Background

Diagnostic real time reverse transcription PCR (rRT-PCR) is usually done using nucleic acid (NA) purified from the sample. In the SARS-CoV-2 pandemic reagents and utensils for NA purification has been in short supply. This has generated interest in methods that eliminate the need for NA purification.

Objectives

To investigate if addition of detergent to rRT-PCR master mix (MM) enabled in-well direct lysis and detection of SARS-CoV-2 in clinical eSwab specimens.

Study design

IGEPAL-CA-630 (IGEPAL) was added to SARS-CoV-2 MM to 0.3 % final concentration and crude sample was added directly to the PCR well containing MM. Cycle of positivity (Cp) and categorical agreement was compared in samples tested in standard rRT-PCR after NA purification and in in-well lysis, direct rRT-PCR.

Results

In-well lysis direct rRT-PCR detected SARS-CoV-2 in 27/30 previously SARS-CoV-2+ samples with an average bias of 3.26 cycles (95 %CI: 0.08–6.43 cycles). All 30 previously test negative samples remained negative when tested in in-well lysis, direct PCR.

Conclusions

Supplementation of detergent to MM was shown to be useful for the detection of SARS CoV-2 in eSwab specimens (COPAN) by direct rRT-PCR without prior NA purification.

中文翻译：

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一种实时RT-PCR快速检测eSwab标本中SARS-CoV-2的孔内直接裂解方法

背景

诊断实时逆转录PCR（rRT-PCR）通常使用从样品中纯化的核酸（NA）进行。在SARS-CoV-2中，用于NA纯化的大流行试剂和器皿供不应求。这引起了对不需要NA纯化的方法的兴趣。

目标

调查rRT-PCR预混液（MM）中添加去污剂是否能在临床eSwab标本中进行孔内直接裂解和SARS-CoV-2的检测。

学习规划

将IGEPAL-CA-630（IGEPAL）添加至SARS-CoV-2 MM至终浓度0.3％，并将粗样品直接添加至含MM的PCR孔中。在纯化NA之后，在标准rRT-PCR和孔内裂解直接rRT-PCR中测试的样品中比较了阳性周期（Cp）和分类一致性。

结果

孔内裂解直接rRT-PCR在27/30个先前的SARS-CoV-2 +样品中检测到SARS-CoV-2，平均偏差为3.26个周期（95％CI：0.08-6.43个周期）。在孔内裂解，直接PCR中测试时，所有30个先前测试的阴性样品均保持阴性。

结论

研究表明，向MM补充洗涤剂可通过直接rRT-PCR检测eSwab标本（COPAN）中的SARS CoV-2，而无需事先纯化NA。