Influence of Different Inactivation Methods on Severe Acute Respiratory Syndrome Coronavirus 2 RNA Copy Number
Influence of Different Inactivation Methods on Severe Acute Respiratory Syndrome Coronavirus 2 RNA Copy Number
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DOI：
10.1128/jcm.00958-20
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发表时间：
2020-08-01
影响因子：
9.4
通讯作者：
Ma, Chaofeng
中科院分区：
文献类型：
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作者：
Chen, Hailong;Wu, Rui;Ma, Chaofeng
2019冠状病毒病（COVID-19）爆发已蔓延至全球，并被定性为大流行。为了保护医学实验室人员免受感染，大多数实验室在检测前都会在临床样本中检测引起COVID-19的病毒，即严重急性呼吸道综合征冠状病毒2（SARS-CoV-2）。然而，灭活对检测结果的影响仍然未知。在这里，我们使用数字PCR检测来确定63个鼻咽拭子样本中的SARS-CoV-2 RNA绝对拷贝数，并评估灭活方法对病毒RNA拷贝数的影响。通过三种不同的方法进行病毒灭活：（i）在室温下与TRlzol LS试剂一起孵育10分钟，（ii）在56 ℃水浴中加热30分钟，和（iii）高温处理，包括在121 ℃高压灭菌20分钟，在100 ℃煮沸20分钟，与原始样品中的RNA量相比，TRlzol处理破坏了47.54%的核衣壳蛋白（N）基因和39.85%的开放阅读框架（ORF）实验室。对于在56 ℃处理30 min的样品，N基因和ORF实验室的拷贝数分别减少了48.55%和56.40%。在80 ℃下加热20 min后，病毒RNA拷贝数下降了50%至66%。在121 ℃下高压灭菌或在100 ℃下煮沸20 min后几乎没有检测到病毒RNA。这些结果表明，灭活减少了可检测的病毒RNA的数量，并可能导致假阴性结果，特别是在弱阳性病例中。因此，建议使用TRlzol试剂而不是热灭活进行样品灭活，因为在检测方法中，TRlzol试剂对RNA拷贝数的影响最小。
The outbreak of coronavirus disease 2019 (COVID-19) has spread across the world and was characterized as a pandemic. To protect medical laboratory personnel from infection, most laboratories inactivate the virus causing COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in clinical samples before testing. However, the effect of inactivation on the detection results remains unknown. Here, we used a digital PCR assay to determine the absolute SARS-CoV-2 RNA copy number in 63 nasopharyngeal swab samples and assess the effect of inactivation methods on viral RNA copy number. Viral inactivation was performed by three different methods: (i) incubation with the TRlzol LS reagent for 10 min at room temperature, (ii) heating in a water bath at 56 degrees C for 30 min, and (iii) high-temperature treatment, including autoclaving at 121 degrees C for 20 min, boiling at 100 degrees C for 20 min, and heating at 80 degrees C for 20 min. Compared to the amount of RNA in the original sample, TRlzol treatment destroyed 47.54% of the nucleocapsid protein (N) gene and 39.85% of open reading frame (ORF) lab. For samples treated at 56 degrees C for 30 min, the copy number of the N gene and ORF lab was reduced by 48.55% and 56.40%, respectively. The viral RNA copy number dropped by 50 to 66% after heating at 80 degrees C for 20 min. Nearly no viral RNA was detected after autoclaving at 121 degrees C or boiling at 100 degrees C for 20 min. These results indicate that inactivation reduced the quantity of detectable viral RNA and may cause false-negative results, especially in weakly positive cases. Thus, use of the TRlzol reagent rather than heat inactivation is recommended for sample inactivation, as the TRlzol reagent had the least effect on the RNA copy number among the tested methods.