Visual Detection of Multiple Nucleic Acids in a Capillary Array

Abstract

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently needed in disease diagnosis, microbial monitoring, genetically modified organism (GMO) detection, and forensic analysis. We have previously described the platform called CALM (Capillary Array-based Loop-mediated isothermal amplification for Multiplex visual detection of nucleic acids). Herein, we describe improved fabrication and performance processes for this platform. Here, we apply a small, ready-to-use cassette assembled by capillary array for multiplex visual detection of nucleic acids. The capillary array is pre-treated into a hydrophobic and hydrophilic pattern before fixing loop-mediated isothermal amplification (LAMP) primer sets in capillaries. After assembly of the loading adaptor, LAMP reaction mixture is loaded and isolated into each capillary, due to capillary force by a single pipetting step. The LAMP reactions are performed in parallel in the capillaries. The results are visually read out by illumination with a hand-held UV flashlight. Using this platform, we demonstrate monitoring of 8 frequently appearing elements and genes in GMO samples with high specificity and sensitivity. In summary, the platform described herein is intended to facilitate the detection of multiple nucleic acids. We believe it will be widely applicable in fields where high-throughput nucleic acid analysis is required.

Mini-Disk Capillary Array Coupling with LAMP for Visual Detection of Multiple Nucleic Acids using Genetically Modified Organism Analysis as an Example

“…Then, the reaction mixture is automatically dispersed into each capillary via capillary forces. There is no need to use extra specific devices or forces for sample dispersal compared with CALM, cLAMP, and microfluidic disk chip, etc. − This sample loading method will decrease cross contamination dramatically compared with the pipetting and injecting modes.…”

“…A capillary-based multiplexed isothermal nucleic acid test was also applied for sexually transmitted disease detection in patients . Recently, we also reported a capillary array-based LAMP (CALM) for multiplex visual detection of seven frequently used transgenic genes/elements with high specificity and sensitivity. , Moreover, Zhou et al developed a centrifuge-based microfluidic disk chip integrated with the LAMP method to detect 10 different bacteria in less than 30 min . However, most of the reported devices or arrays were still only used in the laboratory, which have some disadvantages, such as the complex steps or techniques in device or array production, high cost of production, less reproducibility, etc.…”

Method for the elucidation of LAMP products captured on lateral flow strips in a point of care test for HPV 16

“…The resulting gel does not have the resolution to distinguish between non-specific primer amplification and target-specific amplification. In some POC applications, analysis involves using intercalating dye in situ, with the realtime fluorescence increase observed by eye or using a portable reader [15]. In this embodiment, the user does not have an internal control to detect the presence and amount of nonspecific amplification.…”