Peptide Nucleic Acid (PNA)-Enhanced Specificity of a Dual-Target Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) Assay for the Detection and Differentiation of SARS-CoV-2 from Related Viruses

Abstract

The threat posed by coronaviruses to human health has necessitated the development of a highly specific and sensitive viral detection method that could differentiate between the currently circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other SARS-related coronaviruses (SARSr-CoVs). In this study, we developed a peptide nucleic acid (PNA)-based real-time quantitative polymerase chain reaction (RT-qPCR) assay targeting the N gene to efficiently discriminate SARS-CoV-2 from other SARSr-CoVs in human clinical samples. Without compromising the sensitivity, this method significantly enhanced the specificity of SARS-CoV-2 detection by 100-fold as compared to conventional RT-qPCR. In addition, we designed an RT-qPCR method for the sensitive and universal detection of ORF3ab-E genes of SARSr-CoV with a limit of detection (LOD) of 3.3 RNA copies per microliter. Thus, the developed assay serves as a confirmative dual-target detection method. Our PNA-mediated dual-target RT-qPCR assay can detect clinical SARS-CoV-2 samples in the range of 18.10–35.19 Ct values with an 82.6–100% detection rate. Furthermore, our assay showed no cross-reactions with other coronaviruses such as human coronaviruses (229E, NL63, and OC43) and Middle East respiratory syndrome coronavirus, influenza viruses (Type B, H1N1, H3N2, HPAI H5Nx, and H7N9), and other respiratory disease-causing viruses (MPV, RSV A, RSV B, PIV, AdV, and HRV). We, thus, developed a PNA-based RT-qPCR assay that differentiates emerging pathogens such as SARS-CoV-2 from closely related viruses such as SARSr-CoV and allows diagnosis of infections related to already identified or new coronavirus strains.

Development, evaluation of the PNA RT-LAMP assay for rapid molecular detection of SARS-CoV-2

“…To overcome those issues, we applied dual-labeled PNA as a detection probe in LAMP reaction for fluorescence detection of the amplification product in a real-time manner. PNA has been reported that having superior specificity against its template nucleic acid with its neutrally charged peptide backbone nature which does not have a nonspecific binding affinity with minus charged natural phosphate backbone of the template nucleic acid 22,32 . The un-cleavable peptide backbone also reduces the risk of non-specific signal production as a result of thermal degradation during long-term incubation at elevated temperatures.…”

Emerging Biosensing Technologies for the Diagnostics of Viral Infectious Diseases

“…A unique application incorporating PNAs has recently utilized the hybridization capabilities of the synthesized PNAs as a blocking mechanism to measure human SARS‐CoV‐2 specific gene sequences more sensitively, as compared to bat and other SARS‐related coronaviruses through the conserved sequences of ORF3ab E‐and N‐genes. [ 148 ] This blocking technique aimed to discriminate between SARS‐CoV‐2 and other SARS related‐CoVs in human samples without compromising sensitivity. The sensitivities were significantly increased in both detections when the designed PNAs probe was combined with an RT‐qPCR workflow.…”

“…Lately, many efforts have been directed towards understanding their potential in nucleic acid‐based diagnostics. [ 148,156–158 ]…”

Label free electrochemical DNA biosensor for COVID-19 diagnosis

“…The rigid D-prolyl-2-aminocyclopentanecarboxylic acid (ACPC) backbone [ 21 ] of acpcPNA leads to the stronger binding affinity and sequence specificity toward DNA compared to many systems including the original PNA [ 22 ]. Given the benefits of PNA probe over conventional oligonucleotide probes, it has been used as a probe for RT-LAMP [ 23 ] and RT-PCR [ 24 ] with colorimetric assay for highly specific determination of SARS-CoV-2.…”