Detection Of Cronobacter Sakazakii U Sing Recombinase Polymerase Amplification Combined With Lateral Flow Strip

Cronobacter sakazakii is a food-borne opportunistic pathogen that can cause diseases in different age groups,especially newborns and infants with poor immune function.The national standard method for detection of this bacterium is based on traditional culture,which usually needs 7 days to complete and is time-consuming and laborious.Although the molecular detection method based on polymerase chain reaction(PCR)greatly shortens the detection time and has high sensitivity compared with the traditional culture method,it needs expensive and precise equipment for detection as well as professional personnels.Therefore,it is not easy to be applied in limited conditions.In this study,a rapid detection method for C.sakazakii was developed by combining recombinase polymerase amplification(RPA)with lateral flow strip(LF).The conditions of RPA-LF for detection of C.sakazakii were optimized and the results showed that the optimum amplification conditions were 45℃ for 15 min.Sensiti-vity analysis showed that the detection limit in pure culture was 1.7×102 CFU/mL.Specificity analysis showed that RPA-LF method did not react crossly with other common pathogens.Food samples were used to evaluate the performance of RPA-LF and the results showed that the detection limit of RPA-LF reached 1.7×100 CFU/g after 4 or 6 hours of enrichment.This method is simple,rapid,specific and sensitive for detection of C.sakazakii.It is suitable for fast detection of C.sakazakii in areas with limited conditions and food enterprises.RPA is known as the approach closest to isothermal amplification considering it can be completed in 20 minutes at lower temperatures.In this study,three major enzymes(protein)for RPA amplification were recombinantly expressed in E.coli including Bsu DNA polymerase,T4 UvsX recombinase and T4 gp32 binding protein.The molecular weight of recombinant Bsu DNA polymerase was about 64 kDa and the optimal induction conditions were determined as:0.05 mmol/L IPTG used to induce for 8 hours at 30℃.The purified protein concentration was 0.26 mg/mL.The molecular weight of recombinant T4 UvsX recombinase was about 43 kDa.The optimal induction conditions were determined as:0.005 mmol/L IPTG used to induce for 12 hours at 20℃.The purified protein concentration reached 0.27 mg/mL.The molecular weight of recombinant T4 gp32 binding protein was about 33 kDa and the optimal induction conditions were determined as:0.05 mmol/L IPTG used to induce for 8 hours at 30℃.The purified protein concentration reached 0.20 mg/mL.This study lays a good foundation for the development of rapid detection technology for C.sakazakii is helpful for application of RPA technique in rapid detection of microorganisms.