Detection of SARS-CoV-2 in different human biofluids using the loop-mediated isothermal amplification assay: A prospective diagnostic study in Fortaleza, Brazil

Abstract

We adopted the reverse-transcriptase-loop-mediated isothermal amplification (RT-LAMP) to detect severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) in patient samples. Two primer sets for genes N and Orf1ab were designed to detect SARS-CoV-2, and one primer set was designed to detect the human gene Actin. We collected prospective 138 nasopharyngeal swabs, 70 oropharyngeal swabs, 69 salivae, and 68 mouth saline wash samples from patients suspected to have severe acute respiratory syndrome (SARS) caused by SARS-CoV-2 to test the RT-LAMP in comparison with the gold standard technique reverse-transcription quantitative polymerase chain reaction (RT-qPCR). The accuracy of diagnosis using both primers, N5 and Orf9, was evaluated. Sensitivity and specificity for diagnosis were 96% (95% confidence interval [CI]: 87-99) and 85% (95% CI: 76-91) in 138 samples, respectively. Accurate diagnosis results were obtained only in nasopharyngeal swabs processed via extraction kit. Accurate and rapid diagnosis could aid coronavirus disease 2019 (COVID-19) pandemic management by identifying, isolating, and treating patients rapidly.

Keywords: RT-LAMP; coronavirus; molecular diagnosis.

Figure 1

Reaction specificity and detection limit tests. (A) Representative results of the specificity of the RT‐LAMP reaction using the N5 primer set and control plasmids for the human HRP30 gene, the N gene of SARS‐Cov‐2, and the Orf1ab gene of SARS‐CoV‐2. (B) Representative results of the detection limit of the RT‐LAMP reaction using the N5 primer set and the control plasmid for the SARS‐CoV‐2 N gene. (C) Representative results of the specificity of the RT‐LAMP reaction using the primer set Orf9 and the control plasmids for the human HRP30 gene, the SARS‐Cov‐2 Orf1ab gene and the N gene of SARS‐CoV‐2. (D) Representative results of the detection limit of the RT‐LAMP reaction of the Orf9 primer set and the control plasmid for the SARS‐CoV‐2 Orf1ab gene. (E) Representative results of the specificity of RT‐LAMP reaction using primer set Actin158 and positive control for human Actin gene and control plasmids for SARS‐Cov‐2 gene N and SARS‐CoV‐2 gene Orf1ab. Negative Control, Neg Ctrl; X, empty well. (F) Representative results of specificity of the RT‐LAMP reaction with viral samples cultured in vitro using primer sets N5 and Orf9. RT‐LAMP, reverse‐transcriptase‐loop‐mediated isothermal amplification; SARS‐CoV‐2, severe acute respiratory syndrome coronavirus 2.

Figure 2

Primer sets in silico and in vitro validation. (A) Nucleotide substitution rate of the Orf9 primer set was obtained from the alignment analysis of 3844 Brazilian SARS‐CoV‐2 sequences. (B) Nucleotide substitution rate of the N5 primer set obtained from the alignment analysis of 3844 Brazilian SARS‐CoV‐2 sequences. (C) Consensus sequence of SARS‐CoV‐2 P1 variant at the N5‐LB sequence. (D) Representative limit of detection results of the RT‐LAMP reaction using the N5 primer set in samples of SARS‐CoV‐2, Variant P1, and variant P2 cultured in vitro. (E) Representative results of the detection limit of the RT‐LAMP reaction using the Orf9 primer set in samples of SARS‐CoV‐2, Variant P1, and Variant P2 cultured in vitro. RT‐LAMP, reverse‐transcriptase‐loop‐mediated isothermal amplification; SARS‐CoV‐2, severe acute respiratory syndrome coronavirus 2.

