Taqman多重实时荧光定量PCR检测裸鼠肿瘤转移模型中肿瘤转移率方法的建立

Multiplex Taqman Real-time PCR for Detection of Malignant Tumor Metastasis Rate in Cancer Metastasis Model in Nude Mice

目的:建立基于多重荧光定量PCR技术的动物体内恶性肿瘤转移模型中肿瘤转移率高效检测的方法。方法:以人和小鼠的β2m基因为目标,设计相应的引物和Taqman探针;分别抽提人涎腺腺样囊性癌ACC细胞和小鼠Sp2/0细胞的基因组DNA作为模板,建立两物种双重荧光定量PCR检测方法,且分别以人和鼠定量基因检测为参照考察双重荧光定量PCR方法的特异性、灵敏性和精确性,并与传统转移灶称重计算肿瘤转移率的方法进行比较。结果:双重Taqman实时荧光定量PCR检测方法具有很好的特异性和较高的灵敏度(0.006ng/μl DNA),重复性较好(批间平均CV=0.036),有较强稳定性,比传统称重法更高效,更准确。结论:双重荧光定量PCR能够在同一反应体系下同时对动物组织中人肿瘤转移灶和周围组织进行快速准确的定量检测,计算出肿瘤转移率,具有经济、快速、特异性强等优点,在肿瘤动物模型肿瘤转移率检测方面具有很高的应用价值。

Objective： To make the efficient detection of malignant tumor metastasis rate in cancer metastasis in animal models by use of the multiplex real-time fluorescence quantitative PCR. Methods： The species-specific primers and probes for detection of beta-2-macroglobulin（β2m） gene of human beings and mouse in multiplex real-time fluorescence quantitative PCR assay was designed. Total DNA as templates were extracted respectively from human ACC cells and mouse Sp2/0 cells. The specificity and sensitivity of these primer pairs were separately confirmed using simplex real-time PCR analysis. The specificity, sensitivity and accuracy of the multiplex real-time fluorescence quantitative PCR method were evaluated and compared with the traditional method by weighing tissues of tumor metastasis to calculate the rate of tumor metastasis. Results： The limit of detection （LOD） of the assay was 0.006ng/μl DNA for each target species （1：104 dilution）. Conclusions： This system proved its accuracy, precision and applicability for the detection of malignant tumor metastasis rate in animal models.