CN111500724A - 用于乳腺癌六基因同时检测的引物组与探针组合 - Google Patents
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Abstract
本发明涉及一种用于乳腺癌六基因同时检测的引物组与探针组合，可为临床内分泌药物选择提供客观的检测指标，从而科学、及时地指导患者临床用药。本发明可采用多重RT‑PCR实现检测，特异性强，检测速度快，与基因表达芯片相比费用相对较低。本发明通过检测相关基因的表达水平，量化评估各药物选择的复发风险和治疗获益，协助内分泌治疗药物的选择。本发明设计运用靶向特异性引物组，大幅度提高检测的灵敏度。该检测方法的灵敏度高，特异性好，快速准确，性价比高等优点。
Description
技术领域
本发明属于生物检测技术领域，涉及用于乳腺癌六基因同时检测的引物组与探针组合。
背景技术
乳腺癌已成为全球范围内最常见的恶性肿瘤之一。激素受体阳性乳腺癌是乳腺癌中最常见的类型。内分泌治疗对这类亚型具有显著的治疗效应，但是其耐药性的产生导致患者治疗失败的主要原因。
根据NCCN等治疗指南标准，乳腺癌等激素受体阳性、HER2阴性且无淋巴结转移的乳腺癌患者，首选辅助内分泌治疗。激素受体(ER/PR)可以通过免疫组化(IHC)的方法进行检测，来确定患者是否适合内分泌治疗。
目前临床广泛采用的辅助内分泌治疗药物主要分为以下三类：
(1)选择性雌激素受体调节剂(SERM)，如他莫昔芬(tamoxifen)，托瑞米芬(Toremifene)；
(2)芳香化酶抑制剂(AI)，如依西美坦(Exemestane)、来曲唑(Letrozole)和阿那曲唑(Anastrozole)；
(3)选择性雌激素受体下调剂(SERD)，如氟维司群(Fulvestrant)。此外，mTOR抑制剂，如依维莫司(Everolimus)和CDK4/6抑制剂，包括帕博西尼(Palbociclib)、瑞博细腻(Ribococlib)和玻玛西尼(Abemaciclib)也认为是广义的雌激素受体药物。
如何从现有内分泌治疗药物中选择最适合的一种，目前多依据临床医生的经验，缺乏类似乳腺癌21基因检测类似的客观指标。患者个体化内分泌治疗方案的设计，急需类似客观指标及快速检测方案，来量化评估各药物选择的复发风险和治疗获益。
发明内容
有鉴于此，本发明的目的在于提供用于乳腺癌六基因同时检测的引物组与探针组合，可综合评估患者对内分泌辅助治疗的潜在耐药性，用于辅助肿瘤患者内分泌治疗药物的临床选择。
为达到上述目的，本发明提供如下技术方案：
1、用于乳腺癌六基因同时检测的引物组与探针组合，包含：
(1)引物组：
ESR1正向引物：5’-CGCCGCCTACGAGTTCAA-3’，如SEQ ID NO.1所示；
ESR1反向引物：5’-GGAGACACGCTGTTGAGTGG-3’，如SEQ ID NO.2所示；
ERa36正向引物：5’-AACGCTCTAAGAAGAACA-3’，如SEQ ID NO.5所示；
ERa36反向引物：5’-GTAGGATCATACTCGGAATA-3’，如SEQ ID NO.6所示；
MKI67正向引物：5’-CACCTAAGACCTGAACTA-3’，如SEQ ID NO.7所示；
MKI67反向引物：5’-CCACATGGATTTCTGAAC-3’，如SEQ ID NO.8所示；
KDM5B正向引物：5’-GCATCAAGCAAGAACCTA-3’，如SEQ ID NO.11所示；
KDM5B反向引物：5’-GCCATCACACAACAGTAG-3’，如SEQ ID NO.12所示；
CCND1正向引物：5’-CTCGGTGTCCTACTTCAA-3’，如SEQ ID NO.13所示；
CCND1反向引物：5’-GGTCCAGGTAGTTCATGG-3’，如SEQ ID NO.14所示；
CDK7正向引物：5’-CAGCTAACAAGAATATTTGAAA-3’，如SEQ ID NO.15所示；
CDK7反向引物：5’-AGCACATGGATTAAATAAGAA-3’，如SEQ ID NO.16所示；
(2)探针：
ESR1探针：5’-TGGAGCCGAACGCCGCAGCCT-3’，如SEQ ID NO.21所示；
