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.

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

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.

Comparison of a Real-Time PCR Method with a Culture Method for the Detection of Salmonella enterica Serotype Enteritidis in Naturally Contaminated Environmental Samples from Integrated Poultry Houses

2012 ◽  
Vol 75 (4) ◽  
pp. 743-747 ◽  
Author(s):  
BWALYA LUNGU ◽  
W. DOUGLAS WALTMAN ◽  
ROY D. BERGHAUS ◽  
CHARLES L. HOFACRE
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Salmonella Enteritidis ◽  
Culture Method ◽  
Pcr Assay ◽  
Culture Methods ◽  
The Real ◽  
Selective Enrichment ◽  
Conventional Culture ◽  
Field Samples

Conventional culture methods have traditionally been considered the “gold standard” for the isolation and identification of foodborne bacterial pathogens. However, culture methods are labor-intensive and time-consuming. A Salmonella enterica serotype Enteritidis–specific real-time PCR assay that recently received interim approval by the National Poultry Improvement Plan for the detection of Salmonella Enteritidis was evaluated against a culture method that had also received interim National Poultry Improvement Plan approval for the analysis of environmental samples from integrated poultry houses. The method was validated with 422 field samples collected by either the boot sock or drag swab method. The samples were cultured by selective enrichment in tetrathionate broth followed by transfer onto a modified semisolid Rappaport-Vassiliadis medium and then plating onto brilliant green with novobiocin and xylose lysine brilliant Tergitol 4 plates. One-milliliter aliquots of the selective enrichment broths from each sample were collected for DNA extraction by the commercial PrepSEQ nucleic acid extraction assay and analysis by the Salmonella Enteritidis–specific real-time PCR assay. The real-time PCR assay detected no significant differences between the boot sock and drag swab samples. In contrast, the culture method detected a significantly higher number of positive samples from boot socks. The diagnostic sensitivity of the real-time PCR assay for the field samples was significantly higher than that of the culture method. The kappa value obtained was 0.46, indicating moderate agreement between the real-time PCR assay and the culture method. In addition, the real-time PCR method had a turnaround time of 2 days compared with 4 to 8 days for the culture method. The higher sensitivity as well as the reduction in time and labor makes this real-time PCR assay an excellent alternative to conventional culture methods for diagnostic purposes, surveillance, and research studies to improve food safety.

Sign in / Sign up

Export Citation Format

Share Document