改良固相吸附法在HCV RNA实时荧光定量检测中的应用  

Application of modified solid-phase adsorption method in real-time fluorescence quantification of HCV RNA

作　　者：张宝华[1] 王红 倪二茹[1] 

机构地区：[1]南京军区福州总医院第二住院部检验科,福建福州350003 [2]北京总参管理保障部北极寺门诊部,北京100191

出　　处：《国际检验医学杂志》2014年第5期562-564,共3页International Journal of Laboratory Medicine

摘　　要：目的：通过对固相吸附法的改良，探讨采用固相吸附法提取血清中丙型肝炎病毒RN A在实时荧光定量检测中的应用。方法首先对HCV RNA的提取方法进行改良，对固相载体纳米二氧化硅颗粒表面进行硅醇基化以提高其核酸吸附能力；同时对病毒裂解液进行优化，获得异硫氰酸胍（GuSCN ）的最佳浓度用于 HCV RNA 的提取；最后使用改良方法提取的 HCV RNA进行实时荧光定量PCR实验，并绘制其标准曲线，计算出线性方程，同时检测该提取方法下实时荧光定量PCR对血清样本中HCV RNA的最低检测限并对其检测重复性进行验证。结果纳米二氧化硅颗粒硅醇基化后其吸附核酸的能力显著提高；病毒裂解液中GuSCN的最佳浓度为4．23 mol/L。使用改良方法提取血清样本中的HCV RNA用于实时荧光定量PCR ，获得标准曲线和线性方程，其线性相关系数（R2）达到0．999，检测的线性范围为2．52～5．52 IU/mL ，或者102．52～105．52病原体数/毫升。该方法的检测限达到（2．52＆#177;0．50）IU/mL ，且其检测重复性良好。结论改良后的固相吸附法可以大大降低实时荧光定量PCR的最低检测限，提高了HCV的阳性检测率。该方法操作简单，成本低，可以广泛应用于临床中HCV的检测和其他病毒的核酸检测。Objective To explore the application of adopting the solid phase adsorption method to extract serum hepatitis C virus （HCV） RNA in the real-time fluorescence quantification PCR（ RT-qPCR） by the improvement of solid phase adsorption method . Methods Firstly ,the surface of silica nano-particles was modified with Si-OH for increasing their adsorption ability with HCV RNA .Then the concentration of guanidinium thiocyanate （GuSCN） in the viral lysate was optimized .Thirdly ,the HCV RNA of standard serum samples was extracted using the modified solid-phase adsorption method and then performed in the RT-qPCR .Its standard curve was drawn and the linear equation was calculated .At the same time ,the lowest detection limit of RT-qPCR for de-tecting HCVRNA in serum sample was measured and the detection repeatability was verified .Results The adsorption ability of sil-ica nano-particles for HCV RNA in serum samples was remarkably improved after surface modification with Si-OH .The optimal GuSCN concentration in viral lysates was 4 .23 mol/L .Using the modified method to extract HCV RNA in serum samples was ap-plied in RT-qPCR ,the standard curve and the linear equation were obtained ,the linear correlation coefficient（R2） reached to 0 .999 and the detected linear range was （2 .52 -5 .52） IU/mL or 102 .52 -105 .52 pathogen number/mL .The detection limit of HCV RNA in serum samples was （2 .52 ＆#177; 0 .50）IU/mL with good detection repeatability .Conclusion The modified solid-phase adsorp-tion method can greatly decrease the lowest detection limit of RT-qPCR and increase the positive detection rate of HCV .This meth-od is simple to operate ,has low cost and can be widely applied in the HCV detection and the nucleic acid detection of other viruses in clinic .

关 键 词：纳米二氧化硅 固相吸附 聚合酶链反应 

分 类 号：R450[医药卫生—治疗学]