ERa36探针：5’-CATCCAACAAGGCACTGACCATC-3’，如SEQ ID NO.24所示；
MKI67探针：5’-ACTTGCCTCCTAATACGCCTCTC-3’，如SEQ ID NO.26所示；
KDM5B探针：5’-CTTCATCATTGCCACTGCCACATAA-3’，如SEQ ID NO.28所示；
CCND1探针：5’-TCCTCCTCGCACTTCTGTTCC-3’，如SEQ ID NO.29所示；
CDK7探针：5’-ATCTAGTAAGTCGTCTCCTGCTGC-3’，如SEQ ID NO.31所示；
其中，所述的乳腺癌六基因包括：ESR1、ERa36、MKI67、KDM5B、CCND1、CDK7。
优选的，探针的5’端和3’端分别修饰荧光基团和猝灭基团。
进一步优选的，所述荧光基团选自FAM、VIC、Cy5、YAK中的任一种。
优选的，所述引物组与探针组合还包含：
质控引物ACTB正向引物：5’-GCGTGACATTAAGGAGAAG-3’，如SEQ ID NO.19所示；
质控引物ACTB反向引物：5’-GAAGGAAGGCTGGAAGAG-3’，如SEQ ID NO.20所示；
质控探针ACTB：5’-CGGAACCGCTCATTGCCAAT-3’，如SEQ ID NO.34所示。
进一步优选的，质控探针ATCB的5’端和3’端分别修饰荧光基团和猝灭基团。
更进一步优选的，所述荧光基团选自FAM、VIC、Cy5、YAK中的任一种。
2、上述引物组与探针组合在制备乳腺癌六基因检测试剂盒中的应用。
3、一种乳腺癌六基因检测试剂盒，含有上述引物组与探针。
优选的，所述试剂盒还含有阳性对照、阴性对照和PCR反应液。其中，阴性对照：采用无核酸酶水(DEPC水)；阳性对照：β-actin cDNA质粒标准品(终浓度为5μM)；PCR反应体系：2×RT-PCR Buffer 12.5μl，25×RT-PCR Enzyme Mix 1μl，mRNA模板10μl，引物/探针组合1.5μl(终浓度为0.5μM)。
4、利用上述引物组与探针组合实现的一种乳腺癌六基因同时检测方法，具体步骤如下：
(1)抽提肿瘤组织的核酸提取物(mRNA)，构建25μl反应体系，包括：2×RT-PCRBuffer 12.5μl，25×RT-PCR Enzyme Mix 1μl，mRNA模板10μl，引物/探针组合1.5μl(终浓度为0.5μM)；
(2)PCR反应设置为：50℃15分钟，95℃10分钟，然后40个循环：95℃8秒，60℃35秒，实时检测各通道荧光；
(3)PCR检测结果分析：计算各组Ct值，如果有信号上升且Ct<33，则判定为阳性。否则为阴性。
基因指标检测结果指导药物选择如下：
(A)ESR1或PGR检测阳性患者建议纳入辅助内分泌治疗方案，两者均阴性不建议纳入内分泌治疗；
(B)ERα36检测阴性者，建议采用他莫昔芬等SERM类药物治疗，检测阳性者可参考患者是否绝经和后续检测指标，选择芳香化酶抑制剂(AI)或者选择性雌激素受体下调剂(SERD)。
(C)MKI67检测结果为阴性时，且为绝经患者，建议选择芳香化酶抑制剂(AI)治疗，检测阳性患者依据后续检测结果采用选择性雌激素受体下调剂(SERD)或者CDK4/6抑制剂。
(D)排除SERM类和AI类药物的患者，同时检测KDM5B阴性时，采用选择性雌激素受体下调剂(SERD)氟维司群药物治疗。若KDM5B检测阳性，建议采用化疗。
(E)如果患者CCND1检测阳性且CDK7检测阴性患者可建议选择CDK4/6抑制剂治疗。
本发明的有益效果在于：
针对目前缺乏量化评估针对乳腺癌的内分泌辅助治疗药物选择的客观标准，本发明设计了一组用于乳腺癌六基因同时检测的引物组与探针组合，可为临床内分泌药物选择提供客观的检测指标，从而科学、及时地指导患者临床用药。本发明可采用多重RT-PCR实现检测，特异性强，检测速度快，与基因表达芯片相比费用相对较低。
本发明通过检测相关基因的表达水平，量化评估各药物选择的复发风险和治疗获益，协助内分泌治疗药物的选择。本发明设计运用靶向特异性引物组，大幅度提高检测的灵敏度。该检测方法的灵敏度高，特异性好，快速准确，性价比高等优点。
