Primer exchange reaction-amplified protein-nucleic acid interactions for ultrasensitive and specific microRNA detection

Fang, Min; Liu, Fengfei; Fang, Dan; Chen, Yu; Xiang, Yuanhang; Zhang, Hui; Huang, Minmin; Qin, Xiaojie; Pan, Linghui; Yang, Fan

doi:10.1016/j.bios.2023.115274

Cited by 15 publications

(6 citation statements)

References 49 publications

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“…The HC-ELISA assay is simple and can be adopted into sentinel labs, reference labs and national laboratories for pathogen detection to detect active infection at low cost. As this manuscript was prepared for submission, new reports on the use of S9.6 in diagnostics emerged, supporting our ndings that it can be adopted for detecting any gene or transcript speci c to a disease or pathogen (13)(14)(15).…”

Section: Discussionsupporting

confidence: 65%

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S9.6-based hybrid capture immunoassay for pathogen detection

Bothra,

Perry,

Wei

et al. 2023

Preprint

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The detection of pathogens is critical for clinical diagnosis and public health surveillance. Detection is usually done with nucleic acid-based tests (NATs) and rapid antigen tests (e.g., lateral flow assays [LFAs]). Although NATs are more sensitive and specific, their use is often limited in resource-poor settings due to specialized requirements. To address this limitation, we developed a rapid DNA-RNA Hybrid Capture immunoassay (HC) that specifically detects RNA from pathogens. This assay utilizes a unique monoclonal antibody, S9.6, which binds DNA-RNA hybrids. Biotinylated single-stranded DNA probes are hybridized to target RNAs, followed by hybrid capture on streptavidin and detection with S9.6. The HC-ELISA assay can detect as few as 104 RNA molecules that are 2.2 kb in length. We also adapted this assay into a LFA format, where captured Bacillus anthracis rpoB RNA of 3.5 kb length was detectable from a bacterial load equivalent to 107 CFU per 100 mg of mouse tissue using either HC-ELISA or HC-LFA. Importantly, we also demonstrated the versatility of HC by detecting other pathogens, including SARS-CoV2 and Toxoplasma gondii, showing its potential for broad pathogen detection. Notably, HC does not require amplification of the target nucleic acid and utilizes economical formats like ELISA and LFA, making it suitable for use in sentinel labs for pathogen detection or as a molecular tool in basic research laboratories. Our study highlights the potential of HC as a sensitive and versatile method for RNA-based pathogen detection.

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Section: Discussionsupporting

confidence: 65%

“…Various low-cost techniques, including LFA and ELISA, have been developed to detect disease-speci c nucleic acids (5,10,(13)(14)(15). One example is the HC2-HPV diagnostic assay for human papillomavirus (HPV) infection marketed by Qiagen (Digene), which employs polyclonal antibodies in a "hybrid capture" sandwich ELISA format (16,17).…”

Section: Introductionmentioning

confidence: 99%

S9.6-based hybrid capture immunoassay for pathogen detection

Bothra,

Perry,

Wei

et al. 2023

Preprint

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“…To efficiently bind the capture probe, the prepared WSNs undergo a series of surface modifications (Figure a). WSNs@Ab conjugates were obtained by forming stable amide bonds between the surface-activated carboxyl groups of WSNs, and the amino groups of the S9.6 Ab. − Multicolor QDs were used as fluorescent reporters and labeled to the 5′ end of ssDNA complementary to the target miRNAs. When a mixed solution contained the three miRNAs, the formed double-stranded hybrids (bQDs-ssDNA1/miR-135b, gQDs-ssDNA2/miR-21, and rQDs-ssDNA3/miR-96) can be recognized and captured by S9.6 Ab with high specificity and affinity.…”

Section: Resultsmentioning

confidence: 99%

Amplification-Free Analysis of Bladder Cancer MicroRNAs on Wrinkled Silica Nanoparticles with DNA-Functionalized Quantum Dots

Wang,

Wei,

Shen

et al. 2024

Anal. Chem.

