Effect of PCR amplicon length on suppressing signals from membrane-compromised cells by propidium monoazide treatment

Jopia-Contreras, Paz; Urrutia, Homero; Sossa, Katherine; Nocker, Andreas

doi:10.1016/j.mimet.2011.07.016

Cited by 82 publications

(79 citation statements)

References 29 publications

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“…If so, the interpretation of PCR results obtained from this type of clinical samples has to be interpreted with care and always to observe that the control sample in this case was efficient. Elimination of this residual nucleic acid can be obtained by sample treatment with viability dye, such as propidium monoazide might overcome this limitation [20,21].…”

Section: Discussionmentioning

confidence: 99%

PCR-RFLP as a useful tool for diagnosis of invasive mycoses in a healthcare facility in the North of Brazil

Sousa

Santos

Wanke

et al. 2015

Electronic Journal of Biotechnology

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Background: The incidence of invasive mycoses is increasing worldwide. PCR-RFLP was applied to the identification of 10 reference strains and 90 cultures of agents of invasive mycoses. In addition, the new approach was applied to detect fungal agents in 120 biological samples (blood, cerebrospinal fluid and bone marrow). PCR-RFLP results were compared with the ones obtained with conventional methods (culture, microscopy, and biochemical testing). Results: The assays carried out with the reference strains (Candida albicans, Candida parapsilosis, Candida tropicalis, Candida krusei, Candida guilliermondii, Cryptococcus neoformans, Cryptococcus gattii and Histoplasma capsulatum), demonstrated that the RFLP profiles were correctly predicted by the in silico investigation and allowed unequivocal identification of all chosen reference strains. The PCR-RFLP also identified 90 cultures of agents of invasive mycoses correctly, 2.5 times faster than the conventional assays. Evaluating PCR-RFLP with biological samples it was observed that the PCR was found to be 100% accurate and the RFLP profiles allowed the identification of the etiological agents: C. neoformans (n = 3) and C. gattii (n = 1) in CSF samples, H. capsulatum (n = 1) in bone marrow and C. albicans (n = 2) in blood cultures. The detection and identification by PCR-RFLP were found to be between two to ten times faster than the conventional assays. Conclusion: The results showed that PCR-RFLP is a valuable tool for the identification of invasive mycoses that can be implemented in hospital laboratories, allowing for a high number of clinical analyses per day.

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Section: Discussionmentioning

confidence: 99%

PCR-RFLP as a useful tool for diagnosis of invasive mycoses in a healthcare facility in the North of Brazil

Sousa

Santos

Wanke

et al. 2015

Electronic Journal of Biotechnology

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“…-Photoactivation carried out using high-power halogen lamps ranging from 500 to 750 W [23,27] and samples placed horizontally on ice a distance of 20e30 cm from the light source [19,24,28]. -Amplicon length affect qPCR: longer DNA sequences correlate with a higher probability that the DNA polymerase will encounter modifications in the stretch of DNA that is targeted by the primers which will result in an increased suppression of signals from membrane-compromised cells [24,29,30]. After applying PMA to heat-killed Legionella pneumophila, there was a significant difference in the number of dead cells that was measured when qPCR was based on the amplification of 16SrRNA (454 bp) or 5S rRNA (108 bp) [15].…”

Section: Introductionmentioning

confidence: 99%

Legionella in water samples: How can you interpret the results obtained by quantitative PCR?

Ditommaso

Ricciardi

Giacomuzzi

et al. 2015

Molecular and Cellular Probes

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“…Additional strategies to increase PMA treatment efficiency include incubation at elevated temperature (10°C above the optimal growth temperature) to maximize dye penetration into damaged cells (37) and the amplification of longer DNA sequences (38)(39)(40). The latter strategy is thought to increase the probability that at least one dye molecule would bind to the targeted DNA stretch in damaged cells (38)(39)(40).…”

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Dead or Alive: Molecular Assessment of Microbial Viability

Cangelosi

Meschke

2014

Appl Environ Microbiol

264

207

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Nucleic acid-based analytical methods, ranging from species-targeted PCRs to metagenomics, have greatly expanded our understanding of microbiological diversity in natural samples. However, these methods provide only limited information on the activities and physiological states of microorganisms in samples. Even the most fundamental physiological state, viability, cannot be assessed cross-sectionally by standard DNA-targeted methods such as PCR. New PCR-based strategies, collectively called molecular viability analyses, have been developed that differentiate nucleic acids associated with viable cells from those associated with inactivated cells. In order to maximize the utility of these methods and to correctly interpret results, it is necessary to consider the physiological diversity of life and death in the microbial world. This article reviews molecular viability analysis in that context and discusses future opportunities for these strategies in genetic, metagenomic, and single-cell microbiology.Yet it hath happened that the veritable body without the spirit hath walked.-Ambrose Bierce, The Death of Halpin Frayser M icrobiologists, like characters in zombie fiction, quickly learn the critical importance of distinguishing the living from the dead. In addition to characterizing the numbers and species of microorganisms in samples, it is important to collect data on their physiological states. The most fundamental physiological state of microbial cells is their viability, defined here as the capacity to form progeny. For ecologists, pathobiologists, metagenomicists, food or water safety analysts, infectious-disease clinicians, and virtually every other stripe of microbiologist, the observation of a viable microorganism in a sample means something entirely different from the observation of a dead one. Despite its importance, this distinction remains extremely challenging by current microbiological methods.Microbiological culture meets this requirement, as it selectively detects viable organisms. However, because only a small percentage of species can be cultured, this strategy underestimates microbial diversity (1-6). In contrast, nucleic acid-based methods, ranging from species-specific PCR to metagenomic methods, have greatly advanced our ability to detect diverse microorganisms independently of microbiological culture (7,8). However, these methods provide only limited information on the physiology of microorganisms in samples. They can assess microbial viability retrospectively, by measuring quantitative changes over time, but they cannot discern viability cross-sectionally (in single measurements). Traditional PCR is notoriously poor at differentiating DNA associated with a viable bacterial cell from DNA associated with an inactivated one or from a free DNA fragment. All of these analytes register as "hits" in PCR, despite their very distinct meanings.In order to address this limitation, alternative PCR-based strategies have been developed. This article reviews two complementary strategies. One strategy, term...

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Effect of PCR amplicon length on suppressing signals from membrane-compromised cells by propidium monoazide treatment

Cited by 82 publications

References 29 publications

PCR-RFLP as a useful tool for diagnosis of invasive mycoses in a healthcare facility in the North of Brazil

PCR-RFLP as a useful tool for diagnosis of invasive mycoses in a healthcare facility in the North of Brazil

Legionella in water samples: How can you interpret the results obtained by quantitative PCR?

Dead or Alive: Molecular Assessment of Microbial Viability

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