Optimization of haemolysis in enhanced haemolysis agar (EHA)_a selective medium for the isolation of Listeria monocytogenes
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The presence of Listeria monocytogenes in enrichment media can be masked by faster growth of other Listeria spp. Therefore, enhanced haemolysis agar (EHA) is a good alternative for another isolation media, because the presence of a few L. monocytogenes colonies can be detected in a majority of colonies of other listeriae on the basis of haemolysis. In this study the haemolysis reaction in EHA was optimized. In a collaborative study using reference samples, no significant differences in counts on EHA, Palcam and Oxford agar were shown.Keywords:
Haemolysis
Isolation
To assess the growth and survival of Listeria Monocytogenes in spiked powered milk of different water activity at different temperature.Listeria Monocytogenes was added into powdered milk of different water activity at a density of 103 cfu/g,the spiked powered milk stored at the different temperature,the influence of different temperature and different water activity on the growth and survival of Listeria Monocytogenes in the powered milk was analyzed by counting.The temperature influenced the growth and survival of Listeria Monocytogenes in powered milk of different water activity.The Listeria Monocytogenes in powered milk of low water activity could be maintaining growth activity for a long time at low temperature and the number of Listeria Monocytogenes in powered milk of high water activity could increase.However,the Listeria Monocytogenes growth activity decreased very quickly at high temperature and the cell was sublethal,but the growth activity of sublethal Listeria Monocytogenes could recover in the appropriate condition.The growth activity of Listeria Monocytogenes decreases easily at high temperature.If powdered milk was contaminated with Listeria Monocytogenes,the growth of Listeria Monocytogenes will be controlled when being stored at room temperature(25℃).
Water activity
Chocolate milk
Bacterial growth
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Monitoring of Listeria monocytogenes, the causative agent of listeriosis, in food is inportant for public health. The Korean Food Standards Codex has adopted a ‘zero-tolerance’ policy for L. monocytogenes. The standard detection method of L. monocytogenes is based on enrichment. Thus, proper enrichment methods need to be instituted to ensure quality control of the detection procedures. In this study, the growth of L. monocytogenes and Listeria innocua as a mixed culture in Listeria enrichment broth (LEB) was monitored during artificial contamination of enrichment culture. We confirmed competitive growth or interspecies inhibitory activity of L. monocytogenes and L. innocua. Interspecies growth differences and the inhibitory activity of different inoculation and mixtures L. innocua against L. monocytogenes were examined. The concentration of L. monocytogenes must be 2.0 log CFU/mL or more than L. innocua to grow better than L. innocua. It is known that Listeria spp. and L. monocytogenes show growth difference during LEB, resulting in the risk of false-negative results. The inhibition of L. monocytogenes by L. innocua was always observed when present at lower concentrations. However, it was confirmed that L. innocua suppressed when L. monocytogenes was present at a higher concentration. Therefore if a mixture of Listeria spp. is present, detecting L. monocytogenes is difficult. Thus, a new enrichment broth to improve the detection rate of L. monocytogenes is needed.
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This is to select solid selective culture media suitable for detection of listeria monocytogenes.The verification of culture media in ISO/TS 11133-2 was introduced and modified.Three solid selective culture media were compared.The OXA and listeria CHROMagar had equal effect,while listeria monocytogenes and listeria ivanovii had typical colony on the listeria CHROMagar.It concluded that OXA and listeria CHROMagar have excellent effect and are well suitable for detection of listeria monocytogenes.Their combined application is considered to be more efficacious.
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Listeria infection
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Food microbiology
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We report a duplex real-time PCR-based assay for the simultaneous quantitative detection of Listeria spp. and the food-borne pathogen Listeria monocytogenes. The targets of this single tube reaction were the 23S rDNA and hly genes of Listeria spp. and L. monocytogenes, respectively. Our assay was efficient, 100% selective (i.e., it allowed accurate simultaneous identification of 52 L. monocytogenes and 120 Listeria spp. strains through the FAM-labelled hly and the VIC-labelled 23S rDNA probes, respectively); and had a detection limit of one target molecule in 100% (23S rDNA) and 56% (hly) of the reactions. Simultaneous quantification was possible along a 5-log dynamic range, with an upper limit of 30 target molecules and R2 values >0.995 in both cases. Our results indicate that this assay based on the amplification of the 23S rDNA gene can accurately quantify any mixture of Listeria species and simultaneously unambiguously quantify L. monocytogenes.
23S ribosomal RNA
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We report a duplex real-time PCR-based assay for the simultaneous quantitative detection of Listeria spp. and the food-borne pathogen Listeria monocytogenes. The targets of this single tube reaction were the 23S rDNA and hly genes of Listeria spp. and L. monocytogenes, respectively. Our assay was efficient, 100% selective (i.e., it allowed accurate simultaneous identification of 52 L. monocytogenes and 120 Listeria spp. strains through the FAM-labelled hly and the VIC-labelled 23S rDNA probes, respectively); and had a detection limit of one target molecule in 100% (23S rDNA) and 56% (hly) of the reactions. Simultaneous quantification was possible along a 5-log dynamic range, with an upper limit of 30 target molecules and R2 values > 0.995 in both cases. Our results indicate that this assay based on the amplification of the 23S rDNA gene can accurately quantify any mixture of Listeria species and simultaneously unambiguously quantify L. monocytogenes.
23S ribosomal RNA
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