Pathogenic Bacteria Detection Using RNA-Based Loop-Mediated Isothermal-Amplification-Assisted Nucleic Acid Amplification via Droplet Microfluidics

Abstract

Nucleic acid amplifications, such as polymerase chain reaction (PCR), are very beneficial for diagnostic applications, especially in the context of bacterial or viral outbreaks due to their high specificity and sensitivity. However, the need for bulky instrumentation and complicated protocols makes these methods expensive and slow, particularly for low numbers of RNA or DNA templates. In addition, implementing conventional nucleic acid amplification in a high-throughput manner is both reagent- and time-consuming. We bring droplet-based microfluidics and loop-mediated isothermal amplification (LAMP) together in an optimized operational condition to provide a sensitive biosensor for amplifying extracted RNA templates for the detection of Salmonella typhimurium (targeting the invA gene). By simultaneously performing ∼10⁶ LAMP-assisted amplification reactions in picoliter-sized droplets and applying a new mathematical model for the number of droplets necessary to screen for the first positive droplet, we study the detection limit of our platform with pure culture and real samples (bacterial contaminated milk samples). Our LAMP-assisted droplet-based microfluidic technique was simple in operation, sensitive, specific, and rapid for the detection of pathogenic bacteria Salmonella typhimurium in comparison with well-established conventional methods. More importantly, the high-throughput nature of this technique makes it suitable for many applications in biological assays.

Keywords: LAMP; biosensor; droplet microfluidics; nucleic acid amplification; pathogenic bacteria detection.