Betaine-assisted recombinase polymerase assay with enhanced specificity

Luo, Guangcheng; Yi, Tingting; Jiang, Bibiao; Guo, Xiaolan; Zhang, Guo-Yuan

doi:10.1016/j.ab.2019.03.018

Cited by 35 publications

(26 citation statements)

References 14 publications

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“…S7). RT-RPA also exhibits nonspecific amplification [66][67][68] , and therefore, alternative isothermal amplification technologies, such as RT-LAMP 41,69 , could be implemented to improve specificity. Although RT-LAMP is likely to improve specificity as well as sensitivity, the requirement of two separate reactions remains problematic, increasing the likelihood of contamination due to sample transfers 70 .…”

Section: Discussionmentioning

confidence: 99%

A Sensitive, Rapid, and Portable CasRx-based Diagnostic Assay for SARS-CoV-2

Brogan

Chaverra-Rodríguez

Lin

et al. 2020

Preprint

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Since its first emergence from China in late 2019, the SARS-CoV-2 virus has spread globally despite unprecedented containment efforts, resulting in a catastrophic worldwide pandemic. Successful identification and isolation of infected individuals can drastically curtail virus spread and limit outbreaks. However, during the early stages of global transmission, point-of-care diagnostics were largely unavailable and continue to remain difficult to procure, greatly inhibiting public health efforts to mitigate spread. Furthermore, the most prevalent testing kits rely on reagent- and time-intensive protocols to detect viral RNA, preventing rapid and cost-effective diagnosis. Therefore the development of an extensive toolkit for point-of-care diagnostics that is expeditiously adaptable to new emerging pathogens is of critical public health importance. Recently, a number of novel CRISPR-based diagnostics have been developed to detect COVID-19. Herein, we outline the development of a CRISPR-based nucleic acid molecular diagnostic utilizing a Cas13d ribonuclease derived from Ruminococcus flavefaciens (CasRx) to detect SARS-CoV-2, an approach we term SENSR (Sensitive Enzymatic Nucleic-acid Sequence Reporter). We demonstrate SENSR robustly detects SARS-CoV-2 sequences in both synthetic and patient-derived samples by lateral flow and fluorescence, thus expanding the available point-of-care diagnostics to combat current and future pandemics.

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Section: Discussionmentioning

confidence: 99%

A Sensitive, Rapid, and Portable CasRx-based Diagnostic Assay for SARS-CoV-2

Brogan

Chaverra-Rodríguez

Lin

et al. 2020

Preprint

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show abstract

“…Some typical chemicals such as betaine, DMSO, BSA, dithiothreitol (DTT), and glycerol have been shown to improve the product specificity and/or yield during PCR amplification, especially for the difficult reactions with long or GC-rich template 28, 29, 30, 31, 32 . In addition, betaine was also reported to reduce non-specific product and enhance the amplification efficiency for isothermal RPA reaction 33 . However, betaine addition in LAMP reaction rather play an inhibitory role for target amplification, possibly due to the molecular barrier function of betaine to hinder intermolecular hybridization 38 .…”

Section: Discussionmentioning

confidence: 99%

“…Chemicals such as polyols, polymers, amino acid and their derivatives have been used as solvent additives to stabilize proteins and/or affect solvent properties, thereby influencing the kinetics of reaction chemistry 27 . Researchers have shown that chemical additives such as DMSO, glycerol and betaine can enhance the efficiency and/or specificity of some PCR or isothermal amplification reactions 28, 29, 30, 31, 32, 33 . However, systematic study is still required to examine whether these chemical additives may affect the detection power of CRISPR-based nucleic acid detection, in hope of searching the reaction enhancer for CRISPR diagnostics.…”

Section: Introductionmentioning

confidence: 99%

A Chemical-Enhanced System for CRISPR-Based Nucleic Acid Detection

Zhao¹,

Ma²,

Li³

et al. 2021

Preprint

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The CRISPR-based nucleic acid detection systems such as SHERLOCK, DETECTR and HOLMES have shown great potential for point-of-care testing of viral pathogens, especially in the context of COVID-19 pandemic. Here we optimize several key parameters of reaction chemistry and develop a Chemical Enhanced CRISPR Detection system for nucleic acid (termed CECRID). For the Cas12a/Cas13a-based signal detection phase, we determine buffer conditions and substrate range for optimal detection performance. By comparing several chemical additives, we find that addition of L-proline can secure or enhance Cas12a/Cas13a detection capability. For isothermal amplification phase with typical LAMP and RPA methods, inclusion of L-proline can also enhance specific target amplification as determined by CRISPR detection. Using SARS-CoV-2 pseudovirus, we demonstrate CECRID has enhanced detection sensitivity over chemical additive-null method with either fluorescence or lateral flow strip readout. Thus, CECRID provides an improved detection power and system robustness towards practical application of CRISPR-based diagnostics.

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“…For multiplex RT‐RPA assays, the optimized concentrations of both forward and reverse primers for each virus/viroid were adjusted to the following: 100 n m for ASGV and ASPV; 150 n m for ACLSV; and 200 n m for ApNMV and ASSVd, such that the total oligonucleotide concentration in one reaction was 1500 n m . Moreover, 0.8 m betaine (Sigma Aldrich, UK) was added to the reaction mixture to improve the sensitivity and specificity (Luo et al ., 2019).…”

Section: Experimental Methodsmentioning

confidence: 99%

Field detection of multiple RNA viruses/viroids in apple using a CRISPR/Cas12a‐based visual assay

Jiao

Kong

Han

et al. 2020

Plant Biotechnology Journal

100

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Summary Co‐infection of apple trees with several viruses/viroids is common and decreases fruit yield and quality. Accurate and rapid detection of these viral pathogens helps to reduce losses and prevent virus spread. Current molecular detection assays used for apple viruses require specialized and expensive equipment. Here, we optimized a CRISPR/Cas12a‐based nucleic acid detection platform for the diagnosis of the most prevalent RNA viruses/viroid in apple, namely Apple necrotic mosaic virus (ApNMV), Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV), Apple chlorotic leaf spot virus (ACLSV) and Apple scar skin viroid (ASSVd). We detected each RNA virus/viroid directly from crude leaf extracts after simultaneous multiplex reverse transcription‐recombinase polymerase amplification (RT‐RPA) with high specificity. Positive results can be distinguished by the naked eye via oligonucleotide‐conjugated gold nanoparticles. The CRISPR/Cas12a‐RT‐RPA platform exhibited comparable sensitivity to RT‐qPCR, with limits of detection reaching 250 viral copies per reaction for ASPV and ASGV and 2500 copies for the others. However, this protocol was faster and simpler, requiring an hour or less from leaf harvest. Field tests showed 100% agreement with RT‐PCR detection for 52 samples. This novel Cas12a‐based method is ideal for rapid and reliable detection of apple viruses in the orchard without the need to send samples to a specialized laboratory.

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Betaine-assisted recombinase polymerase assay with enhanced specificity

Cited by 35 publications

References 14 publications

A Sensitive, Rapid, and Portable CasRx-based Diagnostic Assay for SARS-CoV-2

A Sensitive, Rapid, and Portable CasRx-based Diagnostic Assay for SARS-CoV-2

A Chemical-Enhanced System for CRISPR-Based Nucleic Acid Detection

Field detection of multiple RNA viruses/viroids in apple using a CRISPR/Cas12a‐based visual assay

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