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CRISPR-Based Isothermal Next-Generation Diagnostic Method for Virus Detection in Sugarbeet.

ABSTRACT: Rhizomania is a disease of sugarbeet caused by beet necrotic yellow vein virus (BNYVV) that significantly affects sugarbeet yield globally. Accurate and sensitive detection methods for BNYVV in plants and field soil are necessary for growers to make informed decisions on variety selection to manage this disease. A recently developed CRISPR-Cas-based detection method has proven highly sensitive and accurate in human virus diagnostics. Here, we report the development of a CRISPR-Cas12a-based method for detecting BNYVV in the roots of sugarbeet. A critical aspect of this technique is the identification of conditions for isothermal amplification of viral fragments. Toward this end, we have developed a reverse transcription (RT) recombinase polymerase amplification (RPA) for detecting BNYVV in sugarbeet roots. The RT-RPA product was visualized, and its sequence was confirmed. Subsequently, we designed and validated the cutting efficiency of guide RNA targeting BNYVV via in vitro activity assay in the presence of Cas12a. The sensitivity of CRISPR-Cas12a trans reporter-based detection for BNYVV was determined using a serially diluted synthetic BNYVV target sequence. Further, we have validated the developed CRISPR-Cas12a assay for detecting BNYVV in the root-tissue of sugarbeet bait plants reared in BNYVV-infested field soil. The results revealed that BNYVV detection is highly sensitive and specific to the infected roots relative to healthy control roots as measured quantitatively through the reporter signal. To our knowledge, this is the first report establishing isothermal RT-RPA- and CRISPR-based methods for virus diagnostic approaches for detecting BNYVV from rhizomania diseased sugarbeet roots.

SUBMITTER: Ramachandran V

PROVIDER: S-EPMC8297705 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The occurrence of Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) is increasing worldwide, particularly in low- and middle-income countries. In cases where most infections are asymptomatic and remain in a coinfection condition. It may cause missed detections or the severe subsequent symptoms. Accurate diagnosis and convenient screening are essential for disease control. However, molecular diagnostics for CT/NG predominantly rely on polymerase chain reaction (PCR)-based detection, which requires special devices and conditions. Here, we developed an isothermal CRISPR-based CT/NG dual-target detection system by pairing recombinase polymerase amplification (RPA) and CRISPR-Cas12a/13a detection. Multiplex RPA can amplify NG and CT simultaneously, and a single-pot reaction with the Cas12a/13a can be performed for further recognizing the amplified target, driving the cleavage of the corresponding fluorescence reporter separately; thus, the signal can be monitored in different channels. This CRISPR-based CT/NG detection system achieved analytical sensitivities of 10° copies/μL for both synthetic CT/NG dsDNA and exhibited no cross-reaction with other species. In tests on 88 clinical samples, our CRISPR-based assay showed excellent agreement with the commercial assay Roche Cobas 4800 (100% accuracy for NG, 94.32% accuracy for CT, and 97.73% accuracy for CT/NG coinfection) and showed higher positive percent agreement, negative percent agreement, and accuracy than those reported for TaqMan PCR in a previous study. In addition, the results of this CRISPR-based system could be acquired within 75 min. Our isothermal diagnostic method with promising performance provides more convenience than PCR-based methods for detection and screening.IMPORTANCEA method for Neisseria gonorrhoeae (NG)/Chlamydia trachomatis (CT) detection is developed using multiplex-recombinase polymerase amplification and Cas12a/Cas13a. This method can detect NG and CT simultaneously with high sensitivity and specificity. This method has great potential to be further developed into larger-scale screening and point-of-care testing (POCT).

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CRISPR-Based Isothermal Next-Generation Diagnostic Method for Virus Detection in Sugarbeet.

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