CN104164480B - 一种乳腺癌mRNA组合的检测试剂盒 - Google Patents
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Abstract
本发明公开了一种乳腺癌mRNA组合的检测试剂盒,其包括PCR反应缓冲液,靶基因COX‑2、HERV‑K和GAPDH标准扩增产物的梯度稀释液;一对COX‑2特异性的正、反向引物,一条COX‑2特异性的探针;一对HERV‑K特异性的正、反向引物,一条HERV‑K特异性的探针;一对内参基因特异性的正、反向引物,一条内参基因特异性的探针。本试剂盒可通过RT‑PCR技术检测了COX‑2和HERV‑K在正常人和乳腺癌患者外周血单核细胞中的表达量,发现差异的效果显著,特异区分正常及乳腺癌的效果良好;该乳腺癌mRNA组合的检测特异性和灵敏度高、操作简单、取材方便、安全无创伤及易于大量筛查的特点。
Description
技术领域
本发明涉及一种基因检测试剂盒,尤其是一种乳腺癌mRNA组合的检测试剂盒。
背景技术
乳腺癌历来被认为是一种异质性疾病,无论在分子免疫表型、病理组织形态、生物学行为还是对治疗的反应都存在明显的个体差异,这种差异被认为是由于乳腺癌的分子差异(关键基因的异常表达)所造成的。越来越多的研究证实,乳腺癌预后与许多分子标志物密切相关,这为标志物基因分型的研究奠定了分子生物学基础。同时,由于基因芯片及组织微阵列等高通量研究技术的出现,基因分型不仅能反映各型乳腺癌相应的临床特点,更为乳腺癌基因分型的进一步研究提供了技术支持。
乳腺癌发生和转移受环境和遗传因素二者共同作用,复杂的发病机制是长期困扰医学界的一大难题。因此,发现预测和早期诊断乳腺癌发生转移的特异标记物是防治这类重大疾病的关键所在。目前,对乳腺癌的预警和早期诊断的技术主要有以下三种。1.临床体格检查:定期接受临床体格检查是早期发现乳腺癌的有效方法之一。始于1963年纽约的健康保险计划(HIP),历经18年研究发现,接受定期临床体格检查的妇女乳腺癌死亡率比对照组低23%。我国也开展了一系列乳腺癌的预防工作,但是,由于乳腺癌普查耗资巨大,成本/效果比高,因此,我国乳腺癌早期发现主要着眼于自我检查和提高自身保健意识,但患者偶尔触及肿块而就诊,常常为时过晚。2.影像学诊断法:影像学检查是以钼靶X线摄片为主要手段,辅以超声扫描和MRI扫描。钼靶X线摄片最早开始于1960年,在近五十年的发展历程中,对乳腺癌的早期诊断起到重要作用。乳腺组织本身密度小,包括肿瘤在内的很多病变表现为微小钙化。钼靶X线摄片方法能发现细小钙化点,如果表现为泥沙样钙化或细颗粒样钙化,或者每1cm2内<0.5mm的超过5枚则可提示乳腺癌。但对于不典型的病变,尤其是致密型乳腺内的病变及近胸壁的病变易漏诊。2005年国际上乳腺癌诊疗领域的23位专家在肯定钼靶X线摄片技术在乳腺癌早期诊断的重要地位的同时,建议结合超声,MRI和病理检查,进行最佳评价。虽然该方法在发达国家乳腺癌的普查和早期发现中起到很大作用,尤其是乳腺MRI技术的应用,将诊断敏感率提高至77%~100%,但是由于特殊仪器的限制,对诊断技术的高要求和昂贵的价格,使这些方法在发展中国家及地区得不到广泛应用。3.生物靶标早期检测:包括乳腺癌相关基因普查,例如BRCA1和BRCA2的突变分析;乳头溢液特异物检测及血清肿瘤标志物的测定。Her-2/neu癌基因在乳腺癌中过度表达,在血清中可以检测到Her-2蛋白片断。通过酶联免疫反应测定血清Her-2水平,可以作为诊断乳腺癌的指标之一。因此,重点发展生物靶标检测,寻找新的乳腺癌检测靶标并发展为可供临床检测且简单实用的技术对于乳腺癌患者提前预警及采取有效的治疗手段意义重大。
虽然疾病组织mRNA表达谱往往与疾病发病及预后密切相关,但是检测技术复杂、创伤大,难以真正应用于临床诊断,且不同的mRNA对不同疾病的诊断特异性差异较大。
发明内容
本发明要解决的技术问题是提供一种
为解决上述技术问题,本发明所采取的技术方案是:其包括PCR反应缓冲液,靶基因COX-2、HERV-K和GAPDH标准扩增产物的梯度稀释液;一对COX-2特异性的正、反向引物,一条COX-2特异性的探针;一对HERV-K特异性的正、反向引物,一条HERV-K特异性的探针;一对内参基因特异性的正、反向引物,一条内参基因特异性的探针。
