A Molecular Lateral Flow Assay for SARS-CoV-2 Quantitative Detection

Abstract

Since the onset of the SARS-CoV-2 pandemic, several COVID-19 detection methods, both commercially available and in the lab, have been developed using different biomolecules as analytes and different detection and sampling methods with high analytical performance. Developing novel COVID-19 detection assays is an exciting research field, as rapid accurate diagnosis is a valuable tool to control the current pandemic, and also because the acquired knowledge can be deployed for facing future infectious outbreaks. We here developed a novel gold-nanoparticle-based nucleic acid lateral flow assay for the rapid, visual, and quantitative detection of SARS-CoV-2. Our method was based on the use of a DNA internal standard (competitor) for quantification and involved RT-PCR, the hybridization of biotinylated PCR products to specific oligonucleotide probes, and detection with a dual lateral flow assay using gold nanoparticles conjugated to an anti-biotin antibody as reporters. The developed test allowed for rapid detection by the naked eye and the simultaneous quantification of SARS-CoV-2 in nasopharyngeal swabs with high specificity, detectability, and repeatability. This novel molecular strip test for COVID-19 detection represents a simple, cost-effective, and accurate rapid test that is very promising to be used as a future diagnostic tool.

Design and Validation of a Three-Dimensional Printer-Based System Enabling Rapid, Low-Cost Construction of the Biosensing Areas of Lateral Flow Devices for Immunoassays and Nucleic Acid Assays

“…Biotinylated-BSA and oligo­(dT) 30 were again deposited onto the membrane of LFAs using the proposed dispensing system. Three nasopharyngeal samples of healthy individuals and three nasopharyngeal samples of volunteers, which were found positive to SARS-CoV-2 virus using a commercial rapid test with a subsequent molecular PCR test, were subjected to RT-PCR following the protocol previously described . Then, a 1-μL aliquot of each PCR product, corresponding to SARS-CoV-2 DNA sequence, was mixed with 1 pmol of the complementary detection probe tailed with poly­(dA), 1 μL of NaCl 900 mM, and 7 μL of 1× PCR buffer.…”

“…For construction of the CS, the b-BSA solution was deposited using a single pass at 60% printing speed. Meanwhile, functionalized microspheres with (dT) 30 oligonucleotide or specific DNA probes were prepared for construction of the TS as previously reported. ,,,, The functionalized microspheres were loaded into the technical pen and the pipet tip. The TS were finally formed by one-time deposition using the technical pen or three-time deposition using the tip on the membrane with a printing speed of 60%.…”

“…7,8,18−21 Finally, a dual strip was constructed for the simultaneous detection of SARS-CoV-2 amplified DNA sequence and its competitor sequence used for quantification. 22 One of the main aspects of the construction of lateral flow devices is the loading (printing or deposition) of precise volumes and immobilization of capture (bio)molecules onto the membrane as lines or spots followed by drying. The most common immobilization approach is the physical adsorption of the molecules onto the membrane.…”

“…The two products for the two alleles of each gene were differently labeled with digoxigenin or fluorescein and biotin and were detected at two distinct TZs in the same strip by immobilized antidigoxigenin or antifluorescein antibody and streptavidin, respectively. − Multiallele visual detection was achieved for the simultaneous detection of up to five different SNPs (10 alleles) by preparing multiple test spots in a single strip for diagnosis of different diseases/syndromes such as Parkinson, thalassemia, and thrombophilia. Polystyrene microspheres were conjugated to specific oligonucleotides and immobilized onto the membrane at different positions forming up to 10 different test spots. ,,− Finally, a dual strip was constructed for the simultaneous detection of SARS-CoV-2 amplified DNA sequence and its competitor sequence used for quantification …”

Carrageenan from Gigartina skottsbergii: A Novel Molecular Probe to Detect SARS-CoV-2

“…In the last two years, research has sought to develop new molecular strategies for detecting the virus. Recently, a new strip test-based quantitative molecular lateral flow assay for SARS-CoV-2 was developed based on a nucleic acid lateral flow assay of gold nanoparticles [ 7 ]; it proved to be a straightforward and inexpensive method with quick analysis and simultaneous quantification, providing high levels of detection and specificity.…”