Development and Application of a Novel Peptide Nucleic Acid Probe for the Specific Detection of
            <i>Helicobacter pylori</i>
            in Gastric Biopsy Specimens

Guimarães, Nuno; Azevedo, Nuno F.; Figueiredo, Céu; Keevil, C. W.; Vieira, M. J.

doi:10.1128/jcm.00858-07

Cited by 52 publications

(59 citation statements)

References 33 publications

(28 reference statements)

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“…Similarly, PNA also has a restricted flexibility of probe design as PNA cannot be mixed with nucleotide monomers like DNA, LNA, or UNA. In this case, and because there are no guidelines related to the minimum ΔG°, we based our design on our extensive experience in working with these probes (Guimaraes et al 2007;Cerqueira et al 2011Cerqueira et al , 2013. In contrast, LNA, 2′OMe, and UNA bases can be positioned anywhere within an oligonucleotide sequence, which means that the design is much more flexible and that probe fine-tuning is possible (You et al 2006).…”

Section: Probe Designmentioning

confidence: 99%

“…The hybridization procedure for the PNA probe was similar to that used for the LNA probes (Guimaraes et al 2007;Fontenete et al 2013) however using a different type of hybridization buffer as described in Table 2. The hybridization method in suspension for LNA probes (LNA+DNA, LNA+2′ OMe, and LNA+UNA) was based on procedures described by Fontenete et al (Fontenete et al 2013).…”

Section: Optimization Of Probe Hybridization Conditionsmentioning

confidence: 99%

“…However, it has been shown that for DNA sequences, this discrimination is often difficult to achieve (Kubota et al 2006). More recently, other types of synthetic nucleic acids such as peptide nucleic acid (PNA) and locked nucleic acids (LNA) have been used in FISH with very promising results (Guimaraes et al 2007;Mook et al 2007;Tavares et al 2008;Campbell and Wengel 2011;Almeida et al 2013;Cerqueira et al 2013). To understand the specificity of these novel molecules towards DNA and RNA, several thermal dissociation studies have been conducted to analyze the effects of mismatches on duplex stability (Owczarzy et al 2011;Yan et al 2012;Matsumoto et al 2013).…”

Section: Introductionmentioning

confidence: 99%

“…As a case study, we tested the discrimination of the sequences for two closely related Helicobacter spp., Helicobacter pylori and Helicobacter acinonychis (Marshall et al 1984;Eaton et al 1993). In fact, our group has been focused on the development of several FISH methodologies to detect Helicobacter species, namely using peptide nucleic acids (PNA) (Guimaraes et al 2007;Cerqueira et al 2011Cerqueira et al , 2013, locked nucleic acids (LNA), and 2′-O-methyl RNAs (2′OMe) (Fontenete et al 2013). The novel oligonucleotides were purposely designed for a specific and conservative region in the 16S rRNA of H. pylori that is also present, with a single mismatch, in H. acinonychis.…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Probe Designmentioning

confidence: 99%

Section: Optimization Of Probe Hybridization Conditionsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

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

Fontenete

Barros

Madureira

et al. 2015

Appl Microbiol Biotechnol

View full text Add to dashboard Cite

show abstract

“…To overcome these issues, nucleic acid analogues, also known as DNA mimics, have been developed. The peptide nucleic acid (PNA) was the first to be published, in 1991, by Nielsen et al (1991) and since the late 90s has been used in microbial detection (Guimarães et al, 2007;Cerqueira et al, 2011;Almeida et al, 2011;Alves et al, 2014). In this DNA mimic the negatively charged sugar-phosphate backbone of DNA is replaced by a neutral polyamide backbone composed of N-(2-aminoethyl) glycine units.…”

Section: Introductionmentioning

confidence: 99%

Novel strategy to detect and locate periodontal pathogens: The PNA-FISH technique

Mendes

Rocha

Azevedo

et al. 2016

Microbiological Research

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Purpose: We aim to develop peptic nucleic acid (PNA) probes for the identification and localization of Aggregatibacter actinomycetemcomintans and Porphyromonas gingivalis in sub-gingival plaque and gingival biopsies by Fluorescence in situ Hybridization (FISH).Methods: A PNA probe was designed for each microorganism. The PNA-FISH method was optimized to allow simultaneous hybridization of both microorganisms with their probe (PNA-FISH multiplex). After being tested on representative strains of P. gingivalis and A. actinomycetemcomitans, the PNA-FISH method was then adapted to detect microorganisms in the subgingival plaque and gingival samples, collected from patients with severe periodontitis. Results:The best hybridization conditions were found to be 59 • C for 150 min for both probes (PgPNA1007 and AaPNA235). The in silico sensitivity and specificity was both 100% for PgPNA1007 probe and 100% and 99.9% for AaPNA235 probe, respectively. Results on clinical samples showed that the PNA-FISH method was able to detect and discriminate target bacteria in the mixed microbial population of the subgingival plaque and within periodontal tissues.Conclusion: This investigation presents a new highly accurate method for P. gingivalis and A. actino-mycetemcomitans detection and co-location in clinical samples, in just few hours. With this technique we were able to observe spatial distribution of these species within polymicrobial communities in the periodontal pockets and, for the first time with the FISH method, in the organized gingival tissue.2

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Discriminating typical and atypical cystic fibrosis‐related bacteria by multiplex PNA‐FISH

Lopes

Carvalho

Azevedo

2016

Biotech & Bioengineering

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This study aims to report the development of peptide nucleic acid (PNA) probes to specifically detect the cystic fibrosis (CF)-associated traditional and atypical species Pseudomonas aeruginosa and Inquilinus limosus, respectively. PNA probes were designed in silico, developed and tested in smears prepared in phosphate-buffer saline (PBS), and in artificial sputum medium (ASM). A multiplex fluorescent in situ hybridization (FISH) approach using the designed probes was further validated in artificially contaminated clinical sputum samples and also applied in polymicrobial 24 h-old biofilms involving P. aeruginosa, I. limosus, and other CF-related bacteria. Both probes showed high predictive and experimental specificities and sensitivities. The multiplex PNA-FISH assay, associated with non-specific staining, was successfully adapted in the clinical samples and in biofilms of CF-related bacteria, allowing differentiating the community members and inferring about microbial-microbial interactions within the consortia. This study revealed the great potential of PNA-FISH as a diagnostic tool to discriminate between classical and less common CF-associated bacteria, being suitable to further describe species-dependent prevention strategies and deliver more effective target control therapeutics. Biotechnol. Bioeng. 2017;114: 355-367. © 2016 Wiley Periodicals, Inc.

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Development and Application of a Novel Peptide Nucleic Acid Probe for the Specific Detection of Helicobacter pylori in Gastric Biopsy Specimens

Cited by 52 publications

References 33 publications

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

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

Novel strategy to detect and locate periodontal pathogens: The PNA-FISH technique

Discriminating typical and atypical cystic fibrosis‐related bacteria by multiplex PNA‐FISH

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