scholarly journals Screening and Detecting Salmonella in Different Food Matrices in Southern Tunisia Using a Combined Enrichment/Real-Time PCR Method: Correlation with Conventional Culture Method

2017 ◽  
Vol 8 ◽  
Author(s):  
Mariam Siala ◽  
Amina Barbana ◽  
Salma Smaoui ◽  
Salma Hachicha ◽  
Chema Marouane ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Culture Method ◽  
Conventional Culture ◽  
Southern Tunisia ◽  
Pcr Method ◽  
Food Matrices ◽  
Conventional Culture Method

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.

ELIME assay vs Real-Time PCR and conventional culture method for an effective detection of Salmonella in fresh leafy green vegetables

Talanta ◽  
2017 ◽  
Vol 166 ◽  
pp. 321-327 ◽  
Author(s):  
L. Fabiani ◽  
E. Pucci ◽  
E. Delibato ◽  
G. Volpe ◽  
S. Piermarini ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Culture Method ◽  
Conventional Culture ◽  
Conventional Culture Method

scholarly journals Comparison of OmpA Gene-Targeted Real-Time PCR with the Conventional Culture Method for Detection of Acinetobacter baumanii in Pneumonic BALB/c Mice

2019 ◽  
Vol 23 (2) ◽  
pp. 159-164 ◽  
Author(s):  
Niloofar Hassannejad ◽  
Abbas Bahador ◽  
Nasim Hayati Rudbari ◽  
Mohammad Hossein Modarressi ◽  
Kazem Parivar ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Culture Method ◽  
Ompa Gene ◽  
Acinetobacter Baumanii ◽  
Conventional Culture ◽  
Conventional Culture Method

Preliminary Evaluation of Flow-Through Immunocapture followed by Real-Time PCR for the Detection of Salmonella Serovars on Tomato Surfaces within 8 Hours

2006 ◽  
Vol 69 (9) ◽  
pp. 2253-2257 ◽  
Author(s):  
HYUN-GYUN YUK ◽  
BENJAMIN R. WARREN ◽  
KEITH R. SCHNEIDER
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Culture Method ◽  
Preliminary Evaluation ◽  
Conventional Culture ◽  
Significant Difference ◽  
Assay Time ◽  
Flow Through ◽  
Pcr Method ◽  
Salmonella Serovars

This study reports a preliminary evaluation of flow-through immunocapture (FTI) followed by real-time PCR (FTI-PCR) for the detection of Salmonella serovars on tomato surfaces within 8 h. The FTI-PCR method was compared with real-time PCR, direct plating of FTI beads on xylose lysine desoxycholate (XLD), and the conventional culture method for Salmonella found in the U.S. Food and Drug Administration's (FDA) Bacteriological Analytical Manual (BAM). Unwaxed green tomatoes were spot inoculated with a five-serovar Salmonella cocktail on smooth surfaces at levels of 100 to 104 CFU per tomato and washed in lactose broth (LB) using a shake-rub method. The resulting LB rinse was incubated at 37°C for 4 h prior to analysis by FTI-XLD, real-time PCR, or FTI-PCR and for 24 h as the first step in the BAM Salmonella culture method. For FTI-XLD, the observed lowest detection level (LDL) was 4.6 × 101 CFU per tomato. There was no significant difference in performance between the FTI-XLD method and the BAM Salmonella culture method (P &gt; 0.05); however, the FTI-XLD method reduced the overall assay time by 48 h. For real-time PCR and FTI-PCR, the observed LDLs were 4.6 × 101 and 9.2 × 100 CFU per tomato, respectively. The FTI-PCR method was superior to the BAM Salmonella culture method (P &lt; 0.05) for the detection of Salmonella serovars on tomato surfaces and was completed within 8 h.

An Evaluation of Conventional Culture, invA PCR, and the Real-Time PCR iQ-Check Kit as Detection Tools for Salmonella in Naturally Contaminated Premarket and Retail Turkey

2008 ◽  
Vol 71 (2) ◽  
pp. 386-391 ◽  
Author(s):  
CHANTAL W. NDE ◽  
MOHAMED K. FAKHR ◽  
CURT DOETKOTT ◽  
CATHERINE M. LOGUE
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Culture Method ◽  
False Positives ◽  
Poultry Meat ◽  
Processing Plant ◽  
False Negatives ◽  
Conventional Culture ◽  
Total Agreement ◽  
Conventional Culture Method

This study was aimed at comparing the ability of conventional culture, the iQ-Check real-time PCR kit, and invA PCR to detect Salmonella in naturally contaminated premarket and retail turkey parts. Premarket (n = 120) turkey parts collected from a commercial turkey processing plant, and retail turkey parts (n = 138) were examined. Both PCR methods detected a significantly greater (P &lt; 0.05) number of positive samples when compared with the conventional culture method for the premarket turkey parts. The indices of total agreement between the conventional culture method and the iQ-Check kit for the premarket and retail parts were 79.2% (95% CI: 70.8, 86) and 90.6% (95% CI: 84.4, 94.9), respectively. When the conventional culture method was compared with invA PCR for Salmonella detection in the premarket and retail parts, the indices of total agreement were 75.8% (95% CI: 67.2, 83.2) and 84.1% (95% CI: 76.9, 89.7), respectively. The rates of false positives (premarket: 31.9%, retail: 9.7%) and false negatives (premarket: 5.9%, retail: 9.7%) were determined between the culture method and the iQ-Check kit. When invA PCR was compared with the culture method, the rates of false positives (premarket: 37.7%, retail: 11.1%) and false negatives (premarket: 5.9%, retail: 18.3%) were obtained. The higher total agreement and the lower rates of both false positives and false negatives for the iQ-Check kit compared with invA PCR for both premarket and retail turkey parts corroborates the use of the iQ-Check kit as a screening tool for Salmonella in poultry meat.

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