We describe our efforts at developing a one-step quantitative reverse-transcription (qRT)-PCR protocol to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA directly from saliva samples, without RNA purification. We find that both heat and the presence of saliva impairs the ability to detect synthetic SARS-CoV-2 RNA. Buffer composition (for saliva dilution) was also crucial to effective PCR detection. Using the SG2 primer pair, designed by Sigma-Aldrich, we were able to detect the equivalent of 1.7x10⁶ viral copies per mL of saliva after heat inactivation; approximately equivalent to the median viral load in symptomatic patients. This would make our assay potentially useful for rapid detection of high-shedding infected individuals. We also provide a comparison of the PCR efficiency and specificity, which varied considerably, across 9 reported primer pairs for SARS-CoV-2 detection. Primer pairs SG2 and CCDC-N showed highest specificity and PCR efficiency. Finally, we provide an alternate primer pair to use as a positive control for human RNA detection in SARS-CoV-2 assays, as we found that the widely used US CDC primers (targeting human RPP30 ) do not span an exon-exon junction and therefore does not provide an adequate control for the reverse transcription reaction.

中文翻译：

---

SYBR Green一步法qRT-PCR检测唾液中的SARS-CoV-2 RNA

我们描述了我们在开发一步定量逆转录（qRT）-PCR协议以直接从唾液样本中检测严重急性呼吸综合征冠状病毒2（SARS-CoV-2）RNA而不进行RNA纯化的努力。我们发现热量和唾液的存在均损害了检测合成SARS-CoV-2 RNA的能力。缓冲液成分（用于唾液稀释）对于有效的PCR检测也至关重要。使用由Sigma-Aldrich设计的SG2引物对，我们能够检测到1.7x10 ⁶灭活后每毫升唾液的病毒拷贝；大约等于有症状患者的中位病毒载量。这将使我们的测定对于快速检测高流失感染个体具有潜在的实用价值。我们还提供了9个报告的SARS-CoV-2检测引物对中PCR效率和特异性的比较，差异很大。引物对SG2和CCDC-N显示出最高的特异性和PCR效率。最后，由于我们发现广泛使用的美国CDC引物（靶向人RPP30）不跨越外显子-外显子连接，因此我们提供了另一对引物作为SARS-CoV-2分析中人RNA检测的阳性对照。因此不能为逆转录反应提供足够的控制。