基于量子点荧光微球的三段式杂交反应法用于前列腺癌miRNA检测

Detection of three-segment hybridization based onquantum dot fluorescent microspheres for miRNA in prostate cancer

目的建立基于量子点荧光微球的三段式杂交反应(three-segment hybridization,TSH)法并用于前列腺癌(prostate cancer,PC)微小核糖核酸(miRNAs)检测。方法制备二氧化硅微球(Bead)和量子点荧光微球(Qbead),分别经过表面改性及生物偶联寡核苷酸H1和H2制备成为Bead-H1和Qbead-H2;利用Bead和Qbead上偶联的寡核苷酸(H1和H2)和靶标miRNA之间的互补配对建立TSH法,检测产物的荧光强度并建立标准曲线;分别用TSH法和实时荧光定量逆转录PCR(qRT-PCR)检测7例PC患者临床组织样本miR-21的表达水平,比较两者之间的差异性并进行统计学分析。结果建立的TSH法对miR-21的最低检测限为0.16 pmol/L,且具有区分碱基突变的能力;其检测PC患者临床组织样本miR-21结果与qRT-PCR结果的相关性显著(r=0.983,P<0.001)。结论建立的TSH法可用于PC患者miR-21水平检测。

Objective To establish a detectonof three-segment hybridization(TSH)based on quantum dot fluorescent microspheres for miRNA in prostate cancer(PC).Methods The silicon dioxide(SiO_(2))microspheres(Bead)and quantum dot microspheres(Qbead)were prepared.The oligonucleotides H1 and H2 were connected to the Bead and Qbead by surface modification and biocoupling to become Bead-H1 and QBead-H2,respectively.The complementary pairing between the coupled nucleic acids(H1 and H2)and the target miRNA was used to establish theTSH reaction.The fluorescence intensities of the products were measured and the standard curve was drawn.The clinical samples from 7 PC patients were detected by TSH and quantitative real time polymerase chain reaction(qRT-PCR)simultaneously,and the differences of the results between the two detections were compared and analyzed statistically.Results The minimum detection limit of miR-21 using the developed method was 0.16 pmol/L,which was able to distinguish the base mutations.The detection results of PC clinical samples from PC were correlated significantly with the those of qRT-PCR(r=0.983,P<0.001).Conclusion The method established in this study could be used in laboratory for the diagnosis of PC.