PNA-based microbial pathogen identification and resistance marker detection

Smolina, Irina; Miller, Nancy S.; Frank‐Kamenetskii, Maxim D.

doi:10.4161/adna.1.2.13256

Cited by 23 publications

(20 citation statements)

References 26 publications

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“…Collectively, these results suggest successful SNP discrimination within the EBV EBNA-3A gene in BC-1 cells. Such high sequence specificity has been demonstrated earlier in PNA-based detection assays confirming the zero mismatch tolerance of the PNA-RCA approach 19,41 .…”

Section: Resultssupporting

confidence: 74%

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Target DNA detection and quantitation on a single cell with single base resolution

et al. 2013

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In this report, we present a new method for sensitive detection of short DNA sites in single cells with single base resolution. The method combines peptide nucleic acid (PNA) openers as the tagging probes, together with isothermal rolling circle amplification (RCA) and fluorescence-based detection, all performed in a cells-in-flow format. Bis-PNAs provide single base resolution, while RCA ensures linear signal amplification. We applied this method to detect the oncoviral DNA inserts in cancer cell lines using a flow-cytometry system. We also demonstrated quantitative detection of the selected signature sites within single cells in microfluidic nano-liter droplets. Our results show single-nucleotide polymorphism (SNP) discrimination and detection of copy-number variations (CNV) under isothermal non-denaturing conditions. This new method is ideal for many applications in which ultra-sensitive DNA characterization with single base resolution is desired on the level of single cells.

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Section: Resultssupporting

confidence: 74%

“…Previously, we showed that the PNA-RCA approach can be used for fluorescent in situ detection of short single-copy DNA sequences within the bacteria 18,19 and more recently in human cells 20 . Here we demonstrate that this approach can be used in a cells-in-flow format where we are able to detect single base changes.…”

Section: Introductionmentioning

confidence: 99%

Target DNA detection and quantitation on a single cell with single base resolution

et al. 2013

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“…3, 4 One such technique is Rolling-Circle-Amplification (RCA), by which a circular DNA template is converted to a ~10 3 tandem repeat Rolling-Circle-Product (RCP) that can easily be visualized at the single-molecule level. 5, 6 RCA has successfully been employed to detect nucleic-acid sequences 7, 8 , proteins 9, 10 or small-molecules. 11 However, the disadvantage of most published methods is the requirement of extensive sample preparation, and the formation of only one or few RCPs per target molecule.…”

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confidence: 99%

Droplet Microfluidics Platform for Highly Sensitive and Quantitative Detection of Malaria-Causing Plasmodium Parasites Based on Enzyme Activity Measurement

et al. 2012

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We present an attractive new system for the specific and sensitive detection of the malaria causing Plasmodium parasites. The system relies on isothermal conversion of single DNA cleavage-ligation events catalyzed specifically by the Plasmodium enzyme topoisomerase I to micrometer sized products detectable at the single-molecule level. Combined with a droplet-microfluidics Lab-on-a-Chip platform, this design allowed for sensitive, specific and quantitative detection of all human malaria causing Plasmodium species in single drops of unprocessed blood with a detection limit of less than one parasite/μL. Moreover, the setup allowed for detection of Plasmodium parasites in non-invasive saliva samples from infected patients. During recent years malaria transmission has declined worldwide and with this the number of patients with low-parasite density has increased. Consequently, the need for accurate detection of even a few parasites is becoming increasingly important for the continued combat against the disease. We believe that the presented droplet-microfluidics platform, which has a high potential for adaptation to point-of-care setups suitable for low-resource settings may contribute significantly to meet this demand. Moreover, potential future adaptation of the presented setup for the detection of other microorganisms may form the basis for the development of a more generic platform for diagnosis, fresh water- or food quality control or other purposes within applied or basic science.

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“…The PD-loop formation is limited to the preselected 20–30-bp-long target site within dsDNA and is normally unique in the entire genome. 8 − 11 A significant advantage of PNA-based padlock probe design is that the rest of the DNA retains its duplex structure and is inaccessible for probe binding, which greatly reduces background noise. The assembled circular probe serves as a template for rolling circle amplification.…”

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confidence: 99%

Visual Detection of Bacterial Pathogens via PNA-Based Padlock Probe Assembly and Isothermal Amplification of DNAzymes

2014

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We have developed a self-reporting isothermal system for visual bacterial pathogen detection with single base resolution. The new DNA diagnostic is based on combination of peptide nucleic acid (PNA) technology, rolling circle amplification (RCA) and DNAzymes. PNAs are used as exceedingly selective chemical tools that bind genomic DNA at a predetermined sequence under nondenaturing conditions. After assembly of the PNA-DNA construct a padlock probe is circularized on the free strand. The probe incorporates a G-quadruplex structure flanked by nicking enzyme recognition sites. The assembled circle serves as a template for a novel hybrid RCA strategy that allows for exponential amplification and production of short single-stranded DNA pieces. These DNA fragments fold into G-quadruplex structures and when complexed with hemin become functional DNAzymes. The catalytic activity of each DNAzyme unit leads to colorimetric detection and provides the second amplification step. The combination of PNA, RCA, and DNAzymes allows for sequence-specific and highly sensitive detection of bacteria with a colorimetric output observed with the naked eye. Herein, we apply this method for the discrimination of Escherichia coli, Salmonella typhimurium, and Clostridium difficile genomes.

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PNA-based microbial pathogen identification and resistance marker detection

Cited by 23 publications

References 26 publications

Target DNA detection and quantitation on a single cell with single base resolution

Target DNA detection and quantitation on a single cell with single base resolution

Droplet Microfluidics Platform for Highly Sensitive and Quantitative Detection of Malaria-Causing Plasmodium Parasites Based on Enzyme Activity Measurement

Visual Detection of Bacterial Pathogens via PNA-Based Padlock Probe Assembly and Isothermal Amplification of DNAzymes

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