Room Temperature Isothermal Colorimetric Padlock Probe Rolling Circle Amplification for Viral DNA and RNA Detection

Huang, Wilson; Hsu, Hannah; Su, Jimmy; Clapper, Jude; Hsu, Jonathan Y.

doi:10.1101/2020.06.12.128876

Cited by 10 publications

(12 citation statements)

References 27 publications

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“…More recently, a number of nucleic acid isothermal amplification techniques for viral detection have been described, including loop-mediated isothermal amplification (LAMP) [ [13] , [14] , [15] ], using a 4- or 6-primer set and incubated at 65 °C for 30-60 minutes, recombinase-polymerase amplification (RPA) [ 16 ], using multiple enzymes and incubated at 42 °C for 15-20 minutes, and many others [ [17] , [18] , [19] ]. Isothermal assays have been shown to detect SARS-CoV-2 with very high sensitivity and specificity, and underlie several tests recently authorized for emergency use, including the ID NOW from Abbott, and CRISPR-based detection by SHERLOCK [ 20 ] (currently not approved for sale outside the United States).…”

Section: Introductionmentioning

confidence: 99%

Extraction-free RT-LAMP to detect SARS-CoV-2 is less sensitive but highly specific compared to standard RT-PCR in 101 samples

Schellenberg¹,

Ormond²,

Keynan

2021

Journal of Clinical Virology

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The current scale of public and private testing cannot be expected to meet the emerging need for higher levels of community-level and repeated screening of asymptomatic Canadians for SARS-CoV-2. Rapid point-of-care techniques are increasingly being offered to fill the gap in screening levels required to identify undiagnosed individuals with high viral loads. However, rapid, point-of-care tests often have lower sensitivity in practice. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) for SARS-CoV-2 has proven sensitive and specific and provides visual results in minutes. Using a commercially available kit for RT-LAMP and primer set targetting nucleocapsid (N), we tested a blinded set of 101 archived nasopharyngeal (NP) swab samples with known RT-PCR results. RT-LAMP reactions were incubated at 65 °C for 30 minutes, using heat-inactivated nasopharyngeal swab sample in viral transport medium, diluted tenfold in water, as input. RT-LAMP agreed with all RT-PCR defined negatives (N = 51), and all positives with cycle threshold (Ct) less than 20 (N = 24), 65% of positives with Ct between 20-30 (N = 17), and no positives with Ct greater than 30 (N = 9). RT-LAMP requires fewer and different core components, so may not compete directly with the mainline testing workflow, preserving precious central laboratory resources for those with the greatest need. Careful messaging must be provided when using less-sensitive tests, so that people are not falsely reassured by negative results, but this caveat must be weighed against the clear benefits of reliably identifying those with high levels of virus in prioritized samples at the point of care.

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Section: Introductionmentioning

confidence: 99%

Extraction-free RT-LAMP to detect SARS-CoV-2 is less sensitive but highly specific compared to standard RT-PCR in 101 samples

Schellenberg¹,

Ormond²,

Keynan

2021

Journal of Clinical Virology

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“…The method was successful in detecting and differentiating synthetic SARS-CoV-2 RdRp cDNA from synthetic SARS-CoV RdRp cDNA. Another similar approach has also been just reported detecting SARS-CoV-2 synthetic DNA recently [ 140 ]. Although none of the approaches have been tested against clinical samples, RCA can be an advantageous candidate for point of care detection.…”

Section: Advances In Sars-cov-2 Diagnosismentioning

confidence: 98%

Diagnosis for COVID-19: current status and future prospects

Kabir

Ahmed²,

Iqbal

et al. 2021

Expert Review of Molecular Diagnostics

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Introduction: Coronavirus disease 2019 (COVID-19), a respiratory illness caused by novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), had its first detection in December 2019 in Wuhan (China) and spread across the world. In March 2020, the World Health Organization (WHO) declared COVID-19 a pandemic disease. The utilization of prompt and accurate molecular diagnosis of SARS-CoV-2 virus, isolating the infected patients, and treating them are the keys to managing this unprecedented pandemic. International travel acted as a catalyst for the widespread transmission of the virus. Areas covered: This review discusses phenotype, structural, and molecular evolution of recognition elements and primers, its detection in the laboratory, and at point of care. Further, market analysis of commercial products and their performance are also evaluated, providing new ways to confront the ongoing global public health emergency. Expert commentary: The outbreak for COVID-19 created mammoth chaos in the healthcare sector, and still, day by day, new epicenters for the outbreak are being reported. Emphasis should be placed on developing more effective, rapid, and early diagnostic devices. The testing laboratories should invest more in clinically relevant multiplexed and scalable detection tools to fight against a pandemic like this where massive demand for testing exists.

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“…RCA has been used before for detecting single nucleotide polymorphisms [135,136], M. tuberculosis genomic DNA [137], SARS-CoV [138], Ebola, and other pathogens [139]. Recently, a study described a modified RCA assay capable of detecting up to picomolar concentrations of synthetic viral DNA strands of SARS-CoV-2, Influenza A (H1N1pdm09), and Influenza B [140]. As in other nucleic acid amplification methods, RCA output can be detected by measuring fluorescence, by using colorimetric chemistry, or by coupling the results with other readout techniques [139].…”

Section: Rolling Circle Amplification (Rca)mentioning

confidence: 99%

Tracking SARS-CoV-2: Novel Trends and Diagnostic Strategies

et al. 2021

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The COVID-19 pandemic has had an enormous impact on economies and health systems globally, therefore a top priority is the development of increasingly better diagnostic and surveillance alternatives to slow down the spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In order to establish massive testing and contact tracing policies, it is crucial to have a clear view of the diagnostic options available and their principal advantages and drawbacks. Although classical molecular methods such as RT-qPCR are broadly used, diagnostic alternatives based on technologies such as LAMP, antigen, serological testing, or the application of novel technologies such as CRISPR-Cas for diagnostics, are also discussed. The present review also discusses the most important automation strategies employed to increase testing capability. Several serological-based diagnostic kits are presented, as well as novel nanotechnology-based diagnostic methods. In summary, this review provides a clear diagnostic landscape of the most relevant tools to track COVID-19.

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Room Temperature Isothermal Colorimetric Padlock Probe Rolling Circle Amplification for Viral DNA and RNA Detection

Cited by 10 publications

References 27 publications

Extraction-free RT-LAMP to detect SARS-CoV-2 is less sensitive but highly specific compared to standard RT-PCR in 101 samples

Extraction-free RT-LAMP to detect SARS-CoV-2 is less sensitive but highly specific compared to standard RT-PCR in 101 samples

Diagnosis for COVID-19: current status and future prospects

Tracking SARS-CoV-2: Novel Trends and Diagnostic Strategies

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