CN110205369A - 一种用于定量检测STAT3 mRNA水平的引物、探针和试剂盒 - Google Patents
一种用于定量检测STAT3 mRNA水平的引物、探针和试剂盒 Download PDFInfo
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Abstract
本发明涉及生物检测技术领域,具体涉及一种用于定量检测STAT3 mRNA水平的引物、探针和试剂盒,所述引物包括用于扩增STAT3基因的引物F1和引物R1、以及用于扩增对照基因GADPH的引物F2和引物R2。所述探针包括用于检测STAT3基因的探针P1和用于检测对照基因GAPDH的探针P2。所述试剂盒包括上述所述的引物和上述所述的探针。本发明的引物和探针特异性好,检测灵敏精确。本发明的试剂盒使用FAM和VIC双通道可对STAT3 mRNA的表达进行准确的定量分析,具有快速、简便、灵敏度高等检测优点。
Description
技术领域
本发明涉及生物检测技术领域,具体涉及一种用于定量检测STAT3 mRNA水平的引物、探针和试剂盒。
背景技术
手足口病是由肠道病毒感染引起的,以发热和手、足、口腔、臀部等部位出现皮疹、疱疹或疱疹性咽峡炎为主要特征的一种常见于小儿的急性传染病。自2008年安徽省阜阳市出现手足口病暴发疫情以来,发病率呈现逐年上升,目前发病率位居于我国法定报告传染病的首位。手足口病已成为我国亟需解决的重大公共卫生问题。目前对重症HFMD仍没有好的标志物,轻症与重症临床转化的监测与治疗仍然是一个难点,寻找早期识别标志物及有效的靶向治疗手段,是降低该病死亡率的关键。信号传导与转录激活因子(STATs)为一类双功能分子,分子量为84~113kD之间,是一个可与靶基因调控区DNA结合胞质蛋白家族,其可与酪氨酸磷酸化信号偶联,既参与信号传导,又激活基因转录,从而发挥转录调控作用。我们研究发现重症手足口病样本中STAT3蛋白表达明显上调,可以作为重症手足口病早期识别诊断的分子标志物,本发明将荧光定量PCR技术方法,在mRNA水平检测STAT3蛋白表达情况,为重症手足口病的早期诊断提供了准确、快速、高效、敏感的检测方法。
发明内容
为了克服现有技术中存在的缺点和不足,本发明的目的在于提供一种用于定量检测STAT3 mRNA水平的PCR引物,该引物特异性好,检测灵敏精确。
本发明的另一个目的在于提供一种用于定量检测STAT3 mRNA水平的PCR探针,该探针特异性好,检测灵敏精确。
本发明的还一个目的在于提供一种用于定量检测STAT3 mRNA水平的PCR试剂盒,该试剂盒使用FAM和VIC双通道实时定量检测STAT3 mRNA含量,准确性好,灵敏度高。
本发明的目的通过下述技术方案实现:一种用于定量检测STAT3 mRNA水平的PCR引物,所述引物包括用于扩增STAT3基因的引物F1和引物R1、以及用于扩增对照基因GADPH的引物F2和引物R2,所述4条引物的核苷酸序列分别为:
F1:5’-TGTGACACCAACGACCTGC-3'
R1:5’-CCAAACTGCATCAATGAATCTA-3'
F2:5’-CCTGCCAAGTATGATGACATCAAGA-3'
R2:5’-GTAGCCCAGGATGCCCTTTAGT-3'。
本发明的另一个目的通过下述技术方案实现:一种用于定量检测STAT3 mRNA水平的PCR探针,所述探针包括用于检测STAT3基因的探针P1和用于检测对照基因GAPDH的探针P2,所述探针的核苷酸序列分别为:
P1:5’-ACCATTGACCTGCCGATGTCCC-3'
P2:5’-TGGTGAAGCAGGCGGCCGAG-3';
其中,所述探针P1和探针P2的5’端均标记荧光基团,3’端均标记淬灭基团。
优选的,所述探针P1的5’端标记荧光基团FAM,3’端标记淬灭基团TAMRA。
优选的,所述探针P2的5’端标记荧光基团VIC,3’端标记淬灭基团TAMRA。
本发明的还一个目的通过下述技术方案实现:一种用于定量检测STAT3 mRNA水平的PCR试剂盒,所述试剂盒包括上述所述的引物和上述所述的探针。
优选的,所述试剂盒由2×NASBA反应液、5×NASBA反应酶混合液、标准品、阳性质控品及阴性质控品组成。
优选的,所述2×RT-PCR反应液包括如下具体成分:50mM Tris-HCl(pH8.3)、100mMKCl、10mM MgCl2、1.0% Triton X-100、0.5mM dNTP、引物F1和引物R1各2.5μmol/L、引物F2和引物R2各2μmol/L、P1探针0.5μmol/L和P2探针0.4μmol/L。
