Identification of sacbrood virus disease in honeybee, <i style="">Apis mellifera</i> L. by using ELISA and RT-PCR techniques

Abstract

Studies were conducted on the identification of sacbrood virus (SBV) through ELISA and RT-PCR techniques in diseased prepupae of <i style="">Apis mellifera</i> L. collected from Himachal Pradesh, India. The ELISA kit was prepared from antiserum specific to Thai SBV (TSBV). SBV in the crude samples was detected at 1:1000 titre of the kit. Five pairs of oligonucleotide primers were used to target SBV genome through RT-PCR technique. Presence of SBV was confirmed with 3 primers, viz., Sb1 f/r, Sb3 f/r and Sb5 f/r. Amplification of the genome produced distinct bands/amplicons of expected molecular sizes, i.e., approximately 487, 267 and 597 bp. The amplicon Sb3 was sequenced which yielded a fragment of 822 nucleotides length. The nucleotide sequence was submitted to GenBank database, NCBI, USA under accession number EU232663. Phylogenetically, present SBV was found closer to Nepal-1, Nepal-4, followed by India-2, Nepal-2 and Germany-7 strains.