Evaluation of a protective antigen gene based SYBR Green I real time PCR for detection of Bacillus anthracis in field samples

Abstract

In the present study, 27 field materials from different sources were tested in SYBR Green I dye based Real Time PCR optimized using WHO recommended conventional PCR primers targeting Bacillus anthracis protective antigen PA) gene of pX01 plasmid. The assay had detection limit of 0.000002 pg in terms of target DNA quantity and up to 4 copies of pX01 plasmid per reaction, in terms of copy number. Melt curve analysis of PCR products of field samples showed deviation up to 3.2^OC in melting temperature from standard anthrax strain suggestive of possible mutation mutations in PA gene. Targeted region of PA (596 bp) encompasses domain 2, involved in its binding with lethal factor LF) and/or edema factor (EF) and mutation in this region may alter the efficacy of PA to traffic toxins inside the cells and results in differed level of virulence of pathogen. The test is useful not only for detection but may be suggestive of mutation also.