Summary results for the specificity panel.

ObjectiveValidate the performance characteristics of two analyte specific, laboratory developed tests (LDTs) for the quantification of SARS-CoV-2 subgenomic RNA (sgRNA) and viral load on the Hologic Panther Fusion® using the Open Access functionality.MethodsCustom-designed primers/probe sets targeting the SARS-CoV-2 Envelope gene (E) and subgenomic E were optimized. A 20-day performance validation following laboratory developed test requirements was conducted to assess assay precision, accuracy, analytical sensitivity/specificity, lower limit of detection and reportable range.ResultsQuantitative SARS-CoV-2 sgRNA (LDT-Quant sgRNA) assay, which measures intermediates of replication, and viral load (LDT-Quant VLCoV) assay demonstrated acceptable performance. Both assays were linear with an R2 and slope equal to 0.99 and 1.00, respectively. Assay precision was evaluated between 4–6 Log10 with a maximum CV of 2.6% and 2.5% for LDT-Quant sgRNA and LDT-Quant VLCoV respectively. Using negative or positive SARS-CoV-2 human nasopharyngeal swab samples, both assays were accurate (kappa coefficient of 1.00 and 0.92). Common respiratory flora and other viral pathogens were not detected and did not interfere with the detection or quantification by either assay. Based on 95% detection, the assay LLODs were 729 and 1206 Copies/mL for the sgRNA and VL load LDTs, respectively.ConclusionThe LDT-Quant sgRNA and LDT-Quant VLCoV demonstrated good analytical performance. These assays could be further investigated as alternative monitoring assays for viral replication; and thus, medical management in clinical settings which could inform isolation/quarantine requirements.研究目的：本研究旨在验证两款分析物特异性实验室开发检测（Laboratory Developed Tests, LDTs）的性能特征，这两款检测分别用于定量新型冠状病毒（SARS-CoV-2）亚基因组RNA（sgRNA）与病毒载量，实验基于豪洛捷（Hologic）Panther Fusion®平台并使用其开放访问（Open Access）功能完成。 研究方法：针对新型冠状病毒包膜基因（Envelope gene, E）及其亚基因组E设计定制引物与探针组合并完成优化。依据实验室开发检测的相关规范，开展为期20天的性能验证实验，以评估检测的精密度、准确度、分析灵敏度/特异性、检测下限与可报告范围。 研究结果：定量检测新型冠状病毒sgRNA的LDT-Quant sgRNA检测（用于检测病毒复制中间体）与定量病毒载量的LDT-Quant VLCoV检测均表现出可接受的性能。两款检测均呈线性相关，决定系数（R²）与斜率分别为0.99与1.00。精密度评估在4~6 Log10区间内开展，LDT-Quant sgRNA与LDT-Quant VLCoV的最大变异系数分别为2.6%与2.5%。采用新型冠状病毒阳性及阴性人类鼻咽拭子样本验证，两款检测均展现出良好的准确度（Kappa系数分别为1.00与0.92）。常见呼吸道菌群及其他病毒病原体未被检出，且未对两款检测的检测与定量过程造成干扰。基于95%检出率的判定标准，sgRNA检测与病毒载量检测的分析检测下限分别为729拷贝/毫升与1206拷贝/毫升。 研究结论：LDT-Quant sgRNA与LDT-Quant VLCoV检测均展现出优异的分析性能。该两款检测可作为病毒复制监测的替代检测方法开展进一步研究，进而为临床场景中的医疗管理提供依据，助力隔离/检疫相关要求的制定。