Rapid and visual detection of 2019 novel coronavirus (SARS-CoV-2) by a reverse transcription loop-mediated isothermal amplification assay

Abstract

Objective: To evaluate a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and compare it with RT-PCR.

Methods: We designed primers specific to the orf1ab and S genes of SARS-CoV-2. Total viral RNA was extracted using the QIAamp Viral RNA Mini Kit. We optimized the RT-LAMP assay, and evaluated it for its sensitivity and specificity of detection using real-time turbidity monitoring and visual observation.

Results: The primer sets orf1ab-4 and S-123 amplified the genes in the shortest times, the mean (±SD) times were 18 ± 1.32 min and 20 ± 1.80 min, respectively, and 63°C was the optimum reaction temperature. The sensitivities were 2 × 10¹ copies and 2 × 10² copies per reaction with primer sets orf1ab-4 and S-123, respectively. This assay showed no cross-reactivity with 60 other respiratory pathogens. To describe the availability of this method in clinical diagnosis, we collected 130 specimens from patients with clinically suspected SARS-CoV-2 infection. Among them, 58 were confirmed to be positive and 72 were negative by RT-LAMP. The sensitivity was 100% (95% CI 92.3%-100%), specificity 100% (95% CI 93.7%-100%). This assay detected SARS-CoV-2 in a mean (±SD) time of 26.28 ± 4.48 min and the results can be identified with visual observation.

Conclusion: These results demonstrate that we developed a rapid, simple, specific and sensitive RT-LAMP assay for SARS-CoV-2 detection among clinical samples. It will be a powerful tool for SARS-CoV-2 identification, and for monitoring suspected patients, close contacts and high-risk groups.

Keywords: COVID-19; Detection; RT-LAMP; SARS-CoV-2; Visual.

Fig. 1

The most appropriate primers and reaction temperature for the reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay. (A) Most suitable primer set for the RT-LAMP assay to amplify the orf1ab gene. (B) Optimal reaction temperature for the RT-LAMP assay with primer orf1ab-4. (C) Most suitable primer set for the RT-LAMP assay to amplify the S gene. (D) Optimal reaction temperature for the RT-LAMP assay with primer S-123. The reaction volume was 25 μL contained 2 μL RNA template, and the template concentration was 1 ng/μL.

Fig. 2

Sensitivity of the reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay and conventional PCR for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection. (A,B) Sensitivity of the RT-LAMP assay using primer set orf1ab-4 to target the orf1ab gene for SARS-CoV-2 detection. (D,E) Sensitivity of the RT-LAMP assay using primer set S-123 to target the S gene for SARS-CoV-2 detection. (C,F) Sensitivity of the conventional PCR assay targeting the orf1ab and S genes for SARS-CoV-2 detection. The detection was monitored by turbidity using a Loopamp real-time turbidimeter, and was judged by the naked eye depending on a colour change from orange to green. (B), Lanes 1–9: 10⁸, 10⁷, 10⁶, 10⁵, 10⁴, 10³, 10², 10¹ and 10⁰ copies/μL; (E) lanes 1–8: 10⁷, 10⁶, 10⁵, 10⁴, 10³, 10², 10¹ and 10⁰ copies/μL; (C) and (F) lanes 1–8: 10⁸, 10⁷, 10⁶, 10⁵, 10⁴, 10³, 10² and 10¹ copies/μL. In the sensitivity test, 60 min can be used as the cut-off for the visual detection.

Fig. 3

Specificity of the reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection. (A,B) Specificity of the RT-LAMP assay with primer set orf1ab-4 targeted the orf1ab gene for SARS-CoV-2 detection. (C,D) Specificity of the RT-LAMP assay with primer set S-123 targeted the S gene for SARS-CoV-2 detection. The detection was monitored by turbidity using a Loopamp real-time turbidimeter (A, C), and was judged by the naked eye depending on a colour change from orange to green (B, D). S1: human coronavirus (HCoV) -229E-1; S2: HCoV-229E-2; S3: HCoV-NL63-1; S4: HCoV-NL63-2; S5: HCoV-NL63-3; S6: HCoV-OC43-1; S7: HCoV-OC43-2; S8: HCoV-HKU1-1; S9: HCoV-HKU1-2; S10: H3N2-1; S11: H3N2-2; S12: H3N2-3; S13: H3N2-4; S14: H1N1-1; S15: H1N1-2; S16: influenza B-1; S17: influenza B-2; S18: influenza B-3; S19: parainfluenza (PIV) -1-1; S20: PIV-1-2; S21: PIV-2-1; S22: PIV-2-2; S23: PIV-3-1; S24: PIV-3-2; S25: PIV-4-1; S26: PIV-4-2; S27: adenovirus (ADV) -1-1; S28: ADV-1-2; S29: ADV-2-1; S30: ADV-2-2; S31: ADV-3-1; S32: ADV-3-2; S33: ADV-4; S34: ADV-5-1; S35: ADV-5-2; S36: ADV-6; S37: ADV-7-1; S38: ADV-7-2; S39: respiratory syncytial virus (RSV) A-1; S40: RSV A-2; S41: RSV B-1; S42: RSV B-2; S43: human metapneumovirus HMPV-1; S44: HMPV-2; S45: human bocavirus (BoV) -1; S46: BoV-2; S47: rhinovirus (Rh) A-1; S48: Rh A-2; S49: Rh B-1; S50: Rh B-2; S51: Rh C; S52: Mycoplasma pneumoniae (MP) -FH; S53: MP-M129; S54: Haemophilus influenzae; S55: Staphylococcus aureus; S56: Klebsiella pneumoniae; S57: Streptococcus pneumoniae; S58: Pseudomonas aeruginosa; S59: SARS-CoV; S60: Middle East respiratory syndrome-CoV; PC, positive control (pseudo-virus); NC, negative control (distilled water).

figs1

Sequence comparison and gene analysis of the orf1b and S genes.