Development of a RPA-CRISPR-Cas12a Assay for Rapid, Simple, and Sensitive Detection of Mycoplasma hominis

Huang, Yiyan; Xiao, Bin; Deng, Hao; Gong, Kunxiang; Li, Kun; Li, Linhai; Hao, Wenbo

doi:10.3389/fmicb.2022.842415

Cited by 18 publications

(11 citation statements)

References 38 publications

(38 reference statements)

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“…The crRNA-3 recognizes the more accessible terminal of the target DNA, while crRNA-1 or crRNA-2 binds with the middle region of the target DNA. Both similar and opposite conclusions have been reported, which must be clarified with further experiments. , …”

Section: Discussionmentioning

confidence: 61%

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Detection of Frog Virus 3 by Integrating RPA-CRISPR/Cas12a-SPM with Deep Learning

Lei,

Lian,

Zhang

et al. 2023

ACS Omega

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A fast, easy-to-implement, highly sensitive, and point-of-care (POC) detection system for frog virus 3 (FV3) is proposed. Combining recombinase polymerase amplification (RPA) and CRISPR/Cas12a, a limit of detection (LoD) of 100 aM (60.2 copies/μL) is achieved by optimizing RPA primers and CRISPR RNAs (crRNAs). For POC detection, smartphone microscopy is implemented, and an LoD of 10 aM is achieved in 40 min. The proposed system detects four positive animal-derived samples with a quantitation cycle (Cq) value of quantitative PCR (qPCR) in the range of 13 to 32. In addition, deep learning models are deployed for binary classification (positive or negative samples) and multiclass classification (different concentrations of FV3 and negative samples), achieving 100 and 98.75% accuracy, respectively. Without temperature regulation and expensive equipment, the proposed RPA-CRISPR/Cas12a combined with smartphone readouts and artificial-intelligence-assisted classification showcases the great potential for FV3 detection, specifically POC detection of DNA virus.

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Section: Discussionmentioning

confidence: 61%

“…Both similar and opposite conclusions have been reported, which must be clarified with further experiments. 72,73 Another approach involves designing specific RPA primers and screening out the most efficient ones. Different primers influence the amplification efficiency of the RPA reaction.…”

Section: Discussionmentioning

confidence: 99%

Detection of Frog Virus 3 by Integrating RPA-CRISPR/Cas12a-SPM with Deep Learning

Lei,

Lian,

Zhang

et al. 2023

ACS Omega

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“…In addition, if only Cas12a protein is used as a detection tool without relying on the amplication of the target, a minimum detection limit of 3 × 10 7 copies per reaction is required to obtain a detection signal. 29 Herein, both methods can reach a limit of detection (LOD) of 10 3 CFU ml −1 (Fig. 4b and d), revealing the signicance of the RPA reaction in the sensitive detection.…”

Section: Discussionmentioning

confidence: 75%

“…The optimal ratio of protein to gRNA reported in the literature is generally between 1 : 0.5 and 1 : 2. 28,29 In this study, four ratio gradients were set, and the Cas12a protein amount was set to be 20 nM. The optimal protein to gRNA ratio was explored, and the best ratio of 1 : 1 can be seen from Fig.…”

Section: Rpa-cas12a-lft System Construction and Optimizationmentioning

confidence: 99%

Development and evaluation of RPA-NFO-LFT and RPA-Cas12a-LFT systems for the detection of Candida albicans

et al. 2023

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“…2(b)]. The amount of Cas12a and crRNA needed to induce significant green fluorescence were then optimized as other similar research did 32,34,35 . All components of the reaction system remained the same except for these two ingredients mentioned above.…”

Section: Resultsmentioning

confidence: 99%

New and rapid visual detection assay for Trogoderma granarium everts based on recombinase polymerase amplification and CRISPR/Cas12a

Zeng,

Zheng,

Stejskal

et al. 2023

Pest Management Science

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BACKGROUNDKhapra beetle (Trogoderma granarium Everts), one of the most important quarantine pests globally, is capable of causing severe infestation and huge economic loss to stored grain, and its interception rate has increased in major global trade countries over the past few years. However, difficulties remain in distinguishing this species with similar ones. In order to assist border ports and warehouses in khapra beetle's effective rapid identification as well as pest control at the early stages of monitoring or interception, we herein developed a new and rapid visual detection assay for T. granarium based on recombinase polymerase amplification (RPA) and the CRISPR/Cas12a system.RESULTSWe designed and selected the first khapra beetle‐specific RPA primers and crRNA, and optimized the visualization reaction system (Cas12a/CrRNA = 100 nM/500 nM). With only a 37 °C‐heat‐source and a blue light torch, RPA and CRISPR/CAS12a‐based visualization assays can be completed within 40 min to differentiate between khapra beetle and nine similar Dermestidae species. After DNA extraction using a kit (4–5 h) or a simple method (5 min), the specific amplicons were obtained after a 15 min RPA reaction at 37 °C, followed by a 15 min color reaction under 37 °C in dark conditions using a CRISPR/CAS12a system and a fluorescent probe (5′‐FAM/3′‐BHQ1 labeled). This method is ingenious to low levels of DNA (10−1 ng μL−1) and meets the sensitivity requirements for detecting a single khapra beetle's egg (≈0.7 mm).CONCLUSIONOur specificity and sensitivity analysis inferred that the present visualization system is effective to quickly and uniquely detect khapra beetle at room temperature (37 °C), thereby preventing this species before they spread widely. Our study is suitable for being pushed forward in storage pest management, and provides value as a reference for monitoring and identification of other pests. © 2023 Society of Chemical Industry.

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Development of a RPA-CRISPR-Cas12a Assay for Rapid, Simple, and Sensitive Detection of Mycoplasma hominis

Cited by 18 publications

References 38 publications

Detection of Frog Virus 3 by Integrating RPA-CRISPR/Cas12a-SPM with Deep Learning

Detection of Frog Virus 3 by Integrating RPA-CRISPR/Cas12a-SPM with Deep Learning

Development and evaluation of RPA-NFO-LFT and RPA-Cas12a-LFT systems for the detection of Candida albicans

New and rapid visual detection assay for Trogoderma granarium everts based on recombinase polymerase amplification and CRISPR/Cas12a

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