Background

This study aimed to evaluate the impact of four different reverse transcription quantitative PCR (RT-qPCR) master mixes on the performance of SARS-CoV-2 diagnostic PCRs using three primer/probe assays targeting the N gene (A, B and C). The dynamic range and lowest detected quantity was determined using a SARS-CoV-2 partial N gene RNA transcript dilution series (100,000–1 copy/μl) and verified using 72 nose and throat swabs, 29 of which tested positive for SARS-CoV-2 RNA.

Results

Assay C consistently detected the lowest quantity of partial N gene RNA transcript with all mastermixes. The Takara One Step PrimeScript™ III RT-PCR Kit mastermix enabled all primer pairs to detect the entire dynamic range evaluated, with the Qiagen Quantifast and Thermofisher TaqPath 1-Step kits also performing well.

Sequences from all three primer/probe sets tested in this study (assay A, B and C) have 100 % homology to ≥97 % of the of SARS-CoV-2 sequences available up to 31st December 2020 (n = 291,483 sequences).

Conclusions

This work demonstrates that specific assays (in this case assay C) can perform well in terms of dynamic range and lowest detected quantity regardless of the mastermix used. However we also show that, by choosing the most appropriate mastermix, poorer performing primer pairs are also able to detect all of the template dilutions investigated. This work increases the potential options when choosing assays for SARS-CoV-2 diagnosis and provides solutions to enable them to work with optimal analytical sensitivity.

中文翻译：

---

SARS-CoV-2 N 基因实时 RT-PCR 靶标与市售预混液的比较

背景

本研究旨在使用针对 N 基因（A、B 和 C）的三种引物/探针分析来评估四种不同的逆转录定量 PCR (RT-qPCR) 预混液对 SARS-CoV-2 诊断 PCR 性能的影响。动态范围和最低检测量是使用 SARS-CoV-2 部分 N 基因 RNA 转录本稀释系列（100,000–1 拷贝/μl）确定的，并使用 72 个鼻子和喉咙拭子进行验证，其中 29 个样本呈 SARS-CoV-阳性2 核糖核酸。

结果

测定 C 在所有预混液中始终检测到最低量的部分 N 基因 RNA 转录物。Takara One Step PrimeScript™ III RT-PCR Kit mastermix 使所有引物对都能检测评估的整个动态范围，Qiagen Quantifast 和 Thermofisher TaqPath 1-Step 试剂盒也表现良好。

本研究中测试的所有三个引物/探针组（测定 A、B 和 C）的序列与截至 2020 年 12 月 31 日可用的 SARS-CoV-2 序列（n = 291,483 个序列）的 ≥ 97% 具有 100% 的同源性。

结论

这项工作表明，无论使用何种 mastermix，特定检测（在本例中为检测 C）在动态范围和最低检测量方面都能表现良好。然而，我们还表明，通过选择最合适的 mastermix，性能较差的引物对也能够检测所研究的所有模板稀释液。这项工作增加了选择用于 SARS-CoV-2 诊断的检测方法的潜在选择，并提供了使它们能够以最佳分析灵敏度工作的解决方案。