申请人筛选出了一组含有7个基因的检测指标，可以较好的判断肿瘤患者最适合的内分泌治疗药物选择。
(1)ESR1：经典型全长的雌激素受体ERα。检测验证患者的激素受体表达水平，是否适合纳入内分泌治疗；
(2)ERα36：雌激素受体亚型，是导致选择性雌激素受体调节剂(SERM)治疗耐药的关键指标。作为排除SERM治疗方案的标准。
(3)MKI67:即细胞增殖指标ki67。Ki67可以作为芳香化酶抑制剂是否受益的关键指标。
(4)CCND1：即细胞周期蛋白Cyclin D1。该指标阳性可以作为CDK4/6治疗潜在获益的关键指标；
(5)CDK7：即细胞周期蛋白依赖性激酶7。CDK7是ESR1-Y537S蛋白磷酸化相应的关键调节分子。该指标阳性表达，提示患者对CDK4/6治疗无法获益的重要指标。
(6)KDM5B：即赖氨酸脱甲基酶5B。该指标的高表达，与患者氟维司群耐药密切相关，可以作为SERD药物的排除指标。
(7)ACTB：即β-actin作为内参使用，用于分析样本准备情况。
具体实施方式
下面将对本发明的优选实施例进行详细的描述。
实施例1：
引物/探针设计与BLAST优化组合
1.1检索GeneBank，查找7个基因的mRNA序列(ESR1基因：NM_000125.4；ERα36基因：BX640939.1；MKI67基因：NM_001145966.2；KDM2B基因：NM_006618.5；CCND1基因：NM_053056.3；CDK7基因：NM_001799.4；ACTB基因：NM_001101.5)，经序列比对查找mRNA的保守区域片段，使用Beacon Designer软件设计多重RT-PCR的引物，及其相应的Taqman探针(表1)。每个基因分别针对不同位点设计2组引物。
表1.多重RT-PCR引物组合与相应的探针序列
1.2 NCBI网站BLAST分析验证各组引物之间的潜在非特异产物。优化组合排除各组引物之间不出现潜在非特异产物。按照BLAST分析结果，排除各组引物之间不存在交叉影响，获得优化组合的初步结果(分两组四通道检测)：
第一组：引物1(ESR1)、引物2(MKI67)、引物1(CCND1)、引物2(Actin)
第二组：引物1(ER36)、引物2(KDM2B)、引物1(CDK7)、引物2(Actin)。
实施例2：
RT-qPCR实验验证优化组合的应用效果分析
2.1 RNA提取：采用RNAfast200-总RNA极速抽提试剂盒(飞捷生物)。
(1)收集对数期生长的乳腺癌细胞株MFC-7、MDA-MB 231(购于ATCC)和乳腺癌原代培养细胞BR11(西南医院肿瘤中心原代细胞)。先离心收集细胞并去除全部上清，仅留下细胞团块，充分振荡致细胞完全松散(重要)，加入RA1液100uL，立即振荡混匀数秒后瞬时离心，吸取100μL上清液放入1.5mL管中，立即进行提取。
(2)在处理好的样本中，加入RA2液500μl，充分颠倒混匀5-10次(至裂解物澄清)，静置1min。
(3)将样品裂解物全部吸入或倒入内套管中(已插在外套管)，离心1min。
(4)取出内套管，弃去外套管中液体后放回内套管，加入500μl漂洗液RW，离心1min。
(5)重复步骤(3)再洗一次。
(6)取出内套管，弃去外套管中液体，仍然套回内套管，不加洗液，离心1min。
(7)将内套管移入新的1.5ml管中，在膜中央加入洗脱液25-50μl。
(8)室温静置1min，离心1min，获得总RNA。
2.2浓度与纯度检测:
检测仪器：DS11+超微量分光光度计
纯度指标：260/280＝1.8～2.0
2.3 RT-qPCR
采用试剂盒：AgPath-IDTM One-Step RT-PCR Reagents
按照表2优化组合方案，构建25μl的RT-PCR反应体系，如表3所示。
表2.最佳多重RT-PCR引物的组合
表3.RT-PCR反应体系
注：*按照表2优化组合方案，预混相应的引物和探针，4℃储存备用。
2.4热循环反应
具体条件见表4。
表4.热循环反应条件
2.5PCR检测结果分析：计算各组Ct值，如果有信号上升且Ct<33，则判定为阳性。否则为阴性。
2.6引物检测验证分析：阴性对照组未检测到信号，阳性检测指标融合曲线呈现单峰，提示无非特异性产物形成。结合Ct值分析，三组细胞检测结果如表5所示.
