Simultaneous detection of multiple spoilage bacteria using a fluorogenic loop-mediated isothermal amplification-based multi-sample microfluidic chip

Abstract

Spoilage bacteria, which ubiquitously contribute to food spoilage, pose a global public health risk. In this study, we developed a centrifugal microfluidic chip integrating fluorogenic loop-mediated isothermal amplification (LAMP) to detect six important spoilage bacteria: Bacillus cereus, Clostridium botulinum, Photobacterium phosphoreum, Pseudomonas putida, Streptococcus agalactiae, and Shewanella putrefaciens. The LAMP reaction components were pre-mixed (primers pre-embedded in each reaction chamber of the chip) and evenly pipetted into the two inlet holes, enabling the simultaneous analysis of six genetic targets from a single sample. This facilitates the automated detection of up to four samples simultaneously using a single chip. The LAMP reactions were conducted in a 5-μL volume at 65 °C for 60 min, achieving limits of detection ranging from 10−1 to 10−2 pg/μL of bacterial genomic DNA. Almost all coefficients of variation for time-to-positive values consistently remained under 10% when genomic DNA was used at concentrations within the limits of detection for the six bacteria. This demonstrated excellent reproducibility across replicates. The clinical sensitivity and specificity for field sample detection were 93.3% and 97.5%, respectively. This method offers ease of operation, rapid analysis, and minimal reagent usage and has great potential for the on-site detection of multiple spoilage bacteria.