Establishment and clinical evaluation of a ARMS-qPCR-based assay for detection of SLC25A13 c.2T>C mutation
10.3760/cma.j.cn112150-20230804-00059
- VernacularTitle: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
- Author:
Linxuan GUO
1
;
Wenhui WU
;
Cuiyuan PAN
;
Zhanhui ZHANG
;
Long XIE
;
Xiwen JIANG
Author Information
1. 广东药科大学临床医学院，广州 510006
- Keywords:
Amplification-refractory mutation system quantitative real-time polymerase chain reaction;
Peripheral blood;
Citrin deficiency
- From:
Chinese Journal of Preventive Medicine
2024;58(4):539-544
- CountryChina
- Language:Chinese
-
Abstract:
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).
