Cruceriu Daniel, Baldasici Oana, Balacescu Loredana, Gligor-Popa Stefana, Flonta Mirela, Man Milena A, Visan Simona, Vlad Catalin, Trifa Adrian P, Balacescu Ovidiu, Achimas-Cadariu Patriciu
Department of Genetics, Genomics and Experimental Pathology, The Oncology Institute "Prof. Dr. Ion Chiricuta", 34-36, Republicii Street, 400015 Cluj-Napoca, Romania.
Department of Molecular Biology and Biotechnology, "Babes-Bolyai" University, 1, M. Kogălniceanu Street, 400084 Cluj-Napoca, Romania.
Viruses. 2021 May 13;13(5):902. doi: 10.3390/v13050902.
The primary approach to controlling the spread of the pandemic SARS-CoV-2 is to diagnose and isolate the infected people quickly. Our paper aimed to investigate the efficiency and the reliability of a hierarchical pooling approach for large-scale PCR testing for SARS-CoV-2 diagnosis. To identify the best conditions for the pooling approach for SARS-CoV-2 diagnosis by RT-qPCR, we investigated four manual methods for both RNA extraction and PCR assessment targeting one or more of the RdRp, N, S, and ORF1a genes, by using two PCR devices and an automated flux for SARS-CoV-2 detection. We determined the most efficient and accurate diagnostic assay, taking into account multiple parameters. The optimal pool size calculation included the prevalence of SARS-CoV-2, the assay sensitivity of 95%, an assay specificity of 100%, and a range of pool sizes of 5 to 15 samples. Our investigation revealed that the most efficient and accurate procedure for detecting the SARS-CoV-2 has a detection limit of 2.5 copies/PCR reaction. This pooling approach proved to be efficient and accurate in detecting SARS-CoV-2 for all samples with individual quantification cycle (Cq) values lower than 35, accounting for more than 94% of all positive specimens. Our data could serve as a comprehensive practical guide for SARS-CoV-2 diagnostic centers planning to address such a pooling strategy.
控制新冠病毒（SARS-CoV-2）大流行传播的主要方法是快速诊断和隔离感染者。我们的论文旨在研究用于SARS-CoV-2诊断的大规模聚合酶链反应（PCR）检测的分层混合方法的效率和可靠性。为了确定通过逆转录定量聚合酶链反应（RT-qPCR）进行SARS-CoV-2诊断的混合方法的最佳条件，我们使用两种PCR设备和一种用于SARS-CoV-2检测的自动化通量，研究了针对RNA依赖性RNA聚合酶（RdRp）、核衣壳蛋白（N）、刺突蛋白（S）和开放阅读框1a（ORF1a）基因中的一个或多个基因进行RNA提取和PCR评估的四种手动方法。我们考虑了多个参数，确定了最有效和准确的诊断检测方法。最佳混合样本量计算包括SARS-CoV-2的流行率、95%的检测灵敏度、100%的检测特异性以及5至15个样本的混合样本量范围。我们的研究表明，检测SARS-CoV-2最有效和准确的方法的检测限为2.5拷贝/PCR反应。对于所有个体定量循环（Cq）值低于35的样本，这种混合方法在检测SARS-CoV-2方面被证明是有效和准确的，占所有阳性标本的94%以上。我们的数据可以为计划采用这种混合策略的SARS-CoV-2诊断中心提供全面的实用指南。
