Rapid and accurate monkeypox detection with xylenol orange-based colorimetric assay towards point-of-care: insights from clinical samples in Thailand

Abstract

 Background: Monkeypox has been declared a public health emergency, highlighting the need for rapid and accessible diagnostic tools. While quantitative polymerase chain reaction (qPCR) is the gold standard due to its high sensitivity and specificity, it requires specialized equipment and trained personnel, making it unsuitable for point-of-care testing. Antigen test kits (ATKs) are simpler and more affordable but lack sufficient sensitivity for early outbreak detection.

Objective: This study aims to develop an accurate, rapid, and cost-effective alternative for monkeypox detection based on clinical samples in Thailand.

Methods: A novel colorimetric loop-mediated isothermal amplification (LAMP) assay was developed using xylenol orange (XO) dye for direct, naked-eye detection. This approach enhances visual clarity and simplifies result interpretation while eliminating the need for thermal cyclers.

Results: The assay is completed within 75 minutes, demonstrating 100% accuracy and a detection limit of 10 viral copies per reaction—ten times more sensitive than PCR. It also offers affordability at approximately $3 per test, making it suitable for widespread use.

Conclusion: This is the first study to develop and validate a colorimetric LAMP assay using clinical samples from Thai patients. The assay presents a reliable and accessible alternative to qPCR, with potential applications in broader pathogen diagnostics.