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

Sensors ◽  
2021 ◽  
Vol 21 (12) ◽  
pp. 4132
Author(s):  
Jung Ho Kim ◽  
Seokjoon Kim ◽  
Sung Hyun Hwang ◽  
Tae Hwi Yoon ◽  
Jung Soo Park ◽  
...  

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.

Sensors ◽  
2018 ◽  
Vol 18 (7) ◽  
pp. 2277
Author(s):  
Kang-Ho Lee ◽  
Dongkyu Lee ◽  
Jongsu Yoon ◽  
Ohwon Kwon ◽  
Jaejong Lee

A disposable potentiometric sensor was newly developed for the amplification-coupled detection of nucleic acids. The hydrogen-ion is generally released during isothermal amplification of nucleic acids. The surface potential on the oxide-functionalized electrode of the extended gate was directly measured using full electrical circuits with the commercial metal-oxide semiconductor field-effect transistors (MOSFETs) and ring oscillator components, which resulted in cost-effective, portable and scalable real-time nucleic acid analysis. The current-starved ring oscillator changes surface potential to its frequency depending on the square of the variation in pH with a high signal-to-noise ratio during isothermal amplification. The device achieves a conversion rate of 20.5 kHz/mV and a detection resolution of 200 µV for the surface potential. It is demonstrated that the sensor successfully monitors in real-time isothermal amplification of the extracted nucleic acids from Salmonella pathogenic bacteria. The in situ variations in the frequency of the pH-sensitive sensor were compared with the results of both a conventional optical device and pH-meter during isothermal amplification.


Revista Vitae ◽  
2021 ◽  
Vol 28 (3) ◽  
Author(s):  
Laura Carvajal Barbosa ◽  
Diego Insuasty Cepeda ◽  
Andrés Felipe León Torres ◽  
Maria Mercedes Arias Cortes ◽  
Zuly Jenny Rivera Monroy ◽  
...  

BACKGROUND : Biosensing techniques have been the subject of exponentially increasing interest due to their performance advantages such as high selectivity and sensitivity, easy operation, low cost, short analysis time, simple sample preparation, and real-time detection. Biosensors have been developed by integrating the unique specificity of biological reactions and the high sensitivity of physical sensors. Therefore, there has been a broad scope of applications for biosensing techniques, and nowadays, they are ubiquitous in different areas of environmental, healthcare, and food safety. Biosensors have been used for environmental studies, detecting and quantifying pollutants in water, air, and soil. Biosensors also showed great potential for developing analytical tools with countless applications in diagnosing, preventing, and treating diseases, mainly by detecting biomarkers. Biosensors as a medical device can identify nucleic acids, proteins, peptides, metabolites, etc.; these analytes may be biomarkers associated with the disease status. Bacterial food contamination is considered a worldwide public health issue; biosensor-based analytical techniques can identify the presence or absence of pathogenic agents in food. OBJECTIVES: The present review aims to establish state-of-the-art, comprising the recent advances in the use of nucleic acid-based biosensors and their novel application for the detection of nucleic acids. Emphasis will be given to the performance characteristics, advantages, and challenges. Additionally, food safety applications of nucleic acid-based biosensors will be discussed. METHODS: Recent research articles related to nucleic acid-based biosensors, biosensors for detecting nucleic acids, biosensors and food safety, and biosensors in environmental monitoring were reviewed. Also, biosensing platforms associated with the clinical diagnosis and food industry were included. RESULTS: It is possible to appreciate that multiple applications of nucleic acid-based biosensors have been reported in the diagnosis, prevention, and treatment of diseases, as well as to identify foodborne pathogenic bacteria. The use of PNA and aptamers opens the possibility of developing new biometric tools with better analytical properties. CONCLUSIONS: Biosensors could be considered the most important tool for preventing, treating, and monitoring diseases that significantly impact human health. The aptamers have advantages as biorecognition elements due to the structural conformation, hybridization capacity, robustness, stability, and lower costs. It is necessary to implement biosensors in situ to identify analytes with high selectivity and lower detection limits.


The Analyst ◽  
2018 ◽  
Vol 143 (3) ◽  
pp. 639-642 ◽  
Author(s):  
Rui Mao ◽  
Lifei Qi ◽  
Jianjun Li ◽  
Ming Sun ◽  
Zhuo Wang ◽  
...  

A novel nucleic acid isothermal amplification method with high specificity, efficiency and rapidity was developed.


