博尔纳病病毒荧光定量PCR试剂盒扩增缓冲液的优化  

Optimization of PCR buffer for Borna disease virus real-time PCR diagnostic kit

作　　者：金戈[1,2] 徐晓艳[1,2] 张亮[1,2] 马丽华[1,2] 黄荣忠[1,2] 邓婧[1,2] 李文娟[1,2] 房亮 谢鹏[1,2] 

机构地区：[1]重庆医科大学附属第一医院神经内科,重庆400016 [2]重庆医科大学神经科学研究中心重庆市神经生物学重点实验室,重庆400016

出　　处：《第三军医大学学报》2011年第13期1323-1327,共5页Journal of Third Military Medical University

基　　金：国家重点基础研究发展计划(973计划;2009CB918302);重庆市自然科学基金(CSTC2010BB5393);重庆市卫生局医学科学技术研究项目(2010-2-102)~~

摘　　要：目的选择并优化博尔纳病病毒荧光定量PCR TaqMan探针法反应体系中的扩增缓冲液,使PCR反应体系对该病毒p24模板基因的扩增效率得到明显提高。方法通过常规PCR比较选择3种常用酸(盐酸、磷酸和硫酸)所滴定的缓冲液体系,在其基础上选择适合浓度的硫酸铵配制TSS buffer,在TSS buffer中分别加入四甲基氯化铵、TritonX-100、二甲亚砜和甜菜碱这4种常用的PCR添加剂,并确定各自的最佳工作浓度,选择扩增效果较好的添加剂与其他添加剂进行依次组合,寻找最佳的添加剂组合,最后用荧光定量PCR进行对比验证。结果在滴定酸上选择了效果最好的硫酸,配制TSS buffer硫酸铵的最佳终浓度为6 mmol/L。在添加剂的优化组合上,1.5 mol/L甜菜碱、0.15%Triton X-100、8 mmol/L四甲基氯化铵和TSS buffer组合成的TBTT buffer效果最佳,在其与TaKaRa PCR buffer的对比中,TBTT buffer效果明显好于后者。结论 成功研制优化出适合扩增博尔纳病病毒p24模板基因的扩增缓冲液。Objective To select and optimize the PCR buffer for Borna disease virus real-time fluorescence quantitative PCR diagnostic kit in order to significantly improve PCR amplification efficiency to BDV p24 gene.Methods Conventional PCR was used to contrast the PCR buffers which titrated with hydrochloric acid,phosphoric acid and sulfuric acid respectively,and the appropriate concentration of ammonium sulfate was further explored to prepare TSS buffer.Four PCR additives（TMAC,Triton X-100,DMSO and betaine） were added into TSS buffer respectively to determine each optimal working concentration.One appropriate additive was mixed into other additives by turns in order to find out the most effective combination.Finally the efficiency of these different PCR buffers was verified by real-time PCR.Results Compared to other acids,sulfuric acid was more suitable to titrate the buffer.The optimal concentration of ammonium sulfate was identified as 6 mmol/L to prepare TSS buffer.Furthermore,real-time PCR results indicated that TBTT buffer（TSS buffer mixed with 1.5 mol/L betaine,8 mmol/L TMAC,and 0.15% Triton X-100） was the most effective buffer compare to other PCR additive mix and was more effective to amplify p24 gene than Takara PCR buffer.Conclusion The PCR buffer for amplifying BDV p24 gene is successfully optimized.

关 键 词：PCR扩增缓冲液 PCR试剂盒 博尔纳病病毒 

分 类 号：Q93-335[生物学—微生物学] R373.31[医药卫生—病原生物学]