Rapid and enzyme-free nucleic acid detection based on exponential hairpin assembly in complex biological fluids

The Analyst ◽  
2016 ◽  
Vol 141 (10) ◽  
pp. 2883-2886 ◽  
Author(s):  
Cuiping Ma ◽  
Menghua Zhang ◽  
Shan Chen ◽  
Chao Liang ◽  
Chao Shi
Keyword(s):  
Nucleic Acid ◽  
Self Assembly ◽  
Detection Method ◽  
Biological Fluids ◽  
Nucleic Acid Amplification ◽  
Nucleic Acid Detection ◽  
Dna Hairpins ◽  
Fret Pair

Herein, we have developed a rapid and enzyme-free nucleic acid amplification detection method that combined the exponential self-assembly of four DNA hairpins and the FRET pair Cy3 and Cy5.

scholarly journals Contamination-resistant, rapid emulsion-based isothermal nucleic acid amplification with Mie-scatter inspired light scatter analysis for bacterial identification

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Alexander S. Day ◽  
Tiffany-Heather Ulep ◽  
Elizabeth Budiman ◽  
Laurel Dieckhaus ◽  
Babak Safavinia ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acid Amplification ◽  
Nucleic Acid Detection ◽  
Light Scatter ◽  
Emulsion Droplets ◽  
Assay Performance ◽  
Bacterial Adsorption ◽  
Scatter Intensity ◽  
The Impact ◽  
Theory Light

AbstractAn emulsion loop-mediated isothermal amplification (eLAMP) platform was developed to reduce the impact that contamination has on assay performance. Ongoing LAMP reactions within the emulsion droplets cause a decrease in interfacial tension, causing a decrease in droplet size, which results in decreased light scatter intensity due to Mie theory. Light scatter intensity was monitored via spectrophotometers and fiber optic cables placed at 30° and 60°. Light scatter intensities collected at 3 min, 30° were able to statistically differentiate 103 and 106 CFU/µL initial Escherichia coli O157:H7 concentrations compared to NTC (0 CFU/µL), while the intensity at 60° were able to statistically differentiate 106 CFU/µL initial concentrations and NTC. Control experiments were conducted to validate nucleic acid detection versus bacterial adsorption, finding that the light scatter intensities change is due specifically to ongoing LAMP amplification. After inducing contamination of bulk LAMP reagents, specificity lowered to 0% with conventional LAMP, while the eLAMP platform showed 87.5% specificity. We have demonstrated the use of angle-dependent light scatter intensity as a means of real-time monitoring of an emulsion LAMP platform and fabricated a smartphone-based monitoring system that showed similar trends as spectrophotometer light scatter data, validating the technology for a field deployable platform.

scholarly journals Rapid Detection of SARS-CoV-2 Using Reverse transcription RT-LAMP method

2020 ◽  
Author(s):  
Weihua Yang ◽  
Xiaofei Dang ◽  
Qingxi Wang ◽  
Mingjie Xu ◽  
Qianqian Zhao ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Reverse Transcription ◽  
Virus Disease ◽  
Lamp Assay ◽  
Nucleic Acid Amplification ◽  
N Gene ◽  
Nucleic Acid Detection ◽  
Lamp Method ◽  
Rt Pcr ◽  
Rapid Amplification

AbstractCorona Virus Disease 2019 (COVID-19) is a recently emerged life-threatening disease caused by SARS-CoV-2. Real-time fluorescent PCR (RT-PCR) is the clinical standard for SARS-CoV-2 nucleic acid detection. To detect SARS-CoV-2 early and control the disease spreading on time, a faster and more convenient method for SARS-CoV-2 nucleic acid detecting, RT-LAMP method (reverse transcription loop-mediated isothermal amplification) was developed. RNA reverse transcription and nucleic acid amplification were performed in one step at 63 °C isothermal conditions, and the results can be obtained within 30 minutes. ORF1ab gene, E gene and N gene were detected at the same time. ORF1ab gene was very specific and N gene was very sensitivity, so they can guarantee both sensitivity and specificity for SARS-CoV-2. The sensitivity of RT-LAMP assay is similar to RT-PCR, and specificity was 99% as detecting 208 clinical specimens. The RT-LAMP assay reported here has the advantages of rapid amplification, simple operation, and easy detection, which is useful for the rapid and reliable clinical diagnosis of SARS-CoV-2.

