HPV基因芯片检测质控点设计和参数优化研究

Design of Quality Control and Parameter Optimization for HPV Gene Chip Detection

目的:设计人乳头状瘤病毒(HPV)基因芯片的质量控制点,优化质量控制探针和人β珠蛋白基因引物浓度以及HPV引物浓度比例,以提高基因芯片的检测质量。方法:收集临床HPV患者宫颈刮片样本,提取样本HPV58的DNA,用HPV通用引物扩增病毒DNA;利用人β珠蛋白引物扩增样本中的β珠蛋白DNA,将扩增的PCR产物与线性化的p MD18-T载体连接,构建人β珠蛋白和HPV58 L1区DNA质粒,转化大肠杆菌后,进行单克隆培养,获得HPV58样本和人β珠蛋白的DNA模板;以HPV58和人β珠蛋白DNA基因信息设计并制备扩增HPV和人β珠蛋白探针及PCR引物,将人β珠蛋白基因探针固定于基因芯片作为质量控制点(QC),按1∶20、1∶10、1∶5和1∶4比例混合单克隆HPV扩增引物与人β珠蛋白扩增引物,采用PCR反向点杂交技术,对模板进行扩增后,琼脂糖凝胶电泳检测扩增产物的产量,进行基因芯片孵育杂交和显色;用扫描仪扫描芯片,采用Image J软件对各阳性杂交信号点进行信号强度采集,计算最佳的人β珠蛋白扩增引物和HPV扩增引物浓度比值;将QC的探针点样浓度设为0. 1 pmol/L、1 pmol/L、10 pmol/L、50 pmol/L和100 pmol/L,检测信号强度,优化质量控制探针点样浓度。结果:扩增了HPV58病毒DNA和人β珠蛋白的DNA模板,设计了HPV58和人β珠蛋白探针及PCR引物;琼脂糖凝胶电泳检测分析发现,当人β珠蛋白扩增引物与HPV扩增引物的浓度比例为1∶10时,二者同时PCR扩增时,扩增的强度较为一致,得到的产物量相当,用此引物浓度比例进行PCR获得的产物与测试芯片进行杂交,显色的平衡性较好;对QC的点样浓度进行测试发现,当QC点样探针浓度为50 pmol/L时,可以获得最佳的检测效果。结论:通过对β珠蛋白扩增引物与HPV扩增引物的最佳浓度比例和质量控制探针最佳点样浓度的优化,提高了HPV检测点的检测质量。

Objective： To design a quality control point for human papillomavirus （HPV） gene chip, optimize quality control probe, human β-globin gene primer concentrations, and HPV primer concentration ratio to improve the quality of gene chip detection. Methods ： Cervical smear samples were collected from patients with clinical manifestations of HPV related infections, and HPV58 DNA was extracted from the samples. HPV universal primer and the human β-globin primer were used to amplify the HPV58 and β-globin gene respectively. The amplified PCR product was ligated with the linearized pMD18-T vector to construct human β-globin and HPV58 L1 region DNA plasmids, which were transformed into E. coli. After the transformation, monoclonal cultivation was used to obtain DNA templates of HPV58 samples and human β-globin. Amplified HPV, human β-globin probes and PCR primers were designed and prepared using HPV58 and human β-globin DNA gene information. The probes fixed on the gene chip were used as a quality control point （Qc）. Monoclonal HPV amplification primers and human β-globin amplification primers were mixed at a ratio of 1 ： 20, 1 ： 10, 1 ： 5 and 1 ： 4 to amplify the templates with PCR reverse dot blot （RDB） hybridization that the products were detected by agarose gel electrophoresis. Then gene chips were incubated for hybridization and coloration. Scanned the chip with a scanner to collect the signal intensity of each positive hybridization signal point by Image J software to calculate the optimal concentration ratio of primers of human β-globin and HPV, set the QC probe spot concentration to 0.1 pmol/L,1 pmol/L,10 pmol/L,50 pmol/L and 100 pmol/ L, detect signal strength and optimize concentration of the Qc probe. Results： HPV58 DNA and human β-globin were amplified; Probes and PCR primers were designed to detect HPV58 and human β- globin. Agarose gel electrophoresis analysis showed that when the concentration ratio of primers （β- globin/HPV58） is 1： 10, and both were simultaneously PCR amplified, the amplification intensity was relatively uniform, and the obtained products were equivalent. The products obtained by PCR using this primer concentration ratio is hybridized with the test chip, and the color balance is better. The QC spotting concentration was tested and found to be the best when the Qc spotting probe concentration was 50 pmol/L. Conclusion ： The optimization of the quality control points and the concentration ratio of probes of human β-globin and HPV amplification primers improves the detection quality of HPV detection points.