本发明所述COX-2特异性的正向和反向引物的序列分别是5′-CCCGCAGTACAGAAAGTATC-3’和5’-CCATAGAGTGCTTCCAACTC-3’,COX-2特异性的探针序列是5'-TCAGCATAAAGCGTTTGCGGTACTCA-3’;用于HERV-K特异性的正向和反向引物的序列分别是5’-CAAATGGCGTATGTTAACTGACTT-3’和5’-GCCAATCTTTTGGGATCATG-3’,HERV-K特异性的探针序列是5’-CCGTAAACGCCGTAATTCAACCCATG-3’。
本发明所述COX-2特异性的探针和HERV-K特异性的探针的两端均分别结合有荧光发生基团FAM和荧光淬灭基团TAMRA。
本发明所述内参基因为GAPDH。
本发明所述GAPDH的正向和反向引物的序列分别是5'-GCATCCTGGGCTACACTGAG-3’和5'-TCCACCACCCTGTTGCTGTA-3’,GAPDH特异性的探针序列是5'-TCCTCTGACTTCAACAGCGACACCC-3’。
本发明所述GAPDH特异性的探针两端分别结合有荧光发生基团FAM和荧光淬灭基团TAMRA。
本发明所述PCR反应缓冲液为Taqman反应液。
COX-2是前列腺素合成过程中重要的限速酶,也是启动炎症反应的关键酶,它通过促进肿瘤细胞的增殖,抑制凋亡,促进肿瘤新生血管形成等机制,参与肿瘤的发生、发展。目前研究表明,在正常乳腺组织中COX-2不表达或低表达,而在乳腺癌组织中其表达量明显增高。从早期的乳腺增生到转移的发生,COX-2的高表达贯穿于肿瘤发生发展的整个过程。目前研究认为其主要通过促进肿瘤细胞增殖,抑制肿瘤细胞凋亡,增加肿瘤侵袭力,促进肿瘤血管新生等方式发挥作用。
人内源性逆转录病毒(Humanendogenous:etrovimses,HERVs)普遍存在于人类染色体中。HERV-K家族具有完整的逆转录病毒的基因序列,是所有家族中最可能编码完整并可能具有传染性的逆转录病毒目前研究证实,在多种恶性肿瘤中可以检测到HERV-K的表达。HERV-k作为一种逆转录病毒,可能是乳腺癌的一个理想的肿瘤靶点。
采用上述技术方案所产生的有益效果在于:单用COX-2或者是HERV-KmRNA进行乳腺癌的诊断分析,均存在比较高的假阳性和假阴性,诊断准确率不理想,因此,本发明提出乳腺癌mRNA组合检测的理念,将COX-2和HERV-K同时进行测定,用于乳腺癌的诊断,大大提高了乳腺癌诊断的准确率,减少了诊断结果的假阳性和假阴性。
本发明可通过RT-PCR(逆转录-聚合酶链反应)技术检测了COX-2和HERV-K在正常人和乳腺癌患者外周血单核细胞中的表达量,发现差异的效果显著,特异区分正常及乳腺癌的效果良好;该mRNA组合乳腺癌基因标志物的检测特异性和灵敏度高、操作简单、取材方便、安全无创伤及易于大量筛查的特点。
本发明可广泛运用于乳腺癌患者COX-2和HERV-K基因扩增水平的检测,可用于乳腺癌的早期诊断和疗效评价,提高病理检测的可重复性和准确性,避免过度组织活检,更准确的筛查出的乳腺癌患者,早期治疗干预,可以大大地降低医疗成本和费用,减少医疗资源的浪费,延长部分患者生存期,提高患者生存质量。
附图说明
下面结合附图和具体实施方式对本发明作进一步详细的说明。
图1是本发明乳腺癌mRNA组合的ROC曲线图。
具体实施方式
实施例:本乳腺癌mRNA组合的检测试剂盒采用下述检测过程。
1、外周血中总RNA的提取:
(1)EDTA(乙二胺四乙酸)抗凝的全血,5000rpm离心10min,弃血浆,吸取血细胞500μL转移至1.5mL离心管中,再加入红细胞裂解液1mL,充分混匀,置冰上反应15min,5000rpm离心5min;
(2)弃上清液,加入PBS(Phosphate Buffered Saline,磷酸盐缓冲液)1mL,充分混匀,5000rpm离心5min;
(3)操作三次,至上清液透明,弃上清液;
(4)在纯化的血细胞中,加入总RNA提取剂Trizol试剂1mL,充分混匀,室温放置5min;
(5)加入氯仿200μL,充分混匀30s,室温放置3min,10000rpm离心20min;
(6)取上清液500L,至新的PE试管中,加入异丙醇500μL,-80℃放置过夜;
(7)10000rpm离心30min,弃上清液,加入1mL、75vol%乙醇—DEPC溶解,DEPC为焦碳酸二乙酯,充分混匀,7500rpm离心10min;
