Oligonucleotide Microarray For Identification Of Bacteria Basing On The 16SrDNA Gene's Polymorphism

Background Rapid detection and identification of the pathogenic bacteria still remained a perplexed problem in clinical microbiology. Bacterial cultural and physical morphology, nutritional and biochemical coupled with serological tests are major methods to class and identify pathogen in clinical microbiology. The gene diagnosis based on PCR (polymerase chain reaction) is sensitive, fast and brief, which has already been widely used in clinic. Oligonucleotide microarray is the best way to detect the variation of DNA for its high density of probes and strict specificity. The property of variation in bacterial 16SrDNA gene is correlated to their phylogenetic relationship, which were used widely in study of bacterial taxonomy. It is possible to detect pathogen rapidly when we combine microarray to PCR. In this study, we tested the feasibility of identification of frequent pathogenic bacteria basing on the 16SrDNA gene's polymorphism. Objective To establish a sensitive and strict-specificity technical protocol which can detect the polymorphism of 16SrDNA gene on oligonucleotide microarray. Methods 1,Amplify 16SrDNA genes by a pair of universal primers from frequent pathogenic bacteria; Then clone and sequence them to be regarded as standard control. 2,Collect sequence data of frequent bacteria in clinic from the RDP-II database together the result of sequencing above. Then design the probes respectively. 3,Fragment the amplicons of PCR before hybridization. 4,Target sequences labeled by PCR were hybridized with probes on glass slide. Results 1,Using a pair of universal primers, we have cloned the nearly full-length 16SrDNA genes from 22 strains frequent pathogenic bacteria successfully. 2,Found a method to thymidine-base-specific fragmentation of 16SrDNA gene. 3,By such optimization strategies as designing probe, hybridization, labeling and fragmentation, we have constructed a kind of oligonucleotide array which could detect the 16SrDNA gene's polymorphism in some bacteria and established the corresponding technical protocol. Conclusions The universal primers can amplify the nearly full-length 16SrDNA genes from most frequent pathogenic bacteria effectively. By some optimization strategies, such as designing probe, hybridization, labeling and fragmentation, the investigation demonstrated roughly that bacterial 16SrDNA genes could be detect respectively on the oligonucleotide array.