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 > 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 < 0.05) for the detection of Salmonella serovars on tomato surfaces and was completed within 8 h.

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.

Detection of Salmonella by Flow-Through Immunocapture Real-Time PCR in Selected Foods within 8 Hours

2007 ◽  
Vol 70 (4) ◽  
pp. 1002-1006 ◽  
Author(s):  
BENJAMIN R. WARREN ◽  
HYUN-GYUN YUK ◽  
KEITH R. SCHNEIDER
Keyword(s):  
Real Time ◽  
Drug Administration ◽  
Real Time Pcr ◽  
Food And Drug Administration ◽  
Ground Beef ◽  
Culture Method ◽  
Food Samples ◽  
Flow Through ◽  
Pcr Method

This study investigated flow-through immunocapture (FTI), using the Pathatrix device, followed by plating on xylose lysine desoxycholate (XLD) agar (FTI-XLD) or analysis by real-time PCR (FTI-PCR) for the detection of Salmonella on smooth tomato surfaces and in potato salad and ground beef within 8 h. Food samples were inoculated with an appropriate dilution of a five-serovar Salmonella cocktail and enriched for 5 h. Following enrichment, samples were analyzed by the FTIXLD and FTI-PCR methods. Food samples were also analyzed by a modified U.S. Food and Drug Administration Bacteriological Analytical Manual (BAM) Salmonella culture method for comparison. Salmonella inoculated at 100 CFU per tomato or 100 CFU/25 g was detected by the FTI-XLD method in 6, 8, and 4 of 10 samples for tomatoes, potato salad, and ground beef, respectively. Salmonella inoculated at 100 CFU per tomato or 100 CFU/25 g was detected by the FTI-PCR method in 8, 9, and 9 of 10 samples for tomatoes, potato salad, and ground beef, respectively. The FTI-PCR method achieved significantly higher (P < 0.05) detection of Salmonella on tomatoes, whereas the FTI-XLD method achieved significantly lower (P < 0.05) detection of Salmonella in ground beef when compared with the modified BAM Salmonella culture method; however, all other comparisons to the modified BAM method were not significantly different. The FTI-XLD method demonstrated the ability to isolate presumptive Salmonella colonies up to 48 h faster than did the modified BAM Salmonella culture method.

Detection of Salmonella in Swine Fecal Samples by Flow-Through Immunocapture

2010 ◽  
Vol 73 (5) ◽  
pp. 957-959 ◽  
Author(s):  
KEN KATSUDA ◽  
MARIKO KOHMOTO ◽  
OSAMU MIKAMI
Keyword(s):  
Real Time ◽  
Lower Limit ◽  
Real Time Pcr ◽  
The Other ◽  
Preliminary Evaluation ◽  
Culture Methods ◽  
Direct Plating ◽  
Fecal Samples ◽  
Flow Through ◽  
Pcr Method

This study reports the preliminary evaluation of flow-through immunocapture (FTI) followed by real-time PCR (FTI-PCR) for the detection of different serotypes of Salmonella in pig fecal specimens. The FTI-PCR method was compared with the direct plating of FTI beads on xylose lysine desoxycholate agar, real-time PCR, and the conventional culturing methods, Rappaport-Vassiliadis and modified semisolid Rappaport-Vassiliadis. Artificially contaminated swine fecal specimens were used to evaluate and compare these methods. The results of our comparisons indicate that the FTI-PCR, FTI-plating, and modified semisolid Rappaport-Vassiliadis culture methods were accurate, specific, and sensitive. Moreover, FTI-PCR was the fastest method, providing results in fewer than 20 h, as opposed to the 48 to 96 h required for the other methods. Our results indicate that FTIPCR could be a useful tool for detecting Salmonella in swine fecal specimens at a lower limit of 1.0 CFU/g.

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.

A Real-Time PCR Assay for the Detection of Salmonella in a Wide Variety of Food and Food-Animal Matrices†

2007 ◽  
Vol 70 (5) ◽  
pp. 1080-1087 ◽  
Author(s):  
V. M. BOHAYCHUK ◽  
G. E. GENSLER ◽  
M. E. McFALL ◽  
R. K. KING ◽  
D. G. RENTER
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Culture Method ◽  
Pcr Assay ◽  
Culture Methods ◽  
The Real ◽  
Conventional Culture ◽  
Food Animal ◽  
Highly Sensitive ◽  
Field Samples

Conventional culture methods have traditionally been considered the “gold standards” for the isolation and identification of foodborne pathogens. However, culture methods are labor-intensive and time-consuming. We have developed a real-time PCR assay for the detection of Salmonella in a variety of food and food-animal matrices. The real-time PCR assay incorporates both primers and hybridization probes based on the sequence of the Salmonella invA gene and uses fluorescent resonance energy transfer technology to ensure highly sensitive and specific results. This method correctly classified 51 laboratory isolates of Salmonella and 28 non-Salmonella strains. The method was also validated with a large number of field samples that consisted of porcine feces and cecal contents, pork carcasses, bovine feces and beef carcasses, poultry cecal contents and carcasses, equine feces, animal feeds, and various food products. The samples (3,388) were preenriched in buffered peptone water and then selectively enriched in tetrathionate and Rappaport-Vassiliadis broths. Aliquots of the selective enrichment broths were combined for DNA extraction and analysis by the real-time PCR assay. When compared with the culture method, the diagnostic sensitivity of the PCR assay for the various matrices ranged from 97.1 to 100.0%, and the diagnostic specificity ranged from 91.3 to 100.0%. Kappa values ranged from 0.87 to 1.00, indicating excellent agreement of the real-time PCR assay to the culture method. The reduction in time and labor makes this highly sensitive and specific real-time PCR assay an excellent alternative to conventional culture methods for surveillance and research studies to improve food safety.

