PNA for rapid microbiology

Stender, Henrik; Fiandaca, Mark J.; Hyldig‐Nielsen, J. J.; Coull, James

doi:10.1016/s0167-7012(01)00340-2

Cited by 165 publications

(122 citation statements)

References 59 publications

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“…Their unique properties enable the development of assay formats that extend the possibilities of DNA probes (Stender et al 2002). They cannot function as primers for DNA polymerase (Orum et al 1993), but make very useful probes for real-time RT-PCR assays (Ortiz et al 1998, Fiandaca et al 2001.…”

Section: Peptide Nucleic Acidsmentioning

confidence: 99%

Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems

Bustin

2002

Journal of Molecular Endocrinology

2,232

1,674

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The fluorescence-based real-time reverse transcription PCR (RT-PCR) is widely used for the quantification of steady-state mRNA levels and is a critical tool for basic research, molecular medicine and biotechnology. Assays are easy to perform, capable of high throughput, and can combine high sensitivity with reliable specificity. The technology is evolving rapidly with the introduction of new enzymes, chemistries and instrumentation. However, while real-time RT-PCR addresses many of the difficulties inherent in conventional RT-PCR, it has become increasingly clear that it engenders new problems that require urgent attention. Therefore, in addition to providing a snapshot of the state-of-the-art in real-time RT-PCR, this review has an additional aim: it will describe and discuss critically some of the problems associated with interpreting results that are numerical and lend themselves to statistical analysis, yet whose accuracy is significantly affected by reagent and operator variability.

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Section: Peptide Nucleic Acidsmentioning

confidence: 99%

Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems

Bustin

2002

Journal of Molecular Endocrinology

2,232

1,674

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“…In general, nucleic acid mimics can indeed provide better mismatch discrimination than standard DNA probes in FISH, as it has been suggested previously (Stender et al 2002;Guimaraes et al 2007;Mishra et al 2012Mishra et al , 2013. PNA with a low number of bases can broadly provide a good discrimination.…”

Section: Discussionmentioning

confidence: 73%

Mismatch discrimination in fluorescent in situ hybridization using different types of nucleic acids

Fontenete

Barros

Madureira

et al. 2015

Appl Microbiol Biotechnol

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In the past few years, several researchers have focused their attention on nucleic acid mimics due to the increasing necessity of developing a more robust recognition of DNA or RNA sequences. Fluorescence in situ hybridization (FISH) is an example of a method where the use of these novel nucleic acid monomers might be crucial to the success of the analysis. To achieve the expected accuracy in detection, FISH probes should have high binding affinity towards their complementary strands and discriminate effectively the noncomplementary strands. In this study, we investigate the effect of different chemical modifications in fluorescent probes on their ability to successfully detect the complementary target and discriminate the mismatched base pairs by FISH. To our knowledge, this paper presents the first study where this analysis is per-formed with different types of FISH probes directly in biological targets, Helicobacter pylori and Helicobacter acinonychis. This is also the first study where unlocked nucleic acids (UNA) were used as chemistry modification in oligonucleotides for FISH methodologies. The effectiveness in detecting the specific target and in mismatch discrimination appears to be improved using locked nucleic acids 2 (LNA)/2′-O-methyl RNA (2′OMe) or peptide nucleic acid (PNA) in comparison to LNA/DNA, LNA/UNA, or DNA probes. Further, the use of LNA modifications together with 2′OMe monomers allowed the use of shorter fluorescent probes and increased the range of hybridization temperatures at which FISH would work.

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“…PNAs are synthetic DNA mimics made by grafting either natural or non-natural nucleobases onto a repeating backbone of amide-linked N-(2-aminoethyl) glycine (AEG) units (74). Like DNA probes, PNAs hybridize to complementary DNA or RNA sequences via Watson-Crick base pairing, but their uncharged, hydrophobic backbones confer several advantageous properties over DNA-based probes.…”

Section: Biomimeticsmentioning

confidence: 99%

“…(49,75,76). An additional advantage of PNA probes is their capacity for binding to portions of the ribosome that are physically inaccessible to traditional DNA probes, enabling detection of organism-specific diagnostic sequences that are otherwise "buried" in the higher-order structure of the ribosome (49,50,74,77). This latter property stems largely from the fact that PNA probes are typically hybridized under low salt (0 -100 raM NaCl), high temperature (55°C or higher), high pH (pH 9.0) conditions that destabilize the higher order structures of target nucleic acids.…”

Section: Biomimeticsmentioning

confidence: 99%

Methods for Whole Cell Detection of Microorganisms

Brehm-Stecher

2008

ACS Symposium Series

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Microbes are ubiquitous, and can be found occupying nearly every imaginable niche on Earth. These include organic and inorganic surfaces, interfacial boundaries and within macroscopically solid matrices, such as the pore space of rocks. Because phylogenetically divergent microbes may be visually indistinguishable, understanding the species distribution and ecological significance of environmental microbes requires diagnostic tools that extend beyond simple phenotypic description. Methods for microbial diagnostics can be divided into two broad categories: cellular and acellular. Acellular techniques, such as the polymerase chain reaction or certain immunoassay formats may be effective at detecting molecular, structural or biochemical targets associated with specific cell types, but this information is provided out of its "natural", and arguably most meaningful context -that of the individual microbial cell. In contrast, cellular methods have the potential to preserve an abundance of valuable information. Apart from molecular identity, this includes information regarding cell morphology and other physiological characteristics, cell number and distribution within a sample, and physical or spatial associations with other cell types. The aim of this chapter is to provide an overview of whole cell methods for microbial detection, including both existing approaches and those still in development. The tools described here are expected to find wide application for the detection of microbes on surfaces or within complex matrices across a number of parallel or allied fields, including environmental, food and clinical microbiologies.

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PNA for rapid microbiology

Cited by 165 publications

References 59 publications

Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems

Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems

Mismatch discrimination in fluorescent in situ hybridization using different types of nucleic acids

Methods for Whole Cell Detection of Microorganisms

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