Single-strand conformation polymorphism (SSCP) analysis ofListeria monocytogenes iapgene as tool to detect different serogroups

The objectives of this study were to identify the serotypes of Listeria monocytogenes isolated from packaged fresh turkey meat samples marketed in Ankara, Turkey, and to determine the seasonal distribution of the serotypes. Out of 37 L. monocytogenes isolates obtained from 180 turkey meat samples, 19 (51.4%), 10 (27.0%) and 8 (21.6%) were serotyped by multiplex polymerase chain reaction as 4b (or 4d, 4e), 1/2a (or 3a) and 1/2b (or 3b), respectively. Serotypes 1/2a and 4b were dominant in turkey meat samples collected in the summer, and 4b was the most prevalent L. monocytogenes serotype during the winter. Serotype 4b was the most prevalent serotype in turkey meat cuts and legs, whereas 1/2b was the dominant serotype in turkey breast samples. Turkey meat can be a potential risk for public health; therefore, it should be produced under appropriate hygienic and technological conditions and cooked adequately before consumption. PRACTICAL APPLICATIONS Serotyping has been widely used to characterize L. monocytogenes isolates and is important from the epidemiological point of view. Serotypes 1/2a, 1/2b, 1/2c and 4b are the predominant (>95%) serotypes isolated from food samples and human listeriosis cases among 13 L. monocytogenes serotypes. Conventional agglutination and pulsed‐field gel electrophoresis methods have been routinely used for serotyping and characterizing L. monocytogenes but these techniques are today expensive and time consuming. In the present study, rapid and practical multiplex polymerase chain reaction assay was used to differentiate the major L. monocytogenes serotypes (1/2a, 1/2b, 1/2c and 4b) in turkey meat.

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“…In previous PCR‐based studies (Comi et al . 1997; Manzano et al . 1997; Jinneman and Hill 2001), L .…”

Section: Discussionmentioning

confidence: 99%

SEROTYPE DISTRIBUTION OF LISTERIA MONOCYTOGENES ISOLATED FROM TURKEY MEAT BY MULTIPLEX PCR IN TURKEY

Erol¹,

Ayaz

2011

Journal of Food Safety

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“…The LAMP method developed in this study was specific for two stains of L. monocytogenes, and no amplified products were found for 12 non-L. monocytogenes strains. Although only a few of strains were involved, it is considered that the set of primers had high specificity for the iap gene of L. monocytogenes because the selected target gene has been fully approved to be a specific gene of L. monocytogenes by PCR assay and other nucleic acid sequence-based methods (Manzano et al 1996;Cocolin et al 1997;Comi et al 1997;Manzano et al 1997Manzano et al , 2003Hein et al 2001;Kwiatek et al 2003 Shakuntala et al 2006) and has been recommended to be used in foodborne L. monocytogenes detection in many countries and regions (e.g., European ring trial-detection of L. monocytogenes by the PCR). In addition, LAMP assay had higher specificity for the target gene than PCR.…”

Section: Discussionmentioning

confidence: 99%

DEVELOPMENT AND EVALUATION OF A LOOP‐MEDIATED ISOTHERMAL AMPLIFICATION (LAMP) METHOD FOR DETECTING LISTERIA MONOCYTOGENES IN RAW MILK

Wang

Gui-cheng

Ren

et al. 2010

Journal of Food Safety

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The performance of a Loop‐mediated isothermal amplification (LAMP) assay for detecting food‐borne pathogen Listeria monocytogenes was presented in this paper. Three pairs of primers were specially designed for recognizing eight distinct sequences of iap gene (P60 extracellular protein, invasion associated protein IAP). Time and temperature conditions for amplification of L. monocytogenes were optimized to be 40 min at 63C. Detection limit level for artificially contaminated raw milk samples by the LAMP assay was 186 cfu/mL corresponding to 8–10 cells per reaction tube, while the detection level by conventional PCR was 1.86 × 105 cfu/mL. Data on natural raw milk samples indicated that the LAMP method was highly specific and sensitive, giving 91.67% concordance with the ISO 10560 reference method for the samples without enrichment and 100% concordance after enrichment. PRACTICAL APPLICATION The LAMP method in this paper provided a powerful tool with the specificity, sensitivity and rapidity for detection of Listeria monocytogenes in raw milk.

