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利用短串联重复序列标记的多重扩增和荧光检测快速定量混合嵌合体。

Rapid quantification of mixed chimerism using multiplex amplification of short tandem repeat markers and fluorescence detection.

作者信息

Thiede C, Florek M, Bornhäuser M, Ritter M, Mohr B, Brendel C, Ehninger G, Neubauer A

机构信息

Medizinische Klinik und Poliklinik I, Universitätsklinikum Carl Gustav Carus der Technischen Universität, Dresden, Germany.

出版信息

Bone Marrow Transplant. 1999 May;23(10):1055-60. doi: 10.1038/sj.bmt.1701779.

Abstract

Monitoring the engraftment of donor cells after allogeneic blood stem cell transplantation (BSCT) may be important for the early diagnosis of graft failure or relapse of disease. Several techniques have been reported for this purpose. PCR-based assays analyzing polymorphic short tandem repeat (STR) markers are attractive because they are sensitive and can be performed rapidly. The intent of the present study was to test a novel approach for the quantification of mixed chimerism using a commercial multiplex STR assay with fluorescence-based detection for forensic purposes. The feasibility of this assay and the accuracy of quantitative results was tested using serial cell mixtures of unrelated individuals. Sample preparation was optimized to obtain information from minute amounts of starting material, eg from patients with aplasia or from sorted cell populations. Using the STR-PCR, discrimination between donor and recipient was possible in all patients analyzed (n = 25). Cell dilution experiments showed a linear correlation between the cell numbers added and the proportions found, with the limit of detection for a minor cell population being 5%. Comparison of values obtained with standard FISH analysis in patients transplanted from sex-mismatched donors showed an excellent correlation with the STR-PCR results. Taken together, this procedure allows the rapid, versatile and accurate quantification of mixed chimerism, even with minuscule numbers of cells.

摘要

监测异基因造血干细胞移植(BSCT)后供体细胞的植入情况对于早期诊断移植失败或疾病复发可能很重要。为此已报道了几种技术。基于聚合酶链反应(PCR)分析多态性短串联重复序列(STR)标记的检测方法很有吸引力,因为它们灵敏且能快速进行。本研究的目的是测试一种新方法,该方法使用具有荧光检测功能的商业多重STR检测法来定量混合嵌合体,用于法医目的。使用无关个体的系列细胞混合物测试了该检测方法的可行性和定量结果的准确性。优化了样本制备,以便从微量起始材料(如再生障碍性贫血患者或分选的细胞群体)中获取信息。使用STR-PCR,在所有分析的患者(n = 25)中都能够区分供体和受体。细胞稀释实验表明,添加的细胞数量与检测到的比例之间存在线性关系,次要细胞群体的检测限为5%。在接受性别不匹配供体移植的患者中,将STR-PCR结果与标准荧光原位杂交(FISH)分析获得的值进行比较,显示出极好的相关性。综上所述,即使细胞数量极少,该方法也能快速、通用且准确地定量混合嵌合体。

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