摘要
目的评价高分辨熔解(HRM)技术进行单核苷酸多态性(SNP)基因分型的检验性能。方法以SNP位点rs1205C>T和rs4353A>G为例,采用盲法对361例已分型确认样本进行PCR-HRM检测评价其特异性和灵敏度;通过重复试验评价其重复性和再现性。稳健性评价主要考察DNA模板量和扩增循环数对PCR-HRM检测的影响。结果 PCR-HRM法检测361例标本的rs1205C>T和rs4353A>G两个SNP位点,结果除5例样本的熔解曲线发生飘移接受复查外,其余标本均一次直接基因分型;所有标本的最终分型结果完全正确即灵敏度和特异性达到100%。两个位点的10次批内和3次批间重复试验均显示PCR-HRM法的重复性和再现性良好。rs1205C>T和rs4353A>G的野生型与纯合突变型样本熔解峰Tm值的变异系数分别为0.7%与0.8%和0.6%与0.5%;统计学分析显示每个位点的两种纯合型样本Tm值之间均存在显著性差异(P<0.001),即可明确区分不同纯合基因型。DNA模板量的变化(10ng、20ng、50ng和100ng)和扩增循环数的波动(38、40和42次)均对PCR-HRM法正确基因分型影响不大。结论基于小片段扩增子对rs1205C>T和rs4353A>G两个SNP位点进行常规化PCR-HRM法基因分型在方法学上是可靠的,值得推广;同时这一评估模式也适于其他分子诊断方法的评价。
Objective To evaluate the detection performance of genotyping for single nueleotide polymorphism（SNP） using high resolution melting （HRM）technique. Methods As an example of detecting two SNPs of rs1205C 〉T and rs4353A 〉 G,361 previously genotyped samples in the two SNPs were detected based on blind method using PCR-HRM assay to determine its specificity and sensitivity, and then, repeat tests were implemented to assess the repeatability and reproducibility of the analytical method. The evaluation of the test robustness just focuses on the amounts of genetic DNA and the number of amplified cycle in PCR process. Results Besides five samples with abnormal melting curve profile need replication, others had standardized melting profiles for correct genotype analysis to two SNPs of rs1205C 〉 T and rs4353A 〉 G. The genotyped results of 361 subjects were completely consistent compared with the records which implied the specificity and sensitivity of the analytical approach arrived 100% in the study. The coefficient of variance of Tin（melting temperature）between wild genotype and homozygous mutant genotype were 0. 7% and 0. 8% for rs1205C 〉T,0. 6% and 0. 5% for rs4353A 〉 G in nine replications of a intra-run,while the Tm differences between two homozygotes were statistically significant for the two SNPs （P 〈 0. 001 for both）which means it is easy to discriminate homozygous mutant from wild genotype. Although the melting profiles of three times tests had slight shift in three days, the correctness of genotyping was seldomly affected which suggested the assay have high repeatability and reproducibility. Similarly, the changes of DNA template amounts （10 ng,20 ng,50 ng and 100 rig）and PC R cycle numbers （38,40 and 42 cycles）hardly affected the genotyping results which demonstrated the assay has very strong robustness. Conclusions The technique of PCR-HRM based on small amplicons genotyping for rsl205C 〉 T and rs4353A 〉 G polymorphic sites is proved to be highly reliable and worthy of promotion to routine diagnostics, meanwhile, the diagnostic validation strategy can serve as a model for other applications.
出处
《中华临床医师杂志（电子版）》
CAS
2012年第7期I0024-I0028,共5页
Chinese Journal of Clinicians(Electronic Edition)
基金
教育部春晖计划项目(Z2010085)
甘肃省科技支撑项目(090NKCA094)
甘肃省消化系肿瘤重点实验室开放课题基金(lzujbky-2011-t03-01)
关键词
高分辨熔解曲线
基因分型
检验性能
单核苷酸多态性
High resolution melting
Genotyping
Detection performance
Single nucleotide polymorphism
