Application of polymerase chain reaction and restriction endonuclease analysis for the detection and differentiation of turkey-pox and fowl-pox virus infections

Abstract

Polymerase chain reaction (PCR) was standardized for the amplification ofA-type inclusion body (ATI) gene using self-designed primers. Amplification was also achieved by using published primer for 39 K core protein gene ofturkeypox virus (TPV) and fowl-pox virus (FPV). Amplicons of 1.8 Kbp and 800 bp product were produced for ATI and 39 K gene, respectively, in both turkey-pox and fowl-pox virus infected samples. The applicability of the standardized PCR for the detection of FPV and TPV in a variety of experimental samples [infected chorioallantoic membrane (CAM), scabs, lymphocytes and chicken embryo fibroblast (CEF) cell cultures] indicated the usefulness ofthis novel technique for the diagnosis of pox virus infection at molecular level. Restriction endonuclease analysis (REA) revealed that the 800 bp amplicon of 39 K gene in both these viruses has no cleavage sites for Bam HI, Hpa II and Eco RI endonuclease enzymes. Bam HI enzyme cleaved FPV ATI gene amplicon at 2 sites giving 3 fragments of 1660, 100 and 40 bp size, but could not cleave TPV amplicon. Hpa II cleaved TPV ATI gene amplicon at 2 sites giving 3 fragments of 1400, 250 and 150 bp but did not cleave that of FPV. The enzyme Eco RI cleaved the ATI gene ampIicon of both the viruses at 2 sites yielding 3 fragments of 1500, 200 and 100 bp in TPV, and 1110, 530 and 160 bp fragments in FPV. The RE analysis revealed the existence of genomic differences in ATI genes of turkey-pox virus and fowl-pox virus.