Long-term dry storage of enzyme-based reagents for isothermal nucleic acid amplification in a porous matrix for use in point-of-care diagnostic devices

Kumar, Sujatha; Gallagher, Ryan; Bishop, Josh; Kline, Enos; Buser, Joshua R.; Lafleur, Lisa; Shah, Kamal G.; Lutz, Barry; Yager, Paul

doi:10.1039/d0an01098g

Cited by 26 publications

(25 citation statements)

References 45 publications

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“…6 A, we used a 0.5-mL screw-cap Eppendorf tube with a total of three screw caps consisting of the necessary amounts of RNA stabilizing reagent, RNA amplification reagent and RNA detection reagent to run a single test reaction. For long-term storage, co-dried reagents can be stored in the cap using a glass fiber matrix 29 . For saliva collection, a simple method with a SalivaBio Oral Swab (SOS, Salimetrics LLC, USA) was used 30 .…”

Section: Resultsmentioning

confidence: 99%

Development of robust isothermal RNA amplification assay for lab-free testing of RNA viruses

Biyani

Sharma

Kojima

et al. 2021

Sci Rep

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Simple tests of infectiousness that return results in minutes and directly from samples even with low viral loads could be a potential game-changer in the fight against COVID-19. Here, we describe an improved isothermal nucleic acid amplification assay, termed the RICCA (RNA Isothermal Co-assisted and Coupled Amplification) reaction, that consists of a simple one-pot format of ‘sample-in and result-out’ with a primary focus on the detection of low copy numbers of RNA virus directly from saliva without the need for laboratory processing. We demonstrate our assay by detecting 16S rRNA directly from E. coli cells with a sensitivity as low as 8 CFU/μL and RNA fragments from a synthetic template of SARS-CoV-2 with a sensitivity as low as 1740 copies/μL. We further demonstrate the applicability of our assay for real-time testing at the point of care by designing a closed format for paper-based lateral flow assay and detecting heat-inactivated SARS-COV-2 virus in human saliva at concentrations ranging from 28,000 to 2.8 copies/μL with a total assay time of 15–30 min.

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Section: Resultsmentioning

confidence: 99%

Development of robust isothermal RNA amplification assay for lab-free testing of RNA viruses

Biyani

Sharma

Kojima

et al. 2021

Sci Rep

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“…The paper-based approach was inspired by a previous demonstration where hydroxynaphtol blue was used as a fluorescent reporter in a paper-based device [5] . The technique of long-term dry storage of LAMP reagents was adapted from a previous study that highlighted the role of excipients in storage of enzymes [4] . Due to the simplicity and scalability of this device, it can be used in a variety of applications, enabling the detection of nucleic acids from pathogens in a portable field-deployable manner.…”

Section: *Methods Detailsmentioning

confidence: 99%

Fabrication of a paper-based colorimetric molecular test for SARS-CoV-2

et al. 2021

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“…An ideal POC biosensor should be stable in the ambient environment for a long period of time without the need for frozen storage. − Typically, Cas proteins, guide RNA, and ssDNA reporters are preserved at low temperatures to minimize or avoid degradation. For example, the Cas12a (Cpf1) protein in commercial use is stored in a liquid state with 50% glycerol at −20 °C .…”

Section: Reagent Storagementioning

confidence: 99%

Challenges and Opportunities for Clustered Regularly Interspaced Short Palindromic Repeats Based Molecular Biosensing

Bao

Chen

et al. 2021

ACS Sens.

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Clustered regularly interspaced short palindromic repeats, CRISPR, has recently emerged as a powerful molecular biosensing tool for nucleic acids and other biomarkers due to its unique properties such as collateral cleavage nature, room temperature reaction conditions, and high target-recognition specificity. Numerous platforms have been developed to leverage the CRISPR assay for ultrasensitive biosensing applications. However, to be considered as a new gold standard, several key challenges for CRISPR molecular biosensing must be addressed. In this paper, we briefly review the history of biosensors, followed by the current status of nucleic acid-based detection methods. We then discuss the current challenges pertaining to CRISPR-based nucleic acid detection, followed by the recent breakthroughs addressing these challenges. We focus upon future advancements required to enable rapid, simple, sensitive, specific, multiplexed, amplification-free, and shelf-stable CRISPR-based molecular biosensors.

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Long-term dry storage of enzyme-based reagents for isothermal nucleic acid amplification in a porous matrix for use in point-of-care diagnostic devices

Abstract: Nucleic acid amplification test (NAAT)-based point-of-care (POC) devices are rapidly growing for use in low-resource settings. However, key challenges are the ability to store the enzyme-based reagents in dry form...

Cited by 26 publications

References 45 publications

Development of robust isothermal RNA amplification assay for lab-free testing of RNA viruses

Development of robust isothermal RNA amplification assay for lab-free testing of RNA viruses

Fabrication of a paper-based colorimetric molecular test for SARS-CoV-2

Challenges and Opportunities for Clustered Regularly Interspaced Short Palindromic Repeats Based Molecular Biosensing

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