Evaluation of a Rapid and Accessible Reverse Transcription-Quantitative PCR Approach for SARS-CoV-2 Variant of Concern Identification.

The ability to distinguish between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) is of ongoing interest due to differences in transmissibility, responses to vaccination, clinical prognosis, and therapy. Although detailed genetic characterization requires whole-genome sequencing (WGS), targeted nucleic acid amplification tests can serve a complementary role in clinical settings, as they are more rapid and accessible than sequencing in most laboratories. We designed and analytically validated a two-reaction multiplex reverse transcription-quantitative PCR (RT-qPCR) assay targeting spike protein mutations L452R, E484K, and N501Y in reaction 1 and del69-70, K417N, and T478K in reaction 2. This assay had 95 to 100% agreement with WGS for 502 upper respiratory tract swab samples collected between 26 April 2021 and 1 August 2021, consisting of 43 Alpha, 2 Beta, 20 Gamma, 378 Delta, and 59 non-VOC infections. Validation in a separate group of 230 WGS-confirmed Omicron variant samples collected in December 2021 and January 2022 demonstrated 100% agreement. This RT-qPCR-based approach can be implemented in clinical laboratories already performing SARS-CoV-2 nucleic acid amplification tests to assist in local epidemiological surveillance and clinical decision-making.

Multiplex SARS-CoV-2 Genotyping Reverse Transcriptase PCR for Population-Level Variant Screening and Epidemiologic Surveillance.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with concerning phenotypic mutations is of public health interest. Genomic surveillance is an important tool for a pandemic response, but many laboratories do not have the resources to support population-level sequencing. We hypothesized that a nucleic acid amplification test (NAAT) to genotype mutations in the viral spike protein could facilitate high-throughput variant surveillance. We designed and analytically validated a one-step multiplex allele-specific reverse transcriptase PCR (RT-qPCR) to detect three nonsynonymous spike protein mutations (L452R, E484K, N501Y). Assay specificity was validated with next-generation whole-genome sequencing. We then screened a large cohort of SARS-CoV-2-positive specimens from our San Francisco Bay Area population. Between 1 December 2020 and 1 March 2021, we screened 4,049 unique infections by genotyping RT-qPCR, with an assay failure rate of 2.8%. We detected 1,567 L452R mutations (38.7%), 34 N501Y mutations (0.84%), 22 E484K mutations (0.54%), and 3 (0.07%) E484K plus N501Y mutations. The assay had perfect (100%) concordance with whole-genome sequencing of a validation subset of 229 specimens and detected B.1.1.7, B.1.351, B.1.427, B.1.429, B.1.526, and P.2 variants, among others. The assay revealed the rapid emergence of the L452R variant in our population, with a prevalence of 24.8% in December 2020 that increased to 62.5% in March 2021. We developed and clinically implemented a genotyping RT-qPCR to conduct high-throughput SARS-CoV-2 variant screening. This approach can be adapted for emerging mutations and immediately implemented in laboratories already performing NAAT worldwide using existing equipment, personnel, and extracted nucleic acid.

Evaluation of a measles virus multiplex, triple-target real-time RT-PCR in three specimen matrices at a U.S. academic medical center.

BACKGROUND: Measles virus (MeV) is an important cause of acute febrile illness and pediatric mortality globally, with recent U.S. outbreaks associated with under-vaccination. MeV is highly contagious and timely diagnosis is critical to limit spread. RNA detection is the most sensitive method for acute measles diagnosis; however, MeV nucleic acid amplification assays are not widely available. METHODS: We performed a diagnostic accuracy study of a triple-target, real-time RT-PCR (rRT-PCR) assay for simultaneous detection of MeV N, H, and L genes. RESULTS: The MeV triple-target rRT-PCR was tested against serial dilutions (7.0-2.0 log10 copies/mL) of five MeV isolates representing circulating genotypes, and detected 98.7% (74/75) of nasopharyngeal (NP) swab dilutions, 100% (75/75) of plasma dilutions, and 85.3% (64/75) of urine dilutions. MeV RNA detection in urine was markedly improved with the addition of a nucleic acid stabilizing agent. A 95% lower limit of detection (LLOD) of < 3.0 log10 copies/mL was established in each specimen matrix. No cross-reactivity with relevant viruses or interfering substances were identified in specificity studies. The MeV triple-target rRT-PCR detected all three gene targets in a clinical NP swab from an individual with confirmed measles infection. Furthermore, pooled testing from 798 influenza A/B/RSV-negative pediatric NP swabs identified two specimens positive for MeV RNA, confirmed by N gene sequencing to represent shedding of the vaccine-type measles virus. CONCLUSIONS: The MeV triple-target rRT-PCR assay showed high analytic sensitivity across circulating MeV genotypes in three clinically-relevant matrices. Implementation of this assay in the clinical laboratory may facilitate timely diagnosis of acute measles infection and implementation of appropriate infection control interventions.

Strand-Specific Reverse Transcription PCR for Detection of Replicating SARS-CoV-2.

We developed an assay that detects minus-strand RNA as a surrogate for actively replicating severe acute respiratory syndrome coronavirus 2. We detected minus-strand RNA in 41 persons with coronavirus disease up to 30 days after symptom onset. This assay might inform clinical decision-making about patient infectiousness.