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

Summary

Scientists developed a new rapid test to detect a dangerous fungal disease that affects cotton crops. The test combines two advanced molecular techniques (LAMP and CRISPR) to quickly identify the disease-causing fungus in soil samples. The system works in laboratories but can also be used in fields by farmers, taking less than 2 hours to produce results. This breakthrough will help farmers catch and manage the disease early, protecting their crops and improving yields.

Background

Cotton Verticillium wilt is caused by the soil-borne fungus Verticillium dahliae, which survives in soil as microsclerotia for several years. This destructive disease threatens cotton production globally and in China affects approximately 2.5 million hectares annually. Current detection methods have limitations including high false-positive rates and high operational costs.

Objective

To develop an accurate, sensitive, and rapid detection method for V. dahliae from complex soil samples by combining loop-mediated isothermal amplification (LAMP) with CRISPR/Cas12a technology. The study aimed to create both laboratory and field-deployable diagnostic systems for early warning and management of cotton Verticillium wilt.

Results

The LAMP-CRISPR/Cas12a fluorescence system achieved a lower detection limit of approximately 10 fg/μL genomic DNA, detecting as few as two microsclerotia per gram of soil in less than 90 minutes. The field-based lateral flow strip system detected approximately 1 fg/μL genomic DNA and six microsclerotia per gram of soil within 2 hours, demonstrating excellent specificity with no cross-reactivity to other Verticillium species.

Conclusion

This study successfully developed the first LAMP-CRISPR/Cas12a integration method for V. dahliae detection with both laboratory and field-deployable systems. The approach provides a low-cost, sensitive, and user-friendly diagnostic tool suitable for early detection of cotton Verticillium wilt and can be adapted for detection of other soil-borne pathogens.
  • Published in:International Journal of Molecular Sciences,
  • Study Type:Experimental/Diagnostic Development,
  • Source: 10.3390/ijms25105185, PMID: 38791224
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