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“…However, the performance of RTi-PCR and, thus, its detection limit and quantification accuracy, depend on the degree of conservation of the target sequence(s) of the particular microorganism being analyzed. Strains of L. monocytogenes have been divided into three serovar-related homology groups or divisions on the basis of gene sequence diversity (19,26,28,33,(42)(43)(44). To ensure that our hly-and iap-based RTi-PCR assays were appropriate for the precise quantification of an L. monocytogenes strain regardless of its genetic background, we extensively evaluated our oligonucleotides by using a large panel of isolates of serovars representative of the three phylogenetic divisions of the species.…”

Section: Discussionmentioning

confidence: 99%

“…While the hly gene is relatively well conserved in all L. monocytogenes strains, the iap gene is not. Although iap contains conserved portions at the 5Ј and 3Ј ends, its central region is highly variable and contains sequence polymorphisms even among strains of the same serovar (5,26,33).…”

Section: Bacteria Of the Facultative Anaerobic Gram-positive Genusmentioning

confidence: 99%

Quantitative Detection of Listeria monocytogenes and Listeria innocua by Real-Time PCR: Assessment of hly , iap , and lin02483 Targets and AmpliFluor Technology

Rodrı́guez-Lázaro

Hernández

Scortti

et al. 2004

Appl Environ Microbiol

219

109

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We developed and assessed real-time PCR (RTi-PCR) assays for the detection and quantification of the foodborne pathogen Listeria monocytogenes and the closely related nonpathogenic species L. innocua. The target genes were hly and iap for L. monocytogenes and lin02483 for L. innocua. The assays were 100% specific, as determined with 100 Listeria strains and 45 non-Listeria strains, and highly sensitive, with detection limits of one target molecule in 11 to 56% of the reactions with purified DNA and 3 CFU in 56 to 89% of the reactions with bacterial suspensions. Quantification was possible over a 5-log dynamic range, with a limit of 15 target molecules and R 2 values of >0.996. There was an excellent correspondence between the predicted and the actual numbers of CFU in the samples (deviations of <23%). The hly-based assay accurately quantified L. monocytogenes in all of the samples tested. The iap-based assay, in contrast, was unsuitable for quantification purposes, underestimating the bacterial counts by 3 to 4 log units in a significant proportion of the samples due to serovar-related target sequence variability. The combination of the two assays enabled us to classify L. monocytogenes isolates into one of the two major phylogenetic divisions of the species, I and II. We also assessed the new AmpliFluor technology for the quantitative detection of L. monocytogenes by RTi-PCR. The performance of this system was similar to that of the TaqMan system, although the former system was slightly less sensitive (detection limit of 15 molecules in 45% of the reactions) and had a higher quantification limit (60 molecules). Bacteria of the facultative anaerobic gram-positive genusListeria are widely distributed in the environment, particularly the closely related species Listeria monocytogenes and L. innocua. Both of these Listeria spp. are frequently found in food products, where they can grow over a pH range of 4.39 to 9.40, even at refrigeration temperatures. Ingestion of foods contaminated with L. monocytogenes can result in listeriosis, a severe infectious disease characterized by meningoencephalitis, abortion, septicemia, and a high fatality rate (30%). Listeriosis predominantly affects certain risk groups, including pregnant women, newborns, elderly people, and immunocompromised patients. L. innocua, in contrast, is nonpathogenic, and its presence in foods is no hazard to human health (20,25,35,37,41). Human listeriosis outbreaks are most often associated with ready-to-eat food products that are consumed without prior cooking (8, 36). To err on the side of caution, food safety regulations have tended to adopt a zero-tolerance attitude for L. monocytogenes in these products (9). However, as clinical cases of listeriosis are usually associated with high loads of L. monocytogenes (6,8) and as it is difficult to eradicate listeriae from the environment of food-processing plants (11), the International Commission on Microbiological Specification for Foods concluded that 100 CFU of L. monocytogenes per g of food is accepta...

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Single-strand conformation polymorphism (SSCP) analysis ofListeria monocytogenes iapgene as tool to detect different serogroups

Cited by 14 publications

References 18 publications

SEROTYPE DISTRIBUTION OF LISTERIA MONOCYTOGENES ISOLATED FROM TURKEY MEAT BY MULTIPLEX PCR IN TURKEY

SEROTYPE DISTRIBUTION OF LISTERIA MONOCYTOGENES ISOLATED FROM TURKEY MEAT BY MULTIPLEX PCR IN TURKEY

DEVELOPMENT AND EVALUATION OF A LOOP‐MEDIATED ISOTHERMAL AMPLIFICATION (LAMP) METHOD FOR DETECTING LISTERIA MONOCYTOGENES IN RAW MILK

Quantitative Detection of Listeria monocytogenes and Listeria innocua by Real-Time PCR: Assessment of hly , iap , and lin02483 Targets and AmpliFluor Technology

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