Heteromorphic Primers Isothermal Amplification Technology And Its Application In Detection Of Foodborne Pathogens

Foodborne pathogens can cause foodborne diseases.At present,bacterial culture technology is the gold standard for detecting food-borne pathogens,but it is time-consuming and labor-intensive.The specificity of immunological detection methods is greatly improved,but the preparation of specific antibodies is more stringent,increases detection costs,and has limited sensitivity.The new detection technology based on nucleic acid amplification has high sensitivity and high specificity,but it needs to rely on an accurate thermal cycler,complex design and operation,or the length of the amplification product is obviously limited,etc.There is a need to develop sensitive and specific detection techniques that are easy to use,costeffective,and suitable for primary detection.To this end,we developed a new type of heteromorphic primers isothermal amplification technology and applied it to the detection of foodborne pathogens.The main research contents and results are as follows:1.The establishment of heteromorphic primers isothermal amplification technology.Heterogeneous primers are composed of linear primer and hairpin primer.In the presence of the target,a double-hairpin structure can be rapidly formed through the trigger of heteromorphic primers.This structure finally generates double-stranded DNA structures of varying lengths with repetitive sequences through intramolecular polymerization and intermolecular polymerization of primer/hairpin structures.The technical system was constructed with Escherichia coli as the target,and it was found that it not only has high sensitivity and high specificity,but also has a relatively large length range of amplified products,and can amplify products with a length of 60-300 bp.It is not only suitable for detecting general pathogenic microorganisms,but also for detecting pathogenic microorganisms with high mutation frequency.2.Detection of foodborne pathogens in contaminated simulated samples.We added three common foodborne pathogens to sterile milk to establish contamination simulation samples,and found that the heteromorphic primers isothermal amplification technology can achieve high-specificity and high-sensitivity detection of food-borne microorganisms in contaminated simulation samples.At the same time,we use traditional PCR to verify the accuracy of the method.3.Detection of foodborne pathogens in actual samples.The milk samples from cows suspected of having mastitis were tested for foodborne pathogens,and the PCR test was performed simultaneously for verification.The results were compared with veterinary clinical tests,and a visual test was established with the help of fluorescent dyes.We found that the detection results of heteromorphic primers isothermal amplification were the same as the PCR detection results,and were also consistent with the veterinary clinical test results.The milk samples that detected Salmonella and Staphylococcus aureus were all produced from cows suffering from bovine mastitis,it’s show that the method can effectively detect oodborne pathogens.The above results also show that the heteromorphic primers isothermal amplification technology has good reliability and stability.At the same time,the method is simple in design,convenient in operation,and suitable for use at the grassroots level.