Loop-mediated isothermal amplification (LAMP)/Cas12a assay for detection of

Area of Science:

• Plant Pathology
• Molecular Biology
• Biotechnology

Background:

• Bacterial wilt (BW), caused by Ralstonia solanacearum (RS), poses a significant threat to global agriculture.
• Tomato bacterial wilt, specifically by Asian phylotype I of RS, causes substantial economic losses, particularly in southern China.
• Effective control of BW necessitates rapid, sensitive, and accurate methods for RS detection.

Purpose of the Study:

• To develop a novel, rapid, and sensitive detection assay for Ralstonia solanacearum (RS).
• To evaluate the potential of a combined loop-mediated isothermal amplification (LAMP) and CRISPR/Cas12a system for field applications.
• To establish an effective point-of-care test (POCT) for early detection of tomato bacterial wilt.

Main Methods:

• A novel assay was developed by combining loop-mediated isothermal amplification (LAMP) with CRISPR/Cas12a technology.
• A specific CRISPR RNA (crRNA1) targeting the hrpB gene of RS was selected for its high trans-cleavage activity.
• Visual detection methods, including fluorescence and lateral flow strips, were employed for result interpretation.

Main Results:

• The LAMP/Cas12a assay demonstrated high sensitivity and specificity in detecting RS phylotype I.
• A low limit of detection was achieved, with accurate identification of RS in 14 test strains (down to 2.0 × 10^0 copies).
• The assay successfully identified RS in infected tomato stem and soil samples from field sites, validating its practical application.

Conclusions:

• The developed LAMP/Cas12a assay is a highly sensitive and specific tool for detecting Ralstonia solanacearum.
• This assay shows great promise as a point-of-care test (POCT) for field diagnosis of tomato bacterial wilt.
• The rapid (under 2 hours) and equipment-free nature of the assay makes it an effective and inexpensive option for disease monitoring.