A Novel Point Mutation in the N Gene of SARS-CoV-2 May Affect the Detection of the Virus by Reverse Transcription-Quantitative PCR.
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ABSTRACT:
SUBMITTER: Hasan MR
PROVIDER: S-EPMC8092750 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundCycle threshold (Ct) values from SARS-CoV-2 reverse transcription quantitative PCR (RT-qPCR) tests are used to measure viral burden. Calibration to the First WHO International Standard for SARS-CoV-2 RNA may improve quantitative inter-assay agreement.MethodsWHO standard was tested using four emergency use authorized RT-qPCRs to generate calibration curves and evaluate Ct value differences. Harmonization of two assays, Cepheid Xpert Xpress SARS-CoV-2 targeting E and nucleocapsid (N2) [Xpert (E) and Xpert (N2)] and a laboratory-developed test targeting E [LDT (E)], was assessed using 93 positive upper respiratory samples. Platform (target) pairs were compared via Bland-Altman analysis and Passing-Bablok regression.ResultsCt values with the WHO standard were comparable across platforms and targets, except Xpert (N2) for which the mean difference was a median of 3.68 cycles (Interquartile Range, IQR = 3.23 to 3.76 cycles) greater than other platform (target) pairs. Using clinical samples, the mean difference of Xpert (N2) to LDT (E) was 3.64 cycles (95% Confidence Interval, CI =1.51 to 5.76). After calibration, the mean difference of Xpert (N2) to LDT (E) was 0.08 log10 IU/mL (95% CI = -0.56 to 0.71) and the regression was y = 1.00x * 0.08 (95% CI slope = 0.93 to 1.07, 95% CI intercept = 0.28 to 0.42).ConclusionsCalibration to the WHO standard resulted in the harmonization of two RT-qPCR tests, whereas analysis by Ct value alone may have led to erroneous quantitation. Harmonization to the WHO standard has the potential to improve the generalizability of clinical associations with SARS-CoV-2 RNA levels.
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Project description:With the emergence and wide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs), such as the Delta variant (B.1.617.2 lineage and AY sublineage), it is important to track VOCs for sourcing of transmission. Currently, whole-genome sequencing is commonly used for detecting VOCs, but this is limited by the high costs of reagents and sophisticated sequencers. In this study, common mutations in the genomes of SARS-CoV-2 VOCs were identified by analyzing more than 1 million SARS-CoV-2 genomes from public data. Among them, mutations C1709A (a change of C to A at position 1709) and C56G, respectively, were found in more than 99% of the genomes of Alpha and Delta variants and were specific to them. Then, a method using the amplification refractory mutation system combined with quantitative reverse transcription-PCR (ARMS-RT-qPCR) based on the two mutations was developed for identifying both VOCs. The assay can detect as little as 1 copy/μL of the VOCs, and the results for identifying Alpha and Delta variants in clinical samples by the ARMS-RT-qPCR assay showed 100% agreement with the results using sequencing-based methods. The whole assay can be completed in 2.5 h using commercial fluorescent PCR instruments. Therefore, the ARMS-RT-qPCR assay could be used for screening the two highly concerning variants Alpha and Delta by normal PCR laboratories in airports and in hospitals and other health-related organizations. Additionally, based on the unique mutations identified by the genomic analysis, similar molecular assays can be developed for rapid identification of other VOCs. IMPORTANCE The current stage of the pandemic, led by SARS-CoV-2 variants of concern (VOCs), underscores the necessity to develop a cost-effective and rapid molecular diagnosis assay to differentiate the VOCs. In this study, over 1 million SARS-CoV-2 genomic sequences of high quality from GISAID were analyzed and a network of the common mutations of the lineages was constructed. The conserved unique mutations specific for SARS-CoV-2 VOCs were found. Then, ARMS-RT-qPCR assays based on the two unique mutations of the Alpha and Delta variants were developed for the detection of the two VOCs. Application of the assay in clinical samples demonstrated that the current method is a convenient, cost-effective, and rapid way to screen the target SARS-CoV-2 VOCs.
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