罗氏HIV-1定量检测引物SK145和SKCC1B的评估及优化  

Assessment and optimization of Roche SK145 and SKCC1B primers for quantification of human immunodeficiency virus type 1

作　　者：樊路娟 张春霞[1] 徐庆刚[1] 张驰宇[1,2] 

机构地区：[1]江苏大学生命科学研究院,江苏镇江212013 [2]中国科学院上海巴斯德研究所,上海200025

出　　处：《江苏大学学报（医学版）》2014年第1期26-30,共5页Journal of Jiangsu University：Medicine Edition

基　　金：国家自然科学基金资助项目(81071391)

摘　　要：目的:评估罗氏HIV-1检测系统中引物SK145和SKCC1B对不同亚型HIV-1病毒定量检测的广谱性及准确性,并在此基础上对该引物进行优化。方法:下载并分析5个主要HIV-1流行亚型(B、C、A、01_AE、D)在引物SK145和SKCC1B扩增区段的对应序列,从各亚型筛选出所占比例最高的5条序列构建定量标准品。用梯度稀释的HIV-1各亚型标准品来评估引物SK145和SKCC1B的扩增效率。为改善引物SK145和SKCC1B对HIV-1不同亚型扩增的准确性和广谱性,我们重新设计了一对简并引物并分析其对不同亚型病毒的扩增效果。结果:引物SK145、SKCC1B对于B亚型病毒的扩增效果最好,而对非B亚型病毒的扩增效果并不理想,简并引物能显著改善对各亚型HIV-1病毒扩增的效果。结论:罗氏引物SK145和SKCC1B不适用于非B亚型HIV-1病毒载量的定量检测,而优化设计的简并引物能改善对各亚型HIV-1病毒的扩增效率,因而可用于中国等HIV-1多亚型共流行国家的HIV-1病毒载量检测。Objective： To assess and optimise applicability and accuracy of primers SK145 and SKCCI B for different HIV-1 subtypes in viral load quantification assay. Methods： We downloaded and analyzed genomic sequences of five main HIV-1 subtypes （B,C,A,01_AE,D） with the primers SK145 and SKCC1 B target. The top five dominant sequences were selected from each subtype sequences for the construction of quantification standards. We evaluated amplification efficiency of primers SK145 and SKCCIB for serially diluted standards of each HIV-I subtype. To improve the amplified spectrum primers SK145 and SKCCI B for different HIV-1 subtypes, re-designed this primer pair into degenerate primers according to the top dominant sequences of different HIV-1 subtypes. Results： The amplification efficiency of primers SK145 and SKCCI B was best on quantification HIV-1 subtype B, but not ideal for the non-B subtypes, indicating that primers SK145 and SKCC1B were not able to quantify accurately the viral load of non-B HIV-1 subtypes. When the degenerate primers were used, we obtained relatively consistent amplification efficiency for various HIV-1 subtypes. Conclusion： Roche SK145 and SKCC1B primers were unsuitable for viral RNA quantification of HIV-1 non-B subtypes. Compared to Roche SK145 and SKCC1B primers, the optimized degenerate primers can efficiently amplify various HIV-1 subtypes, suggesting that they can be used for HIV-1 viral load assay in China and other countries where various HIV-1 subtypes are co-criculating.

关 键 词：人类免疫缺陷病毒1型 实时荧光定量PCR SK145 SKCC1B 

分 类 号：R512.91[医药卫生—内科学]