Rapid point-of-care detection of SARS-CoV-2 using reverse transcription loop-mediated isothermal amplification (RT-LAMP)

Abstract

Background: Fast, reliable and easy to handle methods are required to facilitate urgently needed point-of-care testing (POCT) in the current coronavirus pandemic. Life-threatening severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread all over the world, infecting more than 33,500,000 people and killing over 1 million of them as of October 2020. Infected individuals without any symptoms might still transfer the virus to others underlining the extraordinary transmissibility of this new coronavirus. In order to identify early infections effectively, treat patients on time and control disease spreading, rapid, accurate and onsite testing methods are urgently required.

Results: Here we report the development of a loop-mediated isothermal amplification (LAMP) based method to detect SARS-CoV-2 genes ORF8 and N directly from pharyngeal swab samples. The established reverse transcription LAMP (RT-LAMP) assay detects SARS-CoV-2 directly from pharyngeal swab samples without previous time-consuming and laborious RNA extraction. The assay is sensitive and highly specific for SARS-CoV-2 detection, showing no cross reactivity when tested on 20 other respiratory pathogens. The assay is 12 times faster and 10 times cheaper than routine reverse transcription real-time polymerase chain reaction, depending on the assay used.

Conclusion: The fast and easy to handle RT-LAMP assay amplifying specifically the genomic regions ORF8 and N of SARS-CoV-2 is ideally suited for POCT at e.g. railway stations, airports or hospitals. Given the current pandemic situation, rapid, cost efficient and onsite methods like the here presented RT-LAMP assay are urgently needed to contain the viral spread.

Keywords: COVID-19; Gene N; No RNA extraction; ORF8; Point-of-care testing; RT-LAMP; Rapid testing; SARS-CoV-2.

Fig. 1

Optimising RT-LAMP temperature. RT-LAMP reactions for ORF 8 (a) and N (b) detection of SARS-CoV-2 were incubated at 63 °C, 65 °C and 67 °C for 35 min. Extracted RNA from SARS-CoV-2 infected Vero cell culture supernatant was tested along with 1/2, 1/4 and 1/16 dilutions in PCR grade water. As negative controls, pooled negatively tested patient RNA (Negative) and water (NTC) were used. All experiments were performed in triplicates three times over. Amplification Plots show representative results of nine measurements per sample

Fig. 2

Optimising pre-treatment of patient’s swab samples for direct usage in RT-LAMP. Positive sample 1–5 and Negative sample 1–5 were pre-heated at 80 °C, 85 °C, 90 °C and 95 °C for 5 min before being directly pipetted into RT-LAMP for SARS-CoV-2 targeting OFR8 (a) and N (b). All experiments were performed three times in duplicates. Amplification Plots show representative results for six measurements per sample

Fig. 3

Suitability of different swab types available for direct SARS-CoV-2 detection in RT-LAMP. Seven of the most commonly used swab types were used to test one SARS-CoV-2 negative person. 10 µL of heat-inactivated virus was added to each swab type. Samples were heated to 90 °C for 5 min before their addition to RT-LAMP reaction detecting ORF8 (a) and N (b). All experiments were performed three times in triplicates. The Amplification plots show representative amplification curves for nine measurements per sample

Fig. 4

Effects of different swab types on RT-qPCR and RT-LAMP results with isolated RNA. Seven of the most commonly used swab types were used to test one SARS-CoV-2 negative person. 10 µL of heat-inactivated virus was added to each swab type. RNA was extracted and purified before being analysed for SARS-CoV-2 in RT-qPCR (Siemens Kit) targeting Orf1a (a) and RT-LAMP targeting ORF8 (b) and N (c)

Fig. 5

Direct detection of SARS-CoV-2 for POCT with the Genie II instrument. Positive samples 1–5 and Negative samples 1–5 were pre-heated at 90 °C for 5 min before being directly pipetted into RT-LAMP for SARS-CoV-2 targeting ORF8 (a) and N (b). All experiments were performed three times. Amplification Plots show representative results for three measurements per sample

Fig. 6

Workflow of the presented RT-LAMP assay testing direct patients’ swab samples for SARS-CoV-2 (MM master mix)