With the improvement of people’s living standards, food safety has attracted more and more social attention. Organisms, especially microorganisms, are the most important factor that affects food safety. There are many methods currently available to detect biological (microbial) factors in the field of food safety, including isothermal amplification technology especially the new techniques of recombinase polymerase amplification (RPA), recombinase-aided amplification (RAA) and enzymatic recombinase amplification (ERA), which is widely used for food safety detection because it can detect biological ingredients rapidly and accurately without relying on complicated instruments. The three isothermal amplification techniques require milder reaction conditions and simpler design of primers than others, despite having similar principles. In this article, we review the principles and concepts of these techniques and their recent application in the rapid detection of food pathogens, summarize their advantages and problems in food inspection, and present future prospects.

For quick and multiple detection of the contamination of foodborne pathogens in infant milk powder, a dual enzymatic recombinase amplification (ERA) method for the rapid detection of Cronobacter, Salmonella, Listeria monocytogenes and Staphylococcus aureus were established in this study. Firstly, specific and efficient primers and probes were selected for these foodborne pathogens. Then, two groups of dual-ERA systems were established and optimized. The minimum detection limit was 10^-2 ng/μL for Salmonella, L. monocytogenes and S. aureus, and 1 ng/μL for Cronobacter. The results of simulated contamination test showed that the four foodborne pathogens could be detected simultaneously after 6 hours of enrichment culture at an initial contamination level of 1 CFU/mL. When this method and real-time polymerase chain reaction (PCR) were used to detect commercial infant milk powder, consistent results were obtained, which confirmed the accuracy and reliability of the dual-ERA method. This method could simultaneously detect the four foodborne pathogens in about 20 minutes. Compared with the traditional method and real-time PCR, the detection efficiency was significantly improved, which is of great significance for promoting the rapid screening of foodborne pathogens.

Norovirus (NoV) is one of the common foodborne viruses, and even a small amount of NoV can lead to infection and illness. However, there are currently no specific therapeutic drugs and vaccines available, making it crucial to establish a rapid detection method for early screening of NoV in food products. This study first wrapped the target genes of GⅠ and GⅡ NoV specified in GB 4789.42 into the capsid protein of bacteriophage MS2 with armored RNA technology, creating recombinant plasmid reference samples containing both GⅠ and GⅡ NoV targets. Next, based on the Enzymatic Recombinase Amplification (ERA) technology, the primers and probes of basic and fluorescence ERA detection were designed for both G Ⅰ and G Ⅱ NoV, and the optimal primers and probes were screened through experiments. Then two visualization methods for GⅠ and GⅡ NoV detection were established, namely ERA chromogenic and fluorescence method, and the results can be observed with the naked eye. Furthermore, the reaction program was optimized, reducing the amplification time to 5 minutes and 8 minutes for both ERA chromogenic and fluorescence methods, respectively. By optimizing the reaction system, the volume was halved, thereby reducing the cost of detection. Under these conditions, the lowest sensitivity was 10^-2、10^-3 ng/μL for the recombinant plasmid reference sample, respectively. Finally, the established visual rapid detection method was applied to the detection of G Ⅰ and G Ⅱ NoV authentic specimens. And the performance parameters of the method were analyzed. The results show that the established GⅠ and GⅡ NoV ERA visual rapid detection method has good specificity, with no cross-amplification with other foodborne viruses, and can detect as low as 10 copies/μL of NoV. It meets the requirements for visual rapid screening of GⅠ and GⅡ NoV. The establishment of this method provides good technical support for the rapid screening and risk monitoring of NoV, and it is of great significance for controlling NoV outbreaks and safeguarding public health.

Three rapid visual methods, namely chromogenic, fluorescence and test strip, for the rapid detection of Burkholderia gladioli pv. cocovenenans in foods were established based on enzymatic recombinase amplification (ERA). Primers and probes were designed and screened based on the bonM gene of B. gladioli pv. cocovenenans. Then the specificity and sensitivity of the three methods were evaluated, as well as their applicability and accuracy in the detection of commercial food samples. The results showed that three strains of B. gladioli pv. cocovenenans, but not other common foodborne pathogens and other B. gladioli strains, were amplified by the three methods, indicating their good specificity. The detection limits of these methods were all 10^-2 ng/μL, and their sensitivity was good. Out of 15 commercial samples, two tested positive by each of these methods with a detection rate of 13.3%. This result was consistent with that of the national standard method, indicating that our methods had good applicability and accuracy. All three methods give results that can be observed by the naked eye after amplification at 37 ℃ for 15 min, which provide a new and simple strategy for the rapid, visual and on-site screening of B. gladioli pv. cocovenenans in foods.