Research On Molecular Detection Techniques Of Listeria Monocytogenes In Cold Meat

Viable but non-culturable (VBNC) is a special existing form of bacteria, whichhas been found among more than60kinds of bacteria until now. The VBNC state is aself-protection strategy of bacteria facing environmental stresses, where the cellsremain viable, but are no longer culturable on standard laboratory media. Undercertain conditions, resuscitation from this state will happen, and the cells will regainculturability. VBNC bacteria can not be detected by traditional culture methods andsome pathogens in the VBNC state even produces toxin, therefore, the VBNC bacteria,especially those VBNC pathogenic bacteria, has be considered to be a potentialthreat to food safety. Listeria monocytogene is one of the typical foodborne pathogens,which can be found in most raw meat and meat product. At present, the growthbehavior of VBNC Listeria monocytogenes is completely unknown, and little relevantresearch has been reported.Listeria monocytogenes about107CFU/mL was selected to be induced into theVBNC state. The Listeria monocytogenes stain under2℃, pH6.0was confirmed tohave entered the VBNC state, by the enumeration of total cells number, viable cellsnumber as well as the culturable cells number. Becides, Changes in cell shape andvolume, and the tendency to be gathering among cells were confirmed by Stainingand microscope observation. After the induction, the normal Listeria monocytogenes,and the VBNC Listeria monocytogenes were incubated at4℃cold meatrespectively, and the growth behavior were monitored. Results obtained indicatedthat normal Listeria monocytogenes incubated in cold meat kept growing at4℃,reaching the platform after13day, and visible colonies of Listeria monocytogeneswere formed on the surface of the meat after the incubation. This study demonstratedthat the VBNC Listeria monocytogenes recovered to be culturable at the third day ofincubation, and a period of14days were needed for the bacteria to reach the platform.After the incubation, a layer of moist, dense pink plaque was found on the surface of the meat. The trends of viable cells number, determined by PMA-qPCR, wereconsistent with the total cells and the culturable cells’ obtained by qPCR and platecounting respectively. However, enumeration results of viable cells acquired byPMA-qPCR appeared to be lower than the enumerations acquired by other threemethods, e.g. after the incubation, viable cells numbers in the two different meatsamples determined by PMA-qPCR were2.9×107CFU/g and1.4×107CFU/g, bothof which were lower than the enumeration by qPCR(5.6×107CFU/g,2.4×107CFU/g), plate counting(5.3×107CFU/g,2.5×104CFU/g) and flow cytometry(5.4×107CFU/g,2.3×107CFU/g). This result might be explained by the membranedamage of viable cells causing by repeated centrifugation during bacteria collectionprocess, as PMA-qPCR method considers cells loss with membrane integrity to bedead.The criteria on cell’s viability is not yet uniform currently. Theoretically,PMA-qPCR detection technology, which distinguishing cell’s viability by membraneintegrity, is a suitable method for viable cells(including VBNC cells) detection.However, for detection of some dead cells with membrane integrity or viable cellswith reversible membrane damage, this method still has flaws.Based on the metabolic activity of esterase in bacteria, a novel thiazole orangefluorescent dye named TOMA was designed and synthesized to develop a new,efficient viable bacteria detecting technology, which determining the cell viabilitythrough biological metabolic activity. The molecule(TOMA) is mainly composed ofthree functional groups, a binding group, a cross-linking group and a biologicalconnecting arm. The molecule structure was characterized by1H NMR andMS(molecular weight=474.6). By laser scanning confocal microscope, strongfluorescence was observed from cells treated with a final concentration of10μg/mLof TOMA, which shows the capabilities of TOMA to penetrate an intact cellmembrane as well as binding to DNA. The ester bond linking on the molecule ofTOMA is sensitive for esterase activity, meaning that they can be hydrolysed by esterase in a short time. Immobilized lipase was utilized in vitro to catalyze TOMA’shydrolysis in solution. The optimal enzyme dosage is1.0U/μmoL TOMA in theexpenriment, and the hydrolysis rate attained to be83%, both of which revealing thatthe desired functionality of the ester bond could be achieved. DNA extracted fromthree different bacteria were amplified by quantitative PCR after treatment withdifferent concentration of TOMA. Results showed that TOMA’s inhibition on DNAamplification was significant with a final concentration of10μg/mL, and theinhibition extent was obviously positive related to the concentration from0to10μg/mL, indicating that the amplification of DNA in PCR reaction could be inhibitedby TOMA. Cells respectively inactivated by heat,70%isopropyl alcohol andultraviolet radiation were detected by PMA-qPCR and TOMA-qPCR, and comparisonbetween the two methods demonstrated that TOMA-qPCR inhibited the DNAamplification of the three inactivated bacteria successfully, while PMA-qPCR failedto monitored the loss of viability caused by UV-exposure. Through the results, weconcluded that TOMA-qPCR would be a good method for viable cells quantitativedetection, as it’s able to monitor the dead cells with/without membrane integrity byeffectively inhibiting their DNA amplification.