Quantitative analysis of lineage-specific chimerism using real-time PCR and single nucleotide polymorphisms
10.3760/cma.j.issn.1009-9921.2012.09.006
- VernacularTitle:基于单核苷酸多态性分型的系列特异嵌合体定量方法的建立
- Author:
Li MA
;
Liping SU
;
Meijing ZHENG
;
Jingjing DUAN
;
Gengqian ZHANG
;
Lei ZHU
- Publication Type:Journal Article
- Keywords:
Polymorphisms,single nucleotide;
Chimerism;
TaqMan probe;
Stem cell transplantation
- From:
Journal of Leukemia & Lymphoma
2012;21(9):531-533
- CountryChina
- Language:Chinese
-
Abstract:
Objective To develop a real-time quantitative PCR method with TaqMan probe to analysis the lineage-specific chimerism based on single nucleotide polymorphisms (SNP).Methods CD3 positive and CD15 positive cells were separated by magnetic cell sorting system from cord blood,and a quantitative method were established using real-time quantitative PCR and SNP.Detect the artificial chimerism and mark the standard curves.The reaction system was optimized,and the sensitivity and specificity were evaluated.Results Separation purity of blood cells by magnetic cell sorting system was up to 94 ％-97 ％.Discrimination between donor and recipient was possible.Dilution experiments of the mock chimerism sample revealed that Ct values correlated linearly with the logarithm of recipient/donor DNA fraction (r ＞ 0.98),and the limit of detection for a minor DNA percentage was under 0.1％,and the specificity was also good.Quantitative analysis of 4 clinical cases in same period were made by real-time fluorescence quantitative SNP-PCR,the fitted rate was 87.50 ％ based on the chimera standard curve calculated for 1 case.Conclusion The method shows high sensitivity and specificity,and will be used to quantify the lineagespecific chimerism after allogeneic hematopoietic stem cell transplantation.
