A misleading false-negative result of
            Pneumocystis
            real-time PCR assay due to a rare punctual mutation: A French multicenter study

A real-time PCR assay was evaluated for the rapid detection (10 h) ofSalmonellain meats using molecular beacon probes available as a commercial kit (iQ-Check, Bio-Rad laboratories). Raw (chicken, pork) and ready-to-eat (RTE) meats were artificially contaminated withSalmonella entericaserovar Typhimurium at the estimated level of 2 to 4 cells per 25 g. After 8 h of pre-enrichment in buffered peptone water, a molecular beacon-based PCR assay was performed to detect contamination in raw and RTE meats. The sensitivity and accuracy of the assay were compared with the conventional USDA microbiological procedure. Comparative evaluation of the USDA procedure with the rapid PCR assay for meat samples (n = 63) revealed 1 false negative pork sample with the PCR assay. All uninoculated controls (n = 34) but one sample were negative by both the 10-h PCR assay and the USDA procedure. Developing rapid pathogen detection methods with shorter pre-enrichment times (8-h) and real-time data monitoring capabilities will benefit the industry in preventing recall of contaminated meats by stopping the contaminated products from being introduced into the marketplace.

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Evaluation of an Immunomagnetic Separation–Real-Time PCR Assay for the Rapid Detection of Salmonella in Meat

Journal of Food Protection ◽

10.4315/0362-028x-69.12.2896 ◽

2006 ◽

Vol 69 (12) ◽

pp. 2896-2901 ◽

Cited By ~ 27

Author(s):

ANGELIKA NOTZON ◽

REINER HELMUTH ◽

JOHANN BAUER

Keyword(s):

Real Time ◽

Real Time Pcr ◽

False Positive Rate ◽

False Negative ◽

False Negative Rate ◽

Reference Method ◽

Immunomagnetic Separation ◽

Pcr Assay ◽

Positive Rate ◽

Meat Samples

The aim of this study was the comparison of an immunomagnetic separation (IMS)–real-time PCR assay for the detection of Salmonella with the cultural reference method according to §35 of the German Law on Food and Commodities (LMBG, L 00.00.20:1998). The IMS–real-time PCR assay includes a nonselective preenrichment step, an IMS, DNA extraction, as well as DNA purification followed by hybridization probe–based real-time PCR analysis. An accurate comparability was achieved, because both methods analyzed the same preenrichment. The evaluation was carried out using both artificially and naturally contaminated meat samples. The IMS–real-time PCR assay provides a result after 12 to 13 h. Compared with the reference method and regarding artificially contaminated meat samples, the IMS–real-time PCR assay achieved a specificity of 80% (false-positive rate of 20%) and a sensitivity of 100% (false-negative rate of 0%). The relative accuracy was 94%. The detection limit of both methods was 10 CFU/25 g. The concordance indexκ defines the statistical accordance, was 0.85 and indicated the agreement of both methods on statistical criteria. Compared to the reference method and analyzing naturally contaminated meat samples (n = 491), the IMS–real-time PCR assay showed a specificity of 99.3% (false-positive rate of 0.7%) and a sensitivity of 83.7% (false-negative rate of 16.3%). The relative accuracy was 98%. The concordance index κ had a value of 0.87 and highlighted the statistical agreement of both methods. In conclusion, the IMS–real-time PCR assay is suitable as specific, sensitive, and rapid screening method for the detection of Salmonella from meat.

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Evaluation of a novel real-time PCR assay for the detection, identification and quantification of Plasmodium species causing malaria in humans

Malaria Journal ◽

10.1186/s12936-021-03842-8 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Kim van Bergen ◽

Toon Stuitje ◽

Robert Akkers ◽

Eric Vermeer ◽

Rob Castel ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Limit Of Detection ◽

False Negative ◽

Performance Characteristics ◽

Analytical Performance ◽

Melting Curve Analysis ◽

Mixed Infections ◽

Plasmodium Ovale ◽

Pcr Assay

Abstract Background The entry of PCR-based techniques into malaria diagnostics has improved the sensitivity and specificity of the detection of Plasmodium infections. It has been shown that humans are regularly infected by at least six different Plasmodium species. The MC004 real-time PCR assay for malaria diagnosis is a novel single-tube assay that has been developed for the purpose of simultaneously detecting all Plasmodium species known to infect humans, and discrimination between Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale wallikeri, Plasmodium ovale curtisi, Plasmodium knowlesi (including differentiation of three strains) and Plasmodium cynomolgi (including differentiation of three strains). Detection and identification of Plasmodium species relies on molecular beacon probe-based melting curve analysis. In addition, this assay might be used to quantify the parasitaemia of at least P. falciparum by calculating the level of parasitaemia directly from the Cq-value. Methods The samples used in this study comprised reference samples, patient samples, and synthetic controls. The following analytical performance characteristics of the MC004 assay were determined: analytical specificity, limit of detection, the ability to detect mixed infections, and the potential to determine the level of parasitaemia of P. falciparum, including assessment of within-run and between-run precisions. Results No false positive or false negative results were observed. The limit of detection of P. falciparum was 1 × 10–3 IU/mL (WHO standard). Mixed infections with P. falciparum and non-falciparum species were correctly identified. A calibration curve could be established to quantify the parasitaemia of at least P. falciparum. The within-run and between-run precisions were less than 20% CV at the tested parasitaemia levels of 0.09%, 0.16%, 2.15% and 27.27%. Conclusion Based upon the analytical performance characteristics that were determined, the MC004 assay showed performance suitable for use in clinical settings, as well as epidemiological studies.