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

2013 ◽  
Vol 59 (2) ◽  
pp. 436-439 ◽  
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.

scholarly journals Virus detection via programmable Type III-A CRISPR-Cas systems

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Sagar Sridhara ◽  
Hemant N. Goswami ◽  
Charlisa Whyms ◽  
Jonathan H. Dennis ◽  
Hong Li
Keyword(s):  
Nucleic Acid ◽  
Detection Method ◽  
Virus Detection ◽  
High Sensitivity ◽  
Nucleic Acid Detection ◽  
Immune Effector ◽  
Type Iii ◽  
System A ◽  
Acid Cleavage

AbstractAmong the currently available virus detection assays, those based on the programmable CRISPR-Cas enzymes have the advantage of rapid reporting and high sensitivity without the requirement of thermocyclers. Type III-A CRISPR-Cas system is a multi-component and multipronged immune effector, activated by viral RNA that previously has not been repurposed for disease detection owing in part to the complex enzyme reconstitution process and functionality. Here, we describe the construction and application of a virus detection method, based on an in vivo-reconstituted Type III-A CRISPR-Cas system. This system harnesses both RNA- and transcription-activated dual nucleic acid cleavage activities as well as internal signal amplification that allow virus detection with high sensitivity and at multiple settings. We demonstrate the use of the Type III-A system-based method in detection of SARS-CoV-2 that reached 2000 copies/μl sensitivity in amplification-free and 60 copies/μl sensitivity via isothermal amplification within 30 min and diagnosed SARS-CoV-2-infected patients in both settings. The high sensitivity, flexible reaction conditions, and the small molecular-driven amplification make the Type III-A system a potentially unique nucleic acid detection method with broad applications.

scholarly journals High-risk HPV nucleic acid detection kit–the careHPV test –a new detection method for screening

2014 ◽  
Vol 4 (1) ◽  
Author(s):  
Hong Ying ◽  
Fang Jing ◽  
Zhao Fanghui ◽  
Qiao Youlin ◽  
Hu Yali
Keyword(s):  
Nucleic Acid ◽  
High Risk ◽  
Detection Method ◽  
Nucleic Acid Detection ◽  
High Risk Hpv

scholarly journals Development of a Portable SPR Sensor for Nucleic Acid Detection

Micromachines ◽  
2020 ◽  
Vol 11 (5) ◽  
pp. 526 ◽  
Author(s):  
Yafeng Huang ◽  
Lulu Zhang ◽  
Hao Zhang ◽  
Yichen Li ◽  
Luyao Liu ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Incident Angle ◽  
Incident Light ◽  
High Sensitivity ◽  
Nucleic Acid Amplification ◽  
Nucleic Acid Hybridization ◽  
Nucleic Acid Detection ◽  
Scanning System ◽  
Label Free ◽  
Spr Sensor

Nucleic acid detection is of great significance in clinical diagnosis, environmental monitoring and food safety. Compared with the traditional nucleic acid amplification detection method, surface plasmon resonance (SPR) sensing technology has the advantages of being label-free, having simple operation, and providing real-time detection. However, the angle scanning system in many SPR angle modulation detection applications usually requires a high-resolution stepper motor and complex mechanical structure to adjust the angle. In this paper, a portable multi-angle scanning SPR sensor was designed. The sensor only uses one stepping motor to rotate a belt, and the belt pulls the mechanical linkages of incident light and reflected light to move in opposite directions for achieving the SPR angle scanning mode that keeps the incident angle and reflected angle equal. The sensor has an angle scanning accuracy of 0.002°, response sensitivity of 3.72 × 10−6 RIU (refractive index unit), and an angle scanning range of 30°–74°. The overall size of the system is only 480 mm × 150 mm × 180 mm. The portable SPR sensor was used to detect nucleic acid hybridization on a gold film chip modified with bovine serum albumin (BSA). The result revealed that the sensor had high sensitivity and fast response, and could successfully accomplish the hybridization detection of target DNA solution of 0.01 μmol/mL.

