荧光实时定量RT-PCR检测DD3mRNA方法的建立

Development of a real-time fluorescent quantitative polymerase chain reaction for detection of differential display code 3 (DD3) mRNA

目的建立荧光实时定量RT—PCR（FQ—RT—PCR）检测前列腺癌（PCa）特异基因DD3mRNA的方法。方法在DD3基因的外显子1和3之间设计一对引物和一条TaqMan—MGB探针，优化反应体系，建立DD3mRNA的FQ—RT—PCR方法，并进行方法学评价。将PCR扩增产物经凝胶电泳纯化后与pBluescript Ⅱ SK载体连接，再将重组质粒转化感受态细胞进行克隆；提取重组质粒在紫外分光光度计上定量，作为定量检测的标准品。结果重组质粒经PCR扩增及序列测定，表明pBluescriptⅡSK—DD3cDNA克隆成功。PCR扩增产物经测序分析证实为DD3 mRNA特异性片段。本法灵敏度为6拷贝／反应，线性范围为6～6×10^8拷贝／反应，相关系数为0．9985，批内、批间变异系数分别为1.0％～2.0％和1、3％～6、6％。结论成功建立了FQ—RT—PCR检测DD3mRNA的方法。为DD3 mRNA作为前列腺癌的诊断指标提供了方法学依据，也为其他肿瘤基因的检测提供了方法学启示。

Objective To establish a real-time fluorescent quantitative reverse transcription-polymerase chain reaction （FQ-RT-PCR） for detection of DD3 mRNA based on Taqman technique. Methods A Taqman-MGB probe and a pair of primers were designed to amplify a fragment of 73 bp between exon 1 and 3 ,then the detection system was developed and evaluated. The PCR products were cloned into the pBluescript II SK vector and transfected into DH5α E. coli cells. The cloned plasmid was used as the standard substance after quantification by ultraviolet spectrophotometer. Results The DD3 standard substance was successfully constructed. The sequencing results confirmed that the PCR product was specific for DD3 mRNA. The parameters for evaluation of the real time FQ-RT-PCR method were as follows. The sensitivity was 6 copies/reaction,the linear range was from 6 to 6 × 10^8 copies/reaction,and the coefficient variation was 1. 0% to 2.0% and 1.3% to 6.6% in intra and inter assay respectively. Conclusions Real-time FQ-RT-PCR for detection of DD3 mRNA was sensitive, specific, rapid and efficient method. Quantitation of DD3 mRNA using real-time FQ-RT-PCR may be used in the diagnosis of prostate cancer and provide some useful information for in gene detection for other tumors.