Isothermal Amplification Detection For Specific Genes Of Bacillus Anthracis

Anthrax is an acute severe infectious disease caused by Bacillus anthracis. Dueto the persistence of spores in the environment, B. anthracis has the potential for useas an agent of bioterrorism. Therefore, detection of B. anthracis is important to theclinical diagnosis of anthrax and anti-bioterrorism. In this study, isothermalamplification methods, including loop-mediated isothermal amplification (LAMP)and strand displacement amplification (SDA), were applied to detect B. anthracis.Ba813 sequence, pag gene and capB gene were chosen as detection targets inLAMP assay of B. anthracis, represent genome and two virulent plasmids pXO1,pXO2 of B. anthracis, respectively. The detection limit of LAMP assay in detecting B.anthracis A16 cultures was 10 spores, which is ten-fold lower than that of multiplexPCR. The specificity of LAMP assay detecting B. anthracis was evaluated bydigestion analysis of LAMP amplification products and LAMP amplification assaysof forty-six strains bacteria. The detection results suggest high specificity. The LAMPassay was further estimated in the application of simulated powder samples. Theamplification products of LAMP assays developed above were detected byelectrophoresis analysis. The fluorescent detection method of LAMP amplificationproducts was also developed in this study with fluorescent dyes EvaGreen,SYBRGold and EB for the detection of target genes Ba813 sequence,pag gene and capBgene, respectively. This method, called multiplex fluorescent LAMP detection of B.anthracis, makes the assay more simple and visual.The SDA detection of B. anthracis was evaluated using pag gene as target andelectrophoresis analysis as detection method of SDA products. As the smear in lane ofSDA products when analyzed by electrophoresis method, the mostly used detection methods of SDA products are fluorescence polarization (FP) and fluorescenceresonance energy transfer (FRET). But these two detection methods both needexpensive fluorescent detection instruments. A new SDA-ELISA method wasdeveloped in this study for simple and visual detection of SDA products usingenzymic reaction. With optimized conditions, this SDA-ELISA method has highspecificity and sensitivity with a detection limit of 10 B. anthracis spores, 10-foldmore sensitive that electrophoresis method.The isothermal detection methods of B. anthracis developed in this study bothhave high specificity and sensitivity. Most important, these methods are preformedunder isothermal condition and do not need complicated instruments. These methodsoffer promising alternatives for B. anthracis detection.