优选的,所述5×RT-PCR反应酶混合液包括4U/mL Taq酶、1.6U/μL MMLV反转录酶、3U/μL RNA酶抑制剂和30mg/mL BSA。
优选的,所述标准品为含有标准品序列的重组pUC57质粒,所述标准品序列为:
GCGCTGCCCCATACCTGAAGACCAAGTTTATCTGTGTGACACCAACGACCTGCAGCAATACCATTGACCTGCCGATGTCCCCCCGCACTTTAGATTCATTGATGCAGTTTGGAAATAAKGGTGAAGGTGCTGAACCCTCAGCAGGAGGGCAGTTTG。
优选的,所述阳性质控品为含有STAT3 mRNA的细胞裂解液样品,阴性质控品为无RNA酶的DEPC-H2O。
本发明的有益效果在于:本发明的引物和探针特异性好,检测灵敏精确。本发明的试剂盒使用FAM和VIC双通道可对STAT3 mRNA的表达进行准确的定量分析,具有快速、简便、灵敏度高等检测优点。
附图说明
图1为标准品的荧光定量PCR检测结果。
图2为标准品的荧光定量PCR检测的标准曲线。
图3为25例临床病例STAT 3基因扩增曲线。
具体实施方式
为了便于本领域技术人员的理解,下面结合实施例及附图1-3对本发明作进一步的说明,实施方式提及的内容并非对本发明的限定。
实施例1试剂盒的设计和组成
本发明的试剂盒由2×NASBA反应液、5×NASBA反应酶混合液、标准品、阳性质控品及阴性质控品组成。
1、引物和探针的设计和合成:
从GenBank中找出STAT 3和GAPDH的保守基因序列,采用Primer Primier 5.0软件分别设计扩增引物和探针序列,确保每一对引物能够特异性扩增出STAT 3和GAPDH。
用于扩增STAT3基因的引物F1和引物R1以及用于扩增对照基因GADPH的引物F2和引物R2的核苷酸序列分别为:
F1:5’-TGTGACACCAACGACCTGC-3'
R1:5’-CCAAACTGCATCAATGAATCTA-3'
F2:5’-CCTGCCAAGTATGATGACATCAAGA-3'
R2:5’-GTAGCCCAGGATGCCCTTTAGT-3'。
用于检测STAT3基因的探针P1和用于检测对照基因GAPDH的探针P2的核苷酸序列分别为:
P1:5’-ACCATTGACCTGCCGATGTCCC-3'
P2:5’-TGGTGAAGCAGGCGGCCGAG-3';
其中,探针P1的5’端标记荧光基团FAM,3’端标记淬灭基团TAMRA;所述探针P2的5’端标记荧光基团VIC,3’端标记淬灭基团TAMRA。
2、反应液及反应酶混合液的配制:
所述2×NASBA反应液及5×NASBA反应酶混合液根据优化试验,确定为如下具体最优配方:50mM Tris-HCl(pH8.3)、100mM KCl、10mM MgCl2、1.0% Triton X-100、0.5mM dNTP、引物F1和引物R1各2.5μmol/L、引物F2和引物R2各2μmol/L、P1探针0.5μmol/L和P2探针0.4μmol/L;5×RT-PCR反应酶混合液包括4U/mL Taq酶、1.6U/μL MMLV反转录酶、3U/μL RNA酶抑制剂和30mg/mL BSA。
3、标准品
标准品为终浓度为1×108 copies/mL的含有标准品序列的重组pUC57质粒,TE溶液溶解(TE溶液的组成为10mmol/L 三(羟甲基)氨基甲烷盐酸盐、1mmol/L 乙二胺四乙酸和水)。重组pUC57质粒插入的标准品序列为:
GCGCTGCCCCATACCTGAAGACCAAGTTTATCTGTGTGACACCAACGACCTGCAGCAATACCATTGACCTGCCGATGTCCCCCCGCACTTTAGATTCATTGATGCAGTTTGGAAATAAKGGTGAAGGTGCTGAACCCTCAGCAGGAGGGCAGTTTG。
4、阳性质控品和阴性质控品
阳性质控品为含有STAT3 mRNA的细胞裂解液样品,阴性质控品为无RNA酶的DEPC-H2O。
实施例2 标准曲线的制备
反应液配制:2×NASBA反应液为10µL×n,5×RT-PCR反应酶混合液为4µL×n,DEPC-H2O为2µL×n。将各组分混匀,每管16 µL分装到八连管中(n为反应管数)。
标准品的梯度稀释:将标准品溶液采用10倍梯度稀释的方法,依次稀释为5×107copies/mL、5×106copies/mL、5×105copies/mL、5×104copies/mL和5×103copies/mL。
