单细胞水平检测von Hippel-Lindau病基因突变的实验研究

目的建立单细胞水平检测vonHippel-Lindau病基因(VHL)突变的实验方法。方法单个淋巴细胞基于多重置换扩增(multiple displacement amplification,MDA)的全基因组扩增后进行常规PCR后测序和实时荧光定量PCR,结合各荧光的终点变化判断对应的等位基因存在与否。结果MDA后单细胞扩增效率为90.91%,污染率为0;通过测序,患者VHL等位基因的脱扣率为26.67%,诊断正确率为73.33%;通过结合相应荧光的终点变化判断患者VHL基因的等位基因的脱扣率为16.67%,正确率为83.33%。结论单细胞MDA后常规PCR后测序及实时荧光定量PCR能够特异、准确地检测单个淋巴细胞的VHL基因型,两者的联合应用有助于提高检测的准确性。

The detection of mutations in VHL gene from a single cell in a patient with von Hippel-Lindau disease

Objective To explore a technology for diagnosing VHL mutations from a single cell and provide experimental evidences for the feasibility of applying technology in detecting genetic mutations from a single cell. Methods After whole-genome amplification (WGA) based on multiple displacement amplication (MDA) for a single cell, we did regular PCR following sequencing and detected the genotypes using the real time PCR based on TaqMan probes. We detected VHL mutations by the different terminal fluorescent changing. Results The rate of amplification for single cell based on MDA was 90.91%. The rate of contamination was 0. After sequencing, the allele drop out (ADO) rate of heterozygotes was 26.67% (8/30); combined with the different terminal fluorescent changing, the rate of ADO of heterozygotes was 16.67%. Conclusion WGA based on MDA for a single cell followed by regular PCR with sequencing and real time PCR can specially and accurately detect the VHL genotypes of single cells.