp. 205-252 » Volume 3, Issue 4 pp. 157-200 » Volume 3, Issue 3 pp. 105-151 » Volume 3, Issue 2 pp. 53-100 » Volume 3, Issue 1 pp. 5-47 » Volume 2, Issue 12 pp. 453-485 » Volume 2, Issue 11 pp. 413-447 » Volume 2, Issue 10 pp. 373-407 » Volume 2, Issue 9 pp. 333-366 » Volume 2, Issue 8 pp. 293-327 » Volume 2, Issue 7 pp. 258-285 » Volume 2, Issue 6 pp. 213-246 » Volume 2, Issue 5 pp. 173-207 » Volume 2, Issue 4 pp. 129-165 » Volume 2, Issue 3 pp. 89-122 » Volume 2, Issue 2 pp. 49-82 » Volume 2, Issue 1 pp. 5-44 » Volume 1, Issue 12 pp. 357-381 » Volume 1, Issue 11 pp. 325-352 » Volume 1, Issue 10 pp. 293-320 » Volume 1, Issue 9 pp. 261-286 » Volume 1, Issue 8 pp. 229-255 » Volume 1, Issue 7 pp. 197-222 » Volume 1, Issue 6 pp. 165-189 » Volume 1, Issue 5 pp. 133-158 » Volume 1, Issue 4 pp. 101-125 » Volume 1, Issue 3 pp. 69-91 » Volume 1, Issue 2 pp. 37-62 » Volume 1, Issue 1 pp. 5-30
| |
Open Access Original Research
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 Malik A Al-Habeeb, M. H. A. Mohamed, Saad Sharawi.
| A B S T R A C T | R E F E R E N C E S | | Abstract
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. Key words: lentogenic, melting temprature, Newcastle virus, syer green I, velogenic
|
| |
| REFERENCES | 1. Miller, P.J., Afonso, C.L., Spackman, E., Scott, M.A., Pedersen, J.C., Senne, D.A., Brown, J.D., Fuller, C.M., Uhart,M.M., Karesh, W.B., Brown, I.H., Alexander, D.J. and Swayne D.E. (2010) Evidence for a New Avian Paramyxovirus Serotype-10 Detected in Rockhopper Penguins from the Falkland Islands. J. Virol., 84 (21): 11496–11504.
[DOI via Crossref]
[Pubmed] [PMC Free Fulltext]
2. Emmerson, P.T. (1994) Newcastle disease virus. In: Webster RG (ed), Encyclopedia of virology. Harcourt Brace and Company, London. 914–919.
3. Aldous, E. W., and Alexander, D. J. (2001) Detection and differentiation of Newcastle disease virus (avian paramyxovirus type 1. Avian Pathol. 30:117–128.
[DOI via Crossref]
[Pubmed]
4. de Leeuw, O., and Peeters. B. (1999) Complete nucleotide sequence of Newcastle disease virus: evidence for the existence of a new genus within the subfamily Paramyxovirinae. J. Gen. Virol. (80):131–136.
[Pubmed]
5. Kolakofsky, D., Roux, L., Garcin, D., and Ruigrok, R.W. (2005) Paramyxovirus mRNA editing, the "rule of six" and error catastrophe: a hypothesis. J Gen Virol 86:1869-1877.
[DOI via Crossref]
[Pubmed]
6. Beard, C.W. and Hanson, R.P. (1984) Newcastle disease. In: Hofstad, M.S., Barnes, H.J., Calnek, B.W., Reid, W.M., Yoder, H.W. (Eds.), Diseases of Poultry, 8th ed. Iowa State University Press, Ames. 452–470.
7. Lamb,R.A., and Kolakofsky, D. (2001) Paramyxoviridae: The Viruses and Their Replication. 4th edition. Hagerstown: Lippincott Williams & Wilkins.
8. Alexander, D. J. and Senne, D. A. (2008) Paramyxoviruses, and Pneumovirus Infections. In : Y. M. Saif, J. R. Glisson, D. J. McDougald, L. K.Nolan, and D. E. Swayne (ed.), Diseases of poultry, 12th ed. Iowa State Press, Ames, IA. 75–100.
9. Dortmans, J.C., Rottier, P.J., Koch, G. & Peeters, B.P. (2010) The viral replication complex is associated with virulence of Newcastle disease virus (NDV). J. Virol., 84 (19): 10113–10120.
[DOI via Crossref]
[Pubmed] [PMC Free Fulltext]
10. Choi,K.S., Lee, E.K., Jeon, W.J. and Kwon, J.H. (2010) Antigenic and immunogenic investigation of the virulence motif of the Newcastle disease virus fusion protein. J. Vet. Sci., 11 (3): 205–211.
[DOI via Crossref]
[Pubmed] [PMC Free Fulltext]
11. Dortmans, J.C., Koch, G., Rottier, P.J. and Peeters, B.P. (2009) Virulence of pigeon paramyxovirus type 1 does not always correlate with the cleavability of its fusion protein. J. Gen. Virol., 90 (Pt 11): 2746–2750.
