Research And Development Of Plasmid DNA Qualitative Reference Material Of Listeria Monocytogenes And Its Application In The Detection Of Tremella

Listeria monocytogenes is a common foodborne pathogen.It is the most virulent Gram-positive non spore bacillus in Listeria.It can cause a variety of diseases,mortality rate is high(20%-30%).It can be detected in cooked food,aquatic products,frozen food and edible fungus.Therefore,rapid detection and diagnosis of L.monocytogenes is great significance for food safety.However,traditional detection method of foodborne pathogens is time-consuming and low detection limit,which is not conducive to rapid detection of pathogens.In recent years,detection technology based on nucleic acid standard substances has been developed for transgenic crops and viruses,which can promote standardization of nucleic acid amplification detection methods,consistency and accuracy of detection results between laboratories.However,in China,development of nucleic acid reference materials is just in its infancy,quantity and variety are not enough for current market and monitoring.The development of plasmid qualitative reference materials for virulence gene detection of L.monocytogenes is even less.In this thesis,the genomic DNA of L.monocytogenes was extracted as a template for PCR amplification of virulence gene primers hly A,prfA1 and prfA2,target fragments of these three virulence genes were recovered.They were cloned into p LB-simple Vector,and recombinant plasmids containing corresponding virulence genes were constructed respectively,the recombinant plasmids were detected by colony PCR and sequencing.The transformed plasmids were extracted and preserved,and a large number of extracted plasmids were lyophilized to make freeze-dried powder,that is to say,the plasmid standard for detection of L.monocytogenes was preliminarily obtained.According to the results of colony PCR and sequencing,the plasmid reference materials containing the virulence genes of hly A,prfA1 and prfA2 were successfully constructed,and its concentration,purity and stability were all up to the requirements.In the long-term stability test,the stability of plasmid reference material is good.And it was found that the sensitivity of PCR was affected by the target gene,and the detection limits of different virulence genes were different.A plasmid standard containing the virulence genes of hly A,prfA1 and prfA2 was constructed as a positive control for the detection of 30 batches of Tremella samples.The results showed that prfA1 and prfA2 was detected in one batch of samples,the detection rate was 3.3%.The positive samples of Tremella were tested by the traditional microbial detection method,but the traditional detection method did not get the target strain,which may be in the presence of environmental stress or dominant strains,the target strain went into dormancy or VBNC state.The strain in this state is not easy to be detected by traditional detection methods,which greatly reduces the detection rate,at the same time,the sensitivity of PCR method is higher than that of traditional detection method.