# A Rapid, Cost-Effective RNA Recovery of Cowpea Mild Mottle Virus (CPMMV) Directly from PCR Tubes Adsorption for Routine-Scale Detection in Soybean

## TL;DR

This paper introduces a low-cost, efficient method for detecting cowpea mild mottle virus in soybean using RNA recovery from PCR tubes.

## Contribution

A novel, cost-effective RNA recovery protocol for CPMMV detection using plastic surface adsorption and optimized reagents.

## Key findings

- The optimized protocol achieved consistent RT-PCR results comparable to standard methods.
- The method reduced costs by 70% compared to Qiagen kits and 90% compared to the Bertheau method.
- The protocol simplifies RNA recovery by eliminating commercial kits and reducing processing steps.

## Abstract

This study describes an optimized plastic surface-based capsid protein adsorption/capturing method for detection of cowpea mild mottle virus (CPMMV) adapted from the direct antigen-capture method reported for the extraction of rose rosette virus (RRV) and other direct virus capturing attempts. Briefly, the method starts with sap incubation, removal of unbound residual tissue and inhibitors by washing, and the viral RNA release using nuclease-free water and heat, in the presence of an RNase inhibitor. The protocol’s efficiency was assessed across different pH conditions, RNaseOUT concentrations, and reverse-transcriptase choices, and its performance was compared with commercial RNA-extraction methods. Three hundred thirty-two positive samples for CPMMV were processed using the optimized protocol (PBS-T, pH 7.4; RNaseOUT at 0.5 U/µL; and M-MLV reverse transcriptase). RT-PCR detection results were consistent with those obtained using the standard method. Cost estimates for tissue trapping indicate reductions of approximately 70% and 90% compared with the Qiagen RNeasy kit (Qiagen, Hilden, Germany) and the Bertheau method, respectively. The tissue-absorption protocol combines simplicity and low cost, making it particularly well suited for field diagnostics; by enabling rapid recovery of viral RNA without commercial kits and substantially reducing processing steps, it represents a practical, cost-effective alternative for routine CPMMV testing.