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Nanditha Shivakumar1, Shylesha Arakalagud Nanjundaiah1, Venkatesan Thiruvengadam1
1ICAR-National Bureau of Agricultural Insect Resources, Bengaluru 560 024, India.
Abstract:
Phenacoccus manihoti Matile-Ferrero (Hemiptera: Pseudococcidae), is an economically important invasive cassava pest responsible for the massive devastation of cassava in Asia and African continent. Initially, identifying this invasive pest posed challenges because it closely resembled native mealybug species. Additionally, the traditional morphological identification process is labor-intensive and time-consuming. Detecting invasive pests at an early stage is crucial, hence development of a rapid detection assay is essential. In the current study, we have developed a simple, rapid, sensitive, and efficient molecular detection assay for P. manihoti based on Recombinase Polymerase Amplification (RPA). The primers for the RPA assay were designed using unique nucleic acid sequences of P. manihoti, and the protocol was standardized. Specificity test demonstrated that the RPA assay could amplify DNA of P. manihoti only, and no amplification was observed in six other mealybug species. The specificity of assay was confirmed using SYBR green-based colorimetric detection and gel electrophoresis where positive samples showed 195 bp amplicon size in P. manihoti samples. The assay successfully amplified P. manihoti DNA in thirty minutes at an annealing temperature of 41° C in a water bath and displayed a sensitivity of 72.5 picograms per microliter. The assay's simplicity, rapidity, and high sensitivity make it a valuable tool for detecting and monitoring P. manihoti in quarantine stations and facilitating in development of a portable diagnostic kit.
Insights
A new molecular detection assay using Recombinase Polymerase Amplification (RPA) rapidly and sensitively identifies the invasive cassava pest, Phenacoccus manihoti. This method aids early detection and monitoring, crucial for agricultural biosecurity.
Area of Science:
- Agricultural Entomology
- Molecular Diagnostics
- Invasive Species Management
Background:
- Phenacoccus manihoti is a significant invasive pest devastating cassava crops in Asia and Africa.
- Traditional identification methods are slow, labor-intensive, and challenging due to species mimicry.
- Early detection is critical for managing invasive pest outbreaks and preventing crop loss.
Purpose of the Study:
- To develop a rapid, sensitive, and efficient molecular detection assay for Phenacoccus manihoti.
- To establish a reliable diagnostic tool for early identification of this invasive mealybug.
- To support quarantine efforts and the development of portable diagnostic kits.
Main Methods:
- Design of specific primers targeting unique nucleic acid sequences of P. manihoti.
- Development and standardization of a Recombinase Polymerase Amplification (RPA) assay protocol.
- Validation of assay specificity, sensitivity, and speed using molecular techniques like SYBR green and gel electrophoresis.
Main Results:
- The RPA assay specifically amplified P. manihoti DNA, showing no cross-amplification with six other mealybug species.
- A distinct 195 bp amplicon was observed for P. manihoti.
- The assay detected P. manihoti DNA within 30 minutes at 41°C with a sensitivity of 72.5 pg/µL.
Conclusions:
- The developed RPA assay is a simple, rapid, and highly sensitive tool for detecting Phenacoccus manihoti.
- This assay is valuable for quarantine stations and in developing portable diagnostic kits for invasive pest monitoring.
- The molecular approach offers a significant improvement over traditional methods for identifying this economically important pest.
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