Rapid and low-cost amplicon visualization for nucleic acid amplification tests using magnetic microbeads

The Analyst ◽  
2021 ◽  
Author(s):  
Michihiko Nakano ◽  
Masafumi Inaba ◽  
Junya Suehiro
Keyword(s):  
Magnetic Field ◽  
Nucleic Acid ◽  
Detection Method ◽  
Low Cost ◽  
Deionized Water ◽  
Nucleic Acid Amplification ◽  
Magnetic Microbeads ◽  
Nucleic Acid Amplification Tests

This study presents a rapid and low-cost amplicon detection method in which amplicons are attached to magnetic microbeads, suspended in deionized water, and subjected to a magnetic field on a...

scholarly journals Rapidemic, a versatile and label-free DNAzyme-based platform for visual nucleic acid detection

2020 ◽  
Author(s):  
Marijn van den Brink ◽  
Sebastian T. Tandar ◽  
Tim A. P. van den Akker ◽  
Sinisha Jovikj ◽  
Violette Defourt ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Infectious Diseases ◽  
Detection Method ◽  
Point Of Care ◽  
Diagnostic Testing ◽  
Nucleic Acid Detection ◽  
Label Free ◽  
Strand Displacement Amplification ◽  
Sequence Detection ◽  
Accessible Point

AbstractIn the last three decades, there have been recurring outbreaks of infectious diseases, brought to light with the recent outbreak of coronavirus disease 2019 (COVID-19). Attempts to effectively contain the spread of infectious diseases have been hampered by the lack of rapidly adaptable, accurate, and accessible point-of-care diagnostic testing. In this study, we present a novel design of a label-free DNAzyme-based detection method called Rapidemic. This assay combines recombinase polymerase amplification (RPA) with linear strand-displacement amplification (LSDA) and guanine-quadruplex (GQ) DNAzyme-catalysed colour-changing reaction. The colorimetry basis of the signal readout omits the need for extensive instrumentation. Moreover, the primer-based sequence detection of RPA gives Rapidemic a potential to be rapidly adapted to target a new sequence. As a proof of concept, we developed the assay to detect isolated genomic DNA of Saccharomyces cerevisiae. The use of low-pH buffers and the optimization of the dilution rates from each preceding reaction to the next showed to be successful strategies to enable visible detection with this method. These findings demonstrate for the first time that a label-free DNAzyme-based detection method can be coupled to RPA and LSDA for nucleic acid detection.

scholarly journals Highly Specific and Sensitive Detection of Yersinia pestis by Portable Cas12a-UPTLFA Platform

2021 ◽  
Vol 12 ◽  
Author(s):  
Yang You ◽  
Pingping Zhang ◽  
Gengshan Wu ◽  
Yafang Tan ◽  
Yong Zhao ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Yersinia Pestis ◽  
Rapid Detection ◽  
Detection Method ◽  
Detection System ◽  
Point Of Care ◽  
High Sensitivity ◽  
Nucleic Acid Detection ◽  
Great Promise ◽  
High Background

The recent discovery of collateral cleavage activity of class-II clustered regularly interspaced short palindromic repeats–CRISPR-associated protein (CRISPR-Cas) makes CRISPR-based diagnosis a potential high-accuracy nucleic acid detection method. Colloidal gold-based lateral flow immunochromatographic assay (LFA), which has been combined with CRISPR/Cas-based nucleic detection, usually associates with drawbacks of relative high background and the subjectivity in naked-eye read-out of the results. Here, we developed a novel system composed of Cas12a-based nucleic acid detection and up-converting phosphor technology (UPT)-based LFA (UPT–LFA), termed Cas12a-UPTLFA. We further demonstrated the utility of this platform in highly sensitive and specific detection of Yersinia pestis, the causative agent of the deadly plague. Due to high infectivity and mortality, as well as the potential to be misused as bioterrorism agent, a culture-free, ultrasensitive, specific, and rapid detection method for Y. pestis has long been desired. By incorporating isothermal recombinase polymerase amplification, the Cas12a-UPTLFA we established can successfully detect genomic DNA of Y. pestis as low as 3 attomolar (aM) and exhibited high sensitivity (93.75%) and specificity (90.63%) for detection of spiked blood samples with a detection limit of 102 colony-forming unit per 100 μl of mouse blood. With a portable biosensor, Cas12a-UPTLFA assay can be operated easily by non-professional personnel. Taken together, we have developed a novel Cas12a-UPTLFA platform for rapid detection of Y. pestis with high sensitivity and specificity, which is portable, not expensive, and easy to operate as a point-of-care method. This detection system can easily be extended to detect other pathogens and holds great promise for on-site detection of emerging infectious pathogens.

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