Detection Method Of Salmonella Typhimurium Based On PCR Nucleic Acid Modification Technology Combined With Nucleic Acid Test Strips

Salmonella as a common zoonotic gram-negative pathogenic bacteria has been broken out in many parts of the world with a high frequency.As one of the most pathogenic pathogens,Salmonella typhimurium often enters the body through the diet to competes nutrients and living spaces with the microbial colonies in the gastrointestinal tract,thereby breaking the original stable living structure of indigenous microorganisms and causing a series of Salmonella poisoning symptoms.In order to reduce the burst rate of infections and economic losses of various countries,PCR modification technology(PMT)and nucleic acid strip(NAS)were combined to complete the POCT and visual detection of Salmonella typhimurium 16 S r DNA/r RNA.The study is as follows:The visual detection of Salmonella typhimurium 16 S r DNA was performed by PMT and NAS.In this study,Biotin-d UTP was used to transform natural d NTP into biotinylated d NTP which was prepared to modify Salmonella 16 S r DNA through the action of enzymatic polymerization,thus multiple biotin molecules were loaded in newly synthesized DNAs.Because of the 5’ extremity of forward primer has been modified Carboxyfluorescein(FAM),each of newly synthesized DNAs carried one FAM and multiple biotin molecules,simultaneously.The FAM/16 S r DNA/Biotin complex was detected by NAS.The results showed that the optimal proportion of biotin-d UTP was 5% in the mixture of d TTP and biotin-d UTP,and the NAS detection effects were clear,intuitive and visible,the detection results were uniform with agarose gel electrophoresis.Based on biotinlynated DNA signal probe and NAS to establish a Salmonella 16 S r RNA visual detection method.Through optimization experiments,we found that the best optimal concentration of the capture probe and bridge probe were repective 0.1 and 0.4 μM,and the incubation time was 4 h.The comparison results of the 16 S r RNA amplification with that of unamplified 16 S r RNA shown that the signal amplified 16 S r RNA detection result was 10 times than that of the non-signal amplified 16 S r RNA.This method was remained a high selectivity for the detection of Salmonella,compared to the common food-borne pathogens of Listeria monocytogenes,Escherichia coli,Campylobacter jejuni,Shigella flexneri,and Staphylococcus aureus.And the limit of detection was 233 CFU·m L-1,the recovery rate was 96.0%~109.1% in milk.In conclusion,the method of foodborne Salmonella typhimurium detection based on enzymemediate PMT and NAS has been successfully established.The method is significantly low-cost,accurate to applied,fast to detect and safe to public health,that not only very suitable for today’s fast-paced and environmentally-friendly society,but greatly provide a POC detection for pathogenic bacteria in poor countries or regions.