Development of rapid qualitative detection DNA microarray platform for Salmonella typhimurium for food safety

摘要

Screening of possible bacteria contamination in food products is one of the measures implemented to avoid potential health hazards.In food industry,standard cell culture technique is widely used to monitor bacterial contamination.However,the main drawback of this technique is its inherited time consumed during the culturing step,which requires about 72 hours.In this research,an alternative DNA microarray technique was developed for a qualitative screening of a possible Salmonella typhimurium contamination in chicken,with the aim of reducing the detection time down to 3 hrs.The identification of S.typhimurium in food samples was carried out by hybridization of the possible contaminant with specific probe immobilized on a biochip.Novel DNA probes were designed as 16-50 base pairs of nucleotides to recognize with specific parts in S.typhimurium genomic.At least two DNA probes were identified as the candidate probes which had the potential to promote the best hybridization.DNA microarray was fabricated by mixing candidate DNA probes with photoactive polymer network,(poly(DMAA-mABP-SSNa)),and was printed onto a plastic substrate by non-contact microspotter.DNA probe was covalently immobilized onto the surface by 254 nm of UV lamp with 1.25 J/cm~2.PCR product functionalized with biotin was hybridized with DNA probe and labelled with streptavidin-cy5.Specific binding yielded fluorescence signal.The intensity image signal was read-out by a fluorescent microarray reader.Two genes specific to S.typhimurium(fimC and invA),were investigated by using specially designed DNA primers and DNA probes.From the assay optimization,it was found that 1 mg/ml of polymer hydrogel concentration,10 μM of DNA probe concentration,10 pg/ml of labelling concentration,and 2 nL of array volume yielded the highest signal intensity.The results were calibrated into CFU/ml(cell forming unit).The system was applied successfully for the detection of S.typhiurium without any contamination.The biochip validation with spiked sample(DNA standard)is currently underway.