Differentiation of infectious bursal disease virus strains using real-time RT-PCR and high resolution melt curve analysis.
Differentiation of infectious bursal disease virus (IBDV) strains is crucial for effective vaccination programs and epidemiological investigations. In this study, a combination of real-time RT-PCR and high resolution melt (HRM) curve analysis was developed for simultaneous detection and differentiation of IBDV strains/isolates. The hypervariable region of VP2 gene was amplified from several IBDV strains and subjected to HRM curve analysis. The method could readily differentiate between classical vaccines/isolates and variants. Analysis of the nucleotide sequence of the amplicons from each strain revealed that each melt curve profile was related to a unique DNA sequence. The real-time RT-PCR HRM curve analysis was also able to differentiate IBDV strains/isolates directly in bursal tissues from field submissions and from vaccinated commercial flocks. The differences between melting peaks generated from IBDV strains were significantly different (P<0.0001) demonstrating the high discriminatory power of this technique. The results presented in this study indicated that real-time RT-PCR followed by HRM curve analysis provides a rapid and robust technique for genotyping IBDV isolates/strains and can contribute to effective control of IBDV outbreaks.
Faculty of Veterinary Science, The University of Melbourne, Werribee, Victoria, Australia. seyedg@unimelb.edu.au
Seyed A
SA
Ghorashi
Denise
D
O'Rourke
Jagoda
J
Ignjatovic
Amir H
AH
Noormohammadi
D000818:Animals; D002645:Chickens / Q000821:virology; D017931:DNA Primers / Q000235:genetics; D007243:Infectious bursal disease virus / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000737:chemistry* / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D044366:Transition Temperature; D014773:Virology / Q000379:methods*
D017931:DNA Primers; D012367:RNA, Viral