Comparative Bioinformatic Analysis Reveals Conserved Regions in SARS-CoV-2 Genome for RAPID Pandemic Response

Abstract

In the face of the SARS-CoV-2 pandemic, characterized by the virus's rapid mutation rates, developing timely and targeted therapeutic and diagnostic interventions presents a significant challenge. This study utilizes bioinformatic analyses to pinpoint conserved genomic regions within SARS-CoV-2, offering a strategic advantage in the fight against this and future pathogens. Our approach has enabled the creation of a diagnostic assay that is not only rapid, reliable, and cost-effective but also possesses a remarkable capacity to detect a wide array of current and prospective variants with unmatched precision. The significance of our findings lies in the demonstration that focusing on these conserved genomic sequences can significantly enhance our preparedness for and response to emerging infectious diseases. By providing a blueprint for the development of versatile diagnostic tools and therapeutics, this research paves the way for a more effective global pandemic response strategy.

Keywords: SARS-CoV-2; bioinformatic analysis; conserved genomic regions; diagnostic assay; point-of-care testing; rapid testing.

Figure 1

Five-Step Strategy for Identification and Primer Set Development. This strategy involves a comprehensive process of sequence analysis, verification, and refinement, culminating in the identification of an optimal primer set (SET1) that produces a specific single amplicon. This streamlined approach ensures the accurate detection of conserved regions within SARS-CoV-2. The red line represents the melting point curve indicating a single amplicon.

Figure 2

Locations of known mutations in SARS-CoV-2 protein variants are unique and are not located within the conserved regions targeted by SET1 primers. In silico verification was conducted to confirm the presence and localization of the SET1 primers (highlighted in yellow) within the protein sequences of selected SARS-CoV-2 variants. A single mutation that differs from the original Wuhan SARS-CoV-2 strain is indicated in red.

Figure 3

Isolation of SARS-CoV-2 Nucleic Acid from Saliva and Optimized qPCR Assay protocol. (A) RNA samples were isolated from 500 microliters of saliva from COVID-19-infected individuals or symptomatic patients. These samples were then mixed with an equal volume of lysis buffer (500 microliters, as shown in Figure 3A—step 1) and 1 milliliter of 100% ethanol (EtOH) (step 2). The mixture was inverted, vortexed, and applied to a preprepared glass wool column (step 3), followed by centrifugation at maximum speed, processing five aliquots of 400 microliters each. The flow-through was discarded. The column was then placed in a new 2 mL test tube and washed twice with 500 mL of 75% EtOH (step 4). RNA was eluted in double-distilled water (18 microliters of DDW preheated to 64 °C) and directly used in the one-step qPCR. Steps 1 and 2 are carried out in the same 2 mL tube. The mixture was transferred into a 0.5 mL tube (red tube), which has a hole made using a 21 G needle (3.81 cm), and placed into a new 2 mL tube (black tube) during loading. The elution of RNA was performed into a new 1.5 mL Eppendorf RNase-free tube. The original Promega one-step protocol was adapted to suit the shortened amplicon produced by SET1 primers. This includes the melting point based on the primer sequences and the elongation time based on the length of the predicted amplicon (B) and compared with the original Allplex Seegen protocol (C).

Figure 4

Detection of the Wuhan SARS-CoV-2 in saliva samples from infected patients. Saliva samples (300 or 500 µL) were collected from SARS-CoV-2 patients with Ct lower than 34 for the E gene (C01, D01, F01, G01 = B02), a SARS-CoV-2 patient with Ct higher than 34 or the E gene (A01 = H01 = C02), and control patients who represent normal and symptomatic flu-like patients who were negative with the Seegene test (B01, E01, A02) and incubated with (+PK) or without (-PK) proteinase K. D02 and E02 are positive and negative control (PC and NC), respectively, provided by the Seegen Allplex TM assay. The Seegene gold-standard protocol involves four different primer sets, each targeting specific regions (E, S, and N genes) of the SARS-CoV-2 viral genome, along with a fourth primer set for a spiked positive control sample (HEX). A qPCR assay with no template was used as a negative control (NC) for the qPCR assay. The interpretation column indicates the call by the gold-standard Seegene platform. ‘NA’ stands for ‘not applicable’, indicating no detected CT value. ‘2019-nCoV Detected’ denotes the declaration of a SARS-CoV-2-positive sample. ‘Negative’ indicates a clear-cut negative sample. ‘2019-nCoV Not Detected’ refers to a borderline sample. ‘Positive control’ denotes an internal positive control amplicon sample provided by the manufacturer, while ‘Negative control’ indicates no template.

Figure 5

Optimization of the RAPID one-step qPCR reagents using SET1 primers on human RNA isolated using the gold-standard Seegene protocol. Graph A and Table E—enzyme comparison, Graph B and Table F—new and old plate from Soroka Hospital, Graph C and Table G—primer dilution, Graph D and Table H—testing new primer mixture. The detailed results, representing each graph (A–D), are depicted in (A–D), respectively. ‘PC’ stands for positive control and denotes a plasmid containing the specific SET1 amplicon. Negative control denotes no nucleic acid input. ‘Undetermined’ indicates a clear-cut negative sample reading by the step-one qPCR machine. Table E contains the samples described in Graph A, Table F describes the samples in Graph B, Table G describes the samples in Graph C, and Table H describes the samples in Graph D.

Figure 5

Optimization of the RAPID one-step qPCR reagents using SET1 primers on human RNA isolated using the gold-standard Seegene protocol. Graph A and Table E—enzyme comparison, Graph B and Table F—new and old plate from Soroka Hospital, Graph C and Table G—primer dilution, Graph D and Table H—testing new primer mixture. The detailed results, representing each graph (A–D), are depicted in (A–D), respectively. ‘PC’ stands for positive control and denotes a plasmid containing the specific SET1 amplicon. Negative control denotes no nucleic acid input. ‘Undetermined’ indicates a clear-cut negative sample reading by the step-one qPCR machine. Table E contains the samples described in Graph A, Table F describes the samples in Graph B, Table G describes the samples in Graph C, and Table H describes the samples in Graph D.

Figure 6

PCR Analysis of RNA Samples from Patients with the Delta Variant using the fully optimized RAPID assay protocol. This table shows results from samples isolated using the Seegene gold-standard method, tested with the standard Seegene PCR test in comparison to our RAPID test before (old protocol) optimization parameters (for details, see Figure 5) and using the improved RAPID assay fully optimized protocol.

Figure 7

RNA isolation of the Omicron SARS-CoV-2 variant in combination with the final optimization of the RAPID test is more sensitive than the swab gold-standard Seegene test used at the Israeli airport. Samples 1 and 1’ (green line) are duplicates of the PCR product of SET1 cloned in the TOPO TA plasmid, serving as positive controls. Sample 2 (red line) is the unknown sample tested for infectivity, which was eventually confirmed to be positive for SARS-CoV-2. Sample 3 (light green line) is the SARS-CoV-2 human RNA isolated and detected using the gold standard test, also serving as a positive control. Sample 4 (dark green line) is the negative control sample with no nucleic acid template.