Detection And Identification Of Food Borne Pathogens Using Universal Oligonucleotides Microarray

The food borne pathogens are the primary reasons that lead to food poisoning and are capable of causing significant illness and represent a serious public healthy threat worldwide. Most of food borne pathogens are difficult to be incubated in the testing laboratories. Current-routine techniques for identification and detection of food borne pathogens are still based on biochemical and physiological methods, Several molecular methods are also used to detect and identify the pathogens such as PCR and southern blotting. The rapid and accurate methods for detection and identification of food borne pathogens are the precondition of preventing and controlling pathogens. In the paper, For the rapid, accurate and high-throughput detection of food borne pathogens, a universal detection and identification system based on oligonucleotides microarray (universal oligonucleotides microarray) was developed.Here in order to prepare a universal oligonucleotides microarray. Firstly we designed the unique antitag probe using the bioinformatics method and synthesized it, then fixed it on the face of modified glass;The eight food borne pathogens were specially amplified using PCR by the different primers, which were Listeria monocytogenes, Staphylococcus aureus, Escherichia coli O157: H7, Salmonella enteritidis, Clostridium perfringens, Yersinia enterocolitica, Shigella flexneri and Bacillus cereus. followed by unique solution-phase hybridization to get probes connecting with fluorescent dye labeled oligos. Then the labeled oligos and prepared universal oligonucleotides microarray were subjected to specific solid-phase hybridization. In this paper we also explored optimization of the following experiment processes: the PCR products purified scheme, the reporter group conducted method and hybridization condition etc. Finally, the sensitivity and specificity of the optimized universal oligonucleotides microarray technologic system were appraised using this eight kinds of pathogenic as the detectedmodel.This research attains the following achievements: (1) The universal oligonucleotides microarray has the virtues of low background and high probed binding efficiency;(2)The method combines the SAP, Exo I and proteinase K to purify the PCR product, determined by our optimal experiment, can obtain the purified product with the short time, low cost and high efficiency;(3) the hybridization temperature is at 60°,the hybridization time is for lh, the reporter group conducted by the LDR technology can gain the strong and stable fluorescent signal;(4) The hybridization results showed this technique could identify and distinguish the eight food borne bacterial, there are no false positive results and cross hybridization. The detection limit of the universal oligonucleotides microarray system were 1.2 X 102cfu/mL of Escherichia coli 0157: H7, 8.5 X 102 cfu/mL of Listeria monocytogenes, 1.8 X 102 cfu/mL of Salmonella enteritidis, 6.2 X 102 cfu/mL of Staphylococcus aureus, 5.7 X 102cfu/mL of Bacillus cereus, 9.3 X 102cfu/mL of Clostridium perfringens, 1.7 X 102cfu/mL of Shigella flexneri, 1.1 X 102cfu/mL of Yersinia enterocolitica.The universal oligonucleotides microarray detection system has good specificity, stability, reliability and clould solve the cross hybridization, The results indicated the detection and identification of the food borne pathogens were accurate, reliable, and feasible. The simultaneously detected method of the eight food borne pathogens was established and provided a firm technical and theoretical basis for the detection and identification of the food borne pathogens in the future with the high accuracy, good sensitivity, high throughput and low cost.