基于RT-PCR法构建新型冠状病毒滴度对应核酸拷贝数的标准曲线

Construction of standard curves for nucleic acid copy numbers corresponding to SARS-CoV-2 titers based on RT-PCR

目的获得反转录-聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)检测的新型冠状病毒滴度与核酸拷贝数的对应关系,以提高检测效率。方法通过RT-PCR法用新型冠状病毒标准品构建循环阈值(CT值)对应核酸拷贝数的标准曲线。按照感染复数(MOI)=0.0001分别接种新型冠状病毒原型株、南非(beta)株、印度(delta)株和奥密克戎株至T75细胞瓶中培养72 h,收获液梯度稀释后利用终点稀释法测定活病毒滴度,清除收获液中残余RNA后,检测各梯度对应病毒核酸拷贝数。结果利用核酸标准物质构建了基于N基因的标准曲线为Y=-3.289X+36.86,基于ORF基因的标准曲线为Y=-3.177X+35.77。残余RNA清除实验结果显示RNase A的加入对于病毒液样品影响显著。对4型别新型冠状病毒收获液检测滴度和拷贝数后,获得原型株基于N基因的滴度对应拷贝数标准曲线为Y=1.039X+3.422,ORF基因为Y=0.9917X+3.209;beta株N基因标准曲线为Y=1.071X+3.316,ORF基因为Y=1.031X+3.447;delta株N基因标准曲线为Y=1.138X+3.410,ORF基因为Y=1.115X+2.991;omicron株N基因标准曲线为Y=1.062X+4.493,ORF基因为Y=1.007X+4.271。结论本研究构建了病毒滴度对应核酸拷贝数的标准曲线,并且具有良好的相关性。相较于常规滴度测定法,本研究建立的新方法极大提高了检测效率和数据准确性,为新型冠状病毒相关实验和数据处理分析提供新的思路与方法。

Objective To quantify the nucleic acid copy numbers corresponding to different levels of SARS-CoV-2 titers detected using reverse transcription-polymerase chain reaction(RT-PCR)assays,so as to improve detection efficiency.Methods A standard curve was constructed by RT-PCR using the SARS-CoV-2 stan⁃dard for the nucleic acid copy numbers corresponding to the cycling threshold(CT).The prototype,South Afri⁃can(beta),Indian(delta)and omicron strains of SARS-CoV-2 were inoculated in T75 cell flasks with multi⁃plicity of infection(MOI)of 0.0001,and incubated for 72 h.The live virus titer was determined using endpoint dilution assay after gradient dilution of the harvested solution,and the nucleic acid copy number of the virus corre⁃sponding to each gradient was measured after removing the residual RNA from the harvested solution.Results The standard curve constructed using nucleic acid standard for N gene was Y=-3.289X+36.86,and the standard curve for the ORF gene was Y=-3.177X+35.77.The results of residual RNA removal assay showed that the addition of RNase A had a significant effect on the viral fluid samples.Based on the titers and copy numbers of the four strains of SARS-CoV-2 tested with the harvested solution,the standard curve for copy numbers corresponding to titers of the prototype strain was Y=1.039X+3.422 for the N gene Y=0.9917X+3.209 for the ORF gene;the standard curve for the beta strain was Y=1.071X+3.316 for the N gene and Y=1.031X+3.447 for the ORF gene;the standard curve for the delta strain was Y=1.138X+3.410 for the N gene and Y=1.115X+2.991 for the ORF gene;the standard curve for the omicron strain was Y=1.062X+4.493 for the N gene and Y=1.007X+4.271 for the ORF gene.Conclusions In this study,a nucleic acid copy number standard curve is constructed for corresponding virus titers with satisfactory correlation.Compared with the conventional titer assay,the novel method established in this study has greatly improved the efficiency and data accuracy,providing a direction for experiment and data analysis in research on SARS-CoV-2.