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Single-Point Mutations in the N Gene of SARS-CoV-2 Adversely Impact Detection by a Commercial Dual Target Diagnostic Assay.

ABSTRACT: Accurate and rapid diagnostic tests are a critical component for the early diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and of the overall control strategy for the current pandemic. Nucleic acid amplification tests are the gold standard for diagnosis of acute SARS-CoV-2 infection, and many real-time PCR diagnostic assays have been developed. Mutations that occur within the primer/probe binding regions of the SARS-CoV-2 genome can negatively impact the performance of diagnostic assays. Here, we report two single-point mutations in the N gene of SARS-CoV-2 associated with N gene target detection failures in the Cepheid Xpert Xpress SARS-CoV-2 assay, the first a C to T mutation at position 29197, found in five patients, and the second a C to T mutation at position 29200, found in eight patients. By sequencing the Xpert amplicons, we showed both mutations to be located within the amplified region of the Xpert N gene target. This report highlights the necessity for multiple genetic targets and the continual monitoring and evaluation of diagnostic assay performance. IMPORTANCE This paper reports the identification of single-point mutations in the N gene of SARS-CoV-2 associated with a gene target failure by the Cepheid Xpert commercial system. In order to determine the mutation(s) responsible for the N gene detection failures, the genomic products from the Cepheid Xpert system were sequenced and compared to whole genomes of SARS-CoV-2 from clinical cases. This report is the first to our knowledge which characterizes the amplified PCR products of the Xpert system, confirming the mutations associated with the gene target failure. The mutations identified have previously been reported.

SUBMITTER: Miller S

PROVIDER: S-EPMC8597629 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which has caused a global pandemic beginning in 2020, can be detected by reverse-transcription polymerase chain reaction (RT-PCR). However, owing to the urgent need for a large number of detection kits, the time spent researching and developing these kits has been shortened during the pandemic, and the kits that are being used commercially have not undergone full and independent evaluation. To ensure the accuracy of SARS-CoV-2 test results, performance verification of commercial Real-Time quantitative PCR (RT-qPCR) kits is required.MethodsThe performance of five commercial RT-qPCR diagnostic kits for SARS-CoV-2 used in China was evaluated using a coronavirus disease 2019 (COVID-19) RNA liquid performance verification reference product-manufactured by Guangzhou Bondson (BDS) Biotechnology Co., Ltd.,Guangzhou, China-that uses droplet digital RT-PCR technology combined with fluorescence quantitative PCR. The five kits of Novel Coronavirus 2019-nCoV nucleic acid detection kit (RT-qPCR method) evaluated were Da An (Da An Gene Co., Ltd. of Sun Yat-sen University), Liferiver (Shanghai ZJ Bio-Tech Co., Ltd.), Kinghawk (Beijing Kinghawk Pharmaceutical Co., Ltd.), eDiagnosis (Wuhan Easy Diagnosis Biomedicine Co., Ltd.), and Maccura (Maccura Biotechnology Co., Ltd.). Performance verification criteria included the coincidence rate, limit of detection (LoD), cross-reactivity, precision, and anti-interference. Finally, through the BDS performance verification reference product kit, clinical samples are used to verify its clinical diagnostic efficacy.ResultsThe coincidence rate was 100% for all kits except for Kinghawk, which was 95%. The LoD for Da An, eDiagnosis and Maccura was 250copies/mL, and it was 1000 copies/ml for Liferiver. Kinghawk was not able to detect its advertised LoD of 500 copies/ml. The cross-reactivity test results were all negative. Moreover, all kits had a coefficient of variation less than 5%; however, Liferiver showed the best precision. Da An, Liferiver, and eDiagnosis showed higher sensitivity to the nucleocapsid (N) gene than they did to the open reading frame (ORF) 1ab genes. Anti-interference results for all five kits were positive. The results of clinical diagnostic efficacy were that the specificity of the four kits was 1.000 (0.877-1.000), the sensitivity of Da An was 1.000 (0.850-1.000), Liferiver was 0.964 (0.798-0.998), Maccura was 0.893 (0.706-0.972), and eDiagnosis was 0.857 (0.664-0.953).ConclusionsAll commercial RT-qPCR diagnostic kits for SARS-CoV-2 passed the BDS performance verification, except for Kinghawk (batch No:20200608113) which failed to detect the LoD of 500 copies/mL. Da An and Liferiver have excellent clinical diagnostic specificity and sensitivity. This study can provide guidance for the selection or optimization of RT-qPCR diagnostic test kits for SARS-CoV-2.

Project description:BackgroundAs vaccines against SARS-CoV-2 are now being rolled out, a better understanding of immunity to the virus, whether from infection, or passive or active immunisation, and the durability of this protection is required. This will benefit from the ability to measure antibody-based protection to SARS-CoV-2, ideally with rapid turnaround and without the need for laboratory-based testing.MethodsWe have developed a lateral flow POC test that can measure levels of RBD-ACE2 neutralising antibody (NAb) from whole blood, with a result that can be determined by eye or quantitatively on a small instrument. We compared our lateral flow test with the gold-standard microneutralisation assay, using samples from convalescent and vaccinated donors, as well as immunised macaques.FindingsWe show a high correlation between our lateral flow test with conventional neutralisation and that this test is applicable with animal samples. We also show that this assay is readily adaptable to test for protection to newly emerging SARS-CoV-2 variants, including the beta variant which revealed a marked reduction in NAb activity. Lastly, using a cohort of vaccinated humans, we demonstrate that our whole-blood test correlates closely with microneutralisation assay data (specificity 100% and sensitivity 96% at a microneutralisation cutoff of 1:40) and that fingerprick whole blood samples are sufficient for this test.InterpretationTaken together, the COVID-19 NAb-testTM device described here provides a rapid readout of NAb based protection to SARS-CoV-2 at the point of care.FundingSupport was received from the Victorian Operational Infrastructure Support Program and the Australian Government Department of Health. This work was supported by grants from the Department of Health and Human Services of the Victorian State Government; the ARC (CE140100011, CE140100036), the NHMRC (1113293, 2002317 and 1116530), and Medical Research Future Fund Awards (2005544, 2002073, 2002132). Individual researchers were supported by an NHMRC Emerging Leadership Level 1 Investigator Grants (1194036), NHMRC APPRISE Research Fellowship (1116530), NHMRC Leadership Investigator Grant (1173871), NHMRC Principal Research Fellowship (1137285), NHMRC Investigator Grants (1177174 and 1174555) and NHMRC Senior Principal Research Fellowships (1117766 and 1136322). Grateful support was also received from the A2 Milk Company and the Jack Ma Foundation.

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Single-Point Mutations in the N Gene of SARS-CoV-2 Adversely Impact Detection by a Commercial Dual Target Diagnostic Assay.

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