Foodborne Pathogenic Bacteria Detection Using Isothermal Amplification Combined With Strips

Many poisoning incidents in the world were caused by Staphylococcus aureus and Listeria monocytogenes,which are great threat of food safety.Traditional methods for detection and identification of bacterial pathogens are time consuming and laborious.Although polymerase chain reaction-based methods are effective,the requirement for sophisticated,expensive equipment and trained personnel greatly limit its applicability in point-of-care detection.In this study,a rapid and visual detection method based on the combination of recombinase polymerase amplification(RPA)and lateral flow(LF)strips was developed for the detection of S.aureus and L.monocytogenes.This method is simple,sensitive,and specific.Recombinant polymerase isothermal amplification is a new amplification method which can amplify target using specific primers at lower temperatures(37℃～-42℃)within 20 min.It is considered as the closest to so-called "isothermal amplification".In this study the specific gene fragments of S.aureus and L:monocytogenes were selected as templates for primers designing.And DNA products labeled with biotin and digoxin were produced by biotin-and digoxin-modified primers.The products were visualized on LF strips after interaction with gold nanoparticle or colored latex microspheres labeled with anti-digoxin antibodies.The RPA-LF method for S.aureus detection was optimized and the results showed that the optimal conditions were determined as amplification for 10 min at 40 ℃,followed by 5 min at room temperature for LF.This method could detect 500 fg genomic DNA and 1.2 × 101 CFU/mL of S.aureus in pure culture.Specificity analysis showed no cross-reaction with S.epidermidis,S.saprophyticus,Salmonella enterica serovar Enteritis and others.In actual food,1.2 × 100 CFU/mL(g)S.aureus could be detected within 4.5 h.The RPA-LF method for L.monocytogenes detection was also optimized and the results showed that the optimal conditions for 10 min at 39℃,followed by 5 min at room temperature for LF detection.This method could detect 300 fg genomic DNA and 1.5 x 101 CFU/mL of L.monocytogenes in pure culture.This RPA-LF method exhibited no cross-reactions with L.grayi,L.seeligeri,S.aureus,E.coli,E.aerogenes,S.choleraesuis,Shigella.Evaluation of the method with food samples indicated that the detection limit was substantially improved to 1.5 × 100 CFU/mL(g)for the original bacterial content in samples after enrichment for 6 h.The advantages of method combining RPA and LF include high rapidness,high specificity and sensitivity.The entire testing process requires only 4.5-6 h using a simple digital water bath to complete the test instead of complex thermal cycling devices and has no requirement of technicians.The method is suitable for rapid detection of pathogens in food industry and remote areas.