Study On DNA Microarray For Vibrio Cholerae Detection

The bacterium Vibrio Cholerae is the causative agent of cholera. Cholera not well controlled can develop into explosive endemic, epidemic and pandemic infection and cause disastrous situation to human health and social stability. Endemic cholera in southern Asia had been documented for more than one thousand years. Seven pandemics have outbroken world wide since 1817, the first 6 pandemics were caused by Classical biotype of 01 sero-group, while the seventh was caused by El Tor biotype. October 1992, a new sero-type of 0139 caused explosive epidemic in India. A number of cholera experts warn that Vibrio Cholerae 0139 may cause the eighth global pandemic. Vibrio Cholerae was used as biological war agents several times since twentieth century. In recent years, rampant terrorists also attempted to use Vibrio Cholerae to conduct bio-terrorism. Therefore, it would be necessary to develop fast and accurate detection method for Vibrio Cholerae as well as other severe infectious agents.DNA microarray is a novel technology emerged from late 1990s. By integrate vast amount genetic information into tiny spaces, biological samples could be detected and analyzed expediently in parallel with enhanced sensitivity and accuracy. Though this technology has advanced quickly, there is yet no report of DNA microarray developed specifically for Vibrio Cholerae detection.In order to develop a practical Vibrio Cholerae detection microarray, we studied and optimized the methods of probes preparation, samples labeling and hybridization dynamics based on lambda phage genomic DNA. In the process of probe preparation, we developed a new and simple technique of GIN-PCR for massive amplification of microarray probes. Concurrently, a novel method of toothpick insertion for DNA recovery from electrophoresis gel was developed.Whole genomic DNA of lambda phage was fragmented with SaulAI digestion. These restriction fragments were cloned into T-vectors and transformed into E.Coli. 697 colonies were identified by colony PCR. These PCR products were electrophoresis analyzed and the interesting band was touched with toothpicks before dipped into a new PCR reaction mixture for a second round PCR amplification. 394 Tarp probes were recovered with this method, and the success rate is 56.5%. A microarray of total 396 samples containing 381 phage Tarp probes, 4 negative controls, 7 blanks and 4 array corner references were micro-grided with Cartesian Microarrayer.The microarray was hybridized with labeled DNA samples derived from the lambda phage genome. The results demonstrated that the hybridization signals reached saturation when probe concentration over 325 ug/mL, and the uniform, steady and regular shaped signal spots indicated that the microarray was successfully manufactured.Based on this platform of the whole genome microarray of lambda phage, a fast protocol for Tarp DNA microarray detection was established. This new method consists of one hour of sample digestion, one hour of adaptor ligation, two hours of PCR amplification and labeling of samples, followed by one hour of array hybridization. The whole protocol can be completed in less than 8 hours.Using the platform and method, a microarray for Vibrio Cholerae detection was developed. Two regions in the Vibrio Cholerae genome harboring pathogenesis elements were selected. These two regions include the CTX genetic element harboring CT gene and VPI pathogenesis island harboring TCP gene. These two regions were chosen for probes preparations. Two pairs of LA PCR primers CTX-U/CTX-L and VPI-U/ VPI-L were designed for amplification of CTX and VPI, respectively. A fragment of 7019bp in CTX and a fragment of 19246bp in VPI were successfully amplified by LA PCR. Finally, a Vibrio Cholerae DNAmicroarray consists of two sub-arrays of 110 CTX samples, 190 VPI samples were fabricated based on GIN-PCR and Tarp methods of probes preparation. This microarray could be used to detect pathogenic presence of Vibrio Cholerae, through detection of the pathogenesis region of CTX genetic element and VPI...