摘要
目的采用不同实时荧光PCR方法检测各类型DNA污染模拟样本,分析检测方法性能,为在新型冠状病毒(简称新冠病毒)核酸检测中鉴别DNA污染提供依据。方法取实验室保存的新冠病毒RNA核酸样本和新冠病毒阳性质控品(含病毒序列的质粒样本),按不同比例混合,并进行梯度稀释,制成模拟样本。分别使用实时荧光RT-PCR、灭活逆转录酶前加样并去除RT步骤的PCR(PCR Set 1)和灭活逆转录酶后加样并去除RT和灭活步骤的PCR(PCR Set 2)三种不同方法,使用同一型号设备对同一批模拟样本进行检测。记录并分析检测结果,确定最佳的DNA污染判断方法。结果新冠病毒RNA核酸样本中,相比实时荧光RT-PCR,PCR Set 1下所有样本的Ct值增高2-4个循环,PCR Set 2中除原始样本N基因阳性外,均为阴性。新冠病毒DNA污染样本三种方法检测Ct值无明显差异。含等量DNA污染的混合污染样本和含梯度DNA污染的混合污染样本三种方法检测显示Ct值存在差异。三种方法检测不同新冠病毒基因,变化规律无明显差异。结论PCR Set 1和PCR Set 2两种方法均可用于鉴别DNA污染,特别是PCR Set 2方法对各浓度的DNA污染均有较好的区分能力。
Objective To use different real-time PCR methods to detect the simulated viral RNA samples contaminated severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),to analyze the performance of the detection methods so as to provide a basis for identifying DNA contamination in SARS-CoV-2 nucleic acid testing.Methods The SARS-CoV-2 viral RNA and the SARS-CoV-2 DNA postive control(plasmid containing virus sequence),which stored in the laboratory,were mixed in different proportions,and then diluted in a gradient dilution to serve as simulated samples.We used three different methods,real-time RT-PCR,PCR with inactivated reverse transcriptase before loading and removal of the RT step(PCR Set 1),and PCR after inactivation of reverse transcriptase loading and removal of RT and inactivation step PCR(PCR Set 2),and utilized the same type of equipment to test the same simulated samples.The test results were analyzed to determine the best DNA contamination judgment method.Results Compared with real-time RT-PCR,the Ct(threshold cycle)value of all samples under PCR Set 1 increased by 2-4 when testing SARS-CoV-2 RNA samples.In PCR Set 2,all samples were negative except the positive N gene of the original sample.There was no significant difference in the Ct value of the three methods for the detection of samples contaminated by SARS-CoV-2 DNA.Three methods of detection of mixed contamination samples containing the same amount of DNA contamination and mixed contamination samples containing gradient DNA contamination showed differences in the Ct values.Similar pattern was observed in three methods when detecting different SARS-CoV-2 genes.Conclusion Both PCR Set 1 and PCR Set 2 methods can be used to identify DNA contamination.In particular,PCR Set 2 has better ability to distinguish DNA contamination at various levels.
作者
梁志超
潘阳
林长缨
李砚萍
李红
王斌
崔淑娟
张代涛
LIANG Zhi-chao;PAN Yang;LIN Chang-ying;LI Yan-ping;LI Hong;WANG Bin;CUI Shu-juan;ZHANG Dai-tao(Beijing Center for Disease Prevention and Control,Beijing Research Center for Preventive Medicine,Beijing 100013,China)
出处
《实用预防医学》
CAS
2022年第9期1064-1067,共4页
Practical Preventive Medicine
基金
首都卫生科研发展专项(首发2021-1G-3012)。
关键词
新型冠状病毒
核酸检测
DNA污染
实时荧光逆转录PCR
severe acute respiratory syndrome coronavirus 2
nucleic acid detection
DNA contamination
real-time RT-PCR