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DuPont Qualicon BAX® System Real-Time PCR Assay for Escherichia coli O157:H7

Journal of AOAC International ◽

10.1093/jaoac/94.4.1117 ◽

2011 ◽

Vol 94 (4) ◽

pp. 1117-1124 ◽

Cited By ~ 1

Author(s):

Frank Burns ◽

Lois Fleck ◽

Bridget Andaloro ◽

Eugene Davis ◽

Jeff Rohrbeck ◽

...

Keyword(s):

Shelf Life ◽

Real Time ◽

Real Time Pcr ◽

False Negative ◽

Method Comparison ◽

Pcr Assay ◽

Culture Methods ◽

Life Study ◽

Test Kit ◽

Consistent Performance

Abstract Evaluations were conducted to test the performance of the BAX® System Real-Time PCR assay, which was certified as Performance Tested MethodSM 031002 for screening E. coli O157:H7 in ground beef, beef trim, spinach, and lettuce. Method comparison studies performed on samples with low-level inoculates showed that the BAX System demonstrates a sensitivity equivalent or superior to the FDA-BAM and the USDA-FSIS culture methods, but with a significantly shorter time to result. Tests to evaluate inclusivity and exclusivity returned no false-negative and no false-positive results on a diverse panel of isolates, and tests for lot-to-lot variability and tablet stability demonstrated consistent performance. Ruggedness studies determined that none of the factors examined affect the performance of the assay. An accelerated shelf life study determined an initial 36 month shelf life for the test kit.

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Assessment of Listeria sp. Interference Using a Molecular Assay To Detect Listeria monocytogenes in Food

Journal of Food Protection ◽

10.4315/0362-028x.jfp-15-122 ◽

2016 ◽

Vol 79 (1) ◽

pp. 138-143 ◽

Cited By ~ 2

Author(s):

SANDRA I. ZITTERMANN ◽

BRENDA STANGHINI ◽

RYAN SOO SEE ◽

ROBERTO G. MELANO ◽

PETER BOLESZCZUK ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Real Time ◽

Real Time Pcr ◽

False Negative ◽

Reference Method ◽

Food Samples ◽

Pcr Assay ◽

The Real ◽

Current Reference ◽

Listeria Species

ABSTRACT Detection of Listeria monocytogenes in food is currently based on enrichment methods. When L. monocytogenes is present with other Listeria species in food, the species compete during the enrichment process. Overgrowth competition of the nonpathogenic Listeria species might result in false-negative results obtained with the current reference methods. This potential issue was noted when 50 food samples artificially spiked with L. monocytogenes were tested with a real-time PCR assay and Canada's current reference method, MFHPB-30. Eleven of the samples studied were from foods naturally contaminated with Listeria species other than those used for spiking. The real-time PCR assay detected L. monocytogenes in all 11 of these samples; however, only 6 of these samples were positive by the MFHPB-30 method. To determine whether L. monocytogenes detection can be affected by other species of the same genus due to competition, an L. monocytogenes strain and a Listeria innocua strain with a faster rate of growth in the enrichment broth were artificially coinoculated at different ratios into ground pork meat samples and cultured according to the MFHPB-30 method. L. monocytogenes was detected only by the MFHPB-30 method when L. monocytogenes/L. innocua ratios were 6.0 or higher. In contrast, using the same enrichments, the real-time PCR assay detected L. monocytogenes at ratios as low as 0.6. Taken together, these findings support the hypothesis that L. monocytogenes can be outcompeted by L. innocua during the MFHPB-30 enrichment phase. However, more reliable detection of L. monocytogenes in this situation can be achieved by a PCR-based method mainly because of its sensitivity.

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Pentaplexed Quantitative Real-Time PCR Assay for the Simultaneous Detection and Quantification of Botulinum Neurotoxin-Producing Clostridia in Food and Clinical Samples

Applied and Environmental Microbiology ◽

10.1128/aem.02490-09 ◽

2010 ◽

Vol 76 (13) ◽

pp. 4387-4395 ◽

Cited By ~ 39

Author(s):

Sebastian Kirchner ◽

K. Melanie Krämer ◽

Martin Schulze ◽

Diana Pauly ◽

Daniela Jacob ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Target Genes ◽

False Negative ◽

Simultaneous Detection ◽

Locked Nucleic Acid ◽

Clinical Samples ◽

Quantitative Real Time Pcr ◽

Pcr Assay ◽

Pcr Assays

ABSTRACT Botulinum neurotoxins are produced by the anaerobic bacterium Clostridium botulinum and are divided into seven distinct serotypes (A to G) known to cause botulism in animals and humans. In this study, a multiplexed quantitative real-time PCR assay for the simultaneous detection of the human pathogenic C. botulinum serotypes A, B, E, and F was developed. Based on the TaqMan chemistry, we used five individual primer-probe sets within one PCR, combining both minor groove binder- and locked nucleic acid-containing probes. Each hydrolysis probe was individually labeled with distinguishable fluorochromes, thus enabling discrimination between the serotypes A, B, E, and F. To avoid false-negative results, we designed an internal amplification control, which was simultaneously amplified with the four target genes, thus yielding a pentaplexed PCR approach with 95% detection probabilities between 7 and 287 genome equivalents per PCR. In addition, we developed six individual singleplex real-time PCR assays based on the TaqMan chemistry for the detection of the C. botulinum serotypes A, B, C, D, E, and F. Upon analysis of 42 C. botulinum and 57 non-C. botulinum strains, the singleplex and multiplex PCR assays showed an excellent specificity. Using spiked food samples we were able to detect between 103 and 105 CFU/ml, respectively. Furthermore, we were able to detect C. botulinum in samples from several cases of botulism in Germany. Overall, the pentaplexed assay showed high sensitivity and specificity and allowed for the simultaneous screening and differentiation of specimens for C. botulinum A, B, E, and F.

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