OBJECTIVES: To detect prfA and hly toxin genes of Listeria monocytogenes using polymerase chain reaction (PCR) and colloidal gold technology. METHODS: L. monocytogenes DNA was extracted by boiling method. With prfA and hly of L. monocytogenes as the target genes, the 5' ends of upstream and downstream primers of prfA gene were labeled with 6-FAM and biotin, and the 5' ends of upstream and downstream primers of hly gene were labeled with digoxin and biotin, respectively, to establish the toxin gene detection method. Using cloning transformation, sequencing analysis, cloning of positive control products, the detection kid was developed and its specificity, sensitivity, reproducibility and stability were tested, followed by verification with sample testing. RESULTS: The concentration of L. monocytogenes DNA extracted by boiling method was 148.81±0.97 ng/μL, and the A₂₆₀/A₂₈₀ ratio ranged from 1.8 to 2.0. The PCR products showed a 100% homology with the gene sequences in GenBank database after cloning, transformation and sequencing. The colloidal gold strip yielded positive results only for L. monocytogenes samples without cross-reactions with Staphylococcus aureus, Escherichia coli or Bacillus cereus, and its minimum detection limit was 10^-2 ng/μL, demonstrating a 10-fold greater sensitivity of the test than agarose gel electrophoresis. The test also showed good reproducibility of the results when performed by different operators with good stability of the test strips after storage for 6 to 12 months. The test results showed that this kit could accurately and quickly detect L.monocytogenes in the test samples. CONCLUSIONS The detection kit developed in this study can simultaneously detect prfA and hly toxin genes of L. monocytogenes with good specificity, sensitivity, reproducibility and stability for use in food safety inspection.
Listeria monocytogenes/isolation & purification* ; Gold Colloid ; Bacterial Toxins/genetics* ; Polymerase Chain Reaction/methods* ; Hemolysin Proteins/genetics* ; Bacterial Proteins/genetics* ; DNA, Bacterial/genetics* ; Food Microbiology ; Heat-Shock Proteins

We aimed to investigate the potential pathogenic profile and antibiotic resistance of Listeria monocytogenes isolated from ready-to-eat food in China. Antimicrobial resistance was determined by broth microdilution following the Clinical and Laboratory Standards Institute protocol. Molecular serotyping, virulence, and resistance genes were identified using PCR. Multi-locus sequence typing was performed on resistant strains. A total of 11.53% (113/980) isolates were resistant, from which 82.3% (93/113) harbored all the virulence genes tested. The resistant strains were subtyped into 18 sequence types (STs), from which ST2, ST5, ST8, and ST9 were involved in listeriosis. This study indicated that several L. monocytogenes isolates from ready-to-eat foods in China have pathogenic potential and are resistant to antibiotics, including antibiotics used as medicines by humans for listeriosis treatment.
Anti-Bacterial Agents ; pharmacology ; China ; epidemiology ; Drug Resistance, Multiple, Bacterial ; Fast Foods ; microbiology ; Food Microbiology ; Listeria monocytogenes ; genetics ; isolation & purification ; pathogenicity ; Listeriosis ; epidemiology ; microbiology ; Multilocus Sequence Typing ; Virulence

OBJECTIVEThe loop-mediated isothermal amplification (LAMP) detection method was applied to detect Listeria monocytogenes in food. The specificity and sensitivity of this method were evaluated through comparing it with Real-time PCR and conventional detection method.

METHODSThe LAMP primers were designed based on hly gene of Listeria monocytogenes. The LAMP method was applied to detect 88 Listeria monocytogenes, 1 reference strain ATCC 15313 of Listeria monocytogenes and 33 non-targets bacteria strains; base-material addition test and practical food samples detection were also conducted. Both of Real-time PCR and ISO 11290-1 methods were used as parallel detection method in addition to LAMP. The three kinds of methods were compared by specificity, sensitivity, detection limit and the detection result of practical food samples.

RESULTSBoth detection results of LAMP and Real-time PCR for 89 Listeria monocytogenes were positive (100%, 89/89), 33 non-targets bacteria strains were negative (100%, 33/33). The sensitivity of LAMP was 2 × 10² CFU/ml, which was consistent with Real-time PCR method (2 × 10² CFU/ml) and better than ISO 11290-1 method (2 × 10² CFU/ml). Base-material addition test result showed that the detection limit of the three kinds of methods were 3 CFU/25 g samples. And the result of practical food samples displayed the same detection rate of 4% in the three methods (2/45).

CONCLUSIONThe LAMP method of Listeria monocytogenes established in this study has good specificity and sensitivity, which can be applied to the rapid detection of Listeria monocytogenes.

