Lateral Flow Biosensor for On-Site Multiplex Detection of Viruses Based on One-Step Reverse Transcription and Strand Displacement Amplification

Abstract

Respiratory pathogens pose a huge threat to public health, especially the highly mutant RNA viruses. Therefore, reliable, on-site, rapid diagnosis of such pathogens is an urgent need. Traditional assays such as nucleic acid amplification tests (NAATs) have good sensitivity and specificity, but these assays require complex sample pre-treatment and a long test time. Herein, we present an on-site biosensor for rapid and multiplex detection of RNA pathogens. Samples with viruses are first lysed in a lysis buffer containing carrier RNA to release the target RNAs. Then, the lysate is used for amplification by one-step reverse transcription and single-direction isothermal strand displacement amplification (SDA). The yield single-strand DNAs (ssDNAs) are visually detected by a lateral flow biosensor. With a secondary signal amplification system, as low as 20 copies/μL of virus can be detected in this study. This assay avoids the process of nucleic acid purification, making it equipment-independent and easier to operate, so it is more suitable for on-site molecular diagnostic applications.

Keywords: AuNPs; NAATs; RT-SDA; lateral flow biosensor; on-site detection.

Figure 1

Principle and workflow of this detection. (A) Specimen was lysed by detergent in the lysis buffer. The yield buffer was directly transferred to the enzyme mixture for SDA amplification. (B) One inverted cup of the amplified product was loaded on the CP1, and 3 drops of chasing buffer were loaded on the SP2 for visual detection. (C) All the materials used in this assay. The lower part of the photo, from left to right are swab, lysis buffer, dropper, inverted cup, enzyme mixture, strip and chasing buffer.

Figure 2

Demonstrates the relationship between different oligonucleotides using influenza A virus detection as an example.

Figure 3

Typical images of the biosensor for viruses detection with different conditions. The capital letter IVA, IVB and S represent influenza virus A-positive samples, influenza virus B-positive samples and SARS-CoV-2-positive samples, respectively. “All” represents the existence of all three kinds of viruses. “No” represents the absence of all three kinds of viruses.

Figure 4

(A) Optimization of the biosensor with different ratio of specific AU probes and EN-prob1. (B) The signal of T-Line of different concentrations of A-prod. (C) Typical images of the biosensor with or without A-SDA primer.

Figure 5

Sensitivity and specificity of the biosensor. Sensitivity for influenza A (A), sensitivity for influenza B (B) and sensitivity for SARS-CoV-2 (C). (D) Typical images of biosensor for different viruses. From left to right are influenza A virus (IVA), influenza B virus (IVB), SARS-CoV-2, respiratory syncytial virus (RSV), mycoplasma pneumoniae (MP), chlamydia pneumoniae (MP), Escherichia coli (E. coli) and yeast.

Figure 6

(A1) Typical images of the biosensor for virus-spiked samples detection. T1-line was specific for influenza A. T2-line was specific for influenza B. T3-line was specific for SARS-CoV-2. Results of inactivated virus diluted in lysis buffer were marked as “B”. Results of inactivated virus diluted in matrix (spiked sample) were marked as “M”. (A2) RT-PCR confirmation of influenza A virus in lysis buffer and spiked samples. 1 & 1′. Carrier RNA, 2. Negative control for PCR, 3 & 3′. Saline, 4 & 4′. 2 × 10¹ copy/μL influenza A virus. 5 & 5′. 1 × 10² copy/μL influenza A virus, 6 & 6′. 5 × 10² copy/μL and 2.5 × 10³ copy/μL influenza A virus, 8. Positive control for PCR. Symbol “ ‘ ” represents that the sample has been treated by RNase A for 30 min. Results of inactivated virus diluted in lysis buffer were marked as ‘B’. Results of inactivated virus diluted in matrix (spiked sample) were marked as “M”.

Figure 7

Typical images of the biosensor for the detection of influenza A virus with different lysis time. The concentration of the target RNA for this experiment was 2.5 × 10³ copy/μL.

Figure 8

Typical images of the biosensor for the detection of influenza A virus with different enzyme mixture. From left to right are freshly made enzyme mixture, freeze-dried amplification tube (45 °C for 4 days) and the enzyme mixture used for freeze-drying (45 °C for 4 days). The concentration of the target RNA used in this experiment was 5 × 10² copy/μL.