Ultrasensitive nucleic acid detection based on phosphorothioated hairpin-assisted isothermal amplification

AbstractHerein, we describe a phosphorothioated hairpin-assisted isothermal amplification (PHAmp) method for detection of a target nucleic acid. The hairpin probe (HP) is designed to contain a 5′ phosphorothioate (PS)-modified overhang, a target recognition site, and a 3′ self-priming (SP) region. Upon binding to the target nucleic acid, the HP opens and the SP region is rearranged to serve as a primer. The subsequent process of strand displacement DNA synthesis recycles the bound target to open another HP and produces an extended HP (EP) with a PS-DNA/DNA duplex at the end, which would be readily denatured due to its reduced thermal stability. The trigger then binds to the denatured 3′ end of the EP and is extended, producing an intermediate double-stranded (ds) DNA product (IP). The trigger also binds to the denatured 3′ end of the IP, and its extension produces the final dsDNA product along with concomitant displacement and recycling of EP. By monitoring the dsDNA products, the target nucleic acid can be identified down to 0.29 fM with a wide dynamic range from 1 nM to 1 fM yielding an excellent specificity to discriminate even a single base-mismatched target. The unique design principle could provide new insights into the development of novel isothermal amplification methods for nucleic acid detection.

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A hairpin probe-mediated isothermal amplification method to detect target nucleic acid

Analytica Chimica Acta ◽

10.1016/j.aca.2020.04.003 ◽

2020 ◽

Vol 1114 ◽

pp. 7-14

Author(s):

Hyo Yong Kim ◽

Jun Ki Ahn ◽

Chang Yeol Lee ◽

Hyun Gyu Park

Keyword(s):

Nucleic Acid ◽

Isothermal Amplification ◽

Hairpin Probe ◽

Amplification Method ◽

Target Nucleic Acid

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Scanning single-molecule counting system for Eprobe with highly simple and effective approach

PLoS ONE ◽

10.1371/journal.pone.0243319 ◽

2020 ◽

Vol 15 (12) ◽

pp. e0243319

Author(s):

Takeshi Hanami ◽

Tetsuya Tanabe ◽

Takuya Hanashi ◽

Mitsushiro Yamaguchi ◽

Hidetaka Nakata ◽

...

Keyword(s):

Nucleic Acid ◽

Single Molecule ◽

Detection System ◽

Signal To Noise Ratio ◽

High Sensitivity ◽

Nucleic Acid Detection ◽

Digital Measurement ◽

Excitation Light ◽

Target Nucleic Acid ◽

Fluorescent Molecules

Here, we report a rapid and ultra-sensitive detection technique for fluorescent molecules called scanning single molecular counting (SSMC). The method uses a fluorescence-based digital measurement system to count single molecules in a solution. In this technique, noise is reduced by conforming the signal shape to the intensity distribution of the excitation light via a circular scan of the confocal region. This simple technique allows the fluorescent molecules to freely diffuse into the solution through the confocal region and be counted one by one and does not require statistical analysis. Using this technique, 28 to 62 aM fluorescent dye was detected through measurement for 600 s. Furthermore, we achieved a good signal-to-noise ratio (S/N = 2326) under the condition of 100 pM target nucleic acid by only mixing a hybridization-sensitive fluorescent probe, called Eprobe, into the target oligonucleotide solution. Combination of SSMC and Eprobe provides a simple, rapid, amplification-free, and high-sensitive target nucleic acid detection system. This method is promising for future applications to detect particularly difficult to design primers for amplification as miRNAs and other short oligo nucleotide biomarkers by only hybridization with high sensitivity.

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Duplex-specific nuclease-mediated target recycling amplification for fluorescence detection of microRNA

Analytical Methods ◽

10.1039/c8ay02265h ◽

2019 ◽

Vol 11 (2) ◽

pp. 200-204 ◽

Cited By ~ 4

Author(s):

Lin Tan ◽

Liu Xu ◽

Jin-Wen Liu ◽

Li-Juan Tang ◽

Hao Tang ◽

...

Keyword(s):

Nucleic Acid ◽

Fluorescence Detection ◽

Low Cost ◽

Isothermal Amplification ◽

Nucleic Acid Detection ◽

Target Recycling ◽

Recycling Amplification

Isothermal amplification techniques for nucleic acid detection have drawn increasing interest recently due to the simplicity and low-cost of instruments.

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IsoPCR: An Analytically Sensitive, Nested, Multiplex Nucleic Acid Amplification Method

Clinical Chemistry ◽

10.1373/clinchem.2012.193664 ◽

2013 ◽

Vol 59 (2) ◽

pp. 436-439 ◽

Cited By ~ 6

Author(s):

Martin Jensen Søe ◽

Mikkel Rohde ◽

Jens Mikkelsen ◽

Peter Warthoe

Keyword(s):

