Abstract:To effectively monitor the characteristic of Acidithiobacillus ferrooxidans ATCC 23270 at the whole-genomic level, a whole-genome 50-mer-based oligonucleotide microarray was developed based on the 3 217 ORFs of A. ferrooxidans ATCC 23270 genome. Based on artificial oligonucleotide probes, the results showed that the optimal hybridization temperature was 45 ℃. Specificity tests with the purified PCR amplifications of 5 genes (Sulfide-quinone reductase, Cytochrome C, Iron oxidase, Mercuric resistance protein, Nitrogenase iron protein) of A. ferrooxidans ATCC 23270 indicated that the probes on the arrays appeared to be specific to their corresponding target genes. Based on the WGA hybridization to global transcriptional difference of A. ferrooxidans ATCC 23270 strains cultured with Fe(Ⅱ) and S(0), the developed 50-mer WGA could be used for global transcriptome analysis of A. ferrooxidans ATCC 23270. The detection limit was estimated to be approximately 5 ng with the genomic DNA, and at 100 ng of the DNA concentration, all of the signals reached the saturation. In addition, strong linear relationships were observed between hybridization signal intensity and the target DNA concentrations (r²=0.977 and 0.992). The results indicated that this technology had potential as a specific, sensitive and quantitative tool for detection and identification of the strain A. ferrooxidans ATCC 23270 at the whole-genome level.

Key words: whole-genome oligonucleotide array(WGA); A. ferrooxidans ATCC 23270; specificity; sensitivity; quantification