Impact of SARS-CoV-2 Mutations on PCR Assay Sequence Alignment

Abstract

Real-time reverse transcription polymerase chain reaction (RT-PCR) assays are the most widely used molecular tests for the detection of SARS-CoV-2 and diagnosis of COVID-19 in clinical samples. PCR assays target unique genomic RNA regions to identify SARS-CoV-2 with high sensitivity and specificity. In general, assay development incorporates the whole genome sequences available at design time to be inclusive of all target species and exclusive of near neighbors. However, rapid accumulation of mutations in viral genomes during sustained growth in the population can result in signature erosion and assay failures, creating situational blind spots during a pandemic. In this study, we analyzed the signatures of 43 PCR assays distributed across the genome against over 1.6 million SARS-CoV-2 sequences. We present evidence of significant signature erosion emerging in just two assays due to mutations, while adequate sequence identity was preserved in the other 41 assays. Failure of more than one assay against a given variant sequence was rare and mostly occurred in the two assays noted to have signature erosion. Assays tended to be designed in regions with statistically higher mutations rates. in silico analyses over time can provide insights into mutation trends and alert users to the emergence of novel variants that are present in the population at low proportions before they become dominant. Such routine assessment can also potentially highlight false negatives in test samples that may be indicative of mutations having functional consequences in the form of vaccine and therapeutic failures. This study highlights the importance of whole genome sequencing and expanded real-time monitoring of diagnostic PCR assays during a pandemic.

Clade-Specific MPXV PCR Assays

“…As demonstrated in previous studies, clade-specific detection and resistance to false negatives due to primer sequence drift are critical to understanding the dimensions of an outbreak and coordinating a robust and effective public health response (26). The combination of algorithms in BioLaboro creates specific primer candidates and reliably assesses their performance in silico prior to in vitro testing which significantly contributes to a rapid diagnostic and public health response.…”

“…Previous work using the BioLaboro pipeline focused primarily on the PSET component to track predicted PCR assay performance over time and debug associated design issues. For example, a previous SARS-CoV-2 study demonstrated the sudden increase in failure rate of several assays related to deletions in the S gene as the virus evolved (26). This motivated the development of a new feature for PSET, which was the inclusion of 5'/3'-sequence context in the primer sequence query to increase local alignment quality and detect additional, potentially deleterious mutations outside of the binding target.…”

Loop-Mediated Isothermal Amplification Assays for the Detection of Antimicrobial Resistance Elements inVibrio cholerae

“…The PCR Signature Erosion Tool (PSET) provides an in silico method for determining primer binding sites relative to taxonomically related sequences and detects mutations in primers used for PCR or qPCR assays (20,21). For this study, we created LAMP assays for V. cholerae detection, beginning with improvements to PSET, and culminating with a robust, quantitative determination of the assay outcome using computer vision.…”

“…The new LAMP workflow module for PSET can generate assays or predict performance with respect to large reference sequence databases, such as the NCBI nucleotide (nt) database. Running the tool periodically can identify potential assay failures as newly evolved sequences are deposited into databases such as GenBank or GISAID (20). Additionally, we developed a LAMP assay design tool based on the open source Primer3 program as an alternative to web-based programs, leveraging multicore architecture.…”