Food Contamination ; analysis ; Food Inspection ; methods ; Food Microbiology ; Listeria monocytogenes ; genetics ; isolation & purification ; Nucleic Acid Amplification Techniques ; methods

Listeria monocytogenes is a pathogenic bacterium, therefore, it is essential for food safety monitoring to establish a rapid and specific detecting method. In this study, immunomagnetic beads and selective medium were combined to detect Listeria monocytogenes at different concentrations (10(1)-10(5) CFU/mL). Other three types of Listeria spp., Staphylococcus aureus and Vibrio parahaemolyticus were also detected to conduct the cross-reaction analysis. Meanwhile, contaminated milk samples were prepared to explore the limit of detection of immunomagnetic beads combining with selective medium. Results showed that Listeria monocytogenes with the concentration of 10(3) CFU/mL and above was successfully detected. Milk samples were detected within 6 hours, with a detection limit of 0.7 CFU/mL. The method developed is capable of detecting milk samples within 30 h, which is 38 h faster compared with national standard method with the same sensitivity.
Bacteriological Techniques ; methods ; Culture Media ; chemistry ; Immunomagnetic Separation ; methods ; Listeria monocytogenes ; growth & development ; isolation & purification ; Sensitivity and Specificity

OBJECTIVETo know the antibiotic resistance and molecular characteristics of Listeria monocytogenes in Shandong province and to study the relationship between antibiotic resistance phenotypes and genome types.

METHODSFrom 2009 to 2010, a total of 80 Listeria monocytogenes isolates were collected from raw meat, cooked meat, aquatic products and other foods in 6 cities of Shandong province. The antibiotic susceptibility was measured by broth microdilution method, PFGE was performed for molecular typing and the relationship between antimicrobial resistance and PFGE patterns was analyzed.

RESULTS16.25% (13/80) of the isolates were drug-resistant. Imipenem resistance was the most prevalent (12.50%, 10/80), followed by tetracycline and doxycycline (3.75%, 3/80 and 2.50%, 2/80). A total of the 80 isolates were subtyped into 9 antibiotic resistance patterns and 34 PFGE types which were largely dominated by the type 17 and 29. Antibiotic resistance pattern A corresponded to 79.41% (27/34) of PFGE types.

CONCLUSIONThe antibiotic resistance of Listeria monocytogenes in Shandong province is serious from 2009 to 2010 and there is no correlation between PFGE types and antibiotic resistance patterns.

Anti-Bacterial Agents ; pharmacology ; Bacterial Typing Techniques ; China ; Drug Resistance, Bacterial ; Food Microbiology ; Listeria monocytogenes ; classification ; drug effects ; isolation & purification ; Microbial Sensitivity Tests

OBJECTIVETo conduct a quantitative risk assessment for Listeria monocytogenes in bulk cooked meat products in China.

METHODSThe data on the contamination level of Listeria monocytogenes in cooked meat products was from national foodborne disease surveillance network, involving a total of 841 samples. All the samples were detected by a qualitative method and 97 samples among them were detected using a quantitative method. The intake data of cooked meats was from Chinese National Nutrition and Health Survey in 2002 and population data in the monitoring sites was collected from National Bureau of Statistics in 2008 to estimate the composition of the population of different ages, which would be the base of assessing the probability of listeriosis in the different subpopulations. Using @Risk software to estimate the risk of listeriosis caused by consuming deli meats for different subpopulation (0 - 4 years old, 5 - 64 years old and 65 years and older) by quantitative risk assessments which involved hazard identification, hazard characterization, exposure assessment and risk characterization and conduct sensitivity analysis.

RESULTSThe contamination level of Listeria monocytogenes in the most of samples (96.08%, 808/841) was less than 3 MPN/g (0.5 lg MPN/g), and the average concentration of Listeria monocytogenes was -0.61 lg CFU/g (90%CI: -1.22 - 0.46 lg CFU/g). Estimated servings of cooked meat consumption for 0 - 4, 5 - 64 and 65 years and older were 5.52 × 10(9), 8.99 × 10(10), 1.01 × 10(10), respectively. Estimated number of cases (median) of listeriosis each year per million people caused by consuming cooked meats in young (0 - 4 years old), intermediate age (5 - 64 years old) and elderly (65 years and older) population were 5.53 × 10(-3), 1.72 × 10(-4), 7.57 × 10(-3), respectively. Results of sensitivity analysis showed that contamination level at retail, serving size of cooked meats, storage time at home, storage temperature and ERG at 5°C were positive factors for the risk of listeriosis (r value was 0.607, 0.408, 0.339, 0.259, 0.183 respectively, P < 0.05).

CONCLUSIONCooked meat products in bulk is a risk food, which could cause listeriosis. Contamination level of Listeria monocytogenes in cooked meat products in bulk is the top risk factor for the listeriosis.

