Direct on-the-spot detection of SARS-CoV-2 in patients

Abstract

Humanity is currently experiencing a global pandemic with devastating implications on human health and the economy. Most countries are gradually exiting their lockdown state. We are currently lacking rapid and simple viral detections, especially methods that can be performed in the household. Here, we applied RT-LAMP directly on human clinical swabs and self-collected saliva samples. We adjusted the method to allow simple and rapid viral detection, with no RNA purification steps. By testing our method on over 180 human samples, we determined its sensitivity, and by applying it to other viruses, we determined its specificity. We believe this method has a promising potential to be applied world-wide as a simple and cheap surveillance test for SARS-CoV-2.

Keywords: Covid-19; Molecular; RT-LAMP; SARS-CoV-2; pandemic; surveillance.

Figure 1.

Protocol adjustment and optimal conditions. (a) Schematic representation of the isothermal colorimetric RT-LAMP reaction. (b) RT-LAMP reaction performed on purified RNA from nasal and throat swabs submerged in UTM buffer. Results from a no-template control (NTC; left), a negative subject (Neg S.; middle), and a positive subject (Pos S.; right) at t = 0 (upper panels) and after a 30-min incubation at 65°C (lower panels) are shown. Three technical replicates of each sample are shown. (c) Representative RT-LAMP test results of clinical diagnostic nasal and throat swabs. Three different negative samples (Neg S.; left), and three different positive samples (Pos S.; right) obtained at t = 0 and t = 30 min were directly tested with no RNA purification step. (d) Comparison of the RT-LAMP method to Ct values obtained by standard RT-qPCR (3 true positive and 2 false negative samples of the 99 samples analyzed are not shown due to inaccessibility to their RT-qPCR Ct values). RT-qPCR negative samples were assigned arbitrary Ct values, for visualization. (e) Classification of true positive (TP), true negative (TN), false positive (FP) and false negative (FN) numbers and rate of RT-LAMP test results, as compared to the standard RT-qPCR test results. (f) Clinical diagnostic nasal and throat swabs tested by two different RT-LAMP protocols. Upper panel, without proteinase K and guanidine hydrochloride. Lower panel, with proteinase K and guanidine hydrochloride. For RT-qPCR positive samples, the Ct value is presented under each sample. The sample to the right is negative. (g) RT-LAMP results for samples from patients confirmed as positive for the following viruses, with the fraction tested indicated: 1–2, HSV; swabs (lysed and inactivated as described in the currently developed protocol). 3, HSV; purified DNA. 4, RSV; purified RNA. 5, Influenza B; RNA. 6, Enterovirus; RNA. 7, RNA extraction from a SARS-CoV-2 positive patient. 8, no template control. Results are shown at t = 0 and 30 min after incubation at 65 ͦC.

Figure 2.

Adjusted RT-LAMP protocol used to test 83 clinical diagnostic nasal and throat swab samples. (a) Distribution of TP, TN, FP, and FN readings and rates of RT-LAMP test results, as compared to standard RT-qPCR results. Boxes from left to right represent results at t = 30, 35, and 40 min, respectively. (b) Bar graph representation of TP, TN, FP, and FN rates shown in (a). (c) Comparison of the RT-LAMP method to Ct values obtained with standard RT-qPCR. (d) Graphical representation of TP rates of RT-LAMP after incubations of 30 min, 35 min and 40 min, compared to RT-qPCR test results separated by Ct value intervals. 29

Figure 3.

Employing the RT-LAMP protocol with saliva samples. (a) RT-LAMP tests were performed on saliva from four volunteers. Each tube represents one tested volunteer. Results attained at t = 0 and t = 35 are shown. Upper panel, RT-LAMP reaction using pop7 primers as a positive control. Middle panel, control for RT-LAMP reaction with no primers. Lower panel, RT-LAMP reaction using SARS-CoV-2 gene N primers. The same samples were analyzed with the conventional hospital RT-qPCR protocol. The RT-qPCR results and Ct values are shown under the relevant samples. (b) Graphical illustration of the potential of RT-LAMP protocol for saliva self-testing.