Broadening the use of virus-mimicking artificial positive controls in plant virus diagnostics

Positive controls are essential for diagnostics yet access to high quality ones remains a persistent bottleneck for assay validation and quality control. Infected tissues are scarce or even regulated, whereas synthetic DNA fragments lack matrix effects and raise contamination risks. Here, we validate the Virus-Mimicking Artificial Positive Control (ViMAPC) platform for 18 viruses, demonstrating platform scalability across multiple virus taxa. ViMAPCs primarily use genus-matched surrogates and built-in size shifts to easily distinguish natural infections (true positives) from false positives due to contamination. Controls were generated via chimeric primer reverse transcription followed by end-point PCR using peer-reviewed protocols. All targets yielded expected amplicons with designed size differentiation and similar amplification behaviour to naturally infected material, with percent differences in amplicon intensity of ≤ 30%. Independent synthesis and validation in two laboratories achieved 100% success with design-to-use timelines of 4-5 days, demonstrating transferability for routine diagnostic workflows. By providing rapid, low-cost controls that mimic natural infection while remaining clearly identifiable, ViMAPCs enable robust diagnostic validation, contamination tracing, and cross-laboratory comparability, supporting diagnostic laboratories in rapid assay deployment for both newly discovered and quarantine viruses.