Discriminative identification of miRNA let-7 family members with high specificity and sensitivity using rolling circle amplification

The MPRP system for SNP discrimination was developed, which showed high specificity and sensitivity for multiplex detection of tumor-related mutations.

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Signal-on electrochemical DNA (E-DNA) sensor for accurate quantification of nicking-assisted rolling circle amplification (N-RCA) products with attomolar sensitivity

Analytical Methods ◽

10.1039/d1ay01664d ◽

2021 ◽

Author(s):

Mengmeng Li ◽

Dandan Li ◽

Guidan Huang ◽

Linying Zhou ◽

Qilin Wen ◽

...

Keyword(s):

Nucleic Acid ◽

Molecular Diagnosis ◽

Rolling Circle Amplification ◽

High Specificity ◽

Dna Sensor ◽

Rolling Circle ◽

Specificity And Sensitivity ◽

Nucleic Acid Analysis ◽

Amplification Technique ◽

Accurate Quantification

Rolling circle amplification (RCA) has become increasingly important amplification technique in nucleic acid analysis, immunoassay, and molecular diagnosis due to its high specificity and sensitivity. However, the accurate quantification of...

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Padlock Probe Assay for Detection and Subtyping of Seasonal Influenza

Clinical Chemistry ◽

10.1373/clinchem.2018.292979 ◽

2018 ◽

Vol 64 (12) ◽

pp. 1704-1712 ◽

Cited By ~ 10

Author(s):

Felix Neumann ◽

Iván Hernández-Neuta ◽

Malin Grabbe ◽

Narayanan Madaboosi ◽

Jan Albert ◽

...

Keyword(s):

Seasonal Influenza ◽

Rolling Circle Amplification ◽

High Specificity ◽

Clinical Samples ◽

Padlock Probe ◽

Rolling Circle ◽

Diagnostic Assays ◽

Padlock Probes ◽

Probe Assay ◽

Digital Quantification

Abstract BACKGROUND Influenza remains a constant threat worldwide, and WHO estimates that it affects 5% to 15% of the global population each season, with an associated 3 to 5 million severe cases and up to 500000 deaths. To limit the morbidity and the economic burden of influenza, improved diagnostic assays are needed. METHODS We developed a multiplexed assay for the detection and subtyping of seasonal influenza based on padlock probes and rolling circle amplification. The assay simultaneously targets all 8 genome segments of the 4 circulating influenza variants—A(H1N1), A(H3N2), B/Yamagata, and B/Victoria—and was combined with a prototype cartridge for inexpensive digital quantification. Characterized virus isolates and patient nasopharyngeal swabs were used for assay design and analytical validation. The diagnostic performance was assessed by blinded testing of 50 clinical samples analyzed in parallel with a commercial influenza assay, Simplexa™ Flu A/B & RSV Direct. RESULTS The assay had a detection limit of 18 viral RNA copies and achieved 100% analytical and clinical specificity for differential detection and subtyping of seasonal circulating influenza variants. The diagnostic sensitivity on the 50 clinical samples was 77.5% for detecting influenza and up to 73% for subtyping seasonal variants. CONCLUSIONS We have presented a proof-of-concept padlock probe assay combined with an inexpensive digital readout for the detection and subtyping of seasonal influenza strains A and B. The demonstrated high specificity and multiplexing capability, together with the digital quantification, established the assay as a promising diagnostic tool for seasonal influenza.

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SCRINSHOT, a spatial method for single-cell resolution mapping of cell states in tissue sections

10.1101/2020.02.07.938571 ◽

2020 ◽

Cited By ~ 1

Author(s):

Alexandros Sountoulidis ◽

Andreas Liontos ◽

Hong Phuong Nguyen ◽

Alexandra B. Firsova ◽

Athanasios Fysikopoulos ◽

...

Keyword(s):

Gene Expression ◽

Single Cell ◽

Rolling Circle Amplification ◽

Marker Gene ◽

Cell Types ◽

Experimental Conditions ◽

Rolling Circle ◽

Padlock Probes ◽

Tissue Sections ◽

Specificity And Sensitivity

AbstractChanges in cell identities and positions underlie tissue development and disease progression. Although, single-cell mRNA sequencing (scRNA-Seq) methods rapidly generate extensive lists of cell-states, spatially resolved single-cell mapping presents a challenging task. We developed SCRINSHOT (Single Cell Resolution INSitu Hybridization On Tissues), a sensitive, multiplex RNA mapping approach. Direct hybridization of padlock probes on mRNA is followed by circularization with SplintR ligase and rolling circle amplification (RCA) of the hybridized padlock probes. Sequential detection of RCA-products using fluorophore-labeled oligonucleotides profiles thousands of cells in tissue sections. We evaluated SCRINSHOT specificity and sensitivity on murine and human organs. SCRINSHOT quantification of marker gene expression shows high correlation with published scRNA-Seq data over a broad range of gene expression levels. We demonstrate the utility of SCRISHOT by mapping the locations of abundant and rare cell types along the murine airways. The amenability, multiplexity and quantitative qualities of SCRINSHOT facilitate single cell mRNA profiling of cell-state alterations in tissues under a variety of native and experimental conditions.

