Identification and differentiation of vaccinal and virulent Newcastle disease virus in Iran by HRM analysis

Subject Areas : Journal of Comparative Pathobiology

Dibazar, Sh., Sheikhi, N.*, Hemmatzadeh, F., Charkhkar, S., Pourbakhsh, A. . ¹

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Received: 2014-03-01 Accepted : 2014-06-26 Published : 2014-09-23

Keywords: HRM Analysis, Newcastle Disease Virus, Vaccine Virus, differentiation, Primer,

Abstract :

To control of outbreaks caused Newcastle disease, it is necessary to distinguish virulent virusesfrom vaccine strains, in minimum possible time and with high accuracy. The aim of this studywas using High-Resolution Melting-Curve Analysis (HRM) for detection and differentiation ofIranian Newcastle disease virus (NDVs) isolates from vaccine strains. In this study, 5 virulentisolates along with 2 vaccine strains, including B1 and Lasota were used. Based on thenucleotide sequence of F gene, 8 primers (A - H) were designed for the analysis of HRM. At thefirst stage, one virulent and 2 vaccine virus was analyzed by 8 primer pairs. Based on thepreliminary results of both RT-PCR HRM, 3 sets of the primers have been selected for finaltesting of the samples. The patterns obtained from wild viruses were compared with vaccinegroup. The melting temperatures for vaccine strains were higher than virulent isolates by A, B,C, F and H primer pairs; despite D, E and G. In this study, B and H primer pairs could separatevaccine strains from each other and from virulent isolates, better than other primers. Based onthese results, HRM analysis and correct primer selection can determinate and differentiatevirulent NDVs from vaccine strains. This technique is able to do this in short time and with highaccuracy in comparison with previous conventional approaches such as pathogenicity indicestests, RT-PCR and nucleotide sequencing.