CN116694642B - 一种不稳定型心绞痛生物标记物及其检测方法、应用和试剂盒 - Google Patents
一种不稳定型心绞痛生物标记物及其检测方法、应用和试剂盒 Download PDFInfo
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Abstract
本发明涉及分子生物技术领域,尤其涉及一种不稳定型心绞痛tsRNA生物标记物及其检测方法、应用和试剂盒。本发明通过研究发现,AS‑tDR‑009512在不稳定型心绞痛患者血浆中的表达水平显著高于非不稳定型心绞痛人群,故可作为辅助诊断不稳定型心绞痛的生物标志物。本发明还提供了含有该AS‑tDR‑009512的扩增引物的试剂盒,该试剂盒能够用于血浆中AS‑tDR‑009512表达量的检测,可用于辅助不稳定型心绞痛的诊断、治疗和预后。
Description
技术领域
本发明涉及分子生物技术领域,尤其涉及一种不稳定型心绞痛tsRNA生物标记物及其检测方法、应用和试剂盒。
背景技术
不稳定型心绞痛(Unstable angina,UA)发病率高、并发症严重,如不及时治疗,患者可能发展为心肌梗死。目前,针对UA的诊断主要依靠临床表现及心电图。生化标志物也可用于辅助诊断不稳定型心绞痛,如肌钙蛋白和肌酸激酶,二者在心肌缺血或心肌梗死时会释放到血液中,使血浆中肌钙蛋白、肌酸激酶的水平升高。但肌钙蛋白升高的原因还包括心肌炎、心包炎、心内膜炎等,肌酸激酶升高的特异性也不强。因此,进一步挖掘UA标志物对于UA患者早期诊断、治疗及预后具有重要价值。
发明内容
针对以上技术问题,本发明提供了一种不稳定型心绞痛tsRNA生物标记物及其检测方法、应用和试剂盒。本发明提供的tsRNA生物标记物在UA患者和非UA人群中的表达水平有显著差异,具有辅助检测UA的潜力,可用于制成检测UA的试剂;本发明提供的试剂盒中含有tsRNA生物标记物特异性引物,可用于检测血浆中的上述tsRNA生物标记物的表达量。
为达到上述发明目的,本发明采用了如下的技术方案:
第一方面,本发明提供一种不稳定型心绞痛tsRNA生物标记物,所述tsRNA生物标记物为AS-tDR-009512,其序列如SEQ ID NO.1所示。
血浆tRNA来源的小RNA(tRNA-derived small RNA,tsRNA)作为一种新型非编码RNA分子,具有含量丰富、存在广泛、性质稳定、组织特异性等特征,具有作为疾病诊断分子的巨大优势,逐渐被人们所挖掘和关注,目前,针对tsRNA与疾病相关性的报道主要涉及癌症、生殖系统疾病、消化道疾病、感染性疾病、代谢性疾病等,其在血管重构性疾病中的诊断作用尚不清楚,且尚无关于将血浆tRNA作为用于辅助诊断UA的诊断标志物的报道。
本发明通过研究发现,AS-tDR-009512靶基因及其富集通路在UA发生、发展中起着关键作用,AS-tDR-009512在增殖型血管平滑肌细胞VSMC(vascular smooth muscle cell,VSMC)中显著上调,VSMC作为血管壁的主要细胞,其异常增殖与动脉粥样硬化、血管狭窄等血管重塑性疾病的发生发展有密切联系,因此,AS-tDR-009512具有作为血管重构性疾病诊断标志物的潜力。本发明经进一步研究发现,AS-tDR-009512在UA患者血浆中的表达水平显著高于健康者,故可作为辅助诊断UA的生物标志物。
SEQ ID NO.1为:GCATTGGTGGTTCAGTGGTAGAATTCTCGCCTCCCACGCGGGAGACCCGGG。
第二方面,本发明提供上述tsRNA生物标记物在制备用于检测不稳定型心绞痛的试剂中的应用。
针对AS-tDR-009512进行引物设计,所得特异性引物即可用于制备检测不稳定型心绞痛的试剂。利用该引物对待测血浆的小RNA进行扩增后,通过检测AS-tDR-009512的表达量,即可初步判断待测血浆是否来自UA患者。
第三方面,本发明提供一种用于检测不稳定型心绞痛的试剂盒,所述试剂盒包括实时荧光定量PCR试剂组,所述实时荧光定量PCR试剂组包括上述tsRNA生物标记物的扩增引物,所述tsRNA生物标记物的正向特异引物序列(如SEQ ID NO.2所示)为:ACCCACGCGGGAGACC。
上述扩增引物能特异地扩增待测血浆中AS-tDR-009512,经PCR扩增后,即可获得待测血浆中AS-tDR-009512的表达量,进而判断待测血浆是否来自于UA患者,对于UA患者的早期诊断、治疗及预后均有重要意义。
优选地,所述试剂盒还包括总sRNA提取试剂组和RNA反转录试剂组。
优选地,所述总sRNA提取试剂组包括细胞裂解液、氯仿、乙醇和漂洗液。
优选地,所述RNA反转录试剂组包括反转录引物、反转录反应缓冲液和反转录酶混合液。
优选地,所述实时荧光定量PCR试剂组还包括内参基因U6扩增引物。
优选地,所述内参基因U6扩增引物的序列(如SEQ ID NO.3~4所示)为:
