Molecular Method of Marek’s Disease Virus Serotype 2 Detection using Polymerase Chain Reaction in Chicken

Background: Marek’s disease is an oncogenic virus that produces malignant lymphomas in chickens. In this study Marek’s disease virus serotype 2 identification using polymerase chain reaction with unique gene. Methods: DNA isolated from MDV infected feather follicles, liver, spleen and commercially available serotype 2 vaccines (SB 1 and HVT combined bivalent, HVT monovalent) by phenol-chloroform DNA isolation method. Serotype 2 specific primers were designed by using primer 3.0 software targeting Long Open Reading Frame (LORF) gene-specific to serotype 2 SB 1 strain. DNA template from vaccines showed amplification of the LORF gene. PCR parameters were optimized and the analysis of sensitivity and specificity of primers was carried out. Result: MDV serotype 2 with an optimized annealing temperature of 53°C with primer concentration of 3 pmol/µl. LORF gene of SB1 of MDV did not cross-react with HVT and MDV 1 strains and also with other oncogenic viruses of Avian Leucosis Virus (ALV) and Reticuloendotheliosis Virus (REV). The sensitivity was analyzed based on the different DNA concentrations and amplification up to 0.5 ng/µl. Screening of field samples of 260 along with three MDV serotype 2 vaccine SB1 strain samples revealed that all the filed samples did not show positivity for MDV serotype 2 whereas in the vaccine strain samples of MDV serotype 2 vaccine SB1 showed positivity by amplification at 270bp and these results indicated that MDV serotype 2 SB1 strain did not cause an outbreak in chicken and might be inferred that no incidence of spontaneous Lymphoid Leucosis-like lymphomas with augmentation property of MDV serotype 2. This serotype 2 specific assay was used to detect Marek’s disease caused by serotype 2 which is commonly used as a vaccine.