单细胞PCR用于HLA配型的研究

Study of single cell PCR for HLA typing

目的研究用筑巢式多重PCR技术对单细胞进行HLA配型，分析影响单细胞PCR扩增的因素。方法首先分别采用不同的细胞裂解方法制备单细胞DNA模板，然后采用多重PCR分别扩增HLA—A，B基因的第2、3外显子和第2内含子区域，HLA—DRB1基因的第2外显子区域，最后根据大量DNA的常规HLA分型结果对单细胞第1轮扩增产物进行第2轮筑巢式序列特异性引物PCR（PCR-SSP）分型。结果酶解法制备单细胞DNA模板效率最高，第1轮扩增成功率为93．3％，而碱裂法和冻融法分别为83．3％和73．3％；采用酶解法制备单细胞DNA模板进行第2轮PCR—SSP分型验证，20份标本扩增成功率为95％，有3份标本只扩增出一条染色体，等位基因脱扣发生率为15％，全部操作可在6h内完成。结论筑巢式多重PCR技术对单细胞HLA分型具有较高的扩增效率，操作时间短，可望应用于妊娠前诊断。

Objective To apply the single cell nested multiplex polymerase chain reaction （PCR） to HLA typing, and analyze the influence factors on the amplification results. Methods Single cell DNA tem- plates were prepared with different methods. The exon 2, 3 and intron 2 of HLA-A, B, and exon 2 of DRB1 were amplified using multiplex PCR. The second round of SSP-PCR HLA typing was carried out according to the large scale routine HLA typing results. Results Enzyme lysis method was the most efficient procedure for preparing the single cell DNA template, with a success rate （SR） of 93.3%, while the SRs of alkali lysis and freezing-thaw lysis methots were 83.3% and 73.3% , respectively. The second round amplification using enzyme lysis and SSP-PCR in 20 samples obtained a 95% success rate and a 15% allele drop out rate. The time for performing the whole procedure was less than 6 hours. Conclusion The modified nested multiplex PCR technique is efficient for single cell HLA typing and might be applied to clinical preimplantation genetic diagnosis.