摘要
目的比较化学合成与体外转录的RNA作为参考品定量检测SIV/SHIV RNA的重复性、稳定性及灵敏度,建立一种实时、灵敏、特异的SIV/SHIV RNA载量检测方法。方法用化学合成的方法合成91个核苷酸(nucleotide,nt)的SIV/SHIV Gag RNA;用带T7启动子的特异性引物逆转录SHIVSF162 P3的基因组RNA,得到带T7启动子的DNA序列后,经体外转录合成385 nt Gag RNA。将两种RNA分别进行10倍系列稀释,通过一步法TaqMan探针荧光定量逆转录PCR(quantitative Real-time reverse transcription PCR;qRT-PCR)测定标准曲线和扩增曲线,10次独立实验重复测量,对比两种RNA的灵敏度和重复性,检测在4℃和25℃分别放置3 h、6 h和9 h后的两种RNA的扩增曲线和Ct值,比较两种RNA的稳定性。结果体外转录RNA组中,相同浓度复孔的扩增曲线重合度高,各浓度Ct值线性关系较好,标准曲线方程为:Y=-3.709x+49.143,R2=0.999,检测灵敏度为5.5×103拷贝/ml,10次重复测量的平均变异系数仅为2.24%;而化学合成RNA组的标准曲线方程为:Y=-4.014x+53.108,R2=0.944,检测灵敏度为5.5×104拷贝/ml,10次重复测量的平均变异系数为5.33%。体外转录RNA组检测结果的稳定性、重复性和灵敏度更高。结论使用体外转录RNA作为参考品,建立的检测方法的灵敏度、特异性、重复性及稳定性均优于化学合成的RNA,用该参考品建立的一步法TaqMan探针qRT-PCR检测方法可用于SIV/SHIV RNA载量分析。
Objective To establish a real-time,sensitive and specific SIV/SHIV RNA load detection method and compare the repeatability,stability and sensitivity of this method with in vitro transcriptions and chemical synthesis of RNA as reference materials.Methods SIV/SHIV Gag 91 nucleotide(nt)RNA was synthesized by chemical synthesis,DNA sequence with T7 promoter was obtained by reverse transcription of SHIVSF162 P3 RNA with T7 promoter specific primer,and Gag 385 nt RNA was synthesized by in vitro transcription.Two kinds of RNA were respectively diluted by 10-fold serial dilution,which was investigated by quantitative Real-time reverse transcription PCR(qRT-PCR).Standard curve and amplification curve were determined by qRT-PCR,and the sensitivity and repeatability of the two references were compared by repeated measurements in 10 independent experiments.Amplification curves and Ct values of the two RNA references were detected at 4℃and 25℃for 3 h,6 h and 9 h,respectively,to compare the stability of the two RNA references.Results In vitro transcribed RNA group,the amplification curves of multiple wells with the same concentration were highly coincident,and Ct values of each concentration had a good linear relationship.The standard curve equation was Y=-3.709x+49.143,R2=0.999.At the same time,the sensitivity of in vitro transcriptions of RNA was 5.5×103copies/ml,and the mean coefficient of variation of 10 repeated measurements was only 2.24%.However,the standard curve for the chemosynthetic RNA group was Y=-4.014x+53.108,R2=0.944,the detection sensitivity was 5.5×104 copies/ml,and the mean coefficient of variation for 10 repeated measurements was 5.33%.The data indicated that the method using in vitro transcribed RNA as reference was more stable,repeatable and sensitive.Conclusions The sensitivity,specificity,repeatability and stability of RNA reference prepared by in vitro transcription method were superior to those of the chemosynthetic RNA reference.The one-step TaqMan probe qRT-PCR method established by this stand
作者
李静
刘颖
李丹
申秀丽
王书晖
Li Jing;Liu Ying;Li Dan;Sheng Xiuli;Wang Shuhui(National Center for AIDS/STD Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China)
出处
《中华实验和临床病毒学杂志》
CAS
CSCD
2023年第6期623-630,共8页
Chinese Journal of Experimental and Clinical Virology
基金
"十三五"国家科技重大专项(2018ZX10731101)
国家重点研发计划(2016YFE0107600)。
关键词
体外转录
化学合成
灵敏度
稳定性
荧光定量PCR
In vitro transcription
Chemical synthesis
Sensitivity
Stability
Fluorescence quantitative PCR
