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Rapid and equipment-free identification of papaya mealybug Paracoccus marginatus based on RPA-CRISPR/Cas12a
Yan-Ting Chen1, Meng-Zhu Shi1,2,3, Yan Chen1
1Fujian Key Laboratory for Monitoring and Integrated Management of Crop Pests, Institute of Plant Protection, Fujian Academy of Agricultural Sciences, Fuzhou, China.
Background:
Paracoccus marginatus has invaded many countries, spreading rapidly and causing significant economic losses to crops. Accurate detection during the monitoring process is critical to prevent its expansion into new areas, therefore it is necessary to develop efficient and reliable detection methods. Traditional detection methods are time-consuming and instrument-dependent owing to the morphological similarities and small sizes of P. marginatus and other mealybugs, therefore establishing an efficient, rapid, and sensitive method for field detection in resource-limited settings is critical.
Results:
A sensitive and rapid detection system was developed to detect P. marginatus using recombinase polymerase amplification (RPA) combined with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a. The RPA-CRISPR/Cas12a assay distinguished P. marginatus from 10 other mealybugs. The entire process can be completed in approximately an hour, and the identification results can be determined by the naked eye using lateral flow strips or a portable mini-UV torch. A method was developed to extract DNA from P. marginatus within 5 min. This method was combined with the RPA-CRISPR/Cas12a assay to achieve rapid and simple detection. In addition, two portable thermos cups with temperature displays were used to maintain the reagents and assay reactions in the field.
Conclusion:
This assay represents the first application of portable and easily available items (mini-UV torch and thermos cup) based on the combination of RPA and CRISPR/Cas12a for rapid pest detection. This method is rapid, highly specific, and instrument-flexible, allowing for the early monitoring of P. marginatus in the field. This study provides guidance for the development of suitable management strategies. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
Insights
A new rapid detection system using recombinase polymerase amplification (RPA) and CRISPR/Cas12a accurately identifies the invasive pest Paracoccus marginatus. This field-deployable method offers a quick and sensitive alternative to traditional pest detection techniques.
Area of Science:
- Agricultural Entomology
- Molecular Diagnostics
- Biotechnology
Background:
- The invasive pest Paracoccus marginatus causes significant crop losses globally.
- Traditional detection methods for P. marginatus are slow, labor-intensive, and require specialized equipment.
- Accurate and rapid field detection is crucial for managing the spread of this invasive mealybug.
Purpose of the Study:
- To develop a rapid, sensitive, and field-deployable detection method for Paracoccus marginatus.
- To overcome the limitations of traditional diagnostic techniques for this invasive pest.
- To enable early detection and monitoring in resource-limited settings.
Main Methods:
- Development of a detection system combining recombinase polymerase amplification (RPA) with CRISPR/Cas12a technology.
- Optimization of a rapid DNA extraction method for P. marginatus (under 5 minutes).
- Utilized portable equipment including thermos cups for temperature control and lateral flow strips or a mini-UV torch for result visualization.
Main Results:
- The RPA-CRISPR/Cas12a assay demonstrated high sensitivity and specificity, distinguishing P. marginatus from 10 other mealybug species.
- The entire detection process, including DNA extraction, was completed in approximately one hour.
- Results were easily interpretable with the naked eye using lateral flow strips or a portable UV torch.
Conclusions:
- This study presents the first use of portable, readily available items (UV torch, thermos cup) for pest detection based on RPA and CRISPR/Cas12a.
- The developed assay is rapid, highly specific, and instrument-flexible, facilitating early field monitoring of P. marginatus.
- The findings provide a foundation for effective pest management strategies against this invasive species.
