Establishment of RAA-CRISPR/Cas12a-LFD-based visual detection of <i>Pseudocryphonectria elaeocarpicola</i>

Huang Can; Wu Haoyu; Xiong Dianguang; Huang Huayi; Tian Chengming

doi:10.12171/j.1000-1522.20240341

Journal of Beijing Forestry University > 2025 > Accepted Manuscript > DOI: 10.12171/j.1000-1522.20240341

Huang Can, Wu Haoyu, Xiong Dianguang, Huang Huayi, Tian Chengming. Establishment of RAA-CRISPR/Cas12a-LFD-based visual detection of Pseudocryphonectria elaeocarpicola[J]. Journal of Beijing Forestry University. DOI: 10.12171/j.1000-1522.20240341

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Establishment of RAA-CRISPR/Cas12a-LFD-based visual detection of Pseudocryphonectria elaeocarpicola

1.
College of Forestry, Beijing Forestry University, Beijing 100083, China
2.
Guangdong Academy of Forestry, Guangzhou 510520, Guangdong, China

More Information

Received Date: October 15, 2024
Revised Date: January 20, 2025
Accepted Date: February 09, 2025
Available Online: February 11, 2025

Graphical Abstract

Abstract

Abstract

Objective
Elaeocarpus spp. stem blight is a new branching disease on Elaeocarpus spp. caused by Pseudocryphonectria elaeocarpicola, for which research is still in its infancy and lacks effective prevention and control measures. The aim of this paper is to establish a rapid and visualized method for the detection of P. elaeocarpicola, which provides an important technical support for the early monitoring and early warning of the disease.
Method
In this study, we utilized PsGti1 gene of P. elaeocarpicola as a target, specifically amplified the target gene by recombinase aided amplification (RAA) reaction, and cut the target and fluorescent probe by CRISPR/Cas12a system, and finally visualized the detection of P. elaeocarpicola by using lateral flow dipstick (LFD).
Result
(1) In this study, the optimal primer pairs for the RAA amplification reaction of the PsGti1 gene of P. elaeocarpicola were screened and obtained. (2) The CRISPR/Cas12a reaction system worked best when the Cas12a and CrRNA concentration ratios were 1 μmol/L and 0.125 μmol/L, respectively. (3) A RAA-CRISPR/Cas12a-LFD visualization detection system was established, which can realize the rapid detection of P. elaeocarpicola under the reaction condition of constant temperature at 37 ℃, and the sensitivity of the detection were 50 fg/μL (gDNA as template) and 20 pg/μL (PsGti1_T-vector as template), respectively.
Conclusion
The RAA-CRISPR/Cas12a-LFD detection system for P. elaeocarpicola established in this study has the advantages of easy-to-reach reaction temperature, high specificity, high sensitivity and ease of operation, which makes it suitable for carrying out the detection of P. elaeocarpicola in the field or in scenarios lacking laboratory testing equipment.
- Pseudocryphonectria elaeocarpicola,
- RAA,
- CRISPR/Cas12a,
- LFD,
- visualization

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References (31)

References

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Establishment of RAA-CRISPR/Cas12a-LFD-based visual detection of Pseudocryphonectria elaeocarpicola