RT-RPA-PfAgo System: A Rapid, Sensitive, and Specific Multiplex Detection Method for Rice-Infecting Viruses

Area of Science:

• Molecular Biology
• Plant Pathology
• Biotechnology

Background:

• CRISPR-Cas biosensors offer rapid, sensitive plant virus detection but face challenges like false positives and limited multiplexing.
• Novel methodologies are needed to improve plant virus diagnostic tools.

Purpose of the Study:

• To develop a novel, highly sensitive, and specific detection system for multiple plant viruses.
• To overcome limitations of existing CRISPR-Cas based biosensors.

Main Methods:

• Integration of reverse transcriptase (RT) with recombinase polymerase isothermal amplification (RPA) and Pyrococcus furiosus Argonaute (PfAgo).
• Simultaneous detection of rice ragged stunt virus (RRSV), rice grassy stunt virus (RGSV), and rice black streaked dwarf virus (RBSDV).

Main Results:

• The RT-RPA-PfAgo system achieved simultaneous detection of three distinct rice viruses.
• Detection limits ranged from 3.13 to 5.13 copies/µL, demonstrating high sensitivity.
• The system showed high specificity, effectively differentiating between the target viruses, with field evaluations aligning with RT-PCR.

Conclusions:

• The RT-RPA-PfAgo system is a robust, versatile, and sensitive method for plant virus diagnostics.
• PfAgo's ability to identify targets via guide DNA, independent of protospacer adjacent motif sequences, offers significant advantages.
• This integrated system holds great potential for practical application in future plant virus diagnostics.