新型环状引物荧光定量PCR检测乳腺癌组织HER2基因mRNA表达方法的建立

Establishment of a novel ring primer real.time PCR for quantification of the mRNA expression levels of HER2 gene

目的研究建立一种基于实时荧光定量PCR技术和新型环状引物的简捷、准确、廉价、易于标准化检测乳腺癌组织人表皮生长因子受体2（HER2）基因mRNA表达水平的方法，为临床上肿瘤个性化分子靶向药物治疗提供用药指导。方法设计HER2和内参基因的新型特异性环状引物，通过Excel在乳腺癌组织标本中随机抽取5份标本检测候选内参基因的表达，同时用geNorm、NormFinder和BestKeeper软件筛选和评估乳腺癌组织中稳定表达的内参基因，设置Mg“浓度和引物浓度梯度对PCR体系进行优化，建立乳腺癌HER2基因mRNA表达水平的新型环状引物EvaGreen染料实时荧光定量逆转录（FQRT）一PCR检测方法（以下简称“自建FQRT。PCR”法），并对其灵敏度、特异性、稳定性进行评价；同时用自建FQRT—PCR法与免疫组织化学（IHC）法平行检测2008至2012年厦门大学附属中山医院普外科收集的55份乳腺癌组织和其中32份同一患者匹配的正常组织（距癌组织1〉5cm）中HER2基因的表达水平，并评价自建FQRT—PCR法对乳腺癌的诊断效能。结果自建FQRT—PCR法对HER2基因和核糖体蛋白L37a（RPL37A）基因的最低检测限均为10^6拷贝／μl，线性范围均为10。～10。拷贝／pJ（r=0．997）；HER2和RPL37A基因的高、低浓度标准品（10^6拷贝／μl和10^1拷贝／μl）批内变异系数（CV）分别为（5．93±0．57）％和（5．11±0．59）％、（2．49±0．81）％和（2．984-0．97）％；批间CV为（5．76±0．58）％和（7．71±0．61）％、（3．75±0．76）％和（4．40±0．96）％。FQRT—PCR法与IHC法平行检测87份乳腺癌组织标本HER2基因表达水平，FQRT—PCR法的敏感度为96．36％（53／55），特异度为78．13％（25／32），阳性预测值为88．33％（53／60），阴性预测值为92．59％（25／27），与IHC检测结果总符合率为89．66％（78／87）。且与IHC法具有较高的一致性（Kappa=0．770，P〉0．05）。结论成功建立了FQRT—PCR检测乳腺癌HER2基因mRNA表达水平的方法，该方法敏感度高、特异度好且快速、价廉，适合临床检验实验室进行肿瘤标本的HER2基因表达检测。

Objective Based on real-time PCR technique and ring primers, to establish a simple, accurate, cost-effective and easily standardized quantitative assay for quantification of HER2 mRNA, and apply to provide medication guidance for clinical tumor personalized molecular targeted therapy. Methods Screening reference gene which was stable expression in breast cancer, and optimizing the PCR reaction system. Then a real-time PCR with Eva Green for quantification of the mRNA expression levels of HER2 gene was developed. The specificity, sensitivity and reproducibility of the method were evaluated 87 specimens including 55 liquid nitrogen-frozen breast cancer tissues and 32 normal tissues were detected by the real-time quantitative reverse transcription （ FQ RT） -PCR and immunohistochemistry （ IHC ）. Results The standard curve of the method indicated a good linear relationship between the Ct value and the template concentration with the correlation coefficient being 0. 997. The linear range of the system was from 101 to 106 eopies/μl and the lower detection limit was 101 eopies/μl. It had a high sensitivity and good specificity. The inter-assay coefficients of variation of HER and RPL37A genes were （5.93 ± 0. 57） % and （5. 11 ± 0. 59） % , （2. 49 ± 0. 81 ） % and （ 2. 98 ± 0. 97 ） % respectively. The intra-assay coefficients of variation were （5.76 ± 0. 58 ） % and （7.71 ± 0. 61 ） % , （ 3.75 ± 0. 76） % and （4. 40 ± 0. 96） % respectively. Using the optimized FQ RT- PCR system, HER2 gene of 87 specimens was quantifieated. The sensitivity of the assay was 96. 36% （53/ 55 ）, the specificity was 78.13% （25/32）, the positive predictive value was 88.33% （53/60）, the negative predictive value was 92. 59% （25/27） , and the total coincidence rate between FQ RT-PCR and IHC was 89.66% （78/87）. The correlation of the results between the FQ RT-PCR and IHC was good （Kappa = 0. 770, P 〉 0. 05 ） . Conclusions The method can quantify the mRNA expression levels of HER2 gene rapidly and cost-effictively with high sensitivity and specificity. It can be applied to clinical molecular diagnosis with attractive prospect.