QUANTITATIVE PATHOGEN DETECTION BASED ON DIGITAL LOOP-MEDIATED ISOTHERMAL AMPLIFICATION USING CROSS-INTERFACE EMULSIFICATION TECHNOLOGY

Abstract

A number of attempts have been made for performing digital PCR1 or isothermal amplifications2 for the absolute quantification of nucleic acids using microfluidic technologies. Here, we describe a portable and easy-to-use digital loop-mediated isothermal amplification (dLAMP) system built on our recently reported cross-interface emulsification (XiE) technology3. XiE is a simple method which generates monodisperse droplets using vibrating capillaries, which can be easily set up and is user-friendly to those who lack microfabrication facilities. A schematic view of the droplet generation by cross-interface emulsification (XiE) is illustrated in Figure 1. The XiE method can generate size-tunable droplet arrays independent of device design. Thousands of nanoliter droplets can be generated in each well of a 96-well plate, and used for digital nucleic acids quantification based on dLAMP. Moreover, droplet arrays with various sizes can further expend the dynamic range of detection based on multivolume digital analysis. We applied this XiE-based dLAMP system in rapid detection of an important infectious pathogens, Mycobacterium bovis (M. bovis), a pathogen which causes bovine tuberculosis (bTB) and human infections. We constructed the mpb70-T1 plasmid which contains unique mpb70 gene of M. bovis4 and used it as the standard to investigate the detection limit of dLAMP (Fig. 2). A good linearity between input templates and measured concentration were obtained. Next, we tested the robustness of dLAMP in direct detection M. bovis in real samples. The plasmids with two concentrations (13fg and 130fg) were added in fetal bovine serum (Fig. 3) with or without dilution, and used as samples to perform dLAMP without further treatment. Our results showed that dLAMP is very robust and succeeded in the amplification of mpb70 gene in fetal bovine serum with 2x dilution, and the results were consistent with water control. This result validated that dLAMP can be directly used to analyze complex samples to avoid loss of nucleic acid targets during purification and further reduce the delay in diagnosis. In summary, our results shows that the XiE-based dLAMP is highly specific and displays comparable sensitivity to real-time PCR (qPCR) and digital PCR (dPCR)5, with reduced detection time of 30 min to 1 hour. Moreover, dLAMP can be used directly for rapid detection of diluted real samples. Therefore, our dLAMP system is especially suitable for environmental and clinical samples with hard-to remove contaminants, and can be widely applied in quantitative and timely diagnosis of infectious diseases.