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SCRINSHOT enables spatial mapping of cell states in tissue sections with single-cell resolution

PLoS Biology ◽

10.1371/journal.pbio.3000675 ◽

2020 ◽

Vol 18 (11) ◽

pp. e3000675

Author(s):

Alexandros Sountoulidis ◽

Andreas Liontos ◽

Hong Phuong Nguyen ◽

Alexandra B. Firsova ◽

Athanasios Fysikopoulos ◽

...

Keyword(s):

Gene Expression ◽

Single Cell ◽

Rolling Circle Amplification ◽

Marker Gene ◽

Cell Types ◽

Experimental Conditions ◽

Rolling Circle ◽

Padlock Probes ◽

Tissue Sections ◽

Specificity And Sensitivity

Changes in cell identities and positions underlie tissue development and disease progression. Although single-cell mRNA sequencing (scRNA-Seq) methods rapidly generate extensive lists of cell states, spatially resolved single-cell mapping presents a challenging task. We developed SCRINSHOT (Single-Cell Resolution IN Situ Hybridization On Tissues), a sensitive, multiplex RNA mapping approach. Direct hybridization of padlock probes on mRNA is followed by circularization with SplintR ligase and rolling circle amplification (RCA) of the hybridized padlock probes. Sequential detection of RCA-products using fluorophore-labeled oligonucleotides profiles thousands of cells in tissue sections. We evaluated SCRINSHOT specificity and sensitivity on murine and human organs. SCRINSHOT quantification of marker gene expression shows high correlation with published scRNA-Seq data over a broad range of gene expression levels. We demonstrate the utility of SCRINSHOT by mapping the locations of abundant and rare cell types along the murine airways. The amenability, multiplexity, and quantitative qualities of SCRINSHOT facilitate single-cell mRNA profiling of cell-state alterations in tissues under a variety of native and experimental conditions.

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Recent advances of fluorescent biosensors based on cyclic signal amplification technology in biomedical detection

Journal of Nanobiotechnology ◽

10.1186/s12951-021-01149-z ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Hongke Qu ◽

Chunmei Fan ◽

Mingjian Chen ◽

Xiangyan Zhang ◽

Qijia Yan ◽

...

Keyword(s):

Signal Amplification ◽

Low Cost ◽

Rolling Circle Amplification ◽

High Sensitivity ◽

High Specificity ◽

Detection Methods ◽

Research Progress ◽

Rolling Circle ◽

Displacement Reactions ◽

Short Time

AbstractThe cyclic signal amplification technology has been widely applied for the ultrasensitive detection of many important biomolecules, such as nucleic acids, proteins, enzymes, adenosine triphosphate (ATP), metal ions, exosome, etc. Due to their low content in the complex biological samples, traditional detection methods are insufficient to satisfy the requirements for monitoring those biomolecules. Therefore, effective and sensitive biosensors based on cyclic signal amplification technology are of great significance for the quick and simple diagnosis and treatment of diseases. Fluorescent biosensor based on cyclic signal amplification technology has become a research hotspot due to its simple operation, low cost, short time, high sensitivity and high specificity. This paper introduces several cyclic amplification methods, such as rolling circle amplification (RCA), strand displacement reactions (SDR) and enzyme-assisted amplification (EAA), and summarizes the research progress of using this technology in the detection of different biomolecules in recent years, in order to provide help for the research of more efficient and sensitive detection methods. Graphical Abstract

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Efficient DNA-assisted synthesis of trans-membrane gold nanowires

Microsystems & Nanoengineering ◽

10.1038/micronano.2017.84 ◽

2018 ◽

Vol 4 (1) ◽

Cited By ~ 3

Author(s):

Maoxiang Guo ◽

Iván Hernández-Neuta ◽

Narayanan Madaboosi ◽

Mats Nilsson ◽

Wouter van der Wijngaart

Keyword(s):

