基于ARMS-qPCR技术检测 SLC25A13基因c.2T>C突变方法的建立及临床验证

Establishment and clinical evaluation of a ARMS-qPCR-based assay for detection of SLC25A13 c.2T>C mutation

建立一种基于荧光定量PCR的扩增阻滞突变系统方法(amplification-refractory mutation system quantitative real-time PCR,ARMS-qPCR)检测 SLC25A13基因c.2T>C突变并验证其诊断性能。根据ARMS-qPCR引物设计原则,针对 SLC25A13的保守序列设计特异性引物,利用人工合成纯合突变型质粒及200份已测序验证的人外周血核酸样本建立 SLC25A13基因c.2T>C突变ARMS-qPCR检测体系及其Sanger测序体系,并应用提取自另外其他200份人外周血的核酸样本验证此体系的诊断效能,所获结果与作为金标准的Sanger测序结果对比,分析2种检测方法的一致性。结果显示,本研究建立的ARMS-qPCR体系可准确区分 SLC25A13基因野生型及携带c.2T>C突变型;利用该方法检测人外周血中 SLC25A13 c.2T>C突变状态,其检测结果与Sanger测序结果一致性为100%。200份外周血样本中,携带 SLC25A13基因c.2T>C突变8份(4%),非携带者192份(96%)。综上,本研究建立的ARMS-qPCR测试能够快速、准确地检测 SLC25A13基因c.2T>C突变,有助于希特林蛋白缺陷病(citrin deficiency,CD)的诊断。

To establish the amplification-refractory mutation system quantitative real-time PCR(ARMS-qPCR)method based on qPCR technique for detecting the c.2T>C mutation of SLC25A13 gene and validate its diagnostic performance.According to the principle of ARMS-qPCR primer design,the specific primers were designed for the conserved sequence of SLC25A13.The c.2T>C mutation ARMS-qPCR detection assay of SLC25A13 gene and the corresponding Sanger sequencing system were established through the use of the synthetic plasmids of homozygous mutation and 200 human peripheral blood specimens which were verified by Sanger sequencing as templates,and the diagnostic efficacy of the qPCR assay was validated by using nucleic acid extracted from another 200 human peripheral blood specimens and the results obtained were compared with the Sanger sequencing results as the gold standard,and the consistency of the two detection methods was analyzed.The results showed that the qPCR assay could accurately identify artificial plasmids carrying different mutations of SLC25A13 gene,and distinguish between wild type SLC25A13 gene and the c.2T>C mutation.This method was used to detect the mutation status of SLC25A13 c.2T>C in human peripheral blood,and the detection results were 100%consistent with the Sanger sequencing results.Among the 200 blood samples,8 samples(4%)carried the c.2T>C mutation of SLC25A13 gene and 192 samples(96%)did not carry it.In conclusion,the ARMS-qPCR test established in this study can quickly,simply and accurately detect the c.2T>C mutation of SLC25A13 gene,which is helpful for the diagnosis of citrin deficiency(CD).