Real-time fluorescence and colorimetric identification of bulbus fritillariae using recombinase assisted loop-mediated isothermal DNA amplification (RALA)

Abstract

Bulbus Fritillariae (BF) is a kind of herbal medicine widely used in many countries including China, Japan, Korea, and so on. Among the known BF medicinal materials, Bulbus Fritillariae cirrhosae (BF cirrhosae) was reported to have the best curative effect. Due to the limited resources of BF cirrhosae, a lot of adulterants have emerged in the market, impairing the market order, resource development, and above all, clinical efficacy. Here, a novel nucleic acid amplification technique, Recombinase Assisted Loop-mediated isothermal DNA Amplification (RALA), was used to establish a real-time fluorescence isothermal molecular authentication method for five commonly used BF drugs. Moreover, this RALA-based assay can also be developed as a colorimetric detection method for on-site detection. Both real-time fluorescence and visual methods could detect as low as 0.1% genuine targets in the mixed samples. In summary, we report an isothermal detection system for five kinds of BF circulating in the market, providing a new choice for the molecular identification of BF drugs and showing promise in the laboratory testing as well as field identification of other herbal medicines.

Advancements and applications of loop-mediated isothermal amplification technology: a comprehensive overview

“…The resulting dumbbell DNA strand initiates LAMP. Wei et al (2022) developed a real-time fluorescence identification system utilizing RALA for the detection of Bulbus Fritillariae , capable of identifying as little as 0.1% of genuine targets in the mixed samples.…”

“…LAMP’s isothermal nature eliminates the need for expensive thermal cyclers, making the technique more accessible, especially in resource-limited settings. Its high sensitivity and rapid amplification capabilities make it particularly useful for detecting low-abundance nucleic acids in clinical, agricultural, and environmental samples ( Wei et al, 2022 ; Jiang et al, 2023 ; Søndreli et al, 2023 ). Furthermore, the method’s ability to provide visual readouts simplifies result interpretation, facilitating its use in field diagnostics and point-of-care testing (POCT).…”