Chemical Sensor ◽

Signal To Noise Ratio ◽

Rolling Circle Amplification ◽

High Specificity ◽

Porous Membrane ◽

Open Circuit ◽

High Signal ◽

Gold Nanowires ◽

Rolling Circle ◽

Electric Circuits

Abstract Whereas electric circuits and surface-based (bio)chemical sensors are mostly constructed in-plane due to ease of manufacturing, 3D microscale and nanoscale structures allow denser integration of electronic components and improved mass transport of the analyte to (bio)chemical sensor surfaces. This work reports the first out-of-plane metallic nanowire formation based on stretching of DNA through a porous membrane. We use rolling circle amplification (RCA) to generate long single-stranded DNA concatemers with one end anchored to the surface. The DNA strands are stretched through the pores in the membrane during liquid removal by forced convection. Because the liquid–air interface movement across the membrane occurs in every pore, DNA stretching across the membrane is highly efficient. The stretched DNA molecules are transformed into trans-membrane gold nanowires through gold nanoparticle hybridization and gold enhancement chemistry. A 50 fM oligonucleotide concentration, a value two orders of magnitude lower than previously reported for flat surface-based nanowire formation, was sufficient for nanowire formation. We observed nanowires in up to 2.7% of the membrane pores, leading to an across-membrane electrical conductivity reduction from open circuit to <20 Ω. The simple electrical read-out offers a high signal-to-noise ratio and can also be extended for use as a biosensor due to the high specificity and scope for multiplexing offered by RCA.

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A Label-Free Fluorescent Sensor Based on the Formation of Poly(thymine)-Templated Copper Nanoparticles for the Sensitive and Selective Detection of MicroRNA from Cancer Cells

Chemosensors ◽

10.3390/chemosensors8030052 ◽

2020 ◽

Vol 8 (3) ◽

pp. 52

Author(s):

Qian Ma ◽

Zhiqiang Gao ◽

Hiranya Dayal ◽

Sam Fong Yau Li

Keyword(s):

Cancer Cells ◽

Copper Nanoparticles ◽

Fluorescent Sensor ◽

Rolling Circle Amplification ◽

High Specificity ◽

Excitation Wavelength ◽

Selective Detection ◽

Label Free ◽

Rolling Circle ◽

Highly Sensitive

In this work, a simple and label-free fluorescence “off” to “on” platform was designed for the sensitive and selective detection of microRNA (miRNA) in cancer cells. This method utilized a padlock DNA-based rolling circle amplification (P-RCA) to synthesize fluorescent poly(thymine) (PolyT) which acted as a template for the synthesis of copper nanoparticles (CuNPs) within 10 minutes under mild conditions. While the repeated PolyT sequence was used as the template for CuNP synthesis, other non-PolyT parts (single strand-DNAs without the capacity to act as the template for CuNP formation) served as “smart glues” or rigid linkers to build complex nanostructures. Under the excitation wavelength of 340 nm, the synthesized CuNPs emitted strong red fluorescence effectively at 620 nm. To demonstrate the use of this method as a universal biosensor platform, lethal-7a (let-7a) miRNA was chosen as the standard target. This sensor could achieve highly sensitive and selective detection of miRNA in the presence of other homologous analogues for the combination of P-RCA with the fluorescent copper nanoparticle. Overall, this novel label-free method holds great potential in the sensitive detection of miRNA with high specificity in real samples.

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Frequent Novel Variations Within MSH2 and MLH1 Genes in a Subset of Iranian Families With Hereditary Non-Polyposis Colorectal Cancer Shadi

ACTA MEDICA IRANICA ◽

10.18502/acta.v57i3.1815 ◽

2019 ◽

Author(s):

Shadi Javan ◽

Alireza Andalib ◽

Ali Hosseini Bereshneh ◽

Mohammad Hassan Emami ◽

Rasul Salehi ◽

...

Keyword(s):

Colorectal Cancer ◽

Mutation Screening ◽

Family Members ◽

High Specificity ◽

Single Strand Conformation Polymorphism ◽

Sequencing Analysis ◽

Mlh1 Gene ◽

Specificity And Sensitivity ◽

Two Samples ◽

Significant Difference

Abstract- Hereditary non-polyposis colorectal cancer (HNPCC) is the most frequent autosomal dominant predisposition for development of colorectal cancer (CRC) caused by germline defects in mismatch repair (MMR) genes. Current study was aimed to find genetic variations in MSH2 and MLH1 genes and their correlation with the serum levels of Carcinoembryonic Antigen (CEA) in seven Iranian HNPCC families. Seven unrelated Iranian families including 11 HNPCC patients and 7 affected family members were selected. They were initially screened for mutations in exons 7 of MSH2 and exon 15 of MLH1 gene through polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP). Positive PCR results were further analyzed through exon sequencing. Serum CEA level was determined using the ELISA test. PCRSSCP was positive in 8 out of 18 patients (44%) for exons 7 of MSH2 gene, whereas two samples (11%) demonstrated to bear a mutation in exon 15 of the MLH1 gene. Sequencing analysis of both amplified exons in positive and negative samples have confirmed no mutation in negative samples while revealed 5 and 7 novel mutations in exons 7 and 15, respectively. The mean serum concentration of CEA had a significant difference between HNPCC patients and their healthy family members. Our results demonstrated that the PCR-SSCP method has high specificity and sensitivity in the first step of mutation screening of HNPCC families. High frequency of novel alterations found in the current assay may revise the mutation screening of MSH2 and MLH1 genes and abet further assessment of their frequency among individual HNPCC patients.

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