内参基因U6的正向引物:CGATACAGAGAAGATTAGCATGGC;
内参基因U6的反向引物:AACGCTTCACGAATTTGCGT。
优选地,所述实时荧光定量PCR试剂组还包括其他可用于荧光定量PCR检测的试剂,如miRcute Plus miRNA Premix(with SYBR&ROX)。
以及,本发明还提供了用上述检测不稳定型心绞痛的试剂盒非诊断目的的检测血浆中AS-tDR-009512表达量的方法,具体包括以下步骤:
提取待测血浆中的总sRNA;
对提取所得总sRNA进行反转录;
用所述荧光定量PCR试剂组对反转录所得产物进行实时荧光定量PCR扩增。
优选地,对提取所得总sRNA进行反转录的反转录体系为:
反转录的条件为:42℃,60min。
优选地,用所述荧光定量PCR试剂组进行扩增的反应体系为:
扩增的反应条件为:95℃ 10min;95℃ 5s,60℃ 20s,72℃ 10s,40个循环。
附图说明
图1为本发明血浆中AS-tDR-009512表达水平在两组人群中的差异(*表示P<0.05);
图2为本发明血浆AS-tDR-009512对UA患者诊断价值的ROC曲线。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合具体实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
实施例1
本发明实施例提供了一种用于检测不稳定型心绞痛的试剂盒,具体包括:
(1)总sRNA提取试剂组:包括细胞裂解液、氯仿、乙醇和漂洗液。
(2)RNA反转录试剂组:反转录引物、反转录反应缓冲液和反转录酶混合液。
(3)荧光定量PCR试剂组,包括tsRNA生物标记物的扩增引物、内参基因U6扩增引物、miRcute增强型miRNA定量预混试剂(含SYBR&ROX),tsRNA生物标记物的扩增引物和内参基因U6的扩增引物的序列为:
tsRNA生物标记物的正向特异引物:ACCCACGCGGGAGACC;
tsRNA生物标记物的反向引物:通用引物;
内参基因U6的正向特异引物:CGATACAGAGAAGATTAGCATGGC;
内参基因U6的反向特异引物:AACGCTTCACGAATTTGCGT。
实施例2
本实施例利用实施例1提供的用于检测不稳定型心绞痛的试剂盒检测血浆中AS-tDR-009512表达量的方法。
1、试验方法
1.1 试验对象
随机选择2021年4月至2021年9月河北医科大学第三医院收治的15例UA患者的留存血液样本,患者年龄48~65岁,平均(58.20±5.10)。选取2021年5月北京铁路局石家庄卫生防疫站体检中心10例健康体检者的留存血液样本作为对照组,体检者年龄54~57岁,平均(55.4±1.02)。UA患者符合《不稳定性心绞痛和非ST段抬高心肌梗死诊断与治疗指南》;对照人群无既往心血管病史,无血缘关系,近期内无外伤史及出血史,排除感染、肿瘤、糖尿病等。两组年龄、体重、收缩压、舒张压均无统计学差异(P> 0.05)。
1.2 主要仪器与试剂
血浆/血浆miRNA提取分离试剂盒(货号:DP503)、miRcute增强型miRNA cDNA第一链合成试剂盒(货号:KR211)、miRcute增强型miRNA荧光定量检测试剂盒(SYBR Green)(货号:FP411)、内参U6(正向特异引物:CGATACAGAGAAGATTAGCATGGC,反向特异引物:AACGCTTCACGAATTTGCGT)、AS-tDR-009512(正向特异引物:ACCCACGCGGGAGACC)、实时定量聚合酶链式反应仪(ABIQ3)。
1.3 血浆中小RNA的提取
取200 μl血浆,向其中加入900 μl裂解液,振荡器充分振荡并混匀30 s,室温下静置5 min。向静置液中加入200 μl氯仿,并充分剧烈振荡15 s,于室温下静置5 min。在12,000 r/min,4℃的条件下离心15 min,将上层水相充分转移至新管后,将2倍体积的无水乙醇缓慢加至上述新管中并进行充分混匀(此时可能会出现沉淀)。将得到的溶液和沉淀一起转入吸附柱,在室温下静置2 min,在12,000 r/min,室温的条件下离心30 s,离心后弃掉流出液。将700 μl去蛋白液加入至吸附柱中,于室温下静置2 min后在12,000 r/min,室温的条件下离心30 s,弃去废液。向吸附柱中加入500 μl漂洗液,于室温下静置2 min后,在12,000 r/min,室温的条件下离心30 s,弃去废液。再次漂洗。在12,000 r/min,室温下离心2min。将吸附柱转移至无核酸酶离心管中,于超净台中干燥10 min,以充分晾干漂洗液。在吸附膜中心位置处加30 μl 无核酸酶H2O,室温静置5 min,在12,000 r/min,室温下离心2min,用上述无核酸酶H2O重复洗脱三次,以增加小RNA浓度。
1.4 tsRNA的qRT-PCR检测