表5.三组细胞检测结果
| 细胞 | ESR1 | ERa36 | MKI67 | CCND1 | KDM2B | CDK7 |
| MFC-7 | + | - | + | - | + | + |
| MDA-MB 231 | - | + | + | + | - | + |
| BR11 | - | + | + | + | + | + |
实施例3：
引物/探针组合的敏感性检测
3.1收集各组基因(ESR1、ERa36、MKI67、CCND1、KDM2B、CDK7)单一RT-PCR实验的产物，采用凝胶电泳，切胶获得相关片段。
制备质量浓度1％琼脂糖凝胶，待凝胶固后放入电泳槽中并倒适量的TAE缓冲液。加入适量的6×Loading Buffer，混匀后即可上样。分别将样品全部加到上样孔中，并点2μlmarker。加样完成后，盖上电泳槽90V恒压电泳，指示剂迁移至凝胶的1/2处，即停止电泳。取出凝胶，置于洗脱液(Elution Buffer)中浸泡20min后凝胶扫描仪下查看电泳结果。在紫外光下，将特异电泳带用刀尽可能切下放入到1.5ml的离心管中，称琼脂糖带的重量。
2.胶回收和DNA纯化
在含琼脂糖带的离心管中分别加入相应比例体积的Buffer GM，65℃水浴中温浴至凝胶完全融化，将离心柱装在收集管内，把获得的DNA熔胶液全部转移至离心柱中，静置1min，12000rpm室温离心2min，弃收集管中的滤液，将离心柱套回内。加700μl已含无水乙醇的Buffer WB至离心柱中。12000rpm室温离心2min。最后弃收集管中的滤液后重新将离心柱放回收集管内。把离心柱装在一个干净的1.5ml离心管上，加入30μl的Elution Buffer到柱基质上，室温放置1min，12000rpm离心1min以洗脱DNA。
3.3酶切目的基因和载体连接反应
10μl pUCm-T载体(Addgene馈赠)加入酶切反应体系中，含如下成分：2μl10×酶切缓冲液，6μl的蒸馏水，Bam HⅠ和HindⅢ(购于New England Biolabs)各1μl。混匀后，37℃酶切反应3h。然后65℃保温10min以终止酶切及防止DNA末端退火，然后冰浴骤冷。连接反应加入4μlPCR产物，1.5μl pUCm-T载体酶切产物，1μl 10×T4连接buffer，0.5μlT4DNA连接酶，加水补足到10μl。轻混反应物并在16℃连接16h。
3.4 DH5α转化并抽提质粒：
将连接产物分别加入到制备好的感受态DH5α细胞(购于上海碧云天生物技术有限公司)里，用移液器轻吸打混匀，冰浴30min。将管放入预加温到42℃的水浴中热激90s，然后冰浴2min。菌液涂板并挑取阳性重组子。根据酶切电泳检测结果，鉴定构建成功的质粒，转移到含LB液体培养基的试管中培养扩增。扩增细菌离心裂解后按照3.1的方案提取质粒。
3.5定量后按照1:1:1:1的体积比，按表2分组混合相应质粒，获得含有相关序列的2组阳性对照样本，并梯度稀释样本。
3.6采用实施例2的RT-qPCR实验方案，检测最佳多重RT-PCR引物的最低检出量。
3.7各基因的最低检出量的实验结果分析如表6所示。
表6.引物最低检出量及特异性分析
| 检测指标 | 最低检出量 | 特异性 | 最低检出量 | 特异性 |
| ESR1 | 100cop/ml | 100％ | 100cop/ml | 100％ |
| ERa36 | 300cop/ml | 100％ | 300cop/ml | 100％ |
| MKI67 | 100cop/ml | 100％ | 100cop/ml | 100％ |
| KDM5B | 100cop/ml | 100％ | 100cop/ml | 100％ |
| CCND1 | 100cop/ml | 100％ | 110cop/ml | 98％ |
| CDK7 | 95cop/ml | 100％ | 100cop/ml | 100％ |
3.8特异性和灵敏度分析，最佳单重RT-PCR引物灵敏度与单一RT-PCR引物不存在显著差异，特异性100％符合。
最后说明的是，以上优选实施例仅用以说明本发明的技术方案而非限制，尽管通过上述优选实施例已经对本发明进行了详细的描述，但本领域技术人员应当理解，可以在形式上和细节上对其作出各种各样的改变，而不偏离本发明权利要求书所限定的范围。
序列表
