To establish a method to identify the authenticity of manuka honey, this study compared different methods of DNA extraction from mānuka honey pollen, and it developed a DNA extraction method for mānuka honey supernatant and a real-time polymerase chain reaction (real-time PCR) method for the detection of plant-derived ingredients in mānuka honey, including plant internal control, mānuka and kānuka. By analyzing the specificity, sensitivity, detection limit and comparing with the method of the New Zealand Ministry for Primary Industries (MPI), the feasibility, accuracy and equivalence of this method were confirmed. The proposed DNA detection method could replace the MPI method, not only shaking off the dependence on kits, but also making up for the deficiency of identifying mānuka honey pollen only. It has an important application value and scientific significance for the identification of mānuka honey.

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.

Meat products, being rich in nutrients, often provide an ideal environment for microbial growth. Even under low temperature storage, most microorganisms remain active and retain partial virulence. Therefore, screening for pathogenic bacteria in meat products is crucial. This study developed a triple-fluorescence ERA (enzymemediated recombinase polymerase amplification) rapid detection method based on enzyme recombinase amplification (ERA) technology. Performance analyses were conducted on the method’s specificity, sensitivity, and detection limits. Additionally, the applicability of the method was validated using commercially available refrigerated meats and compared to the industry standard SN/T 1870―2016 real-time fluorescent polymerase chain reaction (PCR)method. The results show that a triple-fluorescent ERA rapid detection method was successfully constructed for three key pathogens: Staphylococcus aureus, Salmonella and Listeria monocytogenes. After optimization, the method could simultaneously detect the three pathogens within 12 min from a single reaction. The minimum detection limit is 10^-3 ng/µL. In artificially contaminated samples, after 6 hours of pre-enrichment, the method can simultaneously detect contamination levels of 1 CFU/mL for all three pathogens. The sampling results of commercially available refrigerated meat show that the detection rates for Staphylococcus aureus, Salmonella, and Listeria monocytogenes were 19.35%, 12.90%, and 6.45%, respectively, which align with the detection results of the industry standard SN/T 1870―2016. These fully demonstrate the accuracy and applicability of the method. This study not only helps reduce the risk of disease caused by foodborne pathogens but also provides solid technical support for future rapid detection of foodborne pathogens.

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.