Padlock Probe-based Rolling Circle Amplification Lateral Flow Assay for Point-of-Need Nucleic Acid Detection

The Analyst ◽  
2021 ◽  
Author(s):  
Sidhartha Jain ◽  
David S. Dandy ◽  
Brian Geiss ◽  
Charles Henry

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)...

The Analyst ◽  
2015 ◽  
Vol 140 (1) ◽  
pp. 74-78 ◽  
Author(s):  
Xiaoli Zhu ◽  
Chang Feng ◽  
Bin Zhang ◽  
Hui Tong ◽  
Tao Gao ◽  
...  

An isothermal nucleic acid amplification technique termed as netlike rolling circle amplification is proposed. Dense and uniform network morphology of amplified products is first observed, suggesting the ultrahigh amplification efficiency.


2013 ◽  
Vol 142 (1) ◽  
pp. 1-11 ◽  
Author(s):  
J. GRAY ◽  
L. J. COUPLAND

SUMMARYOn 14 January 2013, the US Food and Drug Administration (FDA) announced permission for a multiplex nucleic acid test, the xTAG®Gastrointestinal Pathogen Panel (GPP) (Luminex Corporation, USA), which simultaneously detects 11 common viral, bacterial and parasitic causes of infectious gastroenteritis, to be marketed in the USA. This announcement reflects the current move towards the development and commercialization of detection technologies based on nucleic acid amplification techniques for diagnosis of syndromic infections. We discuss the limitations and advantages of nucleic acid amplification techniques and the recent advances in Conformité Européene – in-vitrodiagnostic (CE-IVD)-approved multiplex real-time PCR kits for the simultaneous detection of multiple targets within the clinical diagnostics market.


2020 ◽  
Vol 11 (3) ◽  
pp. 10559-10571

The early detection of invasive fungal infection (IFD) is significant in order to decrease mortality in susceptible patients. There is, therefore, a need for sensitive and specific fungal species detection assays in a clinical laboratory for early targeted therapy. The isothermal amplification method may be useful for the screening of fungal isolates, especially in resource-poor settings. Therefore, our aim was to review the isothermal nucleic acid amplification methods and their applications in fungal pathogen detection. Out of 50 reported studies, 28, 12, 6, 2, and 2 studies used the isothermal-based assays of a loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), rolling circle amplification (RCA), multiple displacement amplification (MDA) and polymerase Spiral Reaction (PSR), respectively. Thirty-two studies used clinical samples, 18 pure culture, and four environmental samples. The diagnostic accuracy of isothermal nucleic acid amplification testing for pathogenic fungal was reported as high (sensitivity 0.89–1.0 and specificity 0.63–1.0) in all studies irrespective of the sample tested. Although the isothermal-based assays showed high sensitivity and specificity in reported studies, it is still poorer than that of PCR assays. However, improving the assay to make it simpler, more effective, and inexpensive compared with newer PCR methods are still needed.


2020 ◽  
Vol 48 (5) ◽  
pp. e30-e30 ◽  
Author(s):  
Bo Tian ◽  
Gabriel Antonio S Minero ◽  
Jeppe Fock ◽  
Martin Dufva ◽  
Mikkel Fougt Hansen

Abstract False-positive results cause a major problem in nucleic acid amplification, and require external blank/negative controls for every test. However, external controls usually have a simpler and lower background compared to the test sample, resulting in underestimation of false-positive risks. Internal negative controls, performed simultaneously with amplification to monitor the background level in real-time, are therefore appealing in both research and clinic. Herein, we describe a nonspecific product-activated single-stranded DNA-cutting approach based on CRISPR (clustered regularly interspaced short palindromic repeats) Cas12a (Cpf1) nuclease. The proposed approach, termed Cas12a-based internal referential indicator (CIRI), can indicate the onset of nonspecific amplification in an exponential rolling circle amplification strategy here combined with an optomagnetic readout. The capability of CIRI as an internal negative control can potentially be extended to other amplification strategies and sensors, improving the performance of nucleic acid amplification-based methodologies.


Sensors ◽  
2021 ◽  
Vol 21 (2) ◽  
pp. 602
Author(s):  
Sandra Leonardo ◽  
Anna Toldrà ◽  
Mònica Campàs

The easy and rapid spread of bacterial contamination and the risk it poses to human health makes evident the need for analytical methods alternative to conventional time-consuming laboratory-based techniques for bacterial detection. To tackle this demand, biosensors based on isothermal DNA amplification methods have emerged, which avoid the need for thermal cycling, thus facilitating their integration into small and low-cost devices for in situ monitoring. This review focuses on the breakthroughs made on biosensors based on isothermal amplification methods for the detection of bacteria in the field of food safety and environmental monitoring. Optical and electrochemical biosensors based on loop mediated isothermal amplification (LAMP), rolling circle amplification (RCA), recombinase polymerase amplification (RPA), helicase dependent amplification (HDA), strand displacement amplification (SDA), and isothermal strand displacement polymerisation (ISDPR) are described, and an overview of their current advantages and limitations is provided. Although further efforts are required to harness the potential of these emerging analytical techniques, the coalescence of the different isothermal amplification techniques with the wide variety of biosensing detection strategies provides multiple possibilities for the efficient detection of bacteria far beyond the laboratory bench.


Biosensors ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 222
Author(s):  
Chenxin Fang ◽  
Ping Ouyang ◽  
Yuxing Yang ◽  
Yang Qing ◽  
Jialun Han ◽  
...  

A microRNA (miRNA) detection platform composed of a rolling circle amplification (RCA) system and an allosteric deoxyribozyme system is proposed, which can detect miRNA-21 rapidly and efficiently. Padlock probe hybridization with the target miRNA is achieved through complementary base pairing and the padlock probe forms a closed circular template under the action of ligase; this circular template results in RCA. In the presence of DNA polymerase, RCA proceeds and a long chain with numerous repeating units is formed. In the presence of single-stranded DNA (H1 and H2), multi-component nucleic acid enzymes (MNAzymes) are formed that have the ability to cleave substrates. Finally, substrates containing fluorescent and quenching groups and magnesium ions are added to the system to activate the MNAzyme and the substrate cleavage reaction, thus achieving fluorescence intensity amplification. The RCA–MNAzyme system has dual signal amplification and presents a sensing platform that demonstrates broad prospects in the analysis and detection of nucleic acids.


2017 ◽  
Vol 91 ◽  
pp. 136-142 ◽  
Author(s):  
Hai Shi ◽  
Xiaoxia Mao ◽  
Xiaoxia Chen ◽  
Zihan Wang ◽  
Keming Wang ◽  
...  

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Alexander S. Day ◽  
Tiffany-Heather Ulep ◽  
Elizabeth Budiman ◽  
Laurel Dieckhaus ◽  
Babak Safavinia ◽  
...  

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.


Author(s):  
Weihua Yang ◽  
Xiaofei Dang ◽  
Qingxi Wang ◽  
Mingjie Xu ◽  
Qianqian Zhao ◽  
...  

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.


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