STANDARDIZATION OF MULTIPLEX PCR ASSAYS FOR IDENTIFICATION AND VIRULENCE GENE PROFILING OF BRUCELLA ABORTUS

Abstract

Bovine brucellosis, caused principally by Brucella abortus, is endemic in India and causes substantial economic loss. Characterization of Brucella is complicated by the genetic homogeneity of the genus and by the need to distinguish field strains from vaccine strains in routine use. Brucella lacks classical virulence determinants and depends instead on factors mediating intracellular survival. This study standardized a sequential polymerase chain reaction workflow for genus confirmation, species assignment, vaccine strain discrimination, and virulence gene profiling of B. Abortus. Reference strain S99 was characterized phenotypically and biochemically, and genomic DNA was subjected to genus-specific bcsp31 amplification, B. Abortus-specific IS711 amplification, Bruce-ladder multiplex amplification using eight primer pairs, and a four-gene multiplex targeting cyclic beta 1,2-glucan, urease, the type IV secretion system, and outer membrane protein 31. The 16S ribosomal RNA gene was sequenced and compared against reference sequence databases. The strain was catalase, oxidase, and urease positive, reduced nitrate, and showed colony and staining characteristics typical of Brucella. The bcsp31 and IS711 assays amplified products of 223 and 498 base pairs, respectively, confirming the strain as B. Abortus. The 16S ribosomal RNA sequence returned identical similarity scores against nine Brucella species, confirming genus but not species identity. Bruce-ladder amplification produced the five-fragment profile characteristic of B. Abortus field strains, excluding the vaccine strains S19, RB51, and Rev1. The virulence multiplex amplified all four targets, with the 257 base pair omp31 product consistently weaker than the others. The workflow permits genus confirmation, species assignment, vaccine strain discrimination, and virulence gene profiling of B. Abortus from a single DNA preparation. Primer balance requires further optimization, and analytical sensitivity and specificity remain to be determined before diagnostic application to field isolates.