scholarly journals Diversity of KIR genes and their HLA-C ligands in Ugandan populations with historically varied malaria transmission intensity

2021 ◽  
Author(s):  
Stephen Tukwasibwe ◽  
James A. Traherne ◽  
Olympe Chazara ◽  
Jyothi Jayaraman ◽  
John Trowsdale ◽  
...  
Keyword(s):  
Infectious Diseases ◽  
Malaria Transmission ◽  
Real Time ◽  
High Throughput ◽  
Real Time Pcr ◽  
Association Studies ◽  
Transmission Intensity ◽  
Malaria Transmission Intensity ◽  
Significant Difference ◽  
Pcr Method

Abstract Background: Malaria is one of the most serious infectious diseases in the world. The malaria burden is greatly affected by human immunity, and immune responses vary between populations. Genetic diversity in KIR and HLA-C genes, which are important in immunity to infectious diseases, is likely to play a role in this heterogeneity. Several studies have shown that KIR and HLA-C genes influence the immune response to viral infections, but few studies have examined the role of KIR and HLA-C in malaria infection, and these have used low-resolution genotyping. The aim of this study was to determine whether genetic variation in KIR and their HLA-C ligands differ in Ugandan populations with historically varied malaria transmission intensity using more comprehensive genotyping approaches.Methods: High throughput multiplex quantitative real-time PCR method was used to genotype KIR genetic variants and copy number variation and a high-throughput real-time PCR method was developed to genotype HLA-C1 and C2 allotypes for 1,344 participants, aged 6 months to 10 years, enrolled from Ugandan populations with historically high (Tororo District), medium (Jinja District) and low (Kanungu District) malaria transmission intensity. Results: The prevalence of KIR3DS1, KIR2DL5, KIR2DS5 and KIR2DS1 genes was significantly lower in populations from Kanungu compared to Tororo (7.6% vs. 13.2%: p=0.006, 57.2% vs. 66.4%: p=0.005, 33.2% vs. 46.6%: p<0.001 and 19.7% vs. 26.7%: p=0.014 respectively) or Jinja (7.6% vs.18.1%: p<0.001, 57.2% vs. 63.8%: p=0.048, 33.2% vs. 43.5%: p=0.002 and 19.7% vs. 30.4%: p<0.001 respectively). The prevalence of homozygous HLA-C2 was significantly higher in populations from Kanungu (31.6%) compared to Jinja (21.4%), p=0.043, with no significant difference between Kanungu and Tororo (26.7%), p=0.296. Conclusions: The KIR3DS1, KIR2DL5, KIR2DS5 and KIR2DS1 genes may partly explain differences in transmission intensity of malaria since these genes have been positively selected for in places with historically high malaria transmission intensity. The high-throughput multiplex real-time HLA-C genotyping PCR method developed will be useful in disease association studies involving large cohorts.

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.

scholarly journals Diversity of KIR genes and their HLA-C ligands in Ugandan populations with historically varied malaria transmission intensity

2020 ◽  
Author(s):  
Stephen Tukwasibwe ◽  
James A. Traherne ◽  
Olympe Chazara ◽  
Jyothi Jayaraman ◽  
John Trowsdale ◽  
...  
Keyword(s):  
Infectious Diseases ◽  
Malaria Transmission ◽  
Real Time ◽  
High Throughput ◽  
Real Time Pcr ◽  
Association Studies ◽  
Transmission Intensity ◽  
Malaria Transmission Intensity ◽  
Significant Difference ◽  
Pcr Method

Abstract Background: Malaria is one of the most serious infectious diseases in the world. The malaria burden is greatly affected by human immunity, and immune responses vary between populations. Genetic diversity in KIR and HLA-C genes, which are important in immunity to infectious diseases, is likely to play a role in this heterogeneity. Several studies have shown that KIR and HLA-C genes influence the immune response to viral infections, but few studies have examined the role of KIR and HLA-C in malaria infection, and these have used low-resolution genotyping. Our aim was to determine whether genetic variation in KIR and their HLA-C ligands differ in Ugandan populations with historically varied malaria transmission intensity using more comprehensive genotyping approaches. Methods: We used high throughput multiplex quantitative real-time PCR method to genotype KIR genetic variants and copy number variation and developed a high-throughput real-time PCR method to genotype HLA-C1 and C2 allotypes for 1,344 participants, aged 6 months to 10 years, enrolled from Ugandan populations with historically high (Tororo District), medium (Jinja District) and low (Kanungu District) malaria transmission intensity. Results: The prevalence of KIR3DS1, KIR2DL5, KIR2DS5 and KIR2DS1 genes was significantly lower in populations from Kanungu compared to Tororo (7.6% vs. 13.2%: p=0.006, 57.2% vs. 66.4%: p=0.005, 33.2% vs. 46.6%: p<0.001 and 19.7% vs. 26.7%: p=0.014 respectively) or Jinja (7.6% vs.18.1%: p<0.001, 57.2% vs. 63.8%: p=0.048, 33.2% vs. 43.5%: p=0.002 and 19.7% vs. 30.4%: p<0.001 respectively). The prevalence of homozygous HLA-C2 was significantly higher in populations from Kanungu (31.6%) compared to Jinja (21.4%), p=0.043, with no significant difference between Kanungu and Tororo (26.7%), p=0.296. Conclusions: The KIR3DS1 , KIR2DL5, KIR2DS5 and KIR2DS1 genes are potentially beneficial in malaria as these genes have been positively selected for in places with historically high malaria transmission intensity. The high-throughput multiplex real-time HLA-C genotyping PCR method we have developed will be useful in disease association studies involving large cohorts.

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