Abellaneda J M, Martínez-Alarcón L, Quereda J J, Herrero-Medrano J M, Mendonça L, Mrowiec A, García-Nicolás O, Pallarés F J, Ríos A, Muñoz A, Ramírez P, Ramis G
Grupo de Investigación Cría y Salud Animal, Universidad de Murcia, Murcia, Spain.
Grupo de Investigación Cría y Salud Animal, Universidad de Murcia, Murcia, Spain; Departamento de Cirugía, Hospital Clínico Universitario Virgen de la Arrixaca, Murcia, Spain.
Transplant Proc. 2015 Jan-Feb;47(1):132-5. doi: 10.1016/j.transproceed.2014.11.016.
This work was undertaken to evaluate whether a real-time quantitative polymerase chain reaction (qPCR) is as an adequate method for detection and quantification of human-specific DNA elements (Alu gene) in tissues and blood samples of pigs in which human stem cells were engrafted. Real-time qPCR quantification was performed with the use of previously described primers. The human DNA was mixed with different quantities of porcine DNA. The primer concentration and specificity, the qPCR efficiency, the quantification variations due to different porcine DNA concentrations, and the dissociation curve produced by the assay were evaluated. The qPCR proved to be specific, robust, with a reproducible and specific bimodal melting curve. High porcine DNA concentration produced subquantification, especially with low human DNA quantity. However, the assay proved to be useful for the detection of chimeric piglets produced by human cells injected in utero, because the effect caused by the porcine DNA interference was corrected in quantification of human DNA from piglets.
本研究旨在评估实时定量聚合酶链反应（qPCR）是否是检测和定量人干细胞移植猪组织和血液样本中人类特异性DNA元件（Alu基因）的合适方法。使用先前描述的引物进行实时qPCR定量。将人DNA与不同量的猪DNA混合。评估了引物浓度和特异性、qPCR效率、不同猪DNA浓度导致的定量变化以及该检测产生的解离曲线。qPCR被证明具有特异性、稳健性，具有可重复的特异性双峰熔解曲线。高浓度猪DNA会导致定量不足，尤其是在人DNA量较低时。然而，该检测方法被证明可用于检测子宫内注射人细胞产生的嵌合仔猪，因为在仔猪人DNA定量中校正了猪DNA干扰所造成的影响。
