Paper-based Molecular Diagnostics for the Amplification and Detection of Pathogenic Bacteria from Human Whole Blood and Milk Without a Sample Preparation Step

Abstract

We have developed a novel paper-based molecular pathogen diagnostic method by combining the direct loop-mediated isothermal amplification (Direct LAMP) in a paper reactor and an immunochromatographic strip (ICS) detection without any DNA preparation step. The mineral paper is durable, oil- and tear-resistant, and waterproof, so it is a proper material for point-of-care (POC) testing owing to facile chip fabrication, cost-effectiveness, and disposability. The mineral paper was utilized as a substrate to construct the reactor for gene amplification based on the principle of origami without using an expensive and complicated process. In this novel paper reactor, we performed the Direct LAMP reaction, which is a direct isothermal DNA amplification without any sample preparation step such as cell lysis, DNA extraction and purification for amplifying specific target gene. After the Direct LAMP reaction in a paper reactor, a capillary tube was employed to take an appropriate amount (similar to 2 mu L) of amplicons in order to transfer the LAMP product to an ICS. The use of the ICS allowed us to perform the simple and rapid colorimetric detection of the resultant amplified target genes by naked eyes, thereby confirming the presence of the pathogen in a sample in a user-friendly way. In this platform, we could simultaneously identify Staphylococcus aureus and Escherichia coli O157:H7 contaminated in human blood or milk. The whole molecular diagnostic process (the Direct LAMP in a mineral paper plus the ICS detection) could be conducted in a paper substrate, so the proposed method would be ideal for pathogen detection in resourcelimited environments.