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16S rRNA-based probes and polymerase chain reaction method to detect Listeria monocytogenes cells added to foods.

ABSTRACT: A rapid polymerase chain reaction (PCR) method was developed for detection of Listeria monocytogenes in foods. This method used a pair of primers based on a unique region in the 16S rRNA sequence of L. monocytogenes, which were previously reported by us to yield a specific nucleic acid probe. Our method included use of a shorter denaturing time, a shorter annealing time, a rapid transition, and an increase in the number of cycles, resulting in good sensitivity. Just 3 h for PCR plus 1 h for electrophoresis was required. Additional time for DNA isolation and DNA hybridization was not needed. This method detected as few as 2 to 20 CFU of L. monocytogenes in pure cultures and as few as 4 to 40 CFU of L. monocytogenes in inoculated (10(8) CFU), diluted food samples. Seven of eight foods, including four poultry products, gave positive results. Only one food sample, soft cheese, gave interference. An internal probe hybridization test was used to confirm that the PCR products were from L. monocytogenes. A specificity test indicated that this PCR method was positive for all 13 strains of L. monocytogenes tested but negative for the other 6 species of Listeria, including 6 strains of L. innocua, and negative for 17 other gram-positive and gram-negative bacteria tested.

SUBMITTER: Wang RF

PROVIDER: S-EPMC183014 | biostudies-other | 1992 Sep

REPOSITORIES: biostudies-other

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16S rRNA-based probes and polymerase chain reaction method to detect Listeria monocytogenes cells added to foods.

Wang R F RF Cao W W WW Johnson M G MG

Applied and environmental microbiology 19920901 9

A rapid polymerase chain reaction (PCR) method was developed for detection of Listeria monocytogenes in foods. This method used a pair of primers based on a unique region in the 16S rRNA sequence of L. monocytogenes, which were previously reported by us to yield a specific nucleic acid probe. Our method included use of a shorter denaturing time, a shorter annealing time, a rapid transition, and an increase in the number of cycles, resulting in good sensitivity. Just 3 h for PCR plus 1 h for elec ...[more]

PMID: 1444393

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Project description:The prevalence and levels of Listeria monocytogenes in retail raw foods covering most provincial capitals in China were studied with testing of 1036 samples of vegetables, edible mushrooms, raw meat, aquatic products and quick-frozen products from September 2012 to January 2014. The total prevalence of Listeria monocytogenes was 20.0% (207/1036), and the most probable number (MPN) values of 65.7% of the positive samples ranged from 0.3 to 110 MPN/g. Geographical differences were observed in this survey, and the results of both qualitative and quantitative methods indicated that the levels in the samples from North China were higher than those in the samples from South China. A total of 248 isolates were analyzed, of which approximately half belonged to molecular serogroup 1/2a-3a (45.2%), followed by 1/2b-3b-7 (30.6%), 1/2c-3c (16.1%), 4b-4d-4e (5.2%) and 4a-4c (2.8%). Most of the isolates carried hly (100%), inlB (98.8%), inlA (99.6%), inlC (98.0%) and inlJ (99.2%), and 44.8% of the isolates were llsX-positive. Seventeen epidemic clones (ECs) were detected, with 7 strains belonging to ECI (2.8%) and 10 belonging to ECIII (4.03%). Resistance to clindamycin (46.8%) was commonly observed, and 59 strains (23.8%) were susceptible to all 14 tested antibiotics, whereas 84 (33.9%) showed an intermediate level of resistance or were resistant to two or more antibiotics, including 7 multi-resistant strains that exhibited resistance to more than 10 antibiotics. The data obtained in the present study provides useful information for assessment of the possible risk posed to Chinese consumers, and this information will have a significant public health impact in China. Furthermore, the presence of virulence markers, epidemic clones, as well as the antibiotic resistance amongst the isolates strongly implies that many of these strains might be capable of causing listeriosis, and more accurate treatment of human listeriosis with effective antibiotics should be considered. This research represents a more full-scale and systematical investigation of the prevalence of L. monocytogenes in retail raw foods in China, and it provides baseline information for Chinese regulatory authorities that will aid in the formulation of a regulatory framework for controlling L. monocytogenes with the aim of improving the microbiological safety of raw foods.

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16S rRNA-based probes and polymerase chain reaction method to detect Listeria monocytogenes cells added to foods.

Publications

16S rRNA-based probes and polymerase chain reaction method to detect Listeria monocytogenes cells added to foods.

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