scholarly journals Reverse Transcription Lesion-Induced DNA Amplification: An Instrument-Free Isothermal Method to Detect RNA

2020 ◽  
Author(s):  
Bibi Safeenaz Alladin-Mustan ◽  
Jesse Yuzik ◽  
Daria Raquel Queiroz de Almeida ◽  
Yuning Liu ◽  
Yimeng Li ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Room Temperature ◽  
Point Of Care ◽  
Dna Amplification ◽  
Chain Reaction ◽  
Rna Detection ◽  
Isothermal Method ◽  
Enzymatic Amplification ◽  
Point Of Care Diagnostics ◽  
Target Rna

<p>One challenge in point-of-care diagnostics is the lack of room-temperature methods for RNA detection based on enzymatic amplification and visualization steps. Here we perform a reverse transcription ligase chain reaction using our isothermal lesion induced DNA amplification (LIDA) technique that can be tuned to operate at any desired temperature. Using RNA-triggered LIDA, we can detect as little as ~100 attomoles target RNA and can distinguish RNA target from total cellular RNA. Finally, we demonstrate that the resulting DNA amplicons can be detected colorimetrically, also at room temperature, by rapid, target-triggered disassembly of DNA-modified gold nanoparticles. This integrated amplification/detection platform requires no heating or visualization instrumentation, which is an important step towards realizing instrument-free POC testing.</p>

scholarly journals Reverse Transcription Lesion-Induced DNA Amplification: An Instrument-Free Isothermal Method to Detect RNA

2020 ◽  
Author(s):  
Bibi Safeenaz Alladin-Mustan ◽  
Jesse Yuzik ◽  
Daria Raquel Queiroz de Almeida ◽  
Yuning Liu ◽  
Yimeng Li ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Room Temperature ◽  
Point Of Care ◽  
Dna Amplification ◽  
Chain Reaction ◽  
Rna Detection ◽  
Isothermal Method ◽  
Enzymatic Amplification ◽  
Point Of Care Diagnostics ◽  
Target Rna

<p>One challenge in point-of-care diagnostics is the lack of room-temperature methods for RNA detection based on enzymatic amplification and visualization steps. Here we perform a reverse transcription ligase chain reaction using our isothermal lesion induced DNA amplification (LIDA) technique that can be tuned to operate at any desired temperature. Using RNA-triggered LIDA, we can detect as little as ~100 attomoles target RNA and can distinguish RNA target from total cellular RNA. Finally, we demonstrate that the resulting DNA amplicons can be detected colorimetrically, also at room temperature, by rapid, target-triggered disassembly of DNA-modified gold nanoparticles. This integrated amplification/detection platform requires no heating or visualization instrumentation, which is an important step towards realizing instrument-free POC testing.</p>

Achieving room temperature DNA amplification by dialling in destabilization

2015 ◽  
Vol 51 (44) ◽  
pp. 9101-9104 ◽  
Author(s):  
B. Safeenaz Alladin-Mustan ◽  
Catherine J. Mitran ◽  
Julianne M. Gibbs-Davis
Keyword(s):  
Nucleic Acid ◽  
Room Temperature ◽  
Point Of Care ◽  
Dna Amplification ◽  
Heating Element ◽  
Nucleic Acid Sequences

The ability to amplify nucleic acid sequences at room temperature without the need for any heating element has been achieved, which has promise in bio-diagnostics employed at the point of care.

scholarly journals Detecting SARS-CoV-2 at point of care: preliminary data comparing loop-mediated isothermal amplification (LAMP) to polymerase chain reaction (PCR)

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Marc F. Österdahl ◽  
Karla A. Lee ◽  
Mary Ni Lochlainn ◽  
Stuart Wilson ◽  
Sam Douthwaite ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Predictive Value ◽  
Point Of Care ◽  
Isothermal Amplification ◽  
Service Improvement ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Loop Mediated Isothermal Amplification ◽  
Polymerase Chain

Abstract Background A cost effective and efficient diagnostic tool for COVID-19 as near to the point of care (PoC) as possible would be a game changer in the current pandemic. We tested reverse transcription loop mediated isothermal amplification (RT-LAMP), a method which can produce results in under 30 min, alongside standard methods in a real-life clinical setting. Methods This prospective service improvement project piloted an RT-LAMP method on nasal and pharyngeal swabs on 21 residents of a high dependency care home, with two index COVID-19 cases, and compared it to multiplex tandem reverse transcription polymerase chain reaction (RT-PCR). We recorded vital signs of patients to correlate clinical and laboratory information and calculated the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of a single swab using RT-LAMP compared with the current standard, RT-PCR, as per Standards for Reporting Diagnostic Accuracy Studies (STARD) guidelines. Results The novel method accurately detected 8/10 RT-PCR positive cases and identified a further 3 positive cases. Eight further cases were negative using both methods. Using repeated RT-PCR as a “gold standard”, the sensitivity and specificity of a single novel test were 80 and 73% respectively. PPV was 73% and NPV was 83%. Incorporating retesting of low signal RT-LAMP positives improved the specificity to 100%. We also speculate that hypothermia may be a significant early clinical sign of COVID-19. Conclusions RT-LAMP testing for SARS-CoV-2 was found to be promising, fast and to work equivalently to RT-PCR methods. RT-LAMP has the potential to transform COVID-19 detection, bringing rapid and accurate testing to the PoC. RT-LAMP could be deployed in mobile community testing units, care homes and hospitals to detect disease early and prevent spread.

