SARS-CoV-2 RNA identification in nasopharyngeal swabs: issues in pre-analytics
SARS-CoV-2 RNA identification in nasopharyngeal swabs: issues in pre-analytics
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DOI：
10.1515/cclm-2020-0749
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发表时间：
2020-08-01
影响因子：
6.8
通讯作者：
Plebani, Mario
中科院分区：
文献类型：
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作者：
Basso, Daniela;Aita, Ada;Plebani, Mario
目的：建议直接鉴定鼻咽拭子中的SARS-CoV-2 RNA用于诊断新型COVID-19疾病。分析前的决定因素，如取样程序、时间和温度储存条件，可能会影响最终结果。我们的目的是评估采样程序、时间和温度对原始鼻咽拭子储存实时逆转录聚合酶链反应（rRT-PCR）结果的影响。方法：从10名住院COVID-19患者中获得的每个鼻咽拭子被细分为15等份：5份保存在室温下; 5份冷藏（+4 ℃）; 5个立即与提取缓冲液混合，并在+4 ℃下冷藏。每天并持续5天，分析（rRT-PCR）SARS-CoV-2基因E和RNaseP的每个条件的一个等分试样，并比较阈值循环（Ct）。为了评价手工采样，70鼻咽拭子采样两次，由两个不同的操作员，并分别分析一个从其他。在SARS-CoV-2 Ct中未观察到显著的时间或储存依赖性变化。对Ct值小于33的拭子重新采样，结果重复性好（CV=2.9%），Ct值大于33的拭子重复采样结果变异性大（CV=10.3%）。结论：鼻咽拭子保存时间和温度对结果重复性无显著影响。然而，拭子采样是一个关键步骤，特别是在病毒载量低的情况下，可能是诊断错误的潜在来源。
Objectives: The direct identification of SARS-CoV-2 RNA in nasopharyngeal swabs is recommended for diagnosing the novel COVID-19 disease. Pre-analytical determinants, such as sampling procedures, time and temperature storage conditions, might impact on the end result. Our aim was to evaluate the effects of sampling procedures, time and temperature of the primary nasopharyngeal swabs storage on real-time reverse-transcription polymerase chain reaction (rRT-PCR) results.Methods: Each nasopharyngeal swab obtained from 10 hospitalized patients for COVID-19 was subdivided in 15 aliquots: five were kept at room temperature; five were refrigerated (+4 degrees C); five were immediately mixed with the extraction buffer and refrigerated at +4 degrees C. Every day and for 5 days, one aliquot per condition was analyzed (rRT-PCR) for SARS-CoV-2 gene E and RNaseP and threshold cycles (Ct) compared. To evaluate manual sampling, 70 nasopharyngeal swabs were sampled twice by two different operators and analyzed separately one from the other.Results: A total of 6/10 swabs were SARS-CoV-2 positive. No significant time or storage-dependent variations were observed in SARS-CoV-2 Ct. Re-sampling of swabs with SARS-CoV-2 Ct lower than 33 resulted in highly reproducible results (CV=2.9%), while a high variability was observed when Ct values were higher than 33 (CV=10.3%).Conclusions: This study demonstrates that time and temperature of nasopharyngeal swabs storage do not significantly impact on results reproducibility. However, swabs sampling is a critical step, and especially in case of low viral load, might be a potential source of diagnostic errors.