A Novel Technique for Fast Detection of Bacillus Anthracis Spore by Multiplex Pcr



Momenzadeh D¹ ; Parvin S² ; Karami A³ ; Pourali F³ ; Ahmadi A³ ; Heiat M³ ; Kooshki H⁴ ; Bayat N¹ ; Ghasemvand F¹
Source: Journal of Pure and Applied Microbiology Published:2015

Abstract

Bacillus anthracis is a spore-forming bacterium that causes anthrax disease. Its spore resists against physical and chemical agents. Molecular detection of B.anthracis spore requaiers the genomic DNA which is embded in the spores. in this study we developed a rapid detection of spor by Multiplex PCR using rapid physical disruption of spore in a short time without genomic DNA extraction. Genome of non-virulent strain of B.anthracis spores was released by MiniBead Beater-8 system. 3 pairs of primers were used in Multiplex PCR. PCR products were analyzed in agarose gels electrophoresis. Detection limit , specificity and accuracy of the test were determined through various process. This method enables to disrupt spores during 3 minutes and perform detection process with specific primers by Multiplex PCR method. Gel electrophoresis analysis showed 3 fragments of PCR products. digested 1083 bp fragment created 696 bp and 386 bp fragments which confirmed the test accuracy. Detection limit was 7680 spores/ml or 7 spore/µl. The results showed that the optimized physical disruption method has a proper velosity and there is no need for DNA extraction. Finally this method is capable to detect B.anthracis spores with high sensitivity and specificity. © 2015, Journal of Pure and Applied Microbiology. All rights reserved.