Development Of PCR And PCR-ELISA Methods For Listeria Monocytogenes Detection

The genus Listeria is a group of Gram-positive bacteria widely dispersed in the environment and foods,and currently subdivided into six species,L.monocytogenes,L. seeligeri,L.welshimeri,L.innocua,L.ivanovii,and L.grayi.Only L.monocytogenes is a human pathogen and a food-borne pathogen that can cause Listerisis with a high mortality rate.Listeriosis is a clinically serious disease with fatality rate of about 20 percents.Its ability to grow at temperatures as low as 4℃permits multiplication in refrigerated foods. In recent years,L.monocytogenes has become not only an important paradigm for immunological investigation but also an important model system for analysis of the molecular mechanisms of intracellular parasitism.The present method for analysis L. monocytogenes is complex and time consuming,requires 24 and 48 hours of enrichment, followed by a variety of other tests.It is necessary to establish a simple molecular method fits for rapid screen and confirmation of suspected isolates.Listeria monocytogenes strain contain two virulence clusters,Listeria pathogenicity island 1(LIPI-1) and Internalin Island(LIPI-2).LIPI-1 is the Central Virulencee Gene Cluster encode prfA,plcA,hly, mpl,actA,and plcB.In this research,we clone,Sequence,and analysis of the LIPI-1 gene duster from a L.monocytogenes isolate strain XFL0605.Furthermore,a simple PCR and PCR-ELISA method for LM detection was developed.The main research included as below.1.Isolation and biological characterization of Listeria monocytogenes Seven strains of L.monocytogenes were isolated from 156 food samples follows standard method SN/T0148.1-2005.Virulence determination,haemolytic activity detemination, and antibiotic sensitivity test were explored to the isolate L.monocytogenes strain XFL0605 and reference strains CCTCCAB97021 and SC53004.A simple and sensitive ultraviolet spectrophotometry method was established for determining the haemolytic activity of LM strains.The coefficient between SRBC condensity and A540 was 0.99995. The HC₅₀ values of XFL0605,CCTCCAB97021 and C53004 culture supematants were 18.85,21.38,and19.68 respectively,showing a strong eorelationship to the set of LLO concentrations.It showed that the haemolytie activity of the LLO secreted from the 3 different strains was identical which was confirmed by sequences of hly gene encoding the pore-forming cytolysin listeriolysin.While XFL0605 strain,whose LD₅₀ was 2.58×10⁵,was less pathogenic to mice compare to the reference strains.Close relationship between haemolytic activity in the culture supematants and the pH values was found, LLOs were active at pH value of 5.6 while inactive at pH value of 7.0.Antibiotic sensitivity test showed that 3 LM strains were susceptibility to 7 kinds of antimicrobial agents including Ampicillin,Streptomycin,Chloromycetin,Kanamycin,Tetracycline, Norfloxacin and Ciprofloxacin.The spectrophotometric method developed in this study for LLO haemolytic activity testing was direct-viewing and sensitive,and is suitable for testing the expression of hly gene.At the same time,the microtitre plate with serial dilution method was developed and modified which was simple and convenient for LLO haemolytic activity testing.A microtitre plate testing method measure LLO haemolytic ative by serial dilution was developed.2.Development of molecular methods for Listeria monocytogenes detectionTo detect L.monocytogenes contamination in foods,the simple PCR and PCR-ELISA method were developed,according to the published hlyA gene sequence,a forward PCR primers labled with biotin at 5" end and a specific probe labled with digxin were designed by using primer 5.0 software.The PCR-ELISA method which was developed in this study combinated PCR,nucleic acid hybrid reaction and enzyme coloration.Only L. monocytogenes strains were successfully amplified the expected fragment,not from E.coli,Salmonella,L.iuanuii,L.innocua,L.innocua,L.seeligeri,and L.gray were negative. The method was then applied to detect L.monocytogenes in 60 food samples to validate the PCR-ELISA procedures by compareing to the isolate method,which was higher positive detection than SN/T0148.1-2005(devivative from ISO11290-1:2004 ).LM detection can be improved by PCR-ELISA the sensitivity of PCR products 156-folds higher than agarose gel electrophoresis.The system allowed quantitative results to be obtained within 5h after 16h of enrichment in half-Fraser broth and was relatively cost-effective,showing a good potential for routine analytical use.3.Cloning and phylogenetic analysis of LIPI-1 gene cluster from XFL0605 strainThe LIPI-1 gene cluster of Listeria monocytogenes XFL0605 strain was segmentally amplified by PCR and analyses.The result showed that LIPI-1 of i XFL0605 strain was 8558nt(Genbank accesion No.EF661572),contained all the members of the prfA-regulated virulence gene cluster prfA,hly,pleA,plcB,mpl,and actA.Phylogenetic analysis XFL0605 strain and other L.monocytogenes,L.seeligeri and L.ivanovii strains on the base of sequence of the LIPI-1 and prfA,hly,plcA,plcB,mpl,and actA gene individal clearly demonstrated that the evolutionary relationship of strains were difference. It revealed that recombination of LIPI-1 in different assembly process and from different source.Furthermore,the signal peptides and transmember domains and their cleavage positions were prodicted,and investigated the PrfA DNA binding sites of the LIPI-1 virulence genes.After prediction and analysis of signal peptides of the LIPI-1 of XFL0605 strain by the bio-software SignalP3.0,proteins of PlcA,LLO,Mpl,ActA and PlcB have an average of 26.6 amino acids located at the N-terminal region.Only the membrane protein which was essential for bacterial actin-based motility encoded by actA has the transmember domain(578-600 amino acids) in C-terminus region.There were five 14-bp DNA palindromic sequence(PrfA DNA bingding site sequence) that were present in the target gene promoters of prfA,picA,hlyA,actA and actA of XFL0605.All these palindrome domains were the targets for PrfA recognizes and binding.Analysis of hlyA gene and deduced amino acid of the three L.monocytogenes(XFL0605, C53004 and CCTCCAB97021) revealed that the nucleotide homology ranging from 96.9%to 100%,and deduced amino acid sequence homology of over 97.9%.And the three LLOs contained a same 19-amino acid PEST-like sequence (KENSISSMAPPASPPASPK) at the N-terminus region which is essential for the virulence and intracellular compartmentalization of LM,and a same conserved undecapeptide,ECTGLAWEWWR,at the C terminus region which is the characterization of the CDTX toxin.The results supported the conclusion of the previous study of hemolytic activity of the three LM strains.ActA gene of XFL0605 encodes 604 amino acids and contains whole open reading frame but 105bp nucleotides were absent from in proline-rich region,only contains two E/DFPPPPXD/E repeat sequences.We sugest the variation contributes to virulence difference in mice.