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. 2021 Jan 30;41(1):151-156.
doi: 10.12122/j.issn.1673-4254.2021.01.23.

[A non-invasive method for detecting mitochondrial tRNAThr15927G>A mutation]

[Article in Chinese]
Zhining Tang  1 , Xiaowen Tang  1 , Ling Xue  1 , Minxin Guan  1
Affiliations

[A non-invasive method for detecting mitochondrial tRNAThr15927G>A mutation]

[Article in Chinese]
Zhining Tang et al. Nan Fang Yi Ke Da Xue Xue Bao. .

Abstract
in English, Chinese

Objective: To explore the feasibility of detecting maternal hereditary mitochondrial tRNAThr15927G>A (m.15927G>A) mutation using buccal swabs.

Methods: We performed sequence analysis of mitochondrial DNA in blood samples from 2070 cases of maternal hereditary mitochondrial disease in the First Affiliated Hospital of Wenzhou Medical University, and identified 3 patients with m.15927G>A mutation.Buccal swabs and blood samples were obtained from the 3 patients (mutation group) and 3 normal volunteers (control group).After extracting whole genomic DNA from all the samples, the DNA concentration and purity were analyzed.The PCR products were subjected to dot blot hybridization, Southern blot hybridization, and DNA sequencing analysis to verify the feasibility of detecting m.15927G>A mutation using buccal swabs.

Results: There was no significant difference in DNA concentration extracted from buccal swabs and blood samples in either the mutation group or the control group (P > 0.05), but the purity of manually extracted oral mucosa DNA was significantly lower than that of whole blood and oral mucosa DNA extracted using commercial kits (P < 0.05).Dot blot hybridization and Southern blot hybridization both yielded positive results in the control group but negative results in the mutation group.DNA sequencing identified m.15927G>A mutation in all the samples from the mutation group.

Conclusions: Buccal swabs collection accurate is an accurate and sensitive method for the detection of m.15927G>A mutation.

目的: 探索口腔黏膜拭子法在临床上检测母系遗传性线粒体tRNAThr15927G>A (m.15927G>A)突变的可行性。

方法: 对温州医科大学附属第一医院2070例母系遗传性线粒体疾病病例进行测序筛选, 获得3例携带m.15927G>A突变病例(突变组)和3例正常志愿者(对照组)的口腔黏膜样本和血液样本。提取样本全基因组DNA后进行DNA浓度和纯度分析, 再对其PCR产物进行斑点印迹杂、Southern blot印迹杂交和DNA测序分析, 以验证口腔黏膜拭子法检测m.15927G>A突变的可行性。

结果: 突变组和对照组口腔黏膜样本和血液样本提取的DNA浓度无显著差异(P＞0.05), 而手工法提取的口腔黏膜DNA纯度显著低于试剂盒法提取的血液及口腔黏膜DNA纯度(P＜0.05)。斑点印迹杂交和Southern Blot印迹杂交提示对照组均呈阳性结果, 而突变组均呈阴性结果。DNA测序结果示突变组中均可检测到m.15927G>A突变。

结论: 口腔黏膜拭子法是一种准确、灵敏的无创检测m.15927G>A突变的方法, 具有较高的临床应用价值。

Keywords: buccal swab collection; m.15927G>A mutation; mitochondrial mutation; non-invasive detection; tRNAThr15927G>A mutation.

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Figures

1
1
巢式PCR Nested PCR.
2
2
口腔黏膜样本DNA测序峰图 Sanger sequencing of PCR products amplified from genomic DNAextracted from buccal swabs.
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口腔黏膜样本和血液样本的斑点印迹杂交和Southern blot印迹杂交分析 Dot blot hybridization and Southern blot hybridization analysis of buccal swab samples and blood samples. A: Dot blot hybridization analysis; B: Southern Blot hybridization analysis. DNA1: DNA extracted manually from buccal swab samples; DNA2: DNA extracted from buccal swab samples using QIA amp DNA Mini Kits; DNA3: DNA extracted from blood samples using HiPure Tissue DNAMini Kits.
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