A one-step, one-tube real-time RT-PCR based assay with an automated analysis for detection of SARS-CoV-2

Abstract

Early diagnosis of SARS-CoV-2 infected patients is essential to control the dynamics of the COVID-19 pandemic. We develop a rapid and accurate one-step multiplex TaqMan probe-based real-time RT-PCR assay, along with a computational tool to systematically analyse the data. Our assay could detect to a limit of 15 copies of SARS-CoV-2 transcripts-based on experiments performed by spiking total human RNA with in vitro synthesized viral transcripts. The assay was evaluated by performing 184 validations for the SARS-CoV-2 Nucleocapsid gene and human RNase P as an internal control reference gene with dilutions ranging from 1-100 ng for human RNA on a cohort of 26 clinical samples. 5 of 26 patients were confirmed to be infected with SARS-CoV-2, while 21 tested negative, consistent with the standards. The accuracy of the assay was found to be 100% sensitive and 100% specific based on the 26 clinical samples that need to be further verified using a large number of clinical samples. In summary, we present a rapid, easy to implement real-time PCR based assay with automated analysis using a novel COVID qPCR Analyzer tool with graphical user interface (GUI) to analyze the raw qRT-PCR data in an unbiased manner at a cost of under $3 per reaction and turnaround time of less than 2h, to enable in-house SARS-CoV-2 testing across laboratories.

Keywords: COVID qPCR analyzer; COVID-19 detection assay; Diagnostics; Genomics; Graphical user interface (GUI); Infectious disease; Microbial genomics; Molecular biology; Real-time RT- PCR; SARS-CoV-2; Virology.

Figure 1

Sequence based analysis to identify the conserved regions for specific detection of SARS-CoV-2. Representation of the SARS-CoV-2 genome to the scale (adapted from [32]). The marked locations for N1-209 bp, N2-110 bp and N3-132 bp in the SARS-CoV-2 genome are approximated with respect to the genomic co-ordinates. The maps below represent region from the multiple sequence alignment (MSA) of N1 and N2 target templates with the Nucleocapsid (N) gene and N3 target template with the ORF1ab gene, respectively. MSA is performed between the genes from SARS-CoV-2 reference genome NC_045512 (A), isolates of SARS-CoV-2 from Indian samples https://www.ncbi.nlm.nih.gov/nuccore/MT0504932, https://www.ncbi.nlm.nih.gov/nuccore/MT012098 (B,C); SARS-CoV 2002–2004 NC_004718 (D); MERS NC_019843 (E); human coronavirus HKU1 NC_006577 (F); human coronavirus OC43 NC_006213 (G); human coronavirus 229E NC_002645 (H); human coronavirus NL63 NC_005831 (I); and the respective target regions.

Figure 2

qRT-PCR using SYBR Green I dye based detection approach. Real time PCR to determine the sensitivity and specificity of SARS-CoV-2 detection primers of N1, N2, N3 and C1 (RNase P internal control) using SYBR Green I dye. Series of dilutions of each synthetic DNA fragment (1pg, 10pg, 100pg and 1000pg) were used in the background of (a) MCF-7 cell line cDNA (10ng), (b) tongue squamous cell carcinoma (TSCC) sample genomic DNA (10ng), and (c) RNA synthesized by IVT in the background of MCF7 cell line RNA (50ng), for performing real-time PCR. For each primer, Ct values (in triplicates) in different concentrations of synthetic DNA/RNA fragments are plotted in the graph. Specificity of the primers to amplify single amplicon is represented as single peak in melting curves generated by “melting curve genotype analysis” in Roche Light cycler 480 machine.

Figure 3

qRT-PCR using TaqMan probe-based detection approach. Real time PCR to determine the sensitivity and specificity of TaqMan probes and primers to detect the SARS-CoV-2 sequence N1, N2 and RNase P internal control, C1. For each TaqMan probe, Ct values are plotted against varying concentration of synthetic SARS-CoV-2 DNA and RNA templates in the background of (a) TSCC human genomic DNA (10ng), (b) MCF7 cell line total RNA (50ng) and (c) human MCF7 cell line total RNA and TSCC genomic DNA complexity. Specificity of single amplicon was represented in the respective amplification curve indicating 6-FAM/HEX fluorescence signal emitted by dual labelled probes.

Figure 4

Multiplex TaqMan qRT-PCR to detect SARS-CoV-2. Real time PCR to determine the sensitivity of TaqMan probes to detect the SARS-CoV-2 sequences N1, N2 and C1 in a multiplex qRT-PCR. (a) For each TaqMan probe, Ct values are plotted against varying concentration of synthetic SARS-CoV-2 DNA and RNA templates in the background of (a) TSCC human genomic DNA (10ng), (b) human MCF7 cell line total RNA (50ng) and (c) human MCF7 cell line total RNA and TSCC genomic DNA complexity. Specificity of single amplicon was represented in the respective amplification curve indicating 6-FAM/HEX fluorescence signal emitted by dual labelled probes in multiplex PCR.