2016 ◽  
Vol 60 (1) ◽  
pp. 27-35 ◽  
Author(s):  
Pawan Jolly ◽  
Pedro Estrela ◽  
Michael Ladomery

There are an increasing number of applications that have been developed for oligonucleotide-based biosensing systems in genetics and biomedicine. Oligonucleotide-based biosensors are those where the probe to capture the analyte is a strand of deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or a synthetic analogue of naturally occurring nucleic acids. This review will shed light on various types of nucleic acids such as DNA and RNA (particularly microRNAs), their role and their application in biosensing. It will also cover DNA/RNA aptamers, which can be used as bioreceptors for a wide range of targets such as proteins, small molecules, bacteria and even cells. It will also highlight how the invention of synthetic oligonucleotides such as peptide nucleic acid (PNA) or locked nucleic acid (LNA) has pushed the limits of molecular biology and biosensor development to new perspectives. These technologies are very promising albeit still in need of development in order to bridge the gap between the laboratory-based status and the reality of biomedical applications.


2019 ◽  
Vol 17 (30) ◽  
pp. 7222-7227 ◽  
Author(s):  
Rashi Soni ◽  
Deepti Sharma ◽  
A. Murali Krishna ◽  
Jagadeesh Sathiri ◽  
Ashwani Sharma

A Baby Spinach aptamer based minimal-modified sensor (BSMS) detects nucleic acids of potentially any length with high selectivity and specificity, and shows 2.5-fold more fluorescence enhancement compared to the parent aptamer.


2018 ◽  
Vol 54 (75) ◽  
pp. 10562-10565 ◽  
Author(s):  
Xin Ye ◽  
Yang Li ◽  
Lijuan Wang ◽  
Xueen Fang ◽  
Jilie Kong

A novel exonuclease-assisted isothermal amplification to amplify and determine nucleic acids very sensitively and with ultrahigh specificity.


Lab on a Chip ◽  
2015 ◽  
Vol 15 (7) ◽  
pp. 1697-1707 ◽  
Author(s):  
Mark D. Borysiak ◽  
Kevin W. Kimura ◽  
Jonathan D. Posner

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.


2018 ◽  
Vol 14 ◽  
pp. 253-281 ◽  
Author(s):  
Tirayut Vilaivan

Fluorogenic oligonucleotide probes that can produce a change in fluorescence signal upon binding to specific biomolecular targets, including nucleic acids as well as non-nucleic acid targets, such as proteins and small molecules, have applications in various important areas. These include diagnostics, drug development and as tools for studying biomolecular interactions in situ and in real time. The probes usually consist of a labeled oligonucleotide strand as a recognition element together with a mechanism for signal transduction that can translate the binding event into a measurable signal. While a number of strategies have been developed for the signal transduction, relatively little attention has been paid to the recognition element. Peptide nucleic acids (PNA) are DNA mimics with several favorable properties making them a potential alternative to natural nucleic acids for the development of fluorogenic probes, including their very strong and specific recognition and excellent chemical and biological stabilities in addition to their ability to bind to structured nucleic acid targets. In addition, the uncharged backbone of PNA allows for other unique designs that cannot be performed with oligonucleotides or analogues with negatively-charged backbones. This review aims to introduce the principle, showcase state-of-the-art technologies and update recent developments in the areas of fluorogenic PNA probes during the past 20 years.


2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Xiong Ding ◽  
Kun Yin ◽  
Ziyue Li ◽  
Vikram Pandian ◽  
Joan A. Smyth ◽  
...  

Abstract Fluorescence detection of nucleic acid isothermal amplification utilizing energy-transfer-tagged oligonucleotide probes provides a highly sensitive and specific method for pathogen detection. However, currently available probes suffer from relatively weak fluorescence signals and are not suitable for simple, affordable smartphone-based detection at the point of care. Here, we present a cleavable hairpin beacon (CHB)-enhanced fluorescence detection for isothermal amplification assay. The CHB probe is a single fluorophore-tagged hairpin oligonucleotide with five continuous ribonucleotides which can be cleaved by the ribonuclease to specifically initiate DNA amplification and generate strong fluorescence signals. By coupling with loop-mediated isothermal amplification (LAMP), the CHB probe could detect Borrelia burgdorferi (B. burgdorferi) recA gene with a sensitivity of 100 copies within 25 min and generated stronger specific fluorescence signals which were easily read and analysed by our programmed smartphone. Also, this CHB-enhanced LAMP (CHB-LAMP) assay was successfully demonstrated to detect B. burgdorferi DNA extracted from tick species, showing comparable results to real-time PCR assay. In addition, our CHB probe was compatible with other isothermal amplifications, such as isothermal multiple-self-matching-initiated amplification (IMSA). Therefore, CHB-enhanced fluorescence detection is anticipated to facilitate the development of simple, sensitive smartphone-based point-of-care pathogen diagnostics in resource-limited settings.


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