Development Of A PCR Method And Kit For The Detection Of Listeria Monocytogenes

Listeria monocytogenes is an important food-borne pathogen. It iscommonly associated with human listeriosis, the average mortality rate ofwhich is as high as20%~30%. Therefore, it is meaningful to develop aspecific, sensitive and effective detection method to monitor and control thecontamination by L. monocytogenes.It is complicated and time-consuming to detect L.monocytogenes bytraditional microbiological procedures. In vitro amplification of DNA by thepolymerase chain reaction (PCR) has become a potential powerfulalternative in microbiological diagnostics due to its rapidity and accuracy.Currently, the PCR primers mainly derive from the toxin genes of L.monocytogenes and can result in false positive results for poor specificity ofthe target sequence or false negative results due to their absence or defective.With the development of bioinformatics, especially4strains ofL.monocytogenes have been sequenced and published in GenBank, whichwould be a great pool of information resources to mine new specific targetsfor the PCR systems. In this study, potential specific molecular targets for L.monocytogeneswere mined by genomic comparative algorithms and the specific primerswere designed. Three primer sets were determined from potential specificmolecular targets by the evaluation of PCR tests with28L.monocytogenesstrains and60non-L.monocytogenes. Using the primer sets lmo0754F/R, thedetection limit of this PCR system was55copies/PCR with the genomicDNA of L. monocytogenes.Using the primer sets lmo0754, a PCR method incorporated an IACwas developed at an optimal Mg2+concentration of1.5mmol/L and anannealing temperature of56℃. The IAC was constructed with thecomposite primer technique. With the presence of105copies of IACobtained from optimization tests, the detection of the PCR assay was55copies/PCR with L. monocytogenes genomic DNA. Applying this PCRassay to artificially contaminated milk samples, low levels of L.monocytogenes (1to10cfu/mL) were detected after6-9h incubation by onestep enrichment (selective culture enrichment UVM), which showed thatthe PCR method developed in this study was rapid and sensitive.A Real-time PCR detection kit for the rapid detection ofL.monocytogenes was developed. Results from the specificity assays showedthat this kit had high specificity for L.monocytogenes. Results of thesensitivity assays showed that the detection limit of this kit was3.94 fg/reaction with L. monocytogenes genomic DNA. Results of the repetitiveexperiments showed that the average inter-assay CV%of the kit was2.2%and the intra-assay CV%was1.9%. Results of the stability experimentsshowed that the repeated frozen-thaw for40times had no significantinfluence on the activity of this kit and it could be stored at-20℃for1yearaway from light. Therefore, the Real-time PCR detection kit developed in ourstudy is suitable for the rapid detection of L.monocytogenes for its highspecificity, high sensitivity, good reproducibility and strong stability.