一种检测kras基因突变的新型TB-ARMS qPCR方法的建立

Establishment of a novel TB-ARMS qPCR method for kras mutation detection

摘要:

在荧光定量PCR基础上建立一种简单有效并且高度灵敏的TB-ARMS kras基因突变检测方法，并对其检测性能进行评估，探讨其临床应用价值。针对kras基因8种常见的点突变类型，通过设计并优化突变特异性引物、野生型特异性封闭引物并综合应用突变富集扩增反应条件等多种手段，提高点突变检测的灵敏度和特异性，采用已知野生型基因组样品和构建的突变质粒作为标准品，进行方法学评价；通过对临床样本的检测及与现有商品化试剂盒的比较进行性能验证；通过对术前血浆和配对组织样品的对比检测，评估方法是否适用于血液样本的检测。建立了TB-ARMS kras突变检测的新方法，能检测的最低突变率可达到0.01%。通过综合采用野生型特异性封闭引物和突变富集扩增条件等方法证明了其0.01%的突变检测灵敏度。检测准确性优于现有商品化试剂盒，血浆DNA TB-ARMS qPCR检测结果与配对组织DNA测序结果相符合。因此，TB-ARMS kras基因突变检测方法具有广泛的临床应用价值，既适用于临床组织样品的检测，也可应用于液体活检。

Abstract:

A simple, robust and highly sensitive TB-ARMS method based on qPCR technique was developed to detect kras mutations. The technique was evaluated, and its clinical application was investigated. Mutation specific primers for eight common kras mutations and wild type gene targeted blockers were designed and optimized. Moreover, a mutant-enriched condition was used in to improve the sensitivity and specificity of mutation detection. Constructed plasmids carrying mutant kras genes, as well as confirmed wild type genomic DNA, were used as standard samples for evaluation of the methodology. The performance of our new method was validated by comparing the results of our method with that of a commercial kras kit in testing 40 clinical samples. Preoperative plasma samples, as well as paired tissue samples, were tested in parallel for evaluation of its clinical application. We have developed a new TB-ARMS method for kras mutation detection that can detect minor mutant alleles with a frequency as low as 0.01% in a heterogeneous sample. We have successfully demonstrated its 0.01% detection sensitivity with highly specific mutant amplification in conjunction with selective wild type suppression by blocker under a mutant-enriched reaction condition. We also showed that our TB-ARMS method was more accurate than the commercial kras kit, which is widely used presently. Furthermore, we have validated our method as an efficient liquid biopsy method, and the results of the plasma DNA detection with our TB-ARMS method were in consistent with the sequencing results of paired tissue samples. In conclusion, our TB-ARMS qPCR method could be effectively applied in kras mutation test for clinical tissue samples, as well as for liquid biopsy samples such as plasma.