酶消化热裂解法处理血清样本进行RT-PCR检测的研究  

Digested thermal denaturalization method for transcriptase-polymerase chain reaction of serum specimen

作　　者：顾琳[1] 彭晓谋[1] 邓练贤[1] 谢冬英[1] 陈青[1] 

机构地区：[1]中山医科大学附属第三医院传染科病毒性肝炎研究室,510630

出　　处：《广东医学》1999年第5期331-333,共3页Guangdong Medical Journal

基　　金：广东省自然科学基金!970079;广东省卫生厅青年基金!97031

摘　　要：目的探讨相对简便和不易污染的直接热变性处理血清样本进行RT－PCR检测的敏感性和重复性不理想的原因，并对其进行改良。方法对比分析直接热变性上清液中的pH值和蛋白抑制因子对RT－PCR体系的pH值和PCR扩增效果的影响。评价改良方法的性能。结果10份正常血清直接热变性上清的pH值平均为8．96±1．34。与RT－PCRⅠ液和改良RT-PCRⅠ液混合后的pH值分别为8．55±0．53和8．32±0.07。直接热变性上清液对PCR的抑制作用这100－1000倍。这种抑制作用可被加入300μg/mL蛋白酶K消化而完全消除。消化热变性法的敏感性和重复性与传统的抽提法接近，且不易污染。结论pH值波动和蛋白性抑制因子的存在是直接热变性法重复性和敏感性差的重要原因。消化热变性法不仅敏感性高，重复性能与抽提法相媲美，而且简便性和防污染能力更为优越。Objective Direct thermal denaturalization of serum for RT-PCR is relatively slmpler and less subject to contamination.Howere,its sensitivety and stability were not ideal.Methods tThe effects of pH value and protein inhibitingfactors in supernatant of direct thermal denaturalization on pH system and amplifyin reaction were Comparatively analyzed. The property of improved method was evaluated.Results The average pH values in supernatant of directthermal denaturalizaion were 8.96 ± 1.34 among ten normal sera, 8.55 ± 0.53 and 8.32 ± 0.07 when mixed with RT- PCRsystem and improved RT - PCR system respectively. The inhibiting acivity of direct thermal denaturalization on PCR was from100 to 1000 times. This activity could abolish by protease K. The sensitivity and stability of digested thermal denaturalizationwere comparable to those of RNA extraction Conclusion The pH valus variance and existing of protein inhibiting factors arethe importan causes of inferior sensitivity and stability of direct thermal denaturalization. Digested thermal denaturalization arecomparable to RNA extraction in sensitivity and stability, and superior to RNA extraction in simoplification and protection fromcontamination.

关 键 词：聚合酶链反应 HCV-RNA HGV-RNA 肝炎 

分 类 号：R512.630.4[医药卫生—内科学]