反转录体系:总RNA 8 μl(1.5 μg),2×miRNA反转录缓冲液10 μL,miRNA反转录混合酶液2 µl,构成20 μL体系,混匀。其中2×miRNA反转录缓冲液和miRNA反转录混合酶液均来自miRcute增强型miRNA cDNA第一链合成试剂盒(货号:KR211)。反转录程序如下:42℃,60 min,完成miRNA加多聚poly(A)尾反应和反转录反应;95℃,3 min,完成酶失活反应。
qRT-PCR反应体系:2×miRcute增强型miRNA定量预混试剂(含SYBR&ROX)10 μl,tsRNA正向特异引物(10 μmol/L)1 μL,tsRNA反向引物(10 μmol/L)2 μL,U6正向特异引物(10 μmol/L)0.4 μL,U6反向特异引物(10 μmol/L)2 μL,无核酸酶H2O补足至20 μL。其中2×miRcute增强型miRNA定量预混试剂(含SYBR&ROX)和tsRNA反向引物来自miRcute增强型miRNA荧光定量检测试剂盒(SYBR Green)(货号:FP411)。qRT-PCR反应程序如下:95°C预变性10 min;95°C变性5 s,60°C退火20 s,72°C延伸10 s,重复40个循环,然后进行溶解曲线分析。采用相对定量方法(2-ΔΔCt)对qRT-PCR结果进行分析。
1.5 统计学方法
数据分析由SPSS 21.0和GraphPad Prism 8.0.2软件进行处理。除AS-tDR-009512的相对表达量以四分位法表示外,其余数据均以均数(mean)±标准差(S.D.)的形式表示,所有数据均来自三个独立的实验,每个实验重复三次。两组间比较使用秩和检验,P<0.05为差异具有统计学意义。
2、结果
qRT-PCR扩增产物的熔解曲线为单一的峰;对qRT-PCR扩增产物进行测序,所得扩增产物即为目标片段AS-tDR-009512。该结果说明本发明的tsRNA正向特异引物具有良好的特异性。
qRT-PCR结果显示,病例组血浆AS-tDR-009512表达水平较对照组升高了(4.13±4.28)倍,差异具有统计学意义(P< 0.05),如图1所示。
为了进一步评价AS-tDR-009512的诊断价值,本实验对病例组和对照组进行ROC曲线分析。AS-tDR-009512的曲线下面积(Area under the cure,AUC)为0.8067(95% CI:0.6286-0.9848,P= 0.0107),截断值为2.329,敏感性为93.33%,特异性为60.00%,如图2所示。
以上结果表明,AS-tDR-009512可作为辅助诊断UA的血浆诊断生物标志物。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内所作的任何修改、等同替换或改进等,均应包含在本发明的保护范围之内。
Claims (9)
1.一种tsRNA生物标记物在制备用于检测不稳定型心绞痛的试剂中的应用,其特征在于,所述tsRNA生物标记物为AS-tDR-009512,其序列如SEQ ID NO.1所示。
2. 一种tsRNA在制备用于检测不稳定型心绞痛的试剂盒中的应用,其特征在于,所述tsRNA为AS-tDR-009512,其序列如SEQ ID NO.1所示。
3.根据权利要求2所述的应用,其特征在于,所述试剂盒包括实时荧光定量PCR试剂组,所述实时荧光定量PCR试剂组包括所述tsRNA的扩增引物,其正向特异引物序列为:ACCCACGCGGGAGACC。
4.根据权利要求3所述的应用,其特征在于,所述试剂盒还包括总sRNA提取试剂组和RNA反转录试剂组。
5.根据权利要求4所述的应用,其特征在于,所述总sRNA提取试剂组包括细胞裂解液、氯仿、乙醇和漂洗液;和/或
所述RNA反转录试剂组包括反转录引物、反转录反应缓冲液和反转录酶混合液。
6.根据权利要求3所述的应用,其特征在于,所述实时荧光定量PCR试剂组还包括内参基因U6扩增引物。
7.根据权利要求6所述的应用,其特征在于,所述内参基因U6扩增引物的序列为:
内参基因U6的正向引物:CGATACAGAGAAGATTAGCATGGC;
内参基因U6的反向引物:AACGCTTCACGAATTTGCGT。
8.根据权利要求3~7任一项所述的应用,其特征在于,所述试剂盒非诊断目的的检测血浆中AS-tDR-009512表达量的方法具体包括以下步骤:
提取待测血浆中的总sRNA;
对提取所得总sRNA进行反转录;
用所述荧光定量PCR试剂组对反转录所得产物进行实时荧光定量PCR扩增。
9.根据权利要求8所述的应用,其特征在于,用所述荧光定量PCR试剂组进行扩增的反应体系为:
扩增的反应条件为:95℃ 10min;95℃ 5s,60℃ 20s,72℃ 10s,40个循环。
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