标准曲线的制作:取4µL各浓度的标准品稀释液,加入至已装有16µL的反应液中,在罗氏公司的Light Cycler 480 定量PCR仪上进行PCR扩增。PCR反应条件为:50℃,15 min→ 95℃,10 min→(95℃,15 s→ 55℃,45 s)。括号内条件共重复 40 个循环。
根据标准品的荧光定量PCR检测结果(图1)绘制标准曲线,标准曲线如图2所示。横坐标为X,代表标准品起始拷贝数的对数值(Log10),纵坐标为Y,代表Ct值。标准曲线的方程为Y=-3.38X+50.82,相关系数为 0.9975。
本发明的试剂盒使用FAM和VIC双通道可对STAT3 mRNA的表达进行准确的定量分析,具有快速、简便、灵敏度高等检测优点。
上述实施例为本发明较佳的实现方案,除此之外,本发明还可以其它方式实现,在不脱离本发明构思的前提下任何显而易见的替换均在本发明的保护范围之内。
Claims (10)
1.一种用于定量检测STAT3 mRNA水平的PCR引物,其特征在于:所述引物包括用于扩增STAT3基因的引物F1和引物R1、以及用于扩增对照基因GADPH的引物F2和引物R2,所述4条引物的核苷酸序列分别为:
F1:5’-TGTGACACCAACGACCTGC-3'
R1:5’-CCAAACTGCATCAATGAATCTA-3'
F2:5’-CCTGCCAAGTATGATGACATCAAGA-3'
R2:5’-GTAGCCCAGGATGCCCTTTAGT-3'。
2.一种用于定量检测STAT3 mRNA水平的PCR探针,其特征在于:所述探针包括用于检测STAT3基因的探针P1和用于检测对照基因GAPDH的探针P2,所述探针的核苷酸序列分别为:
P1:5’-ACCATTGACCTGCCGATGTCCC-3'
P2:5’-TGGTGAAGCAGGCGGCCGAG-3';
其中,所述探针P1和探针P2的5’端均标记荧光基团,3’端均标记淬灭基团。
3.根据权利要求2所述的一种用于定量检测STAT3 mRNA水平的PCR探针,其特征在于:所述探针P1的5’端标记荧光基团FAM,3’端标记淬灭基团TAMRA。
4.根据权利要求2所述的一种用于定量检测STAT3 mRNA水平的PCR探针,其特征在于:所述探针P2的5’端标记荧光基团VIC,3’端标记淬灭基团TAMRA。
5.一种用于定量检测STAT3 mRNA水平的PCR试剂盒,其特征在于:所述试剂盒包括权利要求1所述的引物和权利要求2-4任一项所述的探针。
6.根据权利要求5所述的一种用于定量检测STAT3 mRNA水平的PCR试剂盒,其特征在于:所述试剂盒由2×NASBA反应液、5×NASBA反应酶混合液、标准品、阳性质控品及阴性质控品组成。
7.根据权利要求6所述的一种用于定量检测STAT3 mRNA水平的PCR试剂盒,其特征在于:所述2×RT-PCR反应液包括如下具体成分:50mM Tris-HCl(pH8.3)、100mM KCl、10mMMgCl2、1.0% Triton X-100、0.5mM dNTP、引物F1和引物R1各2.5μmol/L、引物F2和引物R2各2μmol/L、P1探针0.5μmol/L和P2探针0.4μmol/L。
8.根据权利要求6所述的一种用于定量检测STAT3 mRNA水平的PCR试剂盒,其特征在于:所述5×RT-PCR反应酶混合液包括4U/mL Taq酶、1.6U/μL MMLV反转录酶、3U/μL RNA酶抑制剂和30mg/mL BSA。
9.根据权利要求6所述的一种用于定量检测STAT3 mRNA水平的PCR试剂盒,其特征在于:所述标准品为含有标准品序列的重组pUC57质粒,所述标准品序列为:
GCGCTGCCCCATACCTGAAGACCAAGTTTATCTGTGTGACACCAACGACCTGCAGCAATACCATTGACCTGCCGATGTCCCCCCGCACTTTAGATTCATTGATGCAGTTTGGAAATAAKGGTGAAGGTGCTGAACCCTCAGCAGGAGGGCAGTTTG。
10.根据权利要求6所述的一种用于定量检测STAT3 mRNA水平的PCR试剂盒,其特征在于:所述阳性质控品为含有STAT3 mRNA的细胞裂解液样品,阴性质控品为无RNA酶的DEPC-H2O。
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