title: Development and evaluation of a real-time fluorogenic loop-mediated isothermal amplification assay integrated on a microfluidic disc chip (on-chip LAMP) for rapid and simultaneous detection of ten pathogenic bacteria in aquatic animals.
abstract: Rapid, low-cost, and user-friendly strategies are urgently needed for early disease diagnosis and timely treatment, particularly for on-site screening of pathogens in aquaculture. In this study, we successfully developed a real-time fluorogenic loop-mediated isothermal amplification assay integrated on a microfluidic disc chip (on-chip LAMP), which was capable of simultaneously detecting 10 pathogenic bacteria in aquatic animals, i.e., Nocardia seriolae, Pseudomonas putida, Streptococcus iniae, Vibrio alginolyticus, Vibrio anguillarum, Vibrio fluvialis, Vibrio harveyi, Vibrio parahaemolyticus, Vibrio rotiferianus, and Vibrio vulnificus. The assay provided a nearly-automated approach, with only a single pipetting step per chip for sample dispensing. This technique could achieve limits of detection (LOD) ranging from 0.40 to 6.42pg per 1.414μL reaction in less than 30 min. The robust reproducibility was demonstrated by a little variation among duplications for each bacterium with the coefficient of variation (CV) for time to positive (Tp) value less than 0.10. The clinical sensitivity and specificity of this on-chip LAMP assay in detecting field samples were 96.2% and 93.8% by comparison with conventional microbiological methods. Compared with other well-known techniques, on-chip LAMP assay provides low sample and reagent consumption, ease-of-use, accelerated analysis, multiple bacteria and on-site detection, and high reproducibility, indicating that such a technique would be applicable for on-site detection and routine monitoring of multiple pathogens in aquaculture.
authors: Laboratory of Biochemistry and Molecular Biology, School of Marine Sciences, Ningbo University, Ningbo 315211, PR China; Ningbo Branch of National Engineering Research Center for Beijing Biochip Technology, Ningbo University, Ningbo 315211, PR China.
authors: Qian-Jin
authors: QJ
authors: Zhou
authors: CapitalBio Corporation, 18 Life Science Parkway, Changping District, Beijing 102206, PR China.
authors: Lei
authors: L
authors: Wang
authors: Laboratory of Biochemistry and Molecular Biology, School of Marine Sciences, Ningbo University, Ningbo 315211, PR China; Ningbo Branch of National Engineering Research Center for Beijing Biochip Technology, Ningbo University, Ningbo 315211, PR China. Electronic address: jchen1975@163.com.
authors: Jiong
authors: J
authors: Chen
authors: Laboratory of Biochemistry and Molecular Biology, School of Marine Sciences, Ningbo University, Ningbo 315211, PR China.
authors: Rui-Na
authors: RN
authors: Wang
authors: Laboratory of Biochemistry and Molecular Biology, School of Marine Sciences, Ningbo University, Ningbo 315211, PR China; Ningbo Branch of National Engineering Research Center for Beijing Biochip Technology, Ningbo University, Ningbo 315211, PR China.
authors: Yu-Hong
authors: YH
authors: Shi
authors: Laboratory of Biochemistry and Molecular Biology, School of Marine Sciences, Ningbo University, Ningbo 315211, PR China; Ningbo Branch of National Engineering Research Center for Beijing Biochip Technology, Ningbo University, Ningbo 315211, PR China.
authors: Chang-Hong
authors: CH
authors: Li
authors: Ningbo Branch of National Engineering Research Center for Beijing Biochip Technology, Ningbo University, Ningbo 315211, PR China.
authors: De-Min
authors: DM
authors: Zhang
authors: Ningbo Branch of National Engineering Research Center for Beijing Biochip Technology, Ningbo University, Ningbo 315211, PR China.
authors: Xiao-Jun
authors: XJ
authors: Yan
authors: Center for Disease Control and Prevention of Zhejiang Province, Hangzhou 310051, PR China.
authors: Yan-Jun
authors: YJ
authors: Zhang
mesh_terms: D000818:Animals; D059001:Aquatic Organisms / Q000382:microbiology*; D001419:Bacteria / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D044085:Microfluidics / Q000191:economics / Q000295:instrumentation / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000191:economics / Q000295:instrumentation / Q000379:methods*; D012623:Seawater / Q000382:microbiology*
keywords: Aquaculture; Loop-mediated isothermal amplification; Microfluidic chip; Multiplex detection; Pathogenic bacteria; Real-time detection