RPA-Assisted Cas12a System for Detecting Pathogenic Xanthomonas oryzae, a Causative Agent for Bacterial Leaf Blight Disease in Rice

Buddhachat, Kittisak; Sripairoj, Nattaporn; Ritbamrung, Onchira; Inthima, Phithak; Ratanasut, Kumrop; Boonsrangsom, Thanita; Rungrat, Tepsuda; Pongcharoen, Pongsanat; Sujipuli, Kawee

doi:10.1016/j.rsci.2021.11.005

Cited by 13 publications

(18 citation statements)

References 40 publications

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“…Moreover, incubating reactions for a long-time resulted in cas-gRNA complex binding and cutting of non-speci c targets that presented cleavage uorescence signals of no targets. Consequently, the study for cas12a detection used the various optimal concentration of cas12a:gRNA and the appropriate time for detection (Buddhachat et al, 2022b;Ding et al, 2020a;Mukama et al, 2020). However, the cas12a assay can speci cally detect different DNA within one hour, thereby saving time for detection.…”

Section: Discussionmentioning

confidence: 99%

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RPA coupled with cas12a as a simple, accurate and rapid method for diagnosing rickettsia in dogs

Paenkaew

Jaito

Pradit

et al. 2023

Preprint

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Rickettsial pathogens including Ehrlichia canis and Anaplasma platys are bacteria that cause parasitic infections in dogs such as canine monocytic ehrlichiosis (CME) and canine cyclic thrombocytopenia (CCT), respectively affecting mortality and morbidity worldwide. An accurate, sensitive, and rapid method to diagnose these agents is essential for effective treatment. In this study, a recombinase polymerase amplification (RPA) coupled with CRISPR-Cas12a methods was established to detect E. canis and A. platys infection in dogs based on the 16S rRNA. The optimal condition for DNA amplification by RPA was 37°C for 20 min, followed by CRISPR-Cas12a digestion at 37°C for one hour. RPA coupled with cas12a detection showed no cross-reaction with other parasites, and offered high sensitivity, with a limit of detection at 102 copy numbers of both E. canis and A. platys 1,000 and 100 times higher than agarose gel electrophoresis detection, respectively. The RPA-assisted cas12a assay provides specific, sensitive, rapid, simple, and appropriate detection of rickettsia in canine blood at the point-of-care for diagnostics, disease prevention, and surveillance.

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Section: Discussionmentioning

confidence: 99%

“…Wang et al, 2019). RPAassisted cas12a has been applied in shrimp pathogen diagnosis to detect WSSV (white spot syndrome virus) (Chaijarasphong et al, 2019) and also used for the detection of plant viruses (Aman et al, 2020) and bacteria (Buddhachat et al, 2022a).…”

Section: Introductionmentioning

confidence: 99%

RPA coupled with cas12a as a simple, accurate and rapid method for diagnosing rickettsia in dogs

Paenkaew

Jaito

Pradit

et al. 2023

Preprint

Self Cite

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“…in milk [ 110 ] and 2.5×10 3 CFU/mL for Xanthomonas oryzae pv. oryzae in rice [ 111 ]. Using gold nanoparticles (AuNPs) as the reporter of CRISPR assays allowed colorimetric and on-site detection.…”

Section: Pathogenic Diagnosticsmentioning

confidence: 99%

CRISPR-based nucleic acid diagnostics for pathogens

Yang

Zhang

Teng

et al. 2023

TrAC Trends in Analytical Chemistry

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“…in milk [105] and 2.5×10 3 CFU/mL for Xanthomonas oryzae pv. oryzae in rice [106]. Using gold nanoparticles (AuNPs) as the reporter of CRISPR assays allowed colorimetric visual detection, favoring on-site detection.…”

Section: Bacterial Infection Diagnosismentioning

confidence: 99%

CRISPR-based nucleic acid diagnostics for pathogens

Yang

Zhang

Teng

et al. 2022

Preprint

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Pathogenic infections remain the primary threat for human health, especially the global COVID-19 pandemic. It is important to develop rapid, sensitive and multiplexed tools for detecting pathogens and their mutations, particularly the tailor-made strategies for point-of-care diagnosis allowing for use in resource-constrained settings. The rapidly evolving CRISPR/Cas systems have provided a powerful toolbox for pathogenic diagnostics via nucleic acid tests. In this review, we first describe the resultant promising class 2 (single, multidomain effector) and recently explored class 1 (multisubunit effector complexes) CRISPR tools. We present the diverse engineering nucleic acid diagnostics based on CRISPR/Cas systems for pathogenic viruses, bacteria and fungi, and highlight the application for detecting viral variants and drug-resistant bacteria enabled by CRISPR-based mutation profiling. Finally, we discuss the challenges such as the development of preamplification-free diagnostic assays and present the emerging CRISPR systems and CRISPR cascade that potentially enable multiplexed and preamplification-free pathogenic diagnostics.

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RPA-Assisted Cas12a System for Detecting Pathogenic Xanthomonas oryzae, a Causative Agent for Bacterial Leaf Blight Disease in Rice

Cited by 13 publications

References 40 publications

RPA coupled with cas12a as a simple, accurate and rapid method for diagnosing rickettsia in dogs

RPA coupled with cas12a as a simple, accurate and rapid method for diagnosing rickettsia in dogs

CRISPR-based nucleic acid diagnostics for pathogens

CRISPR-based nucleic acid diagnostics for pathogens

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