Development of a multiplex PCR system with application of IPC (internal positive control) for detecting Bacillus anthracis from environmental samples

Abstract

Background: Bacillus anthracis, the causative agent of anthrax is a major concern of a dangerous and zoonotic disease in medicine and veterinary medicine. The development of rapid, sensitive and simple techniques to detect B.anthracis in suspicious specimens is the important aim of animals and subsequently public health. we described a multiplex PCR system with application of IPC (internal positive control) for detecting virulence markers in B.anthracis. Methods: In PCR assay pag and cap genes located on POX1 and POX2 plasmids were amplified by use of specific primers. In order to control of reaction conditions, we used from PCR products as a templet and designed IPC for pag and cap genes that their length was shorter than product segments. After cloning of PCR products, the sensitivity of the PCR test (limit of detection) was also estimated. Control bacteria of defined species was confirmed by amplification of a fragment of the 16S rRNA gene by using two universal primers. Results: In post-PCR stage 591 and 173 bp fragments of DNA respectively to cap and pag genes recognized in agarose gel. Sensitivity determination assays revealed that this method can detect, 412 and 100 copies as LOD for cap and pag gene respectively in samples.Nonetheless, bands on the results with negative controls, these genes not present in these defined bacteria. Conclusion: In this study, we desigined and optimized a multiplex PCR assay for the detection and characterization of B.anthracis from environmental samples. In addition to we used from IPC to prevention of false negative results and also to detect inhibitory effects of the sample matrix. The PCR system described here can be proposed as rapid, safe, diagnostic and confident method for detecting anthrax. The sensitive and specific nature of this assay provides a valuable tool that can be used for analysing clinical and field samples and for improving our understanding of the ecology and environmental prevalence of B.anthracis in natural foci of disease. Abstracts for SupplementInternational Journal of Infectious DiseasesVol. 14Preview Full-Text PDF Open Archive