(8)弃上清液,室温风干,加入22μL的DEPC水溶解,得总RNA溶液,待用,或-80℃保存。
2、cDNA的合成:
(1)取2μL总RNA溶液,50倍稀释,经紫外分光光度仪测定OD(吸光度),并检测OD260/280≥1.8确定纯度;
(2)取0.2μg总RNA和逆转录试剂,按表1的cDNA合成反应体系,加样进行反应;所述试剂采用德国QIAGEN公司Sensiscript RT Kit,也可用其他公司同类型产品;
表1:cDNA合成的反应体系
反应试剂 | 体积(μL) |
10×Buffer RT | 2 |
dNTP Mix(2.5mM each dNTP) | 2 |
Oligo-dT Primer(10μm) | 2 |
Quant Reverse Transcriptase | 1 |
RNasin-free water | 12 |
样品总RNA(0.2μg) | 1 |
总体积 | 20 |
(3)将反应体系置于37℃孵化60min,cDNA产物-20℃保存。
3、特异基因引物序列:
COX-2正向和反向引物的序列分别是5’-CCCGCAGTACAGAAAGTATC-3’和5'-CCATAGAGTGCTTCCAACTC-3’,COX-2探针序列是5'-TCAGCATAAAGCGTTTGCGGTACTCA-3’,探针的两端分别结合有荧光发生基团FAM和荧光淬灭基团TAMRA;用于HERV-K的正向和反向引物的序列分别是5’-CAAATGGCGTATGTTAACTGACTT-3,和5,-GCCAATCTTTTGGGATCATG-3’,HERV-K探针序列是5'-CCGTAAACGCCGTAATTCAACCCATG-3’,探针的两端分别结合有荧光发生基团FAM和荧光淬灭基团TAMRA;用于GAPDH的正向和反向引物的序列分别是5'-GCATCCTGGGCTACACTGAG-3’和5'-TCCACCACCCTGTTGCTGTA-3’,GAPDH基因的探针的序列是5'-TCCTCTGACTTCAACAGCGACACCC-3’,探针的两端分别结合有荧光发生基团FAM和荧光淬灭基团TAMRA。上述引物DNA由上海生工合成。
4、标准曲线的建立:
标准PCR扩增产物经紫外分光光度计测定其ng/μL的浓度后,按照下面的公式计算其拷贝浓度:copies/μL=[X/(Y×650)]×6.02×1017×K×Z。其中X是经紫外分光光度计测得的ng/μL浓度;Y代表PCR产物的长度;K是比例常数,DNA为0.05、RNA为0.04;Z为稀释倍数。经该算式得到的是PCR产物的copies/μL浓度。对已知浓度的样品以10倍梯度稀释,系列标准品梯度范围为103~109copies/μL,绘制标准曲线。
5、样本定量检测:
以样本cDNA作为模板,采用Taqman反应法,在ABI Prism7300实时荧光PCR扩增仪中进行反应,反应体系及反应条件见表2。PCR的扩增条件为:95℃预变性10min,95℃变性10s,60℃退火30s,72℃扩增30s,进行45个循环。
表2:实时荧光PCR反应体系
反应试剂 | 体积(μL) |
混合缓冲液 | 10 |
上游引物Primer F(10μM) | 0.4 |
下游引物Primer R(10μM) | 0.4 |
探针(2μM) | 1.0 |
cDNA | 1.0 |
RNase-free water | 7.2 |
总体积 | 20 |
注:上述反应试剂采用美国ABI公司Taqman荧光定量试剂盒,也可用其他公司同类型产品。
6、实验方法的重复性:
随机取任一样本,在不同时间重复5次反应,考察批间重复性;随机取任一样本,在一次实验中重复5次同时检测,考察批内重复性,结果见表3。
表3:批内和批间基因重复性
反应次数 | 1 | 2 | 3 | 4 | 5 | RSD(%) |
批间(Ct值) | 31.11 | 31.20 | 31.14 | 32.00 | 31.87 | 1.37% |
批内(Ct值) | 31.90 | 32.16 | 32.03 | 32.25 | 31.83 | 0.54% |
由表3可知,批间基因绝对量的RSD为1.37%,重复性较好;批内基因绝对量的RSD为0.54%,重复性较好;说明此方法的重复性较好,可以进行样本的检测。
