Dataset Information

Development of a New Multiplex Real-Time RT-PCR Assay for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Detection.

ABSTRACT: This research describes the development of a new multiplex real-time RT-PCR test for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with primers designed to amplify a 108 bp target on the spike surface glycoprotein (S gene) and a hydrolysis TaqMan probe designed to specifically detect SARS-CoV-2. The limit of detection (LOD) and clinical performance of this new assay were evaluated. A LOD study with inactivated virus exhibited performance equal to the modified CDC assay, with a final LOD of 1301 ± 13 genome equivalents/mL for the Northwell Health Laboratories laboratory-developed test (NWHL LDT) versus 1249 ± 14 genome equivalents/mL for the modified CDC assay. In addition, a clinical evaluation with 270 nasopharyngeal swab specimens exhibited 98.5% positive percent agreement and 99.3% negative percent agreement compared with the modified CDC assay. The NWHL LDT multiplex design allows testing of 91 patients per plate, versus a maximum of 29 patients per plate on the modified CDC assay, providing the benefit of testing significantly more patients per run and saving reagents, during a time when both of these parameters are critical. The results show that the NWHL LDT multiplex assay performs as well as the modified CDC assay but is more efficient and cost-effective and can be used as a diagnostic assay and for epidemiologic surveillance and clinical management of SARS-CoV-2.

SUBMITTER: Zhen W

PROVIDER: S-EPMC7501774 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Development of a New Multiplex Real-Time RT-PCR Assay for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Detection.

Zhen Wei W Berry Gregory J GJ

The Journal of molecular diagnostics : JMD 20200919 12

This research describes the development of a new multiplex real-time RT-PCR test for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with primers designed to amplify a 108 bp target on the spike surface glycoprotein (S gene) and a hydrolysis TaqMan probe designed to specifically detect SARS-CoV-2. The limit of detection (LOD) and clinical performance of this new assay were evaluated. A LOD study with inactivated virus exhibited performance equal to the modified CDC ass ...[more]

PMID: 32961315

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Project description:Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread worldwide. Many variants of SARS-CoV-2 have been reported, some of which have increased transmissibility and/or reduced susceptibility to vaccines. There is an urgent need for variant phenotyping for epidemiological surveillance of circulating lineages. Whole-genome sequencing is the gold standard for identifying SARS-CoV-2 variants, which constitutes a major bottleneck in developing countries. Methodological simplification could increase epidemiological surveillance feasibility and efficiency. We designed a novel multiplex real-time reverse transcriptase PCR (RT-PCR) to detect SARS-CoV-2 variants with S gene mutations. This multiplex PCR typing method was established to detect 9 mutations with specific primers and probes (ΔHV 69/70, K417T, K417N, L452R, E484K, E484Q, N501Y, P681H, and P681R) against the receptor-binding domain of the spike protein of SARS-CoV-2 variants. In silico analyses showed high specificity of the assays. Variants of concern (VOC) typing results were found to be highly specific for our intended targets, with no cross-reactivity observed with other upper respiratory viruses. The PCR-based typing methods were further validated using whole-genome sequencing and a commercial kit that was applied to clinical samples of 250 COVID-19 patients from Taiwan. The screening of these samples allowed the identification of epidemic trends by time intervals, including B.1.617.2 in the third Taiwan wave outbreak. This PCR typing strategy allowed the detection of five major variants of concern and also provided an open-source PCR assay which could rapidly be deployed in laboratories around the world to enhance surveillance for the local emergence and spread of B.1.1.7, B.1.351, P.1, and B.1.617.2 variants and of four Omicron mutations on the spike protein (ΔHV 69/70, K417N, N501Y, P681H). IMPORTANCE COVID-19 has spread globally. SARS-CoV-2 variants of concern (VOCs) are leading the next waves of the COVID-19 pandemic. Previous studies have pointed out that these VOCs may have increased infectivity, have reduced vaccine susceptibility, change treatment regimens, and increase the difficulty of epidemic prevention policy. Understanding SARS-CoV-2 variants remains an issue of concern for all local government authorities and is critical for establishing and implementing effective public health measures. A novel SARS-CoV-2 variant identification method based on a multiplex real-time RT-PCR was developed in this study. Five SARS-CoV-2 variants (Alpha, Beta, Gamma, Delta, and Omicron) were identified simultaneously using this method. PCR typing can provide rapid testing results with lower cost and higher feasibility, which is well within the capacity for any diagnostic laboratory. Characterizing these variants and their mutations is important for tracking SAR-CoV-2 evolution and is conducive to public infection control and policy formulation strategies.

Dataset Information

Development of a New Multiplex Real-Time RT-PCR Assay for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Detection.

Publications

Development of a New Multiplex Real-Time RT-PCR Assay for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Detection.

Similar Datasets

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