<110> 王强
<120> 用于乳腺癌六基因同时检测的引物组与探针组合
<130> 2020
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Claims (6)
1.用于乳腺癌六基因同时检测的引物组与探针组合，其特征在于，包含：
(1)引物组：
ESR1正向引物：5’-CGCCGCCTACGAGTTCAA-3’，如SEQ ID NO.1所示；
ESR1反向引物：5’-GGAGACACGCTGTTGAGTGG-3’，如SEQ ID NO.2所示；
ERa36正向引物：5’-AACGCTCTAAGAAGAACA-3’，如SEQ ID NO.5所示；
ERa36反向引物：5’-GTAGGATCATACTCGGAATA-3’，如SEQ ID NO.6所示；
MKI67正向引物：5’-CACCTAAGACCTGAACTA-3’，如SEQ ID NO.7所示；
MKI67反向引物：5’-CCACATGGATTTCTGAAC-3’，如SEQ ID NO.8所示；
KDM5B正向引物：5’-GCATCAAGCAAGAACCTA-3’，如SEQ ID NO.11所示；
KDM5B反向引物：5’-GCCATCACACAACAGTAG-3’，如SEQ ID NO.12所示；
CCND1正向引物：5’-CTCGGTGTCCTACTTCAA-3’，如SEQ ID NO.13所示；
CCND1反向引物：5’-GGTCCAGGTAGTTCATGG-3’，如SEQ ID NO.14所示；
CDK7正向引物：5’-CAGCTAACAAGAATATTTGAAA-3’，如SEQ ID NO.15所示；
CDK7反向引物：5’-AGCACATGGATTAAATAAGAA-3’，如SEQ ID NO.16所示；
(2)探针：
ESR1探针：5’-TGGAGCCGAACGCCGCAGCCT-3’，如SEQ ID NO.21所示；
ERa36探针：5’-CATCCAACAAGGCACTGACCATC-3’，如SEQ ID NO.24所示；
MKI67探针：5’-ACTTGCCTCCTAATACGCCTCTC-3’，如SEQ ID NO.26所示；
KDM5B探针：5’-CTTCATCATTGCCACTGCCACATAA-3’，如SEQ ID NO.28所示；
CCND1探针：5’-TCCTCCTCGCACTTCTGTTCC-3’，如SEQ ID NO.29所示；
CDK7探针：5’-ATCTAGTAAGTCGTCTCCTGCTGC-3’，如SEQ ID NO.31所示；
其中，所述的乳腺癌六基因包括：ESR1、ERa36、MKI67、KDM5B、CCND1、CDK7。
2.根据权利要求1所述的引物组与探针组合，其特征在于，探针的5’端和3’端分别修饰荧光基团和猝灭基团。
3.根据权利要求1所述的引物组与探针组合，其特征在于，所述引物组与探针组合还包含：
质控引物ACTB正向引物：5’-GCGTGACATTAAGGAGAAG-3’，如SEQ ID NO.17所示；
质控引物ACTB反向引物：5’-GAAGGAAGGCTGGAAGAG-3’，如SEQ ID NO.18所示；
质控探针ATCB：5’-CGGAACCGCTCATTGCCAAT-3’，如SEQ ID NO.33所示。
4.根据权利要求3所述的引物组与探针组合，其特征在于，质控探针ATCB的5’端和3’端分别修饰荧光基团和猝灭基团。
5.权利要求1～4中任一项所述引物组与探针组合在制备乳腺癌六基因检测试剂盒中的应用。
6.一种乳腺癌六基因检测试剂盒，含有权利要求1～4中任一项所述引物组与探针。
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| WO2018001295A1 (zh) * | 2016-06-30 | 2018-01-04 | 博奥生物集团有限公司 | 分子标志物、内参基因及其应用、检测试剂盒以及检测模型的构建方法 |
| CN109439755A (zh) * | 2018-11-30 | 2019-03-08 | 广东腾飞基因科技股份有限公司 | 一种用于检测乳腺癌相关基因表达水平的试剂盒 |
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