Figure 3

Alternative RNA isolation methodologies for the detection of SARS‐CoV‐2 via RT‐qPCR and RT‐LAMP. (A) Violin plot of CT values obtained from RT‐qPCR of nasopharyngeal swab samples whose RNA was extracted via RNA extraction kit or extracted via the boiling method using primers for the detection of the Orf1ab gene of SARS‐CoV‐2. (B) Box plot of CT values obtained from RT‐qPCR of nasopharyngeal swab samples whose RNA was extracted via RNA extraction kit or extracted via the boiling method using primers for the detection of the N gene of SARS‐CoV‐2. (C) Mean of viral load obtained from RT‐qPCR of nasopharyngeal swab samples extracted via RNA extraction kit or extracted via the boiling method using primers for the detection of the Orf1ab gene of SARS‐CoV‐2. (D) Mean of viral load obtained from RT‐qPCR of nasopharyngeal swab samples whose RNA was extracted via RNA extraction kit or extracted via the boiling method using primers for the detection of the N gene of SARS‐CoV‐2. (E) Box plot of CT values obtained from RT‐qPCR of Saliva, Saline mouth wash, Oropharyngeal swab, and nasopharyngeal swab samples whose RNA was extracted via RNA extraction kit using primers for the detection of the Orf1ab gene of SARS‐CoV‐2. (F) Mean of viral load obtained from RT‐qPCR of Saliva, Saline mouth wash, Oropharyngeal swab, and nasopharyngeal swab samples whose RNA was extracted via RNA extraction kit using primers for the detection of the Orf1ab gene of SARS‐CoV‐2.  CT, cycle threshold; RT‐LAMP, reverse‐transcriptase‐loop‐mediated isothermal amplification; RT‐qPCR, reverse‐transcription quantitative polymerase chain reaction; SARS‐CoV‐2, severe acute respiratory syndrome coronavirus 2.

Figure 4

Representative results of SARS‐CoV‐2 diagnosis via RT‐qPCR and RT‐LAMP from 71 human RNA samples extracted from nasopharyngeal swabs via an automated magnetic bead extraction kit. (A–C) Representative RT‐LAMP results from 24 human nasopharyngeal swabs whose RNA samples were extracted via an automated magnetic bead extraction kit. (D) RT‐qPCR CT values and viral load using Orf1ab SARS‐CoV‐2 primers plotted against RT‐LAMP diagnostic results using Orf9 primer set of nasopharyngeal swabs samples whose RNA samples were extracted via automated magnetic bead extraction kit. (D') Range of RT‐qPCR CT values with a median of all 71 nasopharyngeal swabs. (E) RT‐qPCR CT values and viral load using Orf1ab SARS‐CoV‐2 primers plotted against RT‐LAMP diagnostic results using Orf9 primer set of nasopharyngeal swabs samples whose RNA samples were extracted via automated magnetic bead extraction kit; the X‐axis indicates the maximum CT value of 35 for detection limit evaluation. (F) RT‐qPCR CT values and viral load using N SARS‐CoV‐2 primers plotted against RT‐LAMP diagnostic results using N5 primer set of nasopharyngeal swabs samples whose RNA samples were extracted via automated magnetic bead extraction kit. (F') Range of RT‐qPCR CT values with a median of all 71 nasopharyngeal swabs. (G) RT‐qPCR CT values and viral load using N SARS‐CoV‐2 primers plotted against RT‐LAMP diagnostic results using N5 primer set of nasopharyngeal swabs samples whose RNA samples were extracted via automated magnetic bead extraction kit; the X‐axis indicates the maximum CT value of 35 for detection limit evaluation. ** RT‐LAMP results were given a number from 0 to 2 according to the color of the reaction results. The pink color reactions were assigned the number 0, the orange color reactions were assigned the number 1, and the yellow color reactions were assigned the number 2. Each number and color are shown on the X‐axis on the right. CT, cycle threshold; RT‐LAMP, reverse‐transcriptase‐loop‐mediated isothermal amplification; RT‐qPCR, reverse‐transcription quantitative polymerase chain reaction; SARS‐CoV‐2,  severe acute respiratory syndrome coronavirus 2.

Figure 5

Representative diagnostic results for detection of SARS‐CoV‐2 using the RT‐LAMP kit developed. SARS‐CoV‐2, severe acute respiratory syndrome coronavirus 2.