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Bladder cancer (BC) occurrence and progression are accompanied by alterations in microRNAs (miRNAs) expression levels. Simultaneous detection of multiple miRNAs contributes to the accuracy and reliability of the BC diagnosis. In this work, wrinkled silica nanoparticles (WSNs) were applied as the microreactor for multiplex miRNAs analysis without enzymes or nucleic acid amplification. Conjugated on the surface of WSNs, the S9.6 antibody was adopted as the universal module for binding DNA/miRNA duplexes, regardless of their sequence. Furthermore, single-stranded DNA (ssDNA) was labeled with quantum dots (QDs) for identifying a given miRNA to form QDs-ssDNA/ miRNA, which enabled the specific capture of the corresponding QDs on the wrinkled surface of WSNs. Based on the detection of fluorescence signals that were ultimately focused on WSNs, target miRNAs could be sensitively identified to a femtomolar level (5 fM) with a wide dynamic range of up to 6 orders of magnitude. The proposed strategy achieved high specificity to obviously distinguish single-base mutation sequences and possessed multiplex assay capability. Moreover, the assay exhibited excellent practicability in the multiplex detection of miRNAs in clinical serum specimens.

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“…This hairpin facilitates the continuous growth of a short single-stranded DNA (ssDNA) primer following specific reaction pathways with the assistance of a strand-displacing polymerase. This process enables the stable amplification of target sequences. , Significantly, the production of a new strand in situ occurs only when the appropriate set of PER hairpins and its associated PER primer are present simultaneously. This autonomous process minimizes signal leakage and efficiently mitigates the background response .…”

Section: Introductionmentioning

confidence: 99%

“…This process enables the stable amplification of target sequences. 18 , 19 Significantly, the production of a new strand in situ occurs only when the appropriate set of PER hairpins and its associated PER primer are present simultaneously. This autonomous process minimizes signal leakage and efficiently mitigates the background response.…”

Section: Introductionmentioning

confidence: 99%

Catalytic Assembly of DNAzyme Integrates with Primer Exchange Reaction (CDiPER) for Highly Sensitive Detection of MicroRNA

Zhang,

Li,

Jiang

et al. 2024

ACS Omega

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MicroRNAs (miRNAs) have significant regulatory functions in the modulation of gene expression, making them essential biomarkers for the diagnosis and prognosis of diseases. Nevertheless, the identification of miRNA poses significant difficulty in terms of its low abundance, necessitating sensitive and reliable approaches. Herein, we develop a simple approach, termed Catalytic assembly of DNAzyme integrates with Primer Exchange Reaction (CDiPER), for reliable and sensitive miRNA detection through the target recognition-triggered DNAzyme assembly and primer exchange reaction (PER) strategy. In this method, target miRNA can precisely bind with a specifically designed hairpin probe (H probe) to induce the conformation changes of the H probe, releasing DNAzyme sections to activate the PER process for signal amplification and fluorescence signal production. The established method displays a high dynamic range of over 6 orders of magnitude and a low detection limit of 312 aM. The created method has a number of unique advantages, such as (i) a better sensitivity than existing systems using PER for signal amplification as a result of its integration with the target recognitiontriggered DNAzyme assembly and (ii) streamlined operating procedures. Further, the technology was used to detect the expression of miRNA in collected clinical samples from diabetes mellitus patients, revealing that miRNA was decreased in patients and demonstrating the significant clinical promise of the method.

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Primer exchange reaction-amplified protein-nucleic acid interactions for ultrasensitive and specific microRNA detection

Cited by 15 publications

References 49 publications

S9.6-based hybrid capture immunoassay for pathogen detection

S9.6-based hybrid capture immunoassay for pathogen detection

Amplification-Free Analysis of Bladder Cancer MicroRNAs on Wrinkled Silica Nanoparticles with DNA-Functionalized Quantum Dots

Catalytic Assembly of DNAzyme Integrates with Primer Exchange Reaction (CDiPER) for Highly Sensitive Detection of MicroRNA

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