A filtration-based real-time PCR method for the quantitative detection of viable Salmonella enterica and Listeria monocytogenes in food samples

Abstract Context.—BK virus infections among immunocompromised patients are associated with disease of the kidney or urinary bladder. High viral loads, determined by quantitative polymerase chain reaction (PCR), have been correlated with clinical disease. Objective.—To develop and evaluate a novel method for real-time PCR detection and quantification of BK virus using labeled primers. Design.—Patient specimens (n = 54) included 17 plasma, 12 whole blood, and 25 urine samples. DNA was extracted using the MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche Applied Science, Indianapolis, Indiana); sample eluate was PCR-amplified using the labeled primer PCR method. Results were compared with those of a user-developed quantitative real-time PCR method (fluorescence resonance energy transfer probe hybridization). Results.—Labeled primer PCR detected less than 10 copies per reaction and showed quantitative linearity from 101 to 107 copies per reaction. Analytical specificity of labeled primer PCR was 100%. With clinical samples, labeled primer PCR demonstrated a trend toward improved sensitivity compared with the reference method. Quantitative assay comparison showed an R2 value of 0.96 between the 2 assays. Conclusions.—Real-time PCR using labeled primers is highly sensitive and specific for the quantitative detection of BK virus from a variety of clinical specimens. These data demonstrate the applicability of labeled primer PCR for quantitative viral detection and offer a simplified method that removes the need for separate oligonucleotide probes.

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Quantitative Detection of Campylobacter jejuni on Fresh Chicken Carcasses by Real-Time PCR

Journal of Food Protection ◽

10.4315/0362-028x-70.6.1373 ◽

2007 ◽

Vol 70 (6) ◽

pp. 1373-1378 ◽

Cited By ~ 30

Author(s):

ANNA-CLARA RÖNNER ◽

HANS LINDMARK

Keyword(s):

Detection Limit ◽

Real Time ◽

Dna Extraction ◽

Campylobacter Jejuni ◽

Real Time Pcr ◽

Close Correlation ◽

Quantitative Detection ◽

Quantitative Real Time Pcr ◽

Direct Plating ◽

Pcr Method

Campylobacter jejuni infection is a significant cause of foodborne gastroenteritis worldwide. Consumption and handling of poultry products is believed to be the primary risk factor for campylobacteriosis. Risk assessments require quantitative data, and C. jejuni is enumerated usually by direct plating, which sometimes allows growth of non-Campylobacter bacteria. The objective of the present study was to develop a quantitative real-time PCR method (q-PCR) for enumerating C. jejuni in chicken rinse without a culturing step. The procedure to obtain the template for the PCR assay involved (i) filtration of 10 ml of chicken rinse, (ii) centrifugation of the sample, and (iii) total DNA extraction from the pellet obtained using a commercial DNA extraction kit. The detection limit of the method was comparable to that for plating 100 μl of chicken rinse on modified charcoal cefoperazone deoxycholate agar, and the detection limit could be further improved 10-fold by concentrating the DNA eluate by ethanol precipitation. A close correlation for spiked chicken rinse was obtained for the results of the quantitative real-time PCR method and direct plating (r = 0.99). The coefficient of correlation for the methods was 0.87 when samples from chicken carcasses on the slaughter line were analyzed, whereas a lower correlation (r = 0.76) was obtained when samples from retail carcasses were analyzed. Greater variation in the proportion of dead and/or viable but not culturable Campylobacter types in the retail samples may explain the decreased correlation between the methods. Overall, the new method is simple and fast and the results obtained are closely correlated with those for direct plating for samples containing a low proportion of dead Campylobacter cells.

