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

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. 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.

Respons Imun Ayam Petelur Pascavaksinasi Newcastle Disease dan Egg Drop Syndrome

“…Gejala penyakit ND ditandai dengan kelainan pada saluran pernapasan, saluran pencernaan dan sistem saraf pusat. Tanda klinis penyakit ND tergantung pada strain virus, spesies inang, umur inang, lingkungan dan status kekebalan ayam (Al-Habeeb et al, 2013).…”

Outbreak investigation of Newcastle disease virus from vaccinated chickens in Eritrea

“…Newcastle disease (ND) is a highly contagious and fatal viral disease that affects over 250 species of birds of all age groups (Aldous and Alexander, 2001;Al-Habeeb et al, 2013). It is also called avian paramyxovirus serotype 1 (APMV-1), is a single stranded, enveloped, negative sense RNA virus belonging to the genus Rubulavirus of subfamily Paramyxovirinae and family Paramyxoviridae (Swayne, 2011;Mayo, 2002;OIE, 2012).…”

Status updates of Newcastle disease and amelioration effects of medicinal plants against Newcastle disease virus: A review

“…Genome with length of around 15.2 kb (Zhang et al, 2012) encodes for six structural and two non-structural proteins (Choi et al, 2010). Six proteins, nucleoprotein, large RNA polymerase (L), fusion protein (F), hemagglutinin-neuraminidase, matrix protein (M) and phospho-protein, are encoded in 3' to 5' direction (Al-Habeeb et al, 2013). NP is most important protein which forms the nucleocapsid helical core of NDV and induces antibody production in chickens.…”

“…The clinical signs are dependent upon age and species of the host, viral strain, immune status of the host and environmental conditions (Al-Habeeb et al, 2013). The clinical signs of ND are categorized into reproductive, respiratory, nervous and enteric signs.…”