[DOI via Crossref]
[Pubmed]
12. Fuller, C.M., Brodd, L., Irvine, R.M., Alexander, D.J. and Aldous, E.W. (2010) Development of an L gene real-time reverse-transcription PCR assay for the detection of avian paramyxovirus type 1 RNA in clinical samples. Arch. Virol., 155: 817–823.
[DOI via Crossref]
[Pubmed]
13. Cattoli, G., De battisti, C., Marciano, S., Ormelli, S., Monne, I., Terregino, C. and Capua, I. (2009). False-negative results of a validated real-time PCR protocol for diagnosis of Newcastle disease due to genetic variability of the matrix gene. J. Clin. Microbiol., 47: 3791–3792.
[DOI via Crossref]
[Pubmed] [PMC Free Fulltext]
14. Khan, T.A., Ruec.A., Rehmani, S.F., Ahmed, A., Wasilenko, J.L., Miller, P.J. & Afonso, C.L. (2010) Phylogenetic and biological characterization of Newcastle disease virus isolates from Pakistan. J. Clin. Microbiol., 48(5): 1892–1894.
15. Pham, H.M., Konnai, S., Usui, T., Chang, K.S., Murata, S., Mase, M., Ohashi, K., and Onuma, M. (2005) Rapid detection and differentiation of Newcastle disease virus by real time PCR with melting-curve analysis. Arch. Virol. 150:2429–2438.
[DOI via Crossref]
[Pubmed]
16. Wise, M. G., Suarez, D. L., Seal, B.S., Pedersen, J. C., Senne, D. A., King, D. J., Kapczynski, D. R., and Spackman, E. (2004) Development of a real-time reverse-transcription PCR for detection of Newcastle disease virus RNA in clinical samples. Journal of Clinical Microbiology 42: 329-338.
[DOI via Crossref]
[Pubmed] [PMC Free Fulltext]
17. Wehmann, E., Herczeg, J., Ballagi Pordany, A., and Lomniczi, B. (1997) Rapid identification of Newcastle disease virus vaccine strains La Sota and B1 by restriction site analysis of their matrix gene. Vaccine 15: 1430–1433.
[DOI via Crossref]
18. Chien, L.J., Liao, T.L., Shu, P.Y., Huang, J.H., Gubler, D.J., and Chang, G.J.J. (2006) Developmentof real-time reverse transcriptase PCR assays to detect and serotype dengue viruses. J. Clin. Microbiol. 44:1295–1304.
[DOI via Crossref]
[Pubmed] [PMC Free Fulltext]
19. World Organisation for Animal Health (OIE) (2012) Newcastle disease In: OIE manual of diagnostic tests and vaccines for terrestrial animals. Chapter 2.3.14. World Organisation for Animal Health, ed., Paris, France.
20. Mohamed M.H., Kumar S., Paldurai A., Megahed M.M., Ghanem I.A., Lebdah M.A., and Samal S.K. (2009) Complete genome sequence of a virulent Newcastle disease virus isolated from an outbreak in chickens in Egypt. Virus Genes, 39:234-7.
[DOI via Crossref]
[Pubmed] |
| How to Cite this Article | | Pubmed Style Al-Habeeb MA, Mohamed MHA, Sharawi S. 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. Vet World. 2013; 6(5): 239-243. doi:10.5455/vetworld.2013.239-243
Web StyleAl-Habeeb MA, Mohamed MHA, Sharawi S. 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. www.scopemed.org/?mno=20951 [Access: June 19, 2013]. doi:10.5455/vetworld.2013.239-243
AMA (American Medical Association) StyleAl-Habeeb MA, Mohamed MHA, Sharawi S. 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. Vet World. 2013; 6(5): 239-243. doi:10.5455/vetworld.2013.239-243
Vancouver/ICMJE StyleAl-Habeeb MA, Mohamed MHA, Sharawi S. 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. Vet World. (2013), [cited June 19, 2013]; 6(5): 239-243. doi:10.5455/vetworld.2013.239-243
Harvard StyleAl-Habeeb, M. A., Mohamed, M. H. A. & Sharawi, S. (2013) 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. Vet World, 6 (5), 239-243. doi:10.5455/vetworld.2013.239-243
Turabian StyleAl-Habeeb, Malik A, M. H. A. Mohamed, and Saad Sharawi. 2013. 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, 6 (5), 239-243. doi:10.5455/vetworld.2013.239-243
Chicago StyleAl-Habeeb, Malik A, M. H. A. Mohamed, and Saad Sharawi. "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 6 (2013), 239-243. doi:10.5455/vetworld.2013.239-243
MLA (The Modern Language Association) StyleAl-Habeeb, Malik A, M. H. A. Mohamed, and Saad Sharawi. "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 6.5 (2013), 239-243. Print. doi:10.5455/vetworld.2013.239-243
APA (American Psychological Association) StyleAl-Habeeb, M. A., Mohamed, M. H. A. & Sharawi, S. (2013) 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, 6 (5), 239-243. doi:10.5455/vetworld.2013.239-243
|
|
Become Reviewer
Follow ScopeMed on Twitter