SARS-CoV-2 detection by a clinical diagnostic RT-LAMP assay

Abstract

The ongoing pandemic of SARS-CoV-2 calls for rapid and cost-effective methods to accurately identify infected individuals. The vast majority of patient samples is assessed for viral RNA presence by RT-qPCR. Our biomedical research institute, in collaboration between partner hospitals and an accredited clinical diagnostic laboratory, established a diagnostic testing pipeline that has reported on more than 252,000 RT-qPCR results since its commencement at the beginning of April 2020. However, due to ongoing demand and competition for critical resources, alternative testing strategies were sought. In this work, we present a clinically-validated procedure for high-throughput SARS-CoV-2 detection by RT-LAMP that is robust, reliable, repeatable, specific, and inexpensive.

Keywords: RT-LAMP; SARS-CoV-2; clinical diagnostic.

Figure 1.. Validation of SARS-CoV-2 detection by RT-LAMP targeting the N gene

( A) The N gene of SARS-CoV-2 was targeted using primers designed by Zhang et al. , whilst the internal control implemented utilised primers for 18S rRNA initially published by Lamb et al. . ( B) Upon DNA amplification, a colorimetric pH dye in the reaction mix will change from pink to yellow if the target is present. Four SARS-CoV-2 positive and four SARS-CoV-2 negative patient samples are shown (side view of PCR tubes). ( C–D) Amplification curves from 24 patient samples assessed by SARS-CoV-2 (N gene) RT-LAMP ( C) or 18S ( D) in a Real-Time PCR machine. ( E) Dot plot of the SARS-CoV-2 Ct values from ( C–D) compared to Ct values derived from the Crick’s RT-qPCR diagnostic pipeline using the CE marked BGI kit. Ct values of ‘undetermined’ are plotted as “Ct = 40” (left y-axis) or “Ct = 25” (right y-axis) for illustrative purposes. Data are normalised by assay run/cycles determined by the two methods for comparative purposes. The clinical call from the reference laboratory is indicated above the graph.

Figure 2.. Optimisation of the RT-LAMP assay for accurate detection.

( A–B) 7 samples of water and RNA elution buffer from the CCC pipeline were run 4 independent times by 4 distinct operators by SARS-CoV-2. ( A) or 18S ( B) RT-LAMP. ( C–D) RT-LAMP amplification data from 47 samples of RNA elution buffer using the newly established assay endpoint of 25 min for SARS-CoV-2 ( C) and 20 minutes for 18S ( D). ( E) Dissociation curves are shown for 11 SARS-CoV-2 positive and 11 negative clinical samples along with 12 NTC wells tested by RT-LAMP N gene assay (top) and 18S assay (bottom). ( F) Dot plot of the melting temperatures determined by 94 positive patient samples and 235 negative patient samples tested by RT-LAMP assay. Data are pooled from six separate experiments performed on 3 separate RT-PCR machines. The average melting temperature (Tm) is depicted by a dotted line in ( E) and below the graph in ( F) with the standard of deviation.

Figure 3.. SARS-CoV-2 RT-LAMP is highly specific.

( A) SARS-CoV-2 RT-LAMP was performed on 95 wells of human cell line RNA extracted by the CCC pipeline. ( B) RT-LAMP targeting SARS-CoV-2 (left) or 18S (right) was performed on COVID-19 negative patient samples with positively identified with other viral infections, including human coronaviruses (HCoV), influenzas (Flu), respiratory syncytial virus (RSV), parainfluenzavirus (PIV), adenovirus (Adeno), metapneumovirus (MPV), rhinovirus (Rhino), and human enterovirus (H. Entero). The colorimetric read-out from the actual run is depicted below (wells are visualised from bottom of the plate). NTC (-) and positive control (+). An asterisk denotes a sample that displayed late stage amplification and colour conversion during the 18S analysis.

Figure 4.. SARS-CoV-2 RT-LAMP is highly sensitive, robust, and precise.

( A–B) NIBSC SARS-CoV-2 standard was serially diluted and the indicated number of copies in 4.5 µL was assessed by N gene RT-LAMP. Amplification curves shown with the limit of detection (L.O.D.) determined by the presence or absence of amplification following the depicted dilution in ( A) or via colorimetric read-out ( B). The wells have been visualised from the bottom of the plate. ( C) RNA extracted from laboratory grown SARS-CoV-2 was serially diluted 10-fold and assessed by RT-LAMP in the presence or absence of 1% Triton-X 100. ( D) A COVID-19 positive patient sample was RNA extracted independently 5 times through the CCC pipeline and subjected to 5 independent N gene RT-LAMP reactions. Assay precision for N gene and 18S was determined by calculating the coefficient of variation between Ct values observed (CV).

Figure 5.. Clinical validation of SARS-CoV-2 RT-LAMP.

( A) 37 patient samples were processed in parallel by HSL and the CCC pipeline and interrogated by HSL’s RT-qPCR (N gene) and the CCC’s BGI RT-qPCR (ORF1a) in duplicate. RNA left from the CCC pipeline was assessed in two separate experiments by N gene RT-LAMP. The graph indicates Ct values for HSL and CCC RT-qPCR runs by left y-axis and ‘Ct’ time thresholds for RT-LAMP via the right y-axis. Data are normalised by assay run/cycles determined by the two methods for comparative purposes. The clinical call from the reference laboratory is depicted above the summary table provided below. Positives (P) with low Ct values were reliably detected by RT-LAMP, whilst ‘borderline positive’ (samples with Ct values near the limit of detection of each RT-qPCR assay) were inconsistently detected, displaying no visible amplification at times – negative (N). ( B) 71 VTM samples from HSL were RNA-extracted through the CCC pipeline and interrogated by HSL RT-qPCR (45 cycles), or by two independent N gene RT-LAMP experiments (25 minutes). ( A–B) RT-LAMP assays were performed with 3 µL of sample RNA.