基于荧光探针溶解曲线分析技术的HPA 1～5,15等位基因检测方法的建立

Establishment of detecting method of HPA1-5,15 allele based on fluorescence probe melting curve analysis

目的结合多重聚合酶链式反应（PCR）和荧光探针溶解曲线单核苷酸多态性（SNP）分型技术,建立一种方便准确的HPA等位基因分型方法。方法针对HPA 1~5,15这6种常见的HPA亚型等位基因设计多重PCR引物及SNP探针,通过多重PCR同时扩增6个等位基因目标片段并利用荧光探针的溶解温度变化区分不同SNP位点。结果多重PCR可同时扩增6个等位基因的目标片段,并通过溶解峰的Tm值变化区分SNP位点,结果与PCR-SSP位点相比较有相同的准确度,且操作更为简便,无需PCR后续电泳步骤,减少污染风险。结论成功建立了一种结合多重PCR和荧光探针溶解曲线分析技术的HPA 1~5,15等位基因检测方法。

Objective Establish a convenient and accurate HPA allele discriminate method based on multiplex PCR and fluorescent probe melting curve analysis SNP discrimination technique. Methods Designing Multiplex PCR primers and SNP probes targeting 6 frequent HPA sub-types including HPA1,2,3,4,5 and 15. Amplifying target sequences of 6 HPA alleles simultaneously by multiplex PCR and discriminate SNPs by Tm changes of fluorescent probes. Results Multiplex PCR could amplifying target sequences of 6 alleles simultaneously and discriminate SNPS by Tm changes of melting peaks,the accuracy is the same as PCR - SSP method and the procedure is more convenient without after - PCR electro-phoresis step,thus reduce the contamination risk. Conclusion Successfully establish an HPA1 - 5,15 alleles detection methods combining multi-plex PCR and fluorescent probe melting curve analysis technique.