三种核酸提取方法在2019新型冠状病毒检测中的比较  

Comparison of three nucleic acid extraction methods in the detection of 2019 novel coronavirus

作　　者：陈雄毅[1] 周怡兴[2] 陈世准[2] 陈平[1] 李成德[1] 岑丽莲[1] CHEN Xiong-yi;ZHOU Yi-xing;CHEN Shi-zhun(Clinical laboratory,Zhaoqing first people's hospital,Guangdong Zhaoqing 526040)

机构地区：[1]肇庆市第一人民医院检验科,广东肇庆526040 [2]肇庆市第一人民医院感染疾病科,广东肇庆526040

出　　处：《医学检验与临床》2021年第11期12-16,共5页Medical Laboratory Science and Clinics

摘　　要：目的:比较三种核酸提取方法在2019新型冠状病毒检测中的效果,为优化2019新型冠状病毒检测效果提供依据。方法:选用2020年2~3月新冠肺炎确诊患者的咽拭子共50份,作为研究对象。并选阳性标本一例,进行1:2、1:4、1:8、1:16、1:32、1:64、1:128、1:256比例稀释,获得不同浓度的新冠病毒标本。分别以柱提法、磁珠法和浓缩法三种方法提取病毒核酸(包括ORF1ab基因和N基因),并进行荧光RT-PCR扩增,根据扩增结果对三种方法的核酸提取情况进行比较。结果:用三种核酸提取方法对新冠病毒核酸ORF1ab基因的提取检出率和对实验室确认阳性检出率有统计学差异,磁珠法最佳(P<0.05),对新冠病毒核酸N基因的提取检出率无统计学差异(P>0.05)。在稀释倍数为1:64的阳性标本,三种提取方法提取到的ORF1ab基因Ct结果有统计学差异(P<0.05),而提取到的N基因Ct结果无统计学差异(P>0.05);当稀释倍数为1:128时,浓缩法提取不到ORF1ab基因和N基因,而柱提法和磁珠法比较均有统计学差异(P<0.05);当稀释倍数为1:256时,磁珠法仍可以提取到N基因。结论:磁珠法是快捷有效的核酸提取方法,即使在高倍稀释的情况下也能够达到较高的核酸提取率,并能实现自动化,避免了手工提取的误差,能为临床诊治提供一个快速准确的检测手段,能适应新冠肺炎疫情大规模的筛查需求。Objective:The effects of three nucleic acid extraction methods in the detection of 2019 novel coronavirus were compared to provide a basis for optimizing the detection effect of 2019 novel coronavirus.Methods:A total of 50 samples of pharyngeal swabs from patients diagnosed with COVID-19 from February toMarch in 2020 were selected as the study subjects.A positive specimen was selected and diluted in proportion to 1:2,1:4,1:8,1:16,1:32,1:64,1:128 and 1:256 to obtain new coronavirus specimens of different concentrations.Three methods were used to extract viral nucleic acid(including ORF1ab gene and N gene)by centrifugal column method,paramagnetic particle method and concentration method,and fluorescence RT-PCR was performed.The nucleic acid extraction of the three methods was compared according to the amplification results.Results:There was a statistical difference between the detection rate of ORF1ab gene of new coronavirus nucleic acid and the positive detection rate of laboratory confirmation by three nucleic acid extraction methods(P<0.05).There was no statistical difference in the detection rate of new coronavirus nucleic acid N gene(P>0.05).In the positive samples with dilution ratio of 1:64,there was a statistical difference in the Ct results of the extracted ORF1ab gene by the three extraction methods(P<0.05),while therewas no statistical difference in the Ct results of the extracted N gene(P>0.05).When the dilution ratiowas 1:128,the ORF1ab gene and N gene could not be extracted by the concentration method,while there were statistical differences between centrifugal column method and paramagnetic particle method(P<0.05).When the dilution ratio is 1:256,N gene can still be extracted by paramagnetic particle method.Conclusion:Paramagnetic particle method is fast and effective nucleic acid extraction method,and even in the case of high dilution can achieve high nucleic acid extraction yield,and can realize automation,avoids the error of the extracted by manual.It can provide a rapid and accurate detection met

关 键 词：2019新型冠状病毒 柱提法 磁珠法 浓缩法 RT-PCR 

分 类 号：S85[农业科学—兽医学]