Evaluation of simple nucleic acid extraction methods for the detection of SARS-CoV-2 in nasopharyngeal and saliva specimens during global shortage of extraction kits
Evaluation of simple nucleic acid extraction methods for the detection of SARS-CoV-2 in nasopharyngeal and saliva specimens during global shortage of extraction kits
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DOI：
10.1016/j.jcv.2020.104519
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发表时间：
2020-08-01
影响因子：
8.8
通讯作者：
Kai-Wang To, Kelvin
中科院分区：
文献类型：
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作者：
Chu, Allen Wing-Ho;Chan, Wan-Mui;Kai-Wang To, Kelvin
背景资料：在当前COVID-19大流行期间，核酸提取试剂盒的严重短缺是检测能力的关键限制因素。目的：本研究比较了使用不同简单核酸提取方法对鼻咽和唾液标本进行SARS-CoV-2 RT-PCR的结果。研究设计：检索了50份先前检测为SARS-CoV-2阳性的鼻咽拭子和唾液标本。比较了三种不同的核酸提取方法。第一种方法包括将标本与蛋白酶K一起孵育，然后在98 ℃下热处理5分钟（PKH）;第二种方法包括在98 ℃下热处理5分钟，不进行蛋白酶K预孵育（仅加热）;第三种方法不涉及预处理步骤（直接）。结果：PKH法检测SARS-CoV-2 RT-PCR的阳性率（80%）明显高于单纯加热法（58%; P = 0.001）和直接法（56%; P = 0.002）。PKH的中位Ct值（中位Ct：37.0，IQR 31.7-40）显著早于单纯热疗（中位Ct：40，IQR 36.2-41; P < 0.0001）和直接热疗（中位Ct：37.5; IQR 33.9-41.0; P = 0.0049）。亚组分析显示，PKH法对唾液和唾液标本的检出率、检出限和Ct值均高于其他两组。结论：PKH法可提高RT-PCR法对SARS冠状病毒2型的检出率，是一种在无商品化核酸提取试剂盒时可替代的核酸提取方法。
Background: The severe shortage of nucleic acid extraction kits during the current COVID-19 pandemic represents a key limiting factor in testing capacity.Objectives: This study compared the results of SARS-CoV-2 RT-PCR using different simple nucleic acid extraction methods on nasopharyngeal and saliva specimens.Study design: Fifty nasopharyngeal swab and saliva specimens previously tested positive for SARS-CoV-2 were retrieved. Three different methods of nucleic acid extraction were compared. The first method involves incubating the specimen with proteinase K, and then heat treatment at 98 degrees C for 5 min (PKH); the second method involves heat treatment at 98 degrees C for 5 min without proteinase K pre-incubation (heat only); the third method involves no pre-processing steps (direct). The products from all 3 methods were tested by SARS-CoV-2 RT-PCR.Results: PKH had significantly higher positive rate in SARS-CoV-2 RT-PCR (80 %) than those of heat only (58 %; P = 0.001) or direct (56 %; P = 0.002). The median Ct value was significantly earlier for PKH (median Ct: 37.0, IQR 31.7-40) than that of heat only (median Ct: 40, IQR 36.2-41; P < 0.0001) and direct (median Ct, 37.5; IQR 33.9-41.0; P = 0.0049). Subgroup analysis showed that PKH had higher detection rate, lower limit of detection and earlier Ct values than the other two groups for both NPS and saliva specimens.Conclusions: PKH pre-processing resulted in the highest detection rate of SARS-CoV-2 by RT-PCR, and represents an alternative method for nucleic acid extraction when commercial extraction kits are not available.