Standardization of diagnostic PCR for the detection of foodborne pathogens

Malorny, Burkhard; Tassios, Panayotis T.; Rådström, Peter; Cook, Nigel; Wagner, Martin; Hoorfar, Jeffrey

doi:10.1016/s0168-1605(02)00322-7

Cited by 262 publications

(160 citation statements)

References 24 publications

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“…Alternatively, rapid and versatile nucleic acid‐based techniques that detect specific DNA and RNA genes have been used to identify and quantify bacteria in environmental samples (Malorny et al ., 2003; Yanez et al ., 2011). Despite the advantages of culture‐independent methods, the limitation of molecular assessment (especially for DNA‐based methods) is the possible overestimation of viable cell densities because DNA can persist for an extended period after cell death in environments (Rudi et al ., 2005).…”

Section: Introductionmentioning

confidence: 99%

Molecular viability testing of viable but non‐culturable bacteria induced by antibiotic exposure

Lee

Bae

2017

Microbial Biotechnology

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SummaryNucleic acid amplification‐based methods are limited by their inability to discriminate between viable and dead cells. To overcome this drawback, propidium monoazide (PMA) combined with qPCR has been used to differentiate viable from nonviable cells in environmental samples. However, assessing bacterial physiology using PMA‐qPCR remains a challenge due to its incapability of detecting metabolic activities, leading to overestimation of the viable bacteria population under an inactivation condition (e.g. antibiotic treatments). A recent advanced technique to amplify ribosomal RNA precursors (pre‐rRNA) has been shown to detect viable cells because pre‐rRNAs are intermediates in rRNA synthesis. This study investigated the effect of different types of antibiotics on the bacterial viability or viable but non‐culturable (VBNC) state using both PMA‐qPCR and pre‐rRNA analyses with Pseudomonas aeruginosa. This study demonstrated that P. aeruginosa was more sensitive to colistin than it was to carbenicillin, gentamicin and levofloxacin. We could discriminate VBNC P. aeruginosa cells using PMA‐qPCR when antibiotic pressure induced the VBNC state. Also, pre‐rRNA was able to distinguish viable cells from colistin‐inactivated bacteria cells, and it could detect the presence of VBNC and persister cells. Our results showed that these two molecular methods could successfully eliminate false‐positive signals derived from antibiotics‐inactivated cells.

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Section: Introductionmentioning

confidence: 99%

Molecular viability testing of viable but non‐culturable bacteria induced by antibiotic exposure

Lee

Bae

2017

Microbial Biotechnology

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“…The detection limit, LoD, was determined according to the International Conference on Harmonization (ICH) standards, which defines the LoD as 3 standard deviations of the negative control, implying that the probability of false positive is small 50 (1%) and that of a false negative is 50% for a sample that has a concentration at the LoD. 34,35 The limit of quantification, LoQ, was calculated as 10 standard deviation of the negative control.…”

Section: Detection and Quantification Limitmentioning

confidence: 99%

“…T he optical 45 setup and electronics are aligned in the cover by springs inside the enclosure, which force the parts against reference components. In this study, temperature control units, as well as automated data analysis methods were developed and integrated into the LabReader, in order to enable automated DNA 50 amplification, readout and analysis (see SI). Further, the LabReader was made compatible with a plastic PCR tube as a sample chamber rather than with a glass cuvette (Figure S-8) and it can be operated with a reduced sample volume of ≥40µl to minimize reagent costs.…”

Section: Dna Purification In the Labtubementioning

confidence: 99%

“…15,19 In this paper, we introduce the LabSystem, a semi-automated 45 and frugal DNA p urification, amplification and readout testing system for diagnostic and quality control applications (Figure 1). The LabSystem consists of the LabT ube, a disposable cartridge for automation of DNA purification inside laboratory centrifuges, and the LabReader, a low-cost, handheld UV/Vis reader, to 50 amplify, readout and analyze the purified DNA. The LabReader is low-cost, flexible and achieves high sensitivity and specificity by the simultaneous readout of four synchronous optical channels and continuous data readout during temperature cycling.…”

Section: Introductionmentioning

confidence: 99%

“…41,43 The achieved detection limits of the LAMP -LabSystem are similar to commercially available rapid detection methods for food samples, which vary between 10 2 -10 4 CFU/ml for most PCR-based systems. 11,50 …”

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confidence: 99%

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A versatile-deployable bacterial detection system for food and environmental safety based on LabTube-automated DNA purification, LabReader-integrated amplification, readout and analysis

Hoehl

Bocholt

Kloke³

et al. 2014

Analyst

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Contamination of foods is a public health hazard that episodically causes thousands of deaths and sickens millions worldwide. T o ensure food safety and quality, rapid, low-cost and easy-to-use detection methods are desirable. Here, the LabSystem is introduced for integrated, automated DNA purification, 10 amplification and detection. It consists of a disposable, centrifugally -driven DNA purification platform (LabT ube) and the subsequent amplification and detection in a low-cost UV/vis-reader (LabReader). For demonstration of the LabSystem in the context of food safety, purification of Escherichia coli (nonpathogenic E. coli and pathogenic verotoxin-producing E. coli (VT EC)) in water and milk, and the product-spoiler Alicyclobacillus acidoterrestris (A. acidoterrestris) in apple juice was integrated and 15 optimized in the LabT ube. Inside the LabReader, the purified DNA was amplified, readout and analyzed using both qualitative isothermal loop-mediated DNA amplification (LAMP) and quantitative real-time PCR. For the LAMP -LabSystem, the combined detection limits for purification and amplification of externally lysed VT EC and A. acidoterrestris is 10 2 -10 3 cell-equivalents. In the PCR-LabSystem for E. coli cells, the quantification limit is 10 2 cell-equivalents including LabT ube-integrated lysis. T he 20 demonstrated LabSystem only requires a laboratory centrifuge (to operate the disposable, fully closed LabT ube) and the low-cost LabReader for DNA amplification, readout and analysis. Compared with commercial DNA amplification devices, the LabReader improves sensitivity and specificity by the simultaneous readout of four wavelengths and the continuous readout during temperature cycling. T he use of a detachable eluate tube as an interface affords semi-automation of the LabSystem, which does not 25 require specialized training. It reduces hands-on time from about 50 to 3 min with only two handling steps: sample input and transfer of the detachable detection tube.

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