Development Of A Universal Microarray System For The Detection Of Pathogenic Bacteria

Objective: The potential of DNA microarray technology in high-throughput detection of bacteria is widely acknowledged but has not been fully realized yet. For the rapid and efficient detection of biological agents and clinical infectious bacteria, a 16S rDNA oligonucleotides microarray based detection and identification system was developed.Methods: Oligonucleotides probes complementary to species-specific 16S rDNA regions of targeted bacteria were designed and evaluated by hybridization of different remotely related as well as closely related reference strains. The hybridization and washing conditions were optimized according to a microarray containing 26 species-specific probes. A newly formatted microarray was constructed containing the selected probes and controls. The chromosome DNA of some bacteria was amplified and labeled using a pair of universal primers targeted to 16S rDNA, and then the PCR products were applied to the oligonucleotides microarray. A series of specific hybridization patterns corresponding to each species or genus were obtained. Samples that were unknown were hybridized to the microarray, then the results were compared to the species- or genus-specific patterns and the identities were concluded according to the value of the correlation coefficient. Specificity of the oligonucleotides microarray detection system was validated by hybridization to a set of reference nucleic acids from pure cultures. Sensitivity test was performed and the capability of the microarray system in mixed samples was checked by hybridization to a few artificial samples that consist of two or three kinds of chromosome DNA.Results: 4 probes targeted to each bacteria were designed respectively, i.e. 316 probesfor 79 bacteria all together. Probes were validated against more than 300 strains covering 135 species. 12 most heavily cross-hybridized probes, 60 meaningless probes, i.e. never score positive during the former experiments, and 63 probes that is repetitive to each other at the beginning of the probes' designation were discarded as the result. 181 effective probes were confirmed in the end.The feasibility of the 16S rDNA oligonucleotides microarray system was checked according to a microarray containing 26 species-specific probes. The hybridization and washing conditions were optimized, the hybridization time was shortened. A new method was confirmed for the judgment of positive or negative probes based on the relative signal intensity by comparing several common methods used frequently now. The identities of samples were deduced according to the oligonucleotides profiles, not the so-called specific probes. The result suggests that it is possible to realize the high-throughput detection and identification of pathogenic bacteria by the oligonucleotides microarray. The judgment method of signals guaranteed the stability of the identification result, making it less influenced by the difference of the samples and the hybridization processes. The specificity, reproducibility and reliability were then confirmed by hybridization to a set of pure cultures of targeted species. To find out suitable controls that can be used later, 4 probes were introduced in the microarray to be validated, and the result indicated that EUB338 ACTCCTACGGGAGGCAGC could be a perfect positive control, AGAATATGGCGGCGATGCT rooted in A phage was unfitted for the negative control, but poly(T)49 and 50% DMSO were suited for the negative control and the blank control, respectively. All the signal intensities on their positions were as low as enough comparing with the local background.A newly formatted microarray was constructed containing EUB338, poly(T)49, 50% DMSO and the 181 specific probes. Chromosome DNA of 126 species of bacteria was amplified and labeled using a pair of universal primers targeted to 16S rDNA, and then PCR products were applied to the oligonucleotides microarray. 62 specific hybridization patterns corresponding to each species or genus were obtained. Specificity of the microarray system was verified by hybridization t...