Long-term dry storage of enzyme-based reagents for isothermal nucleic acid amplification in a porous matrix for use in point-of-care diagnostic devices.

Nucleic acid amplification test (NAAT)-based point-of-care (POC) devices are rapidly growing for use in low-resource settings. However, key challenges are the ability to store the enzyme-based reagents in dry form in the device and the long-term stability of those reagents at elevated temperatures, especially where ambient temperatures could be as high as 45 °C. Here, we describe a set of excipients including a combination of trehalose, polyethylene glycol and dextran, and a method for using them that allows long-term dry storage of enzyme-based reagents for an isothermal strand displacement amplification (iSDA) reaction in a porous matrix. Various porous materials, including nitrocellulose, cellulose, and glass fiber, were tested. Co-dried reagents for iSDA always included those that amplified the ldh1 gene in Staphylococcus aureus (a polymerase and a nicking enzyme, 4 primers, dNTPs and a buffer). Reagents also either included a capture probe and a streptavidin-Au label required for lateral flow (LF) detection after amplification, or a fluorescent probe used for real-time detection. The reagents showed the best stability in a glass fiber matrix when stored in the presence of 10% trehalose and 2.5% dextran. The reagents were stable for over a year at ∼22 °C as determined by lateral flow detection and gel electrophoresis. The reagents also exhibited excellent stability after 360 h at 45 °C; the assay still detected as few as 10 copies of ldh1 gene target by lateral flow detection, and 50 copies with real-time fluorescence detection. These results demonstrate the potential for incorporation of amplification reagents in dry form in point-of-care devices for use in a wide range of settings.

Disposable Autonomous Device for Swab-to-Result Diagnosis of Influenza.

A prototype of a self-contained, automated, disposable device for chemically amplified protein-based detection of influenza virus from nasal swab specimens was developed and evaluated in a clinical setting. The device required only simple specimen manipulation without any dedicated instrumentation or specialized training by the operator for interpretation. The device was based on a sandwich immunoassay for influenza virus nucleoprotein; it used an enzyme-labeled antibody and a chromogenic substrate to provide an amplified visible signal, in a two-dimensional paper network format. All reagents were stored within the device. Device performance was assessed at Seattle Children's Hospital; clinical staff collected nasal swab samples from 25 patients and then operated test devices on site to detect influenza A and B in those specimens. The total test time from device initiation to result was approximately 35 min. Device performance for influenza A detection was ∼70% accurate using in-house qRT-PCR influenza A as a gold-standard comparison. The ratio of valid to total completed device runs yielded a success rate of 92%, and the negative predictive value for both the influenza A and B assay was 81%. The ability to diagnose respiratory infections rapidly and close to the patient was well received by hospital staff, inspiring further optimization of device function.