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 h. The entire CHA-LFIA detection method, from nasopharyngeal sampling to obtaining test results, takes less than 90 min. 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 2000 copies/mL) alternative to the SARS-CoV-2 RT-PCR assay, with 100 % positive and negative predictive agreements.

Keywords: Catalytic hairpin assembly reaction; Lateral flow immunoassay strip; Nucleic acid test; Rapid diagnostic test; sars-cov-2.

Fig. 1

Overview of the CHA-LFIA method for SARS-CoV-2 viral RNA detection.

Fig. 2

Schematic of the CHA-LFIA method for SARS-CoV-2 viral RNA detection. (A) The CHA reaction without and with addition of target RNA; (B) The LFIA strip used to detect digoxigenin-biotin double-labeled H1-H2 hybrid duplexes. SA, streptavidin; AF647, Alexa Fluor 647.

Fig. 3

Selected target sequences for specific SARS-CoV-2 detection and their relative positions.

Fig. 4

Thermodynamic analysis of the equilibrium concentrations of H1-H2 hybrid complexes at different temperatures with equal initial concentrations (100 nM) of the H1 probe, H2 probe, and target RNA.

Fig. 5

Analysis of the CHA reaction by native PAGE. Lane 1: H1 probe; lane 2: H2 probe; lane 3: H1 probe and H2 probe; lane 4: annealed H1 and H2; lane 5: H1 probe, H2 probe, and target RNA; lane 6: H1, H2, and non-target RNA. The concentration of all components was 50 nM.

Fig. 6

Optimization of the CHA reaction. (A) Optimization of the H2 concentration (50–200 nM) at a constant H1 concentration (50 nM) in a 2 h reaction at 50 °C; (B) Reaction temperature optimization using a 2 h reaction with 50 nM of H1 and 150 nM of H2; (C) Optimization of the CHA reaction time at 50 °C with 50 nM of H1 and 150 nM of H2; (D) Optimization of the concentrations of H1 and H2 in a 30 min reaction at 50 °C.

Fig. 7

Fluorescence kinetics of the CHA-LFIA method with various concentrations of target RNA in an oropharyngeal swab sample healthy person.

Fig. 8

Sequence specificity analysis of the CHA-LFIA method. The results are the average of three independent trials. SM, single-mismatched target RNA; DM, double-mismatched target RNA.