A comparison between a PCR method and a conventional culture method for detecting pathogenic Yersinia enterocolitica in food

1996 ◽  
Vol 81 (3) ◽  
pp. 303-308 ◽  
Author(s):  
S. Thisted Lambertz ◽  
A. Ballagi-Pordány ◽  
A. Nilsson ◽  
P. Norberg ◽  
M.-L. Danielsson-Tham
Keyword(s):  
Yersinia Enterocolitica ◽  
Culture Method ◽  
Conventional Culture ◽  
Pcr Method ◽  
Pathogenic Yersinia ◽  
Conventional Culture Method

scholarly journals High Prevalence of yadA-Positive Yersinia enterocolitica in Pig Tongues and Minced Meat at the Retail Level in Finland

1999 ◽  
Vol 62 (2) ◽  
pp. 123-127 ◽  
Author(s):  
MARIA FREDRIKSSON-AHOMAA ◽  
SEBASTIAN HIELM ◽  
HANNU KORKEALA
Keyword(s):  
Yersinia Enterocolitica ◽  
Culture Method ◽  
Selective Enrichment ◽  
Conventional Culture ◽  
High Prevalence ◽  
Retail Outlets ◽  
Polymerase Chain ◽  
Meat Samples ◽  
Minced Meat ◽  
Conventional Culture Method

Polymerase chain reaction (PCR) was used to determine the prevalence of yadA-positive Yersinia enterocolitica in pig tongues and minced meat at the retail level in Finland and to confirm the yadA-positive Y. enterocolitica isolates recovered from the same samples using the conventional culture method. A total of 51 pig tongues purchased at 12 retail outlets and 255 minced meat samples purchased at 40 retail outlets in the Helsinki area were studied. The prevalence of Y. enterocolitica carrying the yadA gene was 92% in pig tongues and 25% in minced meat using PCR and 78% in tongues and 2% in minced meat with the culture method. The prevalence of yadA-positive tongues was higher (98%) when both PCR- and culture-positive results were included because Y. enterocolitica carrying the yadA gene could also be isolated in three PCR-negative tongue samples. In the minced meat samples, all PCR-negative samples were also culture-negative. With the culture method, 66 of 80 yadA-positive isolates in 38 tongues and all yadA-positive isolates (4) in four minced meat samples were recovered after selective enrichment. A total of 92 isolates of Y. enterocolitica bioserotype 4/O:3 in tongues and 5 isolates in minced meat were found, of which 13% in tongues and 20% in minced meat did not carry the yadA gene.

Evaluation of a Combined Culture and PCR Method (NMKL-163A) for Detection of Presumptive Pathogenic Yersinia enterocolitica in Pork Products

2007 ◽  
Vol 70 (2) ◽  
pp. 335-340 ◽  
Author(s):  
SUSANNE THISTED LAMBERTZ ◽  
KARIN GRANATH ◽  
MARIA FREDRIKSSON-AHOMAA ◽  
KARL-ERIK JOHANSSON ◽  
MARIE-LOUISE DANIELSSON-THAM
Keyword(s):  
Yersinia Enterocolitica ◽  
Nested Pcr ◽  
Buoyant Density ◽  
Culture Method ◽  
Pcr Primers ◽  
Sample Treatment ◽  
Pork Sausage ◽  
Density Centrifugation ◽  
Pcr Method ◽  
Pathogenic Yersinia

A combined culture and PCR method for detection of pathogenic Yersinia enterocolitica in food (NMKL-163A) was evaluated by testing samples of artificially and naturally contaminated pork. The performance of the pre-PCR sample treatment, buoyant density centrifugation, was first compared with two commercially available methods (DNeasy tissue kit and PrepMan). We found that similar sensitivity was reached (i.e., 25 CFU/g of food was detected by single PCR) with the buoyant density centrifugation and the DNeasy Tissue kit when tested on overnight enrichments. However, the DNeasy tissue kit was superior when tested on nonenriched homogenates; the detection limit was 25 CFU/g in minced beef by single PCR and 25 CFU/g in sausage by nested PCR. We then analyzed 100 raw minced pork samples. Thirty-five tested positive for presumptive pathogenic Y. enterocolitica when analyzed by the NMKL-163A method, whereas none tested positive when analyzed in parallel by a standard culture method (ISO 10273). We also analyzed 97 samples of cold-smoked pork sausage, of which approximately 11% tested positive by the NMKL-163A method. This study showed that sensitivities such as those obtained by nested PCR were required for detection of the pathogen in naturally contaminated samples, and therefore the nested PCR primers, which are included in the NMKL-163A method only as an option, need to be validated and applied routinely.