Adolescent ; Adult ; Aged ; Child ; Child, Preschool ; Colony Count, Microbial ; Food Contamination ; analysis ; Food Microbiology ; Humans ; Infant ; Listeria monocytogenes ; isolation & purification ; Meat Products ; microbiology ; Middle Aged ; Models, Theoretical ; Risk Assessment ; Software ; Young Adult

OBJECTIVETo investigate the occurrence of important foodborne pathogens in shellstock Pacific oysters in the food markets in South China.

METHODSFrom July 2007 to June 2008, retail oysters were collected in different seasons from South China and analyzed for the prevalence and levels of Listeria monocytogenes, Vibrio vulnificus and Vibrio parahaemolyticus.

RESULTSNone of L. monocytogenes could be detected in any of the 202 oyster samples tested, while E vulnificus and V. parahaemolyticus could be detected in 67 (54.9%) and 109 (89.3%) of the 122 oyster samples analyzed, respectively, with an MPN (most probable number) value greater than or equal to 3. V. vulnificus and V. parahaemolyticus with a more than 102 MPN/g were found in 36 (29.5%) and 59 (48.4%) of the 122 oyster samples, respectively. The tdh and trh genes were detected in 4 (0.3%) and 8 (0.6%) of the 1 349 V parahaemolyticus isolates, respectively. Of the 122 samples, 4 (3.3%) was positive for either tdh or trh. The levels of V. vulnificus and total V. parahaemolyticus in oysters in South China varied in different seasons.

CONCLUSIONV. vulnificus and pathogenic V. parahaemolyticus are frequently found in oysters in south China, which may pose a potential threat to public health. Data presented here will be useful for the microbiological risk assessment in oysters in China.

Animals ; China ; Commerce ; Food Microbiology ; Listeria monocytogenes ; isolation & purification ; Ostreidae ; microbiology ; Vibrio parahaemolyticus ; isolation & purification ; Vibrio vulnificus ; isolation & purification

OBJECTIVETo study the virulent gene prevalence of foodborne Listeria monocytogenes (LM) isolated from China.

METHODS78 LM isolates derived from raw meat, cooked food, aquatic products and vegetables of 13 provinces and cities.LM isolates were investigated for prevalence of virulence genes (LIPI-1 (prfA, plcA, hly, mpl, actA, plcB); LIPI-2 (inlA, inlB), and iap) by PCR method.

RESULTS87.2% (68/78) of the isolates were prfA positive, 98.7% (77/78) of the isolates were plcA, actA and plcB positive, 97.4% (76/78) of the isolates were hly positive, 87.2% (68/78) of the isolates were mpl positive, 92.3% (72/78) of the isolates were inlA positive, 100% (78/78) of the isolates were inlB positive, 98.7% (77/78) of the isolates were iap positive. Among 21 virulent gene negative isolates, there was 7 isolates lack of two or more virulence genes. The rate of virulence genes deletion isolates from cooked meat was 31.3% (10/32), the rate of virulence genes deletion isolates from raw meat was 16.1% (5/31), the rate of virulence genes deletion isolates from vegetables was 36.4% (4/11) and rate of virulence genes deletion isolates from seafood was 50% (2/4). No significant difference was found (χ(2) = 3.721, P > 0.05). The virulence gene array-1 strains were dominant among these isolates.

CONCLUSIONAmong 78 LM isolates, prevalent of virulent genes were different except inlB, virulence genes of LIP-1 were deleted prevalently among isolates, virulence gene deletion patterns were diverse.

China ; epidemiology ; Food Contamination ; analysis ; Food Microbiology ; Foodborne Diseases ; epidemiology ; microbiology ; Listeria monocytogenes ; genetics ; isolation & purification ; pathogenicity ; Listeriosis ; epidemiology ; microbiology ; Virulence Factors ; genetics

Bacteremia ; etiology ; Female ; Humans ; Infant, Newborn ; Listeria monocytogenes ; isolation & purification ; Male

OBJECTIVETo analysis risk from Listeria monocytogenes in deli meats and vegetable salads.

METHODSUse Risk Ranger which is a software programme developed by the University of Hobart, Australia and answer 11 questions on affecting the risk from hazards in the specific foods by combining data from national foodborne diseases surveillance network and some references to make semi-quantitative risk assessment for the specific food.

RESULTSRelative risk from Listeria monocytogenes in deli meats and vegetable salads is 61 and 52, respectively. Incidence of listeriosis caused by deli meats-Listeria monocytogenes pairs and vegetable salads-Listeria monocytogenes pairs is 5.4 and 0.2 cases per million people, respectively. Risk from the former is 32 times than that from the latter. By changing the selection for some risk factors in the model, it was known that the risks from two food-hazard combinations could decrease 10 times, if taking necessary actions after processing.

CONCLUSIONDeli meats is a kind of high risk food for listeriosis.

Food Microbiology ; Listeria monocytogenes ; isolation & purification ; Meat Products ; microbiology ; Risk Assessment ; Vegetables ; microbiology