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ISOLATION OF LISTERIA MONOCYTOGENES DNA FROM MEAT PRODUCTS FOR QUANTITATIVE DETECTION BY REAL-TIME PCR

Journal of Rapid Methods & Automation in Microbiology ◽

10.1111/j.1745-4581.2006.00058.x ◽

2006 ◽

Vol 14 (4) ◽

pp. 395-404 ◽

Cited By ~ 12

Author(s):

DAVID RODRÍGUEZ-LÁZARO ◽

MARTA HERNÁNDEZ

Keyword(s):

Listeria Monocytogenes ◽

Real Time ◽

Real Time Pcr ◽

Meat Products ◽

Quantitative Detection

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Rapid detection of Listeria monocytogenes in raw milk and soft cheese by a redox potential measurement based method combined with real-time PCR

Acta Veterinaria Hungarica ◽

10.1556/avet.2014.013 ◽

2014 ◽

Vol 62 (3) ◽

pp. 304-316 ◽

Cited By ~ 1

Author(s):

Orsolya Erdősi ◽

Katalin Szakmár ◽

Olivér Reichart ◽

Zsuzsanna Szili ◽

Noémi László ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Redox Potential ◽

Real Time ◽

Real Time Pcr ◽

Rapid Detection ◽

Raw Milk ◽

Food Samples ◽

Potential Measurement ◽

Effective Manner ◽

Soft Cheese

The incidence of outbreaks of foodborne listeriosis has indicated the need for a reliable and rapid detection of the microbe in different foodstuffs. A method combining redox potential measurement and real-time polymerase chain reaction (PCR) was developed to detectListeria monocytogenesin artificially contaminated raw milk and soft cheese. Food samples of 25 g or 25 ml were homogenised in 225 ml of Listeria Enrichment Broth (LEB) with Oxford supplement, and the redox potential measurement technique was applied. ForListeriaspecies the measuring time was maximum 34 h. The absence ofL. monocytogenescould reliably be proven by the redox potential measurement method, butListeria innocuaandBacillus subtiliscould not be differentiated fromL. monocytogeneson the basis of the redox curves. The presence ofL. monocytogeneshad to be confirmed by real-time PCR. The combination of these two methods proved to detect < 10 cfu/g ofL. monocytogenesin a cost- and time-effective manner. This method can potentially be used as an alternative to the standard nutrient method for the rapid detection ofL. monocytogenesin food.

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

Applied and Environmental Microbiology ◽

10.1128/aem.70.3.1366-1377.2004 ◽

2004 ◽

Vol 70 (3) ◽

pp. 1366-1377 ◽

Cited By ~ 169

Author(s):

David RodrÃ¯Â¿Â½guez-LÃ¯Â¿Â½zaro ◽

Marta HernÃ¯Â¿Â½ndez ◽

Mariela Scortti ◽

Teresa Esteve ◽

JosÃ¯Â¿Â½ A. VÃ¯Â¿Â½zquez-Boland ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Real Time ◽

Real Time Pcr ◽

Target Genes ◽

Dynamic Range ◽

Significant Proportion ◽

Quantitative Detection ◽

Target Sequence ◽

Food Borne ◽

Highly Sensitive

ABSTRACT We developed and assessed real-time PCR (RTi-PCR) assays for the detection and quantification of the food-borne 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).

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

Journal of Food Protection ◽

10.4315/0362-028x-70.4.1002 ◽

2007 ◽

Vol 70 (4) ◽

pp. 1002-1006 ◽

Cited By ~ 22

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.

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Quantitative Detection of Listeria monocytogenes in Biofilms by Real-Time PCR

Applied and Environmental Microbiology ◽

10.1128/aem.71.4.2190-2194.2005 ◽

2005 ◽

Vol 71 (4) ◽

pp. 2190-2194 ◽

Cited By ~ 42

Author(s):

Morgan Guilbaud ◽

Pierre de Coppet ◽

Fabrice Bourion ◽

Cinta Rachman ◽

Hervé Prévost ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Real Time ◽

Real Time Pcr ◽

Quantitative Method ◽

Sybr Green ◽

Sybr Green I ◽

Quantitative Detection ◽

Pcr Assay

ABSTRACT A quantitative method based on a real-time PCR assay to enumerate Listeria monocytogenes in biofilms was developed. The specificity for L. monocytogenes of primers targeting the listeriolysin gene was demonstrated using a SYBR Green I real-time PCR assay. The number of L. monocytogenes detected growing in biofilms was 6 × 102 CFU/cm2.

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