Loop-mediated isothermal amplification (LAMP)/Cas12a assay for detection of Ralstonia solanacearum in tomato

Abstract

Introduction: Bacterial wilt (BW) caused by the aerobic, Gram-negative pathogenic species Ralstonia solanacearum (RS) is a major disease impacting commercial agriculture worldwide. Asian phylotype I of RS is the cause of tomato bacterial wilt, which has caused severe economic losses in southern China for many years. An urgent priority in control of bacterial wilt is development of rapid, sensitive, effective methods for detection of RS.Methods: We describe here a novel RS detection assay based on combination of loop-mediated isothermal amplification (LAMP) and CRISPR/Cas12a. crRNA1, with high trans-cleavage activity targeting hrpB gene, was selected out of four candidate crRNAs. Two visual detection techniques, involving naked-eye observation of fluorescence and lateral flow strips, were tested and displayed high sensitivity and strong specificity.Results and Discussion: The LAMP/Cas12a assay accurately detected RS phylotype Ⅰ in 14 test strains, and showed low detection limit (2.0 × 100 copies). RS in tomato stem tissue and soil samples from two field sites with suspected BW infection was identified accurately, suggesting potential application of LAMP/Cas12a assay as point-of-care test (POCT). The overall detection process took less than 2 h and did not require professional lab equipment. Our findings, taken together, indicate that LAMP/Cas12a assay can be developed as an effective, inexpensive technique for field detection and monitoring of RS.

CRISPR–Cas12a-Mediated Hue-Recognition Lateral Flow Assay for Point-of-Need Detection of Salmonella

“…In comparison with previously reported isothermal nucleic acid tests that only tell yes/no (Table S3), the present CHLFA provides a more accurate semiquantitative outcome, enabling the differentiation of subtle changes in the pathogen load during infection and treatment. Moreover, the CHLFA eliminates the need to exchange the positions of the T and C lines, thereby mitigating false positives associated with conventional tests utilizing a turn-on mode. − …”

Rapid and Sensitive Detection of Verticillium dahliae from Soil Using LAMP-CRISPR/Cas12a Technology

“…However, combining LAMP with the CRISPR system (LAMP-CRISPR/cas12a) can effectively mitigate this limitation. The combination of LAMP and CRISPR/Cas12a technology has been widely applied in the detection of plant pathogens [26][27][28][29][30], indicating that this technology has become quite mature.…”

Development of a reverse transcription loop-mediated isothermal amplification based clustered regularly interspaced short palindromic repeats Cas12a assay for duck Tembusu virus

“…Subsequently, the Cas12a performs the collateral cleavage on nonspecific single-stranded DNA (ssDNA) (e.g., the fluorescent ssDNA reporter HEX-N12-BHQ1) when the Cas12a, crRNA, and target DNA form a ternary complex ( Gootenberg et al, 2018 ; Li et al, 2018b ). Based on this collateral cleavage activity, a number of Cas12a-based molecular diagnostic methods coupled with isothermal amplification techniques containing RPA, RCA, RAA and LAMP have been developed to detect pathogens, including RNA viruses, DNA viruses and bacteria ( Li et al, 2018b ; Xu et al, 2020 ; Mayuramart et al, 2021 ; Chen et al, 2022 ; Qin et al, 2022 ; de Sousa et al, 2023 ; Fan et al, 2023 ; Mao et al, 2023 ; Wu et al, 2023 ; Zhao et al, 2023 ). For instance, an RT-RAA-based CRISPR-Cas12a assay was established to detect SARS-CoV-2 and influenza viruses, and the assay was effective for screening of these viruses ( Mayuramart et al, 2021 ).…”