DNA Microarray Preparation Of Streptococcus Suis Serotype 2 And Its Application On Temperature-induced Transcriptome

Streptococcus suis （S. suis） is an important zoonotic pathogen. Streptococcus suis serotype 2 （SS2） is the most virulent and the most frequently isolated serotype among the 35 serotypes. Since 1968, SS2 have caused more than 200 cases of severe human infection worldwide, with the hallmarks of meningitis, septicemia and arthritis, etc. However, two recent outbreaks of SS2 epidemic in 1998 and 2005, China, appeared to be a more invasive deep-tissue infection with streptococcus toxic shock syndrome （STSS）, showed the agent could be an emerging SS2 virulent variant. Up to now, the molecular pathogenesis of this new emerging pathogen is still poorly understood.Based on the genomic sequences of SS2, a batch of whole-genome DNA microarray was developed and evaluated chip quality with strains 05ZY and 1940. The DNA microarray was used to investigate transcriptome analysis of SS2 response to temperature shift, laying the foundation for genomics studies of SS2. The research contents and results were as follow.1. Preparation of SS2 DNA microarryA total number of 2192 open reading frames （ORFs） were chosen according to the annotation of the genomic sequences of SS2. Specific primer pairs of every gene were designed and diluted properly, amplified by PCR and purified. Then the purified products were transported into 384-well plate. Finally, the products were spotted onto the CSS-1000 silylated glass slides by robotically spotted DNA microarray technology. The DNA microarray was dried at room temperature for 12h. The results showed that 2156 genes wene successfully amplified by PCR and a batch of DNA microarray of SS2 was developed.2. Evaluation of SS2 DNA microarryTo evaltuate DNA microarray quality, gene transcriptome technology was used in the experiment, reference strain was S.suis 05ZY, test strain was S.suis 1940. The experimental result indicated that the effect of chip hybridization and signal were clear, the number of gene deletion was few, and spots of chip were corresponding correctly to serial number of whole-genome. Microarray data and quantitative RT-PCR results showed a good correlation （r=0.9018）. 3. Transcriptome analysis of temperature-induced SS2Above-mentioned whole-genome DNA microarray was used to investigate transcriptome analysis of two temperature-induced pairs, 29℃（test condition） vs 37℃（reference condition） and 40℃（test condition） vs 37℃（reference condition）, obtained differentially express microarray data unit represent temperature stimulon.First all, srtain 05ZY resuscitated in THB medium and inoculated at 37℃, then test condition and reference condition were grown to OD_600nm=0.8 in another fresh medium. Bacteria were harvested by centrifugation for tatol RNA isolation, and reverse transcribed to cDNA, respectively. cDNA was labeled by Cy3 or Cy5 monofunctional dye, the Cy3 and Cy5 reaction mixtures were combines, then the mixture was applied to the microarray slide. The slides were hybrized in a hybrization incubator. Every resulting slide was scanned by a microarray scanner and exported microarray data. Finally, to confirm the microarray data, qRT-PCR was utilized to determine the transcription changes of 30 genes that were identified at two different incubation temperatures.The results were as follow. Among 29℃vs 37℃temperature stimulon. 81 genes were differentially expressed, 34 of those genes were down-regulated, 47 genes were up-regulated; among 29℃vs 37℃temperature stimulon, 43 genes were differentially expressed, 14 genes were decreased expression, 29 genes were increased expression. There was a strong positive correlation （r =0.9013） between the data obtained by the two techniques. These genes involved in many virulent-associated factors, immunogenicity gene and metabolic regulation factors etc.