Padlock probe-based rolling circle amplification lateral flow assay for point-of-need nucleic acid detection

Jain, Sidhartha; Dandy, David S.; Geiss, Brian J.; Henry, Charles S.

doi:10.1039/d1an00399b

Cited by 35 publications

(20 citation statements)

References 35 publications

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“…This can be caused by the limited availability of the biotin-primer. Another possible mechanism can be due to the larger sizes of RCA products formed by extremely high amounts of target DNA, which are entangled and difficult to flow through the membrane [ 37 ]. The signals for the control lines gradually decreased to 57% upon increasing target amounts to 167 fmol, and plateaued or slightly increased at higher target amounts (57–66%).…”

Section: Resultsmentioning

confidence: 99%

A Lateral Flow Assay for Nucleic Acid Detection Based on Rolling Circle Amplification Using Capture Ligand-Modified Oligonucleotides

et al. 2022

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We introduce a lateral flow assay (LFA) integrated with a modified isothermal nucleic acid amplification procedure for rapid and simple genetic testing. Padlock probes specific for the target DNA were designed for ligation, followed by rolling circle amplification (RCA) using capture ligand-modified oligonucleotides as primers. After hybridization with detection linker probes, the amplified target DNA is flowed through an LFA membrane strip for binding of gold nanoparticles as the substrate for colorimetric detection. We established and validated the “RCA-LFA” method for detection of mecA, the antibiotic resistance gene for methicillin-resistant Staphylococcus aureus (MRSA). The assay was optimized using various concentrations of primers and probes for RCA and LFA, respectively. The sensitivity was determined by performing RCA-LFA using various amounts of mecA target DNA, showing a detection limit of ~ 1.3 fmol. The specificity of the assay was examined using target DNAs for other resistance genes as the controls, which demonstrated positive detection signals only for mecA DNA, when added either individually or in combinations with the control targets. Furthermore, applying the RCA-LFA method using specifically designed probes for RNA-dependent RNA polymerase (RdRp) and receptor binding domain (RBD) gene for SARS-CoV-2, which demonstrated feasibility of the method for viral gene targets. The current method suggests a useful platform which can be universally applied for various nucleic acid targets, allowing rapid and sensitive diagnosis at point-of-care. Supplementary Information The online version contains supplementary material available at 10.1007/s13206-022-00080-1.

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Section: Resultsmentioning

confidence: 99%

A Lateral Flow Assay for Nucleic Acid Detection Based on Rolling Circle Amplification Using Capture Ligand-Modified Oligonucleotides

et al. 2022

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“…Then, the exonuclease is added to cleave the primers to generate a complete circular template for later amplification. [76][77][78] 2.2.2 Dumbbell DNA loop. Compared with the formation of the padlock DNA loop, the formation of the dumbbell DNA loop does not need to add additional primers (as shown in Fig.…”

Section: Guoqiang LImentioning

confidence: 99%

“…Then, the exonuclease is added to cleave the primers to generate a complete circular template for later amplification. 76–78…”

Section: Mechanism and Classification Of Rca Technologymentioning

confidence: 99%

Recent advances in biological detection with rolling circle amplification: design strategy, biosensing mechanism, and practical applications

Gao

Huang²,

Wang

et al. 2022

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“…However, SERS-LFA biosensors still cannot realize the ultrasensitive detection for some low concentration targets. It is pretty necessary to combine the SERS-LFA biosensor with a signal amplification strategy, such as hybridization chain reaction (HCR), rolling circle amplification (RCA), and catalytic hairpin assembly (CHA) (Li et al, 2010;Huang et al, 2021;Jain et al, 2021). Although all of these methods could greatly enhance the signal intensity, HCR suffered from laborious labeling techniques and was environmentally sensitive.…”

Section: Introductionmentioning

confidence: 99%

SERS Based Lateral Flow Assay for Rapid and Ultrasensitive Quantification of Dual Laryngeal Squamous Cell Carcinoma-Related miRNA Biomarkers in Human Serum Using Pd-Au Core-Shell Nanorods and Catalytic Hairpin Assembly

Niu

et al. 2022

Front. Mol. Biosci.

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Non-invasive early diagnosis is of great significant in disease pathologic development and subsequent medical treatments, and microRNA (miRNA) detection has attracted critical attention in early cancer screening and diagnosis. However, it was still a challenge to report an accurate and sensitive method for the detection of miRNA during cancer development, especially in the presence of its analogs that produce intense background noise. Herein, we developed a surface-enhanced Raman scattering (SERS)–based lateral flow assay (LFA) biosensor, assisted with catalytic hairpin assembly (CHA) amplification strategy, for the dynamic monitoring of miR-106b and miR-196b, associated with laryngeal squamous cell carcinoma (LSCC). In the presence of target miRNAs, two hairpin DNAs could self-assemble into double-stranded DNA, exposing the biotin molecules modified on the surface of palladium (Pd)–gold (Au) core–shell nanorods (Pd-AuNRs). Then, the biotin molecules could be captured by the streptavidin (SA), which was fixed on the test lines (T1 line and T2 line) beforehand. The core–shell spatial structures and aggregation Pd-AuNRs generated abundant active “hot spots” on the T line, significantly amplifying the SERS signals. Using this strategy, the limits of detections were low to aM level, and the selectivity, reproducibility, and uniformity of the proposed SERS-LFA biosensor were satisfactory. Finally, this rapid analysis strategy was successfully applied to quantitatively detect the target miRNAs in clinical serum obtained from healthy subjects and patients with LSCC at different stages. The results were consistent with the quantitative real-time PCR (qRT-PCR). Thus, the CHA-assisted SERS-LFA biosensor would become a promising alternative tool for miRNAs detection, which showed a tremendous clinical application prospect in diagnosing LSCC.

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Padlock probe-based rolling circle amplification lateral flow assay for point-of-need nucleic acid detection

Abstract: Sensitive, reliable and cost-effective detection of pathogens has wide ranging applications in clinical diagnostics and therapeutics, water and food safety, environmental monitoring, biosafety and epidemiology. Nucleic acid amplification tests (NAATs)...

Cited by 35 publications

References 35 publications

A Lateral Flow Assay for Nucleic Acid Detection Based on Rolling Circle Amplification Using Capture Ligand-Modified Oligonucleotides

A Lateral Flow Assay for Nucleic Acid Detection Based on Rolling Circle Amplification Using Capture Ligand-Modified Oligonucleotides

Recent advances in biological detection with rolling circle amplification: design strategy, biosensing mechanism, and practical applications

SERS Based Lateral Flow Assay for Rapid and Ultrasensitive Quantification of Dual Laryngeal Squamous Cell Carcinoma-Related miRNA Biomarkers in Human Serum Using Pd-Au Core-Shell Nanorods and Catalytic Hairpin Assembly

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