Journal of Pure and Applied MicrobiologyVol. 9 No. Special Edition May. 2015

A Novel Technique for Fast Detection of Bacillus anthracis Spore by Multiplex PCR

Daruosh Momenzadeh1, Sharam Parvin2, Ali Karami3*, Fateme Pourali3, Ali Ahmadi3, Mohammad Heiat3, Hamid Kooshki4, Neda Bayat1 and Fariba Ghasemvand1

1Brain and Spinal injury Research Center, Tehran University of Medical Sciences, Tehran, Iran. 2Chemical Injuries Research Center, Baqiyatallah University of Medical Sciences Tehran, Iran. 3Research Center of Molecular Biology, Baqiyatallah University of Medical Sciences Tehran, Iran. 4Nanobiotechnology Research Center, Baqiyatallah University of Medical Sciences Tehran, Iran.

Received on 08 January 2015 and accepted on 24 March 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/Ál. 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.

Keywords : Detection- Bacillus anthracis - Spore - Multiplex PCR.