Nucleocapsid (N) Gene Mutations of SARS-CoV-2 Can Affect Real-Time RT-PCR Diagnostic and Impact False-Negative Results

Abstract

The current COVID-19 pandemic demands massive testing by Real-time RT-PCR (Reverse Transcription Polymerase Chain Reaction), which is considered the gold standard diagnostic test for the detection of the SARS-CoV-2 virus. However, the virus continues to evolve with mutations that lead to phenotypic alterations as higher transmissibility, pathogenicity or vaccine evasion. Another big issue are mutations in the annealing sites of primers and probes of RT-PCR diagnostic kits leading to false-negative results. Therefore, here we identify mutations in the N (Nucleocapsid) gene that affects the use of the GeneFinder COVID-19 Plus RealAmp Kit. We sequenced SARS-CoV-2 genomes from 17 positive samples with no N gene detection but with RDRP (RNA-dependent RNA polymerase) and E (Envelope) genes detection, and observed a set of three different mutations affecting the N detection: a deletion of 18 nucleotides (Del28877-28894), a substitution of GGG to AAC (28881-28883) and a frameshift mutation caused by deletion (Del28877-28878). The last one cause a deletion of six AAs (amino acids) located in the central intrinsic disorder region at protein level. We also found this mutation in 99 of the 14,346 sequenced samples by the Sao Paulo state Network for Pandemic Alert of Emerging SARS-CoV-2 variants, demonstrating the circulation of the mutation in Sao Paulo, Brazil. Continuous monitoring and characterization of mutations affecting the annealing sites of primers and probes by genomic surveillance programs are necessary to maintain the effectiveness of the diagnosis of COVID-19.

Detection of SARS-CoV-2 Omicron variant (B.1.1.529) infection of white-tailed deer

“…Notably, the N dropout was only observed with the TaqPath assay and not in the OPTI Medical assay, which targets two separate regions in the N gene. N gene dropouts in RT-PCR assays have been reported with earlier SARS-CoV-2 VoCs 43,44 and may therefore have the potential to not only affect assay performance but also might lead to false-negative results. Hence, further studies on the performance of current widely used RT-PCR assay are urgently warranted, since the occurrence of both S and N dropouts or amplification failures may compromise detection of continually emerging variants that are increasingly being observed in clinical samples.…”

“…VoCs 43,44 and may therefore have the potential to not only affect assay performance but also might lead to false-negative results. Hence, further studies on the performance of current widely used RT-PCR assay are urgently warranted, since the occurrence of both S and N dropouts or amplification failures may compromise detection of continually emerging variants that are increasingly being observed in clinical samples.…”

Molecular detection of omicron SARS-CoV-2 variant is achieved by RT-LAMP despite genomic mutations

“…(2,12,13) Lesbon et al (2) reported that high viral load samples (Ct < 33) could be detected when targeting E and RdRp genes but failed when considering N gene as target, even the latter being considered the most sensitive region for RT-qPCR. (2) In addition, the mutation C26340U at E gene was associated with a failure on detecting SARS-CoV-2 by RT-qPCR, (13) compromising RT-qPCR-based SARS-CoV-2 detection kits such as GeneFinder TM CO-VID-19 Plus RealAmp Kit and cobas ® SARS-CoV-2 test. (2,12,13) These evidences lead us to wonder if omicron genomic polymorphisms would affect molecular detection by RT-LAMP.…”

“…(1) Omicron has mutations in genomic coding regions to structural (envelope, nucleocapsid, membrane, spike) and nonstructural proteins that can impair Coronavirus disease (COVID-19) molecular diagnosis based on reverse transcription quantitative polymerase chain reaction (RT-qPCR) tests, leading to misdiagnosis and allowing virus spreading through false negative results. (1,2,3) As an alternative to RT-qPCR, reverse transcription loop-mediated isothermal amplification (RT-LAMP) has been used with success to detect SARS-CoV-2 RNA from nasopharyngeal swabs and saliva samples. (4) During RT-LAMP reaction, the amplification of genetic material occurs at constant temperature (65ºC) without the need for sophisticated thermal cyclers as in RT-qPCR.…”

Improving the specificity of nucleic acid detection with endonuclease-actuated degradation

“…Future directions include extension to other formats such as portable diagnostic devices 35 and further optimization of the protocol for patient-derived samples to achieve clinical-grade diagnostic testing for COVID-19 using SENTINEL. The emergence of new COVID-19 variants may present new challenges to nucleic acid detection with respect to increased false negatives 36 , but SENTINEL is poised to address these challenges by leveraging capacity for single nucleotide discrimination to distinguish between different virus variants.…”