Rapid point-of-care testing for SARS-CoV-2 virus nucleic acid detection by an isothermal and nonenzymatic Signal amplification system coupled with a lateral flow immunoassay strip

Abstract

An outbreak of a new coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), began in December 2019. Accurate, rapid, convenient, and relatively inexpensive diagnostic methods for SARS-CoV-2 infection are important for public health and optimal clinical care. The current gold standard for diagnosing SARS-CoV-2 infection is reverse transcription-polymerase chain reaction (RT-PCR). However, RTPCR assays are designed for use in well-equipped laboratories with sophisticated laboratory infrastructure and highly trained technicians, and are unsuitable for use in under-equipped laboratories and in the field. In this study, we report the development of an accurate, rapid, and easy-to-implement isothermal and nonenzymatic signal amplification system (a catalytic hairpin assembly (CHA) reaction) coupled with a lateral flow immunoassay (LFIA) strip-based detection method that can detect SARSCoV-2 in oropharyngeal swab samples. Our method avoids RNA isolation, PCR amplification, and elaborate result analysis, which typically takes 6–8 hours. The entire CHA-LFIA detection method, from nasopharyngeal sampling to obtaining test results, takes less than 90 minutes. Such methods are simple and require no expensive equipment, only a simple thermostatically controlled water bath and a fluorescence reader device. We validated our method using synthetic oligonucleotides and clinical samples from 15 patients with SARS-CoV-2 infection and 15 healthy individuals. Our detection method provides a fast, simple, and sensitive (with a limit of detection (LoD) of 2,000 copies/mL) alternative to the SARS-CoV-2 RT-PCR assay, with 100% positive and negative predictive agreements.

The accuracy of novel antigen rapid diagnostics for SARS-CoV-2: a living systematic review and meta-analysis

“…After removing duplicates, 8,921 articles were screened, and 266 papers were considered eligible for full-text review. Of these, 148 were excluded because they did not present primary data [13,19–131] or the Ag-RDT was not commercially available [16,132–164], leaving 133 studies to be included in the systematic review (Fig 1) [4,165–296].…”

Facile, Rapid, and Low-Cost Detection for Influenza Viruses and Respiratory Syncytial Virus Based on a Catalytic DNA Assembly Circuit

“…The construction of the test strip was based on our previous study. 34 Briefly, the strip is composed of a sample pad, conjugation pad, nitrocellulose membrane with a test line (T), a control line (C), and an absorbent pad. The conjugation pad was prepared with polyethylene (PE) nanoparticles labeled with streptavidin (SA) and fluorophore Alexa Fluor 647.…”

Recent Progress on Rapid Lateral Flow Assay-Based Early Diagnosis of COVID-19

“…Moreover, qRT-PCR combined with LFAs could simultaneously detect RdRp, ORF3a, and N genes of SARS-CoV-2, with a detection limit of 10 copies per test for each gene ( Yu et al, 2020 ). Furthermore, next-generation sequencing ( Wu et al, 2021 ), a microfluidic-integrated RPA ( Liu D. et al, 2021 ), and a catalytic hairpin assembly enzyme-free signal amplification reaction ( Zou et al, 2021 ) coupled with LFAs techniques have been exploited as POCT for rapid diagnosis.…”