Nucleic Acid ◽

Candida Glabrata ◽

Simultaneous Detection ◽

Lamp Assay ◽

Isothermal Amplification ◽

Qpcr Assay ◽

Nucleic Acid Amplification ◽

Molecular Diagnostic ◽

Nucleic Acid Detection ◽

Amplification Method

BACKGROUND Nucleic acid tests that can simultaneously detect multiple targets with high sensitivity, specificity, and speed are highly desirable. To meet this need, we developed a new approach we call the isoPCR method. METHODS The isoPCR method is a 2-stage nested-like nucleic acid amplification method that combines a single multiplex preamplification PCR with subsequent distinct detection of specific targets by use of isothermal amplification. We compared isoPCR to nested quantitative PCR (qPCR), loop-mediated isothermal amplification (LAMP), and nested LAMP (PCR followed by LAMP), for detection of DNA from Candida glabrata. We evaluated the method's multiplex capability for detecting low copy numbers of pathogens commonly involved in sepsis. RESULTS IsoPCR provided detection of 1 copy of Candida glabrata, an LOD that was 5-fold lower than a nested qPCR assay (5 copies), while the amplification time was simultaneously halved. Similarly, the LOD for isoPCR was lower than that for a LAMP assay (1000 copies) and a nested LAMP assay (5 copies). IsoPCR required recognition of 6 regions for detection, thereby providing a theoretically higher specificity compared to nested qPCR (4 regions). The isoPCR multiplexing capability was demonstrated by simultaneous detection of 4 pathogens with individual LODs of 10 copies or fewer. Furthermore, the specificity of isoPCR was demonstrated by successful pathogen detection from samples with more than 1 pathogen present. CONCLUSIONS IsoPCR provides a molecular diagnostic tool for multiplex nucleic acid detection, with an LOD down to 1 copy, high theoretical specificity, and halving of the amplification time compared to a nested qPCR assay.

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A biosensor for the detection of single base mismatches in microRNA

Chemical Communications ◽

10.1039/c5cc04706d ◽

2015 ◽

Vol 51 (78) ◽

pp. 14597-14600 ◽

Cited By ~ 22

Author(s):

Jieon Lee ◽

Ginam Park ◽

Dal-Hee Min

Keyword(s):

Graphene Oxide ◽

Nucleic Acid ◽

Nucleic Acid Detection ◽

Single Base ◽

Target Probe

Graphene oxide enables highly sequence specific nucleic acid detection by selectively removing the signal from a mismatched target/probe duplex.

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A Novel Single Fluorophore-Labeled Double-Stranded Oligonucleotide Probe for Fluorescence-Enhanced Nucleic Acid Detection Based on the Inherent Quenching Ability of Deoxyguanosine Bases and Competitive Strand-Displacement Reaction

Journal of Fluorescence ◽

10.1007/s10895-011-0935-y ◽

2011 ◽

Vol 22 (1) ◽

pp. 43-46 ◽

Cited By ~ 3

Author(s):

Yingwei Zhang ◽

Jingqi Tian ◽

Hailong Li ◽

Lei Wang ◽

Xuping Sun

Keyword(s):

Nucleic Acid ◽

Oligonucleotide Probe ◽

Nucleic Acid Detection ◽

Displacement Reaction ◽

Strand Displacement ◽

Double Stranded Oligonucleotide ◽

Strand Displacement Reaction

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NAIL: Nucleic Acid detection using Isotachophoresis and Loop-mediated isothermal amplification

Lab on a Chip ◽

10.1039/c4lc01479k ◽

2015 ◽

Vol 15 (7) ◽

pp. 1697-1707 ◽

Cited By ~ 32

Author(s):

Mark D. Borysiak ◽

Kevin W. Kimura ◽

Jonathan D. Posner

Keyword(s):

Nucleic Acid ◽

Nucleic Acids ◽

Mobile Phone ◽

Isothermal Amplification ◽

Nucleic Acid Detection ◽

Complex Matrices ◽

Loop Mediated Isothermal Amplification

The NAIL device integrates isotachophoresis and loop-mediated isothermal amplification (LAMP) with mobile phone detection to extract, amplify, and detect nucleic acids from complex matrices in less than one hour.

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Three-Way Junction-Induced Isothermal Amplification with High Signal-to-Background Ratio for Detection of Pathogenic Bacteria

Sensors ◽

10.3390/s21124132 ◽

2021 ◽

Vol 21 (12) ◽

pp. 4132

Author(s):

Jung Ho Kim ◽

Seokjoon Kim ◽

Sung Hyun Hwang ◽

Tae Hwi Yoon ◽

Jung Soo Park ◽

...

Keyword(s):

Nucleic Acid ◽

Nucleic Acids ◽

Pathogenic Bacteria ◽

High Selectivity ◽

Isothermal Amplification ◽

Rna Aptamers ◽

High Signal ◽

Fluorescence Signal ◽

Enhanced Fluorescence ◽

Target Nucleic Acid

The consumption of water and food contaminated by pathogens is a major cause of numerous diseases and deaths globally. To control pathogen contamination and reduce the risk of illness, a system is required that can quickly detect and monitor target pathogens. We developed a simple and reproducible strategy, termed three-way junction (3WJ)-induced transcription amplification, to detect target nucleic acids by rationally combining 3WJ-induced isothermal amplification with a light-up RNA aptamer. In principle, the presence of the target nucleic acid generates a large number of light-up RNA aptamers (Spinach aptamers) through strand displacement and transcription amplification for 2 h at 37 °C. The resulting Spinach RNA aptamers specifically bind to fluorogens such as 3,5-difluoro-4-hydroxybenzylidene imidazolinone and emit a highly enhanced fluorescence signal, which is clearly distinguished from the signal emitted in the absence of the target nucleic acid. With the proposed strategy, concentrations of target nucleic acids selected from the genome of Salmonellaenterica serovar Typhi (S. Typhi) were quantitatively determined with high selectivity. In addition, the practical applicability of the method was demonstrated by performing spike-and-recovery experiments with S. Typhi in human serum.

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