Pichon Jean-Philippe, Bonnaud Bertrand, Cleuziat Philippe, Mallet François
Unité Mixte de Recherche 2714, CNRS-bioMérieux, IFR128 BioSciences Lyon-Gerland, ENS-Lyon 46 allée d'Italie, 69364 Lyon cedex 07, France.
Nucleic Acids Res. 2006 Mar 22;34(6):e46. doi: 10.1093/nar/gkl086.
Human endogenous retroviruses (HERVs) can be divided into distinct families of tens to thousands of paralogous loci. The expression of HERV elements has been detected in all tissues tested to date, particularly germ cells, embryonic tissues and neoplastic tissues. Hence, the study of HERV expression could represent added value in cancer diagnosis. We developed a quantitative assay combining a multiplex degenerate PCR (MD-PCR) amplification, based on the relative conservation of the pol genes, and a colorimetric Oligo Sorbent Array (OLISA). Nine HERV families were selected and amplification primers and capture probes were designed for each family. The features required to achieve efficient amplification of most of the elements of each HERV family and balanced co-amplification of all HERV families were analyzed. We found that MD-PCR reliability, i.e. equivalence of amplification and dose-effect relationship, relied on the adjustment of three critical parameters: the primer degeneracy, the relative concentration of each primer and the total amount of primers in the amplification mixture. The analysis of tumoral versus normal tissues suggests that this assay could prove useful in tumor phenotyping.
人类内源性逆转录病毒（HERVs）可分为由数十至数千个旁系同源基因座组成的不同家族。迄今为止，在所有检测的组织中均检测到HERV元件的表达，尤其是生殖细胞、胚胎组织和肿瘤组织。因此，对HERV表达的研究可能在癌症诊断中具有附加价值。我们开发了一种定量检测方法，该方法结合了基于pol基因相对保守性的多重简并PCR（MD-PCR）扩增和比色寡核苷酸吸附阵列（OLISA）。选择了9个HERV家族，并为每个家族设计了扩增引物和捕获探针。分析了实现每个HERV家族大多数元件高效扩增以及所有HERV家族平衡共扩增所需的特征。我们发现MD-PCR的可靠性，即扩增的等效性和剂量效应关系，依赖于三个关键参数的调整：引物简并度、每个引物的相对浓度以及扩增混合物中引物的总量。肿瘤组织与正常组织的分析表明，该检测方法可能在肿瘤表型分析中有用。
