Qing FanID, Zhixun Xie *, You Wei, Yanfang Zhang, Zhiqin Xie, Liji Xie,Jiaoling Huang, Tingting Zeng, Sheng Wang, Sisi Luo, Meng Li
Guangxi Key Laboratory of Veterinary Biotechnology, Key Laboratory of China(Guangxi)-ASEAN Cross-Border Animal Disease Prevention and Control, Ministry of Agriculture and Rural Affairs of China, Guangxi Veterinary Research Institute, Nanning, Guangxi, China
PLoS ONE 17(12): e0278451.https://doi.org/10.1371/journal.pone.0278451
Abstract
Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification technique that can be used to amplify target genes at a constant temperature, and it has several advantages, including convenience, specificity and sensitivity. However, due to the special interpretation methods of this technology for reaction results, all the previously reported LAMP detection methods have been restricted to identifying a single target, which limits the application of this technology. In this study, we modified conventional LAMP to include a quencher-fluorophore composite probe complementary to the F1c segment of the inner primer FIP; upon strand separation, a gain in the visible fluorescent signal was observed.The probes could be labeled with different fluorophores, showing different colors at the corresponding wavelengths. Therefore, this multiplex LAMP (mLAMP) assay can simultaneously detect 1–3 target sequences in a single LAMP reaction tube, and the results are more accurate and intuitive. In this study, we comprehensively demonstrated a single-reaction mLAMP assay for the robust detection of three cattle viruses without nonspecific amplification of other related pathogenic cattle viruses. The detection limit of this mLAMP assay was as low as 526–2477 copies/reaction for the recombinant plasmids. It is expected that this mLAMP assay can be widely used in clinical diagnosis.