Prevalence and Antimicrobial Susceptibility of Salmonella Isolated from a Variety of Raw Meat Sausages in Gaborone (Botswana) Retail Stores

2012 ◽  
Vol 75 (4) ◽  
pp. 637-642 ◽  
Author(s):  
RONALD GAELEKOLWE SAMAXA ◽  
MAITSHWARELO IGNATIUS MATSHEKA ◽  
SUNUNGUKO WATA MPOLOKA ◽  
BERHANU ABEGAZ GASHE
Keyword(s):  
Antimicrobial Susceptibility ◽  
Salmonella Enterica ◽  
Antimicrobial Agents ◽  
Culture Method ◽  
Multidrug Resistant ◽  
Pcr Assay ◽  
Raw Meat ◽  
Conventional Culture ◽  
The Difference ◽  
Conventional Culture Method

The objective of the study was to provide baseline data on the prevalence and antimicrobial susceptibility of Salmonella in different types of raw meat sausages directly accessible to the consumers in Gaborone, Botswana. A total of 300 raw sausages comprising 79 beef, 78 pork, 72 chicken, and 71 mutton samples were concurrently analyzed for the presence of Salmonella using a conventional culture method and a validated PCR method. The PCR assay results were in full concordance with those of the conventional culture method for the detection of Salmonella. Sixty-five (21.7%) of 300 samples were positive for Salmonella by both the conventional culture method and PCR assay. Even though more chicken samples contained Salmonella than did any other sausage type, the difference in the presence of Salmonella among the four sausages types was not significant. Eleven serotypes were identified, and Salmonella enterica subsp. salamae II was most prevalent in all the sausage types. Beef sausages generally had higher mesophilic bacterial counts than did the other three sausage types. However, higher microbial counts were not reflective of the presence of salmonellae. Susceptibility of the Salmonella enterica serotypes to 20 antimicrobial agents was determined, and Salmonella Muenchen was resistant to the widest array of agents and was mostly isolated from chicken sausages. Regardless of the meat of origin, all 65 Salmonella isolates were resistant to at least four antimicrobial agents: amikacin, gentamicin, cefuroxime, and tombramycin. This resistance profile group was the most common in all four sausage types, comprising 90% of all Salmonella isolates from beef, 71% from pork, 63% from mutton, and 35% from chicken. These results suggest that raw sausages pose a risk of transmitting multidrug-resistant Salmonella isolates to consumers.

scholarly journals Detection and typing of vancomycin-resistance genes of enterococci from clinical and nosocomial surveillance specimens by multiplex PCR

2001 ◽  
Vol 126 (3) ◽  
pp. 357-363 ◽  
Author(s):  
J. J. LU ◽  
C. L. PERNG ◽  
T. S. CHIUEH ◽  
S. Y. LEE ◽  
C. H. CHEN ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Resistance Genes ◽  
Culture Method ◽  
Vancomycin Resistance ◽  
Conventional Culture ◽  
Multiplex Pcr Assay ◽  
Vancomycin Resistant ◽  
Surveillance Specimens ◽  
Conventional Culture Method

Ninety-three clinical isolates of vancomycin-resistant enterococci (VRE) collected from nine hospitals in Taiwan were examined for the presence of vanA, vanB, vanC1, or vanC2/vanC3 genes by a multiplex PCR. Forty-seven of these VRE isolates were vanA positive, 1 contained both vanC1 and vanA, 40 harboured vanB, 2 were vanC1, and 3 were identified to be vanC2/vanC3. Twenty-four vanA isolates were sensitive to teicoplanin and thus did not have a typical VanA phenotype. Five isolates with the VanC phenotype harboured vanB. None of the 40 clinically isolated vancomycin-susceptible E. faecium or E. faecalis and the vancomycin-resistant Leuconostoc and Pediococcus isolates were positive for any of the van genes. While performing nosocomial surveillance, VRE were isolated from 47 of 467 rectal swabs by culture. Compared with the conventional culture method, the sensitivity and specificity of the multiplex PCR for detecting and identifying vancomycin-resistance genes in enterococci directly from culture-positive broth were 97·9% and 100%, respectively. The results suggest that genotypic characterization of vancomycin-resistance is necessary for all clinical VRE isolates and that the multiplex PCR assay can be an alternative method for this purpose.

scholarly journals Limitations of Using Propidium Monoazide with qPCR to Discriminate between Live and Dead Legionella in Biofilm Samples

2014 ◽  
Vol 7 ◽  
pp. MBI.S17723 ◽  
Author(s):  
Michael J. Taylor ◽  
Richard H. Bentham ◽  
Kirstin E. Ross
Keyword(s):  
Public Health ◽  
Real Time ◽  
Real Time Pcr ◽  
Culture Method ◽  
Propidium Monoazide ◽  
Factors Affecting ◽  
Conventional Culture ◽  
Target Dna ◽  
The Relationship ◽  
Conventional Culture Method

