Microarray construction of antimicrobial resistance genes from Salmonella

Abstract

A glass-based microarray was developed to detect 11 antimicrobial resistance genes that confer resistance to aminoglycosides, tetracyclines, sulfonamides, and chloramphenicols. DNA samples were arrayed with 220 μm diameter pins at a spacing distance 1000 μon silane-coated (amine) glass slides under condition of 25 ℃, 65% relative humidity. DNA microarray was dried at room temperature for 6 h, rehydrated over a 60℃ water bath for 30 s and dried on a heating block at 80℃ for 10 s. Target DNA on the microarray was fixed for 10 min by UV cross-linking at 254 nm. Hybridized microarray was scanned at 10 μm with photomultiplier tube (PMT) at 532 nm (Cy3) with laser power 90% and PMT gain (PMTG) 85%. Hybridization dynamics of the microarray were examined by using tetA gene. As results, target DNA concentration of 100 ng·μL-1 was used for microarray construction, the optimal concentration of probe was 3600 pg·μL-1, and the sensitivity of the microarray was 3 pg·μL-1. Prehybridization of the microarray was carried out at 44℃ for 2 h to block nonspecific binding of probe(s), followed by hybridization at 52℃ for 6 h. When the signal-to-noise (S/N) was ≥1.5 and median pixel intensity was ≥1000, a spot was scored as positive. The microarray can detect 11 antimicrobial resistance genes at the same time with high specificity and repeatability.

Keywords

• <italic>Salmonella</italic>
• antimicrobial resistance gene
• microarray（or gene chip）
• detection