Study On DNA Microarray Detecting Common Bacterial Pathogens Of Weaning Pigs

Object Study on 16S rRNA gene based DNA microarray detecting common bacterial pathogens of weaning pigs.Methods 1) The 12 bacteria were chosen as targets and their 16S rRNA gene sequences were downloaded from GenBank. Biological software Biosun2.0, DNAStar and Primer5.0 were used for setting up database and conducting phylogenetic analysis, followed by searching candidate oligo probes at the variable regions and designing universal primers at the conservative regions. 2) The conventional PCR method was established and target fragments from 11 out of 12 bacteria were amplified and sequenced after cloning into TA vector. 3) Conducting asymmetric PCR, using the PCR products as target and hybridize with candidate oligo probes, to select ideal probes with species specificity and strong signals. 4) Optimize asymmetric PCR and microarray, such as concentration ratio of asymmetric PCR primers, concentration of primer labeled with Cy3, Taq polymerase concentration, Mg²⁺ concentration, annealing temperature and concentration of hybridization buffer. 5) Determine cutoff value of each probe by testing hybridization…. of homologous or positive samples, negative samples and blank samples. 6) Testing sensitivity of the microarray system using serial diluted DNA as PCR templates.Results 1) Seven oligo probes have been achieved from 49 candidates and a DNA microarray detecting B. bronchiseptica,P. multocida,Cl. perfringens type C,S. epidermidis,H.parasuis,S. suis.and M. hyopneumoniae has been primarily accomplished with 2 pairs of universal primers used. The rest of 5 bacteria failed to be identified by the genchip as no suitable distinguishable probes have been found in the 16S rRNA gene sequence to separate between A. pleuropneumoniae and A. suis, E.coli and S. enterica; while no C. jejuni culture or DNA was available at the time. 2) The optimal asymmetric PCR and hybridization conditions are: the concentration ratio of forward primer to reverse primer labeled with Cy3 is 1:30, and the final concentration of reverse primer is 0.7μM, the concentration of Mg²⁺ is 1.0mmol/L, the Taq polymerase concentration is 2 U/reaction, annealing temperature is 55℃, concentration of hybridization buffer is 6×. 3) The cutoff value of each probe has been calculated and the fomular used for cutoff calculation is background value of mean average of negative plus blank + 3SD. 4) The detection limit of the microarray is approximately 102pg DNA template in a PCR.Conclusion In the present study a 16S rRNA gene based DNA microarray has been developed for detection and identification of major bacterial agents in weaning pigs. Further work has been focused on looking for probes of leftover bacteria and validate the microarray with clinical samples.