Accurately quantifying Legionella for regulatory purposes to protect public health is essential. Real-time PCR (qPCR) has been proposed as a better method for detecting and enumerating Legionella in samples than conventional culture method. However, since qPCR amplifies any target DNA in the sample, the technique's inability to discriminate between live and dead cells means that counts are generally significantly overestimated. Propidium monoazide (PMA) has been used successfully in qPCR to aid live/dead discrimination. We tested PMA use as a method to count only live Legionella cells in samples collected from a modified chemostat that generates environmentally comparable samples. Counts from PMA-treated samples that were pretreated with either heat or three types of disinfectants (to kill the cells) were highly variable, with the only consistent trend being the relationship between biofilm mass and numbers of Legionella cells. Two possibilities explain this result: 1. PMA treatment worked and the subsequent muted response of Legionella to disinfection treatment is a factor of biofilm/microbiological effects; although this does not account for the relationship between the amount of biofilm sampled and the viable Legionella count as determined by PMA-qPCR; or 2. PMA treatment did not work, and any measured decrease or increase in detectable Legionella is because of other factors affecting the method. This is the most likely explanation for our results, suggesting that higher concentrations of PMA might be needed to compensate for the presence of other compounds in an environmental sample or that lower amounts of biofilm need to be sampled. As PMA becomes increasingly toxic at higher concentrations and is very expensive, augmenting the method to include higher PMA concentrations is both counterproductive and cost prohibitive. Conversely, if smaller volumes of biofilm are used, the reproducibility of the method is reduced. Our results suggest that using PMA is not an appropriate method for discriminating between live and dead cells to enumerate Legionella for regulatory purposes.

Rapid, Specific Detection of Salmonella Enteritidis in Pooled Eggs by Real-Time PCR

2004 ◽  
Vol 67 (5) ◽  
pp. 864-869 ◽  
Author(s):  
K. H. SEO ◽  
I. E. VALENTIN-BON ◽  
R. E. BRACKETT ◽  
P. S. HOLT
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Salmonella Enteritidis ◽  
Culture Method ◽  
Specific Detection ◽  
Culture Methods ◽  
Conventional Culture ◽  
Low Concentrations ◽  
Pcr Method ◽  
Group D

An assay was developed for the specific detection of Salmonella Enteritidis in eggs with the use of an application of the fluorogenic 5′ nuclease assay (TaqMan). In this assay, a segment of the gene sefA specific to Salmonella group D strains such as Salmonella Enteritidis was used. The amplification of the target gene products was monitored in real-time by incorporating a fluorescent dye–labeled gene-specific probe in the PCR reaction. This method correctly detected and distinguished Salmonella Enteritidis from nearly 50 of non–group D Salmonella and other non- Salmonella strains. Detection of the sefA gene was linear for DNA extracted from approximately 102 to 109 CFU/ml in phosphate-buffered saline and 103 to 108 CFU/ml in raw egg. In two trials, when applied to detection of Salmonella Enteritidis in homogenized egg pools and compared with conventional culture methods, the newly developed PCR method yielded a 100% correlation with results obtained by a conventional culture method. However, the PCR method required only 2 days, compared to the 5 days required by the culture method. The sensitivity of this assay was approximately less than 1 CFU/600 g of egg pool. The real-time PCR assay proved to be a rapid, highly sensitive test for detection and quantification of low concentrations of Salmonella Enteritidis in egg samples.

scholarly journals Detection of Vibrio cholerae O1 and O139 in Environmental Water Samples by an Immunofluorescent-Aggregation Assay

2010 ◽  
Vol 76 (16) ◽  
pp. 5520-5525 ◽  
Author(s):  
Duochun Wang ◽  
Xuebin Xu ◽  
Xiaoling Deng ◽  
Changyi Chen ◽  
Baisheng Li ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Real Time ◽  
Water Samples ◽  
Real Time Pcr ◽  
High Specificity ◽  
Culture Method ◽  
Environmental Water ◽  
Estuarine Water ◽  
Conventional Culture ◽  
Conventional Culture Method

ABSTRACT Environmental waters are an important reservoir for Vibrio cholerae, and effective surveillance of the pathogen can help to warn of and prevent infection with this potentially fatal pathogen. An immunofluorescent-aggregation (IFAG) assay to detect V. cholerae O1 and O139 was established and evaluated with estuarine water samples. The practical application of this assay was compared with the conventional culture method and real-time PCR. The IFAG method had a sensitivity of 103 CFU/ml for detection of V. cholerae O1 and O139 strains in a suspension containing 10 different species of enterobacterial strains (total, 105 CFU/ml). Ten fluorescent bacterial aggregate colonies were randomly picked and tested positive in serum agglutination tests for the V. cholerae O1 and O139 strains, showing a high specificity. The enrichment broths of 146 samples of estuarine water were tested, and the percentage positive by the IFAG assay was 19.9% (29/146), which was significantly higher than that of the conventional culture method (10.3%, 15/146; P < 0.01) but lower than that of real-time PCR (29.5%, 43/146; P < 0.01). The coincidence rates of real-time PCR and IFAG detection were decreased with the reduction of the V. cholerae concentration. The IFAG method, with a high specificity and a relatively high sensitivity, may be used for detection and isolation of V. cholerae in environmental water samples.

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