scholarly journals Handyfuge-LAMP: low-cost and electricity-free centrifugation for isothermal SARS-CoV-2 detection in saliva

2020 ◽  
Author(s):  
Ethan Li ◽  
Adam Larson ◽  
Anesta Kothari ◽  
Manu Prakash
Keyword(s):  
Point Of Care ◽  
Low Cost ◽  
Lamp Assay ◽  
Sample Collection ◽  
Rna Detection ◽  
Open Hardware ◽  
Point Of Care Diagnostics ◽  
Simple Protocol ◽  
Specialized Equipment ◽  
The Cost

AbstractPoint of care diagnostics for COVID-19 detection are vital to assess infection quickly and at the source so appropriate measures can be taken. The loop-mediated isothermal amplification (LAMP) assay has proven to be a reliable and simple protocol that can detect small amounts of viral RNA in patient samples (<10 genomes per μL) (Nagamine, Hase, and Notomi 2002). Recently, Rabe and Cepko at Harvard published a sensitive and simple protocol for COVID-19 RNA detection in saliva using an optimized LAMP assay (Rabe and Cepko, 2020).This LAMP protocol has the benefits of being simple, requiring no specialized equipment; rapid, requiring less than an hour from sample collection to readout; and cheap, costing around $1 per reaction using commercial reagents. The pH based colorimetric readout also leaves little ambiguity and is intuitive. However, a shortfall in many nucleic acid-based methods for detection in saliva samples has been the variability in output due to the presence of inhibitory substances in saliva. Centrifugation to separate the reaction inhibitors from inactivated sample was shown to be an effective way to ensure reliable LAMP amplification. However, a centrifuge capable of safely achieving the necessary speeds of 2000 RPM for several minutes often costs hundreds of dollars and requires a power supply.We present here an open hardware solution- Handyfuge - that can be assembled with readily available components for the cost of <5 dollars a unit and could be used together with the LAMP assay for point of care detection of COVID-19 RNA from saliva. The device is then validated using the LAMP protocol from Rabe and Cepko. With the use of insulated coolers for reagent supply chain and delivery, the assay presented can be completed without the need for electricity or any laboratory scale infrastructure.

scholarly journals Evaluation of commercially available immuno-magnetic agglutination and enzyme-linked immunosorbent assays for rapid point-of-care diagnostics of COVID-19

2020 ◽  
Author(s):  
Maria Engel Moeller ◽  
Jeppe Fock ◽  
Pearlyn Pah ◽  
Antia De La Campa Veras ◽  
Melanie Bade ◽  
...  
Keyword(s):  
Point Of Care ◽  
Enzyme Linked Immunosorbent Assay ◽  
Plasma Samples ◽  
Igg Antibodies ◽  
Chain Reaction ◽  
Point Of Care Test ◽  
Point Of Care Diagnostics ◽  
Polymerase Chain ◽  
Respiratory Coronavirus ◽  
Immunomagnetic Assay

Introduction: Coronavirus Disease 2019 (COVID-19) is caused by severe acute respiratory coronavirus-2 (SARS-CoV-2). Fast, accurate and simple blood-based assays for quantification of anti-SARS-CoV-2 antibodies are urgently needed to identify infected individuals and keep track of the spread of disease. Methods: The study included 35 plasma samples from 22 individuals with confirmed COVID-19 by real time reverse transcriptase polymerase chain reaction and 40 non COVID-19 plasma samples. Anti-SARS-CoV-2 IgM/IgA or IgG antibodies were detected by a microfluidic quantitative immunomagnetic assay (IMA)(ViroTrack Sero COVID IgM+IgA/IgG Ab, Blusense Diagnostics, Denmark) and by enzyme-linked immunosorbent assay ((ELISA) (EuroImmun Medizinische Labordiagnostika, Germany). Results: Of the 35 plasma samples from the COVID-19 patients, 29 (82.9%) were positive for IgA/IgM or IgG by IMA and 29 samples (82.9%) were positive by ELISA. Sensitivity for only one sample per patient was 68% for IgA+IgM and 73% IgG by IMA and 73% by ELISA. For samples collected 14 days after symptom onset, the sensitivity of both IMA and ELISA was around 90%. Specificity of the IMA reached 100% compared to 95% for ELISA IgA and 97.5% for ELISA IgG. Conclusion: IMA for COVID-19 is a rapid simple-to-use point of care test with sensitivity and specificity similar to a commercial ELISA.

Plasmonic and label-free real-time quantitative PCR for point-of-care diagnostics

The Analyst ◽  
2021 ◽  
Author(s):  
Padideh Mohammadyousef ◽  
Miltiadis Paliouras ◽  
Mark Trifiro ◽  
Andrew Kirk
Keyword(s):  
Pathogen Detection ◽  
Point Of Care ◽  
Medical Community ◽  
Molecular Diagnostic ◽  
Label Free ◽  
Chain Reaction ◽  
Real Time Quantitative Pcr ◽  
Point Of Care Diagnostics ◽  
Polymerase Chain ◽  
Infectious Pathogen

In response to the world’s medical community need for accurate and immediate infectious pathogen detection, many researchers have focused on adapting the standard molecular diagnostic method of polymerase chain reaction...

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