Study On The Detection Of Six Food-borne Bacterial Pathogens By PCR And Multiplex PCR Detection Kit

The food security is a major public health problem attracting more and more attention in the world. It is associated closely with not only people's health but also the national development. Bacterial food poisoning is the main factor which causes the food-borne diseases. The traditional detection of pathogenic bacterium was trivial and time-consuming, and can not satify with detection in practice.The monoplex PCR and multiplex PCR detection of six food-borne pathogenic bacteria Salmonella spp., Listeria monocytogenes, Staphylococcus aureus, Shigella spp., Escherichia coli O157:H7 and Bacillus cereus, were developed on the basis of specific primers, optimization of DNA extracted methods and PCR reaction conditions. All these six pairs of primers were species-specific and did not interfere with each other. DNA sequencing showed that the sequences of PCR products had more than 99% identities with expected sequences. The sensitivity of PCR reaction reached a level of pg DNA. These results demonstrated the developed PCR assay had high specificity and sensitivity for detection of six food-borne pathogenic bacteria. Milk samples artificially inoculated with these six pathogenic bacteria, raw-beef and 200 samples collected from market were detected by multiplex PCR using traditional detection method as control. The result showed that the multiplex PCR method was simple and rapid, the whole detection time was less than 24 hours and the sensitivity was as low as 1 CFU/g. Compared to traditional method, the false negative rate of multiplex PCR was 0 and the false positive rate was below 2% in 200 samples. Therefore, the multiplex PCR method can be used as the supplement of traditional method to increase the accuracy and sensitivity of detection. In addition, the multiplex PCR detection kit with high stability and practicability was developed on the basis of the multiplex PCR method and solidified technology. It can be stored at room temperature for at least three months without affecting the detection results. Thus, it will be wildly applied in the fields of food sanitation detection, clinical inspection and so on.