title: A rapid method of accurate detection and differentiation of Newcastle disease virus pathotypes by demonstrating multiple bands in degenerate primer based nested RT-PCR.
abstract: A rapid and accurate method of detection and differentiation of virulent and avirulent Newcastle disease virus (NDV) pathotypes was developed. The NDV detection was carried out for different domestic avian field isolates and pigeon paramyxo virus-1 (25 field isolates and 9 vaccine strains) by using APMV-I "fusion" (F) gene Class II specific external primer A and B (535bp), internal primer C and D (238bp) based reverses transcriptase PCR (RT-PCR). The internal degenerative reverse primer D is specific for F gene cleavage position of virulent strain of NDV. The nested RT-PCR products of avirulent strains showed two bands (535bp and 424bp) while virulent strains showed four bands (535bp, 424bp, 349bp and 238bp) on agar gel electrophoresis. This is the first report regarding development and use of degenerate primer based nested RT-PCR for accurate detection and differentiation of NDV pathotypes by demonstrating multiple PCR band patterns. Being a rapid, simple, and economical test, the developed method could serve as a valuable alternate diagnostic tool for characterizing NDV isolates and carrying out molecular epidemiological surveillance studies for this important pathogen of poultry.
authors.affiliation: Avian Diseases Section, Division of Pathology, Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India.
authors.forename: P A
authors.initials: PA
authors.lastname: Desingu
authors.affiliation: Avian Diseases Section, Division of Pathology, Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India.
authors.forename: S D
authors.initials: SD
authors.lastname: Singh
authors.affiliation: Avian Diseases Section, Division of Pathology, Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India.
authors.forename: K
authors.initials: K
authors.lastname: Dhama
authors.affiliation: Division of Medicine, Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India. Electronic address: vinodhkumar.rajendran@gmail.com.
authors.forename: O R Vinodh
authors.initials: OR
authors.lastname: Kumar
authors.affiliation: Division of Pathology, Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India.
authors.forename: R
authors.initials: R
authors.lastname: Singh
authors.affiliation: Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India.
authors.forename: R K
authors.initials: RK
authors.lastname: Singh
mesh_terms: D017931:DNA Primers / Q000235:genetics*; D005838:Genotype; D060005:Genotyping Techniques / Q000379:methods*; D008969:Molecular Sequence Data; D009522:Newcastle disease virus / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D013997:Time Factors
chemical_list: D017931:DNA Primers; D012367:RNA, Viral
keywords: Degenerate primer; NDV pathotyping; Nested RT-PCR; Newcastle disease virus