A contamination-free and visual method using LAMP (loop-mediated isothermal amplification) and molecular beacon for sensitive and specific detection of rainbow trout (Oncorhynchus mykiss)

Abstract

Fish species adulteration is a global issue, negatively affecting consumer's rights, public health and marine conservation. LAMP (loop-mediated isothermal amplification) has been widely used for fish species identification, while the commonly-used detection techniques are often time-consuming, prone to carry-over contamination, and sequence-independent. This work selected rainbow trout as a case study, and aimed to develop a novel LAMP assay, combing with a sequence-specific MB (molecular beacon) probe and a simple and portable end-point visual detection device, for rapid detection of rainbow trout with great specificity and superb sensitivity.With the results of the present study, a cost-effective and reliable strategy for the selection of MB's stem-loop structure based on the fluorescence analysis was proved. The molecular beacon LBP-4-1 in the final protocol is characterized by the low background signal and high amplification efficiency. The visual MB-LAMP assay, after optimization, has proved its specificity and applicability, and can detect rainbow trout DNA as little as 0.1 pg in 25 min. Moreover, the risk of cross-contamination can be greatly reduced by the omission of lid-opening, and with the use of a novel end-point visual device, the detection system could be used to screen rainbow trout in the field, without the constraints of complex instruments and other factors. Therefore, the present study offers a simple, fast, accurate method for rainbow trout screening, which could support the identification of authentic aquatic products and help control rainbow trout adulteration.