Study On DNA Microarray For Detection Of Bacillus Anthracis

Anthrax is a bacterial disease caused by the spore-forming Bacillus anthracis, a Gram-positive, rod-shaped bacterium. It is primarily a disease of herbivores and humans can contract anthrax directly or indirectly from infected animals or from occupational exposure to contaminated animal products. Its spore can also infect human by the way of respiratory tract, digestive tract and skin touch. Because of the resistance of spores in environment, it is believed that B. anthracis should be easier to be stored, transported and disseminated artificially. Extensive investigation has been reported on the possibility that anthrax used as a biological weaponry agent. Therefore, it is important to find quick ways detecting and identifying the presence of B. anthracis. DNA microarray or the DNA chip technology is a newly developed novel approach, which should provide new method for B. anthracis diagnosis more efficiently.Sets of PCR primers were designed and used to amplify the specific genes including pagA, lef, cya that are present on pXOl and CapB, which is present on pX02. Both the plasmids and the four specific genes have been used for the design and development of DNA microarray probes. These DNA sequences were digested with restriction endonuclease Sau3kl. Then the resulting fragments were cloned into the pMD18-T vector. To identify the positive clones, we used a pair of specific vector primers to amplify the inserted fragments. In order to reduce the redundant sequences in pMD18-T vector which the first PCR products contained, we designed another pair of primers that were beside the cloning site in pMD18-T vector to perform the secondary PCR. 294 cloned probes were collected and a DNA microarray for detecting B. anthracis was prepared by printing the probes on a super-amine modified glassslide.In addition, according to the sequence information of pX01 and pX02 and the designing rules of oligonucleotide probes, 60mer oligonucleotide probes were also designed and synthesized. DMSO was used as spotting solution and these probes were printed on a poly-L-lysin modified DAKO glass slide. So the oligo microarray for detecting B. anthracis was prepared.Total DNA of Bacillus thuringiensis, Bacillus cereus, Bacillus subtilis and human blood were isolated and labeled with Cy3 by using Restriction-Display labeling method. These samples were hybridized with DNA microarray and the intensities of hybridization signal fluorescence were recorded. It was demonstrated that both format microarrays could detect specifically the presence of the B. anthracis. Hybridization conditions were further optimized. The results showed that labeled fluorescence by the method of Restriction-Display could enhance the fluorescent signals. PCR products mixed with EDTA could also enhance the signal-to-noise ratio. Furthermore, the specificity of DNA microarray can be increased under the high stringent condition including elevated hybridization temperature, decreased salt ionic concentration in the hybridization mixture and shortened hybridization time.