6、结果与讨论:
用上述方法可检测得到乳腺癌患者和正常人组血液样本中靶基因COX-2、HERV-K的表达量(与内参进行归一化),靶基因在患者血液中的含量显著升高,结果见表4所示。
表4:COX-2和HERV-K基因的表达情况
基因 | 对照组 | 乳腺癌组 | 显著性 |
COX-2 | 0.16±0.22 | 1.46±2.25 | P=0.001 |
HERV-K | 0.14±0.18 | 0.63±1.31 | P=0.03 |
使用COX-2和HERV-K组合的基础是确定其准确度,即能正常的将一种情况与另一种区分开来,例如正常人和病人。其中,受试者工作特征(Receiver operatincharacteristic,ROC)是用以评价标志物准确度的一种重要工具。可以得到的两个主要指标包括:灵敏度(sensitivity),或真阳性率,评价其在选择特定疾病病人中的表现。在筛选测试中一般要求有高灵敏度以排除没有疾病的人;特异性(specificity),或真阴性率,指示其在正确选择没有疾病的人中的能力。在诊断中一般要求有高特异性以获得较低的假阳性率。本试剂盒检测时,对COX-2和HERV-K组合首先建立logistic回归模型(Logit(P)=12.454(COX-2)+4.104(HERV-K)-2.377),得到联合预测因子。预测因子的表达式为:Lβ(Y)=(COX-2)+0.330(HERV-K),将COX-2、HERV-K测定结果带入方程,求得各预测因子,并以其为分析指标,建立图1所示的双正态模型ROC曲线,确定标志物在区分正常人和乳腺癌患者中的能力;其中横坐标为1-特异性,即假阳性率,纵坐标为灵敏度,即真阳性率。整合标志物组的灵敏度和特异性分别为94.74%和90.00%,且整合基因标志物组的ROC曲线下面积为0.96,且P<0.0001,有统计学意义说明诊断准确性较高,具有临床诊断价值,而且相比单指标诊断,COX-2和HERV-K联合诊断提高了诊断效能。此外,该整合基因标志物组也可考虑作为未来药物研发的治疗靶点。
Claims (5)
1.一种乳腺癌mRNA组合的检测试剂盒,其特征在于:其包括PCR反应缓冲液,靶基因COX-2、HERV-K和内参基因GAPDH标准扩增产物的梯度稀释液;一对COX-2特异性的正、反向引物,一条检测外周血单核细胞中COX-2特异性的探针;一对HERV-K特异性的正、反向引物,一条检测外周血单核细胞中HERV-K特异性的探针;一对内参基因GAPDH特异性的正、反向引物,一条检测外周血单核细胞中内参基因特异性的探针;预测因子的表达式为:Lβ(Y)=(COX-2)+0.330(HERV-K);
所述COX-2特异性的正向和反向引物的序列分别是5′-CCCGCAGTACAGAAAGTATC-3’和5’-CCATAGAGTGCTTCCAACTC-3’,COX-2特异性的探针序列是5’-TCAGCATAAAGCGTTTGCGGTACTCA-3’;用于HERV-K特异性的正向和反向引物的序列分别是5’-CAAATGGCGTATGTTAACTGACTT-3’和5’-GCCAATCTTTTGGGATCATG-3’,HERV-K特异性的探针序列是5’-CCGTAAACGCCGTAATTCAACCCATG-3’。
2.根据权利要求1所述的乳腺癌mRNA组合的检测试剂盒,其特征在于:所述COX-2特异性的探针和HERV-K特异性的探针的两端均分别结合有荧光发生基团FAM和荧光淬灭基团TAMRA。
3.根据权利要求1所述的乳腺癌mRNA组合的检测试剂盒,其特征在于:所述GAPDH的正向和反向引物的序列分别是5’-GCATCCTGGGCTACACTGAG-3’和5’-TCCACCACCCTGTTGCTGTA-3’,GAPDH特异性的探针序列是5’-TCCTCTGACTTCAACAGCGACACCC-3’。
4.根据权利要求3所述的乳腺癌mRNA组合的检测试剂盒,其特征在于:所述GAPDH特异性的探针两端分别结合有荧光发生基团FAM和荧光淬灭基团TAMRA。
5.根据权利要求1、2所述的乳腺癌mRNA组合的检测试剂盒,其特征在于:所述PCR反应缓冲液为Taqman反应液。
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