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

Konry, Tania; Lerner, Adam; Yarmush, Martin L.; Smolina, Irina

doi:10.1142/s2339547813500088

Cited by 8 publications

(4 citation statements)

References 47 publications

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“…We validated our method by applying it to the detection of clinically relevant pathogens. Our previous studies provided strong evidence that the PD-loop design has a single-base selectivity. ,, We first tested the sequence-specificity of this method on the target site within the RNA polymerase gene that differs between Escherichia coli and Bacillus subtillis genomes by only 2 nucleotides. We performed the entire assay on E. coli and B. subtilis genomic DNA using this target site.…”

Section: Resultsmentioning

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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Section: Resultsmentioning

confidence: 99%

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

2014

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“…The potential of RCA in diagnostic testing has not been fully explored, particularly in applications such as viral detection and diagnostics. We previously developed an RCA-based bioassay for the fluorescent detection of DNA and proteins, demonstrating high sensitivity and a low limit of detections, but have not explored this method for viral detection [ 22 , 23 ]. Recently, RCA has been utilized to enhance immunofluorescence sensitivity to visualize SARS-CoV-2 localization in human tissue samples [ 24 ].…”

Section: Introductionmentioning

confidence: 99%

A Novel Rolling Circle Amplification-Based Detection of SARS-CoV-2 with Multi-Region Padlock Hybridization

et al. 2022

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SARS-CoV-2 has remained a global health burden, primarily due to the continuous evolution of different mutant strains. These mutations present challenges to the detection of the virus, as the target genes of qPCR, the standard diagnostic method, may possess sequence alterations. In this study, we develop an isothermal one-step detection method using rolling circle amplification (RCA) for SARS-CoV-2. This novel strategy utilizes a multi-padlock (MP-RCA) approach to detect viral-RNA via a simplified procedure with the reliable detection of mutated strains over other procedures. We designed 40 padlock-based probes to target different sequences across the SARS-CoV-2 genome. We established an optimal one-step isothermal reaction protocol utilizing a fluorescent output detected via a plate reader to test a variety of padlock combinations. This method was tested on RNA samples collected from nasal swabs and validated via PCR. S-gene target failure (SGTF)-mutated strains of SARS-CoV-2 were included. We demonstrated that the sensitivity of our assay was linearly proportional to the number of padlock probes used. With the 40-padlock combination the MP-RCA assay was able to correctly detect 45 out 55 positive samples (81.8% efficiency). This included 10 samples with SGTF mutations which we were able to detect as positive with 100% efficiency. We found that the MP-RCA approach improves the sensitivity of the MP-RCA assay, and critically, allows for the detection of SARS-CoV-2 variants with SGTF. Our method offers the simplicity of the reaction and requires basic equipment compared to standard qPCR. This method provides an alternative approach to overcome the challenges of detecting SARS-CoV-2 and other rapidly mutating viruses.

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“…It has been widely used in food safety, clinical diagnosis, and environmental monitoring because of its high specificity, easy in vitro synthesis and modification, etc . In recent years, the aptamer is usually combined with isothermal amplification to amplify the detecting signal of the target. − Also, researchers have tried to develop the cascade signal amplification strategy, achieving ultrasensitive detection. − RCA is often used as a part of cascade amplification due to its high specificity and strong amplification. − …”

Section: Introductionmentioning

confidence: 99%

Ultrasensitive Detection of 17β-Estradiol (E2) Based on Multistep Isothermal Amplification

Wang

Peng²,

Wu³

et al. 2021

Anal. Chem.

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17β-Estradiol (E2) can cause an adverse effect on the human endocrine system even at the nanomolar level. Measurements of very low levels of E2 remain a critical challenge due to insufficient sensitivity. In this study, a multistep isothermal amplification fluorescence strategy was constructed, which could realize the exponential amplification of target E2. Specifically, strand displacement reaction (SDA), rolling circle amplification (RCA), and multiprimed rolling circle amplification (MRCA) were combined in a series to quantify trace complementary strand of E2 (cDNA). The E2 aptamer and cDNA were hybridized and modified on the magnetic beads. E2 could bind to its aptamer and cause the release of the cDNA. Then, cDNA would combine with the template DNA, initiating the SDA−RCA−MRCA. The molecular beacons, possessing low background signal, whose fluorescence was quenched in the state of chain folding, could be specifically recognized by the long single-stranded DNA (L-ssDNA) generated by the multistep isothermal amplification triggered by cDNA, and then the fluorescence of the molecular beacons could be restored. Therefore, the E2 could be quantitatively detected by the recovery fluorescence intensity. The fluorescence value showed a good linear relationship with the concentration of E2 in the range of 0.001836−183.6 nM, and the limit of detection (LOD) was as low as 63.09 fM. In addition, the recovery rates of this method spiked in milk and water were 80.8−107.0%, respectively. This method has the advantage of multistep isothermal amplification to obtain abundant fluorescence signals, which may provide a new possibility for highly sensitive detection of other small-molecule targets.

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

Cited by 8 publications

References 47 publications

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

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

A Novel Rolling Circle Amplification-Based Detection of SARS-CoV-2 with Multi-Region Padlock Hybridization

Ultrasensitive Detection of 17β-Estradiol (E2) Based on Multistep Isothermal Amplification

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