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. 2020 Sep 15;117(37):22727-22735.
doi: 10.1073/pnas.2014739117. Epub 2020 Aug 31.

Rapid isothermal amplification and portable detection system for SARS-CoV-2

Affiliations

Rapid isothermal amplification and portable detection system for SARS-CoV-2

Anurup Ganguli et al. Proc Natl Acad Sci U S A. .

Abstract

The COVID-19 pandemic provides an urgent example where a gap exists between availability of state-of-the-art diagnostics and current needs. As assay protocols and primer sequences become widely known, many laboratories perform diagnostic tests using methods such as RT-PCR or reverse transcription loop mediated isothermal amplification (RT-LAMP). Here, we report an RT-LAMP isothermal assay for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus and demonstrate the assay on clinical samples using a simple and accessible point-of-care (POC) instrument. We characterized the assay by dipping swabs into synthetic nasal fluid spiked with the virus, moving the swab to viral transport medium (VTM), and sampling a volume of the VTM to perform the RT-LAMP assay without an RNA extraction kit. The assay has a limit of detection (LOD) of 50 RNA copies per μL in the VTM solution within 30 min. We further demonstrate our assay by detecting SARS-CoV-2 viruses from 20 clinical samples. Finally, we demonstrate a portable and real-time POC device to detect SARS-CoV-2 from VTM samples using an additively manufactured three-dimensional cartridge and a smartphone-based reader. The POC system was tested using 10 clinical samples, and was able to detect SARS-CoV-2 from these clinical samples by distinguishing positive samples from negative samples after 30 min. The POC tests are in complete agreement with RT-PCR controls. This work demonstrates an alternative pathway for SARS-CoV-2 diagnostics that does not require conventional laboratory infrastructure, in settings where diagnosis is required at the point of sample collection.

Keywords: COVID-19 diagnostics; RT-LAMP; SARS-CoV-2; point-of-care; smartphone reader.

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Conflict of interest statement

Competing interest statement: W.P.K. is a cofounder and Chief Scientist at Fast Radius Inc., where the additively manufactured cartridge was produced. B.T.C. serves as a consultant to and has financial interests in Reliant Immune Diagnostics.

Figures

Fig. 1.
Fig. 1.
Validation of three LAMP primer sets for four different SARS-CoV-2 gene targets. (A) Workflow for the detection of SARS-CoV-2 using our portable POC device. (B) SARS-CoV-2 genome outline and four gene targets for primer design. (C) Comparison of positive amplification threshold time for four genes (500 copies per μL, n = 3) using primer set 3 for gene Orf 1a, primer set 2 for gene S, primer set 2 for gene Orf 8, and primer set 1 for gene N. (D) Amplification threshold times (n = 3) for detection of different concentration of genomic RNA using primer set 3 for gene Orf 1a, primer set 2 for gene S, primer set 2 for gene Orf 8, and primer set 1 for gene N. The best detection limit was 50 copies per μL attained using gene N primer set 1. The bar graphs show mean and SD.
Fig. 2.
Fig. 2.
Detection of SARS-CoV-2 virus from mock NP swab samples transported to VTM. (A) Process flow for viral detection from a mock NP swab. A swab is inserted into a tube with virus-spiked nasal fluid and absorbs the fluid. After vigorously mixing the swab in 100 μL or 500 μL of VTM, an aliquot of the VTM sample is thermally lysed at 95 °C for 1 min. The RT-LAMP reagents are added to the lysed viral sample, and the reaction is conducted at 65 °C for 60 min. (B) Amplification threshold times (n = 3) for viral detection in a 16-μL reaction with 12.5% and 50% VTM sample per reaction from a 500-μL VTM sample.
Fig. 3.
Fig. 3.
Detection of SARS-CoV-2 virus from VTM clinical samples. (A) Process flow for viral detection from VTM clinical samples. The sample is collected from the patient using an NP swab. After the sample is transferred to the VTM (step 2), an RT-PCR test is performed, and the results are used as control. The discarded VTM is frozen for transfer and storage. After thaw, aliquots are thermally lysed (step 3b) before the RT-LAMP is conducted (65 °C, 60 min). RT-LAMP pathway does not require of RNA extraction. (BD) Assessment of clinical samples (n = 4). VTM samples from 10 SARS-CoV-2−positive and 10 SARS-CoV-2−negative patients (as judged by RT-PCR control test with RNA extraction) were analyzed using the developed RT-LAMP assay. (B) Undetermined Ct values are plotted at 1. (C and D) Raw fluorescence data (n = 4) for SARS-CoV-2 detection from VTM clinical samples.
Fig. 4.
Fig. 4.
Additively manufactured microfluidic cartridge and handheld POC instrument. (A) Diagram of the microfluidic diagnostic cartridge used for rapid detection of SARS-CoV-2 in VTM. The fluid inlet ports mate with syringes that inject either RT-LAMP reagents or thermally lysed patient sample into a 3D serpentine mixing region before filling the amplification and diagnostic region. (B) Photographs of the disposable microfluidic cartridge. (C) The 3D scans of the microfluidic cartridge and magnified view of the detection pools in the amplification and diagnostic region of the cartridge. (D) Photograph of the instrument used for rapid detection. (E) Schematic of the handheld POC instrument showing components in an exploded view. A smartphone images the cartridge, while isothermal heating and illumination are battery powered. Optical components integrated with the instrument match the excitation and emission characteristics of the fluorescent signal.
Fig. 5.
Fig. 5.
Rapid detection of SARS-CoV-2 in VTM clinical samples using an additively manufactured microfluidic cartridge and handheld POC instrument. (A) Fluorescence intensities of real-time RT-LAMP on the additively manufactured amplification chip at different time points. The developed device can clearly differentiate all of the positives samples from the negatives as fast as in 30 min (n = 6). (B) A t test shows that the fluorescence intensity of the positive samples (P) is statistically significant in comparison with the negative samples (N). (C) ROC curves analyzed for the four time conditions. For each time point, the positive samples were analyzed against the negative samples. (D) Fluorescence images of real-time RT-LAMP SARS-CoV-2 analysis at the endpoint of detection on the additively manufactured amplification chip.

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