Author(s): Shikha Bishnoi, Sitaram, Mukani Kumari, Kritika Dhial, Rakesh Ranjan, Brij Nandan Shringi, Narendra Singh Rathore, Jai Prakash Kachhawa and Amit Kumar Pandey

Abstract: The prompt diagnosis and treatment of viral disease in dairy and poultry animals has always been a great challenge before a clinician. Any delay in such operations may lead to economic losses and destruction to dairy, poultry related food industries and pose a threat to food security in the country. In recent times, the Lumpy Skin Disease (LSD) has become rampant and caused havoc in several Asian countries and northern states of India with loss of more than Rs. 600 Cr. The disease is caused by Lumpy Skin Disease Virus (LSDV) from Poxviridae family and Capripox virus genus, with other related viruses namely, Sheep Pox and Goat Pox viruses. It is transmitted through several arthropods viz. biting flies, mosquitoes and ticks. It is marked with high fever of 41 °C, inflammation in superficial lymph nodes and distinct scab lesions on skin. The clinical diagnosis of LSDV relies on ELISA, Indirect Fluorescent Antibody Test (IFAT), AGID, virus confirmation by transmission electron microscopy, virus isolation and PCR. OIE has recommended PCR based method for screening of LSD diagnosis. The present study was envisaged to investigate an alternate PCR based diagnostic strategy with improved sensitivity and specificity. The RNA Polymerase subunit gene (RPO30) has been selected for the investigation. In this regard, the target nucleotide sequence of 602 bp length has been utilized for designing of two primers namely, SR01 and SR02. The SR01 was found to be capable of screening of Capripox viruses. The nucleotide alignment of RNA polymerase gene from other Capripox viruses reveals significant differences at several positions. Therefore, such non-conserved portion was utilized for primer designing with the objective to differentiate among closely related members of Capripox viruses. The PCR reaction was optimized for amplification of the gene with annealing temperature of 66 °C. No PCR amplification was observed with GPV, SPV and CPV as source of target DNA. The validation of the method was conducted on DNA isolated from sixteen scab samples. The developed method offers highest sensitivity, specificity with excellent positive predictive and negative predictive value for LSDV diagnosis. The PCR experiment with serially diluted LSDV DNA template showed decent limit of detection (LOD) of 50.7 pg.

DOI: 10.33545/26174693.2024.v8.i1Sm.486