Research On New Methods For Typing And Detection Of Listeria Monocytogenes

Listeria monocytogenes is a common food-borne pathogenic microorganism with strong pathogenicity.Symptoms pose a fatal threat to the health of people with low immunity,such as young children,pregnant women,and the elderly,with a combined fatality rate of 25% to 30%.It is one of the key monitoring food-borne pathogens in the world.The existing detection methods for Listeria monocytogenes are very diverse,among which nucleic acid detection has the characteristics of low cost,high sensitivity,rapidity,and high throughput,and its applications are very wide.The most commonly used method in the field of Listeria monocytogenes typing is the serotyping scheme,including lineage I(1/2b,3b,7,4b,4d,4e),lineage II(1/2a,3a,1/2c,3c),lineage III(4a,4c),of which 1/2a,1/2b,1/2c,and 4b are the four most important serotypes(accounting for more than 95%).Distinguishing the four main serotypes is the main purpose of typing.The general method at this stage is to combine the specific genes of different serotypes to establish a multiple polymerase chain reaction(PCR)to distinguish the four main serotypes.As the speed of food circulation becomes faster,the rapid and immediate detection of the bacteria is of great significance to prevent the spread of the bacteria.The research on the typing of the bacteria is very important for the traceability of the bacteria and providing information about the location about the source of infection.This study focuses on the typing and detection methods of the bacteria.This project has developed a universally applicable method for mining specific single nucleotide polymorphism(SNP)from the genome database,and applied it to the mining of the specific SNP of Listeria monocytogenes.Through a comparative analysis of the genomes of253 known serotypes,it is found that the genome of Listeria monocytogenes contains a large number of specific SNPs,which have been sorted into six types: 1/2a-3a,1/2b-3b-7,1/2c-3c,lineage I,lineage II,and lineage III.At the same time,the SNP distribution of seven housekeeping genes in the Multilocus sequence typing(MLST)scheme was analyzed,and five types of lineage I,lineage II,lineage III,1/2c-3c,and 1/2b-7 were found.These specific SNPs established a connection between ST type and serotype and made it possible to identify the serotype and ST type at the same time.In order to establish a method for distinguishing the four main serotypes,a large number of genes containing specific SNPs were screened,and it was found that lmo1441 contained highly conserved and specific lineage I and lineage II specific SNPs.And lmo1076 contains highly conserved and specific SNPs of type 1/2c-3c and type1/2b-3b-7.Taking these two genes as targets for amplification,two specific primers and one universal primer were designed to distinguish the two types with a single gene,and then the positive control target prf A was introduced to establish an allele-specific multiplex PCR.The allele-specific multiplex PCR method can distinguish the four main serotypes of Listeria monocytogenes with only 8 primers.Comparing the typing results with the current general serotyping method using 60 food-borne samples,it shows that the agreement rate is 100%.By comparing the genomes of 253 Listeria monocytogenes and 540 non-Listeria monocytogenes species,8 specific genes of Listeria monocytogenes were screened.lmo2733 regarded as the new target of Listeria monocytogenes was used to establish a PCR detection method,and verify the specificity and reliability of the target with 9 strains of non-Listeria monocytogenes and 10 strains of Listeria monocytogenes.A loop-mediated isothermal amplification(LAMP)method was established based on the new target.By adding hydroxynaphthol blue(HNB)to the reaction system to visualize the test results,the positive samples were sky blue after the reaction,and the negative samples were maintained violet after the reaction.The detection limit is 2 pg of genomic DNA.This research was a systematic study that focused on the serotyping and detection of Listeria monocytogenes.A method for mining specific SNPs was developed and the four main serotypes of Listeria monocytogenes can be distinguished based on SNPs,which was different from the mainstream genotyping method,and can be applied to the typing of other species based on its universality.lmo2733 was screened as a new detection target of Listeria monocytogenes,based on which,a visualable detection method of LAMP banding with HNB was established.This method is economical and efficient and has potential application value.