低拷贝病毒RNA捕获及多重实时荧光定量PCR检测  

RNA Capture and Multiplex real-time Fluorescence Quantitative PCR Detection of Low Copy Virus

作　　者：余佳 李晓宁[1] 肖君华[1] 李凯[1] 周宇荀[1] YU Jia;LI Xiao-ning;XIAO Jun-hua;LI Kai;ZHOU Yu-xun(School of Chemistry and Bioengineering,Donghua University,Shanghai,201620,China)

机构地区：[1]东华大学化学化工与生物工程学院,上海201620

出　　处：《现代生物医学进展》2022年第20期3806-3812,共7页Progress in Modern Biomedicine

基　　金：中央高校基本科研业务费专项基金项目(2232020G-04);中央高校基本科研业务费学科交叉重点计划项目(2232020A-07)。

摘　　要：目的:为提高COVID-19检测灵敏度,减少新冠患者临床假阴性检测结果,本研究建立了一种SARS-CoV-2低拷贝假病毒RNA捕获方法。方法:利用链霉亲和素磁珠,设计并合成生物素探针捕获SARS-CoV-2假病毒RNA;对磁珠用量、生物素探针浓度和洗脱次数等捕获条件进行了优化;参考WHO发布的序列,针对SARS-CoV-2病毒基因组的ORF1ab基因和N基因序列合成引物和TaqMan探针,对捕获的SARS-CoV-2假病毒RNA进行反转录实时荧光定量PCR检测。采用一步法和分步法检测、单重和多重实时荧光定量PCR检测,并分别比较检测结果。结果:本研究设计的生物素探针能富集到距离探针结合位点至少1.8 K处的序列,在生物素探针浓度12.5μM,磁珠的工作浓度333.33μg/mL时,合并RNA的两次洗脱液,对低拷贝病毒RNA的捕获效率最高。研究得到对低拷贝病毒RNA分步法比一步法、多重比单重实时荧光定量PCR检测更灵敏,进行分步法、多重实时荧光定量PCR检测限低于10 copies/μL,组间和重复实验组之间CT值变异系数均小于1%。所有阴性对照样品在检测中均为阴性。结论:本研究优化的链霉亲和素磁珠-生物素探针捕获法是富集SARS-CoV-2低拷贝假病毒RNA的有效方法,其抽提RNA病毒的结果优于一些商业化试剂盒,分步法、多重实时荧光定量PCR对SARS-CoV-2低拷贝假病毒RNA实现了高效检测,这为临床检测低拷贝RNA病毒提供了一种参考方案。Objective:To improve the detection sensitivity of COVID-19 and reduce the clinical false negative results,this paper constructed a SARS-CoV-2 RNA capture method which can extract low copy RNA.Methods:The biotin probe in this study was designed based on the conserved region of the SARS-CoV-2 genome sequence.Streptavidin magnetic beads-biotin probe capture method was used to capture SARS-CoV-2 pseudovirus RNA by optimizing capture conditions such as magnetic beads concentration,biotin probes concentration and the number of RNA elution times.SARS-CoV-2 pseudovirus was detected by reverse transcription real-time fluorescence quantitative PCR with reference to the SARS-CoV-2 ORF1ab and N gene primers and TaqMan probes published by WHO.This study used one step and step-by-step real-time fluorescence quantitative PCR detection,single and multiple real-time fluorescence quantitative PCR,and the detection results were compared respectively.Results:The biotin probe can capture the RNA sequence at least 1.8K away from the probe binding site in this study.When the biotin probe concentration was 12.5μM and the concentration of magnetic beads was 333.33μg/mL,combined with the twice eluent of RNA,the capture efficiency was the highest for low copy SARS-CoV-2 pseudovirus RNA.The study results showed that the step-by-step method was more sensitive than one-step method and two TaqMan probes were more sensitive than one TaqMan probe for real-time fluorescence quantitative PCR,and the minimum detection limit of the step-by-step and multiple real-time fluorescence quantitative PCR was 10 copies/μL.The intra-group and repeated experimental groups coefficient of variation was less than 1%.All negative control samples were negative in the study.Conclusions:The streptavidin magnetic beads-biotin probe capture method optimized in this study was an effective method to capture SARS-CoV-2 low copy pseudovirus RNA,the results of RNA extraction were better than some commercial kits,step-by-step and multiple real-time fluorescence quantitati

关 键 词：SARS-CoV-2假病毒 链霉亲和素磁珠 生物素探针 多重实时荧光定量PCR 

分 类 号：Q-33[生物学] Q78Q813