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

Zhiyu Fan1, Yuxia Mei1, Jiawei Xing1

  • 1State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, China.

Frontiers in Bioengineering and Biotechnology
|June 7, 2023
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Summary

A new assay combines loop-mediated isothermal amplification (LAMP) and CRISPR/Cas12a for rapid detection of Ralstonia solanacearum (RS), the bacterium causing tomato bacterial wilt. This method offers a sensitive, field-deployable tool for early disease identification.

Keywords:
CRISPR/Cas12aLAMPRalstonia solanacearumbacterial wilt (BW)detection

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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.