CN116732165A - 结核诊断circRNA标志物、引物及其应用 - Google Patents
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Abstract
本发明公开了一种结核诊断circRNA标志物、引物及其应用。本发明筛选到两条在活动性肺结核患者PBMC中显著高表达的circRNA,即:hsa_circ_0002371,其转录区域位于10号染色体,由CCAR1基因的外显子20和21的反向剪接形成;hsa_circ_0007460,其转录区域位于1号染色体,由GOSR1基因的外显子6~9的反向剪接形成。本发明还公开了基于circRNA标志物的结核诊断试剂盒中的引物;利用本发明的circRNA标志物能够有效区分未感染人群与活动性肺结核患者,为研究活动性肺结核的发病机制提供新的思路,也为活动性肺结核的早期诊断提供了新的靶标。
Description
技术领域
本发明涉及生物技术领域,具体地说,涉及一种结核诊断circRNA标志物、引物及其应用。
背景技术
结核病(Tuberculosis,TB)是由结核分枝杆菌(Mycobacterium tuberculosis,Mtb)引起的严重呼吸道传染病。
结核感染的早期诊断对于控制该疾病的传播至关重要。目前,细菌培养和涂片镜检仍是结核病临床诊断中最广泛使用的工具,但是它们耗时、灵敏度差。因此,迫切需要寻找结核病特异性的生物标志物,建立一种快速、灵敏、高效的肺结核诊断方法,以防止结核病的广泛传播。
环状RNA(Circular RNA,circRNA)是真核生物中共价闭合的内源性分子,缺乏5'-帽子和3'-poly(A)尾巴,大多数由已知的蛋白质编码基因表达,具有组织特异性和细胞特异性。由于具有共价闭合环状的特性,且能耐受RNase R的降解,circRNA可以作为肿瘤、心血管疾病和其他疾病的潜在生物标志物。
很多研究发现circRNA的失调和Mtb感染有关,其或许可以作为TB诊断的新型生物标志物。Zhuang Z-G等研究表明,hsa_circ_0005836可作为结核病诊断新的潜在生物标志物。Huang Z等发现hsa_circ_0043497和hsa_circ_0001204在TB患者的单核细胞衍生巨噬细胞中的表达显着增加。Zhang X-L等研究表明hsa_circ_0028883对结核病诊断具有潜在价值,是一种潜在可靠的生物标志物。上述研究表明,circRNA可能在TB中发挥重要作用,并具有作为结核病诊断的新型生物标志物的巨大潜力。
发明内容
本发明所要解决的技术问题是提供一种结核诊断circRNA标志物、引物及其应用;本发明首次公开了两个circRNA作为活动性肺结核病特异性标志物,具有很高的稳定性;本发明利用临床上比较容易直接获得的全血样本,分离其中的PBMC,结合设计的引物对进行RT-qPCR验证circRNA的差异表达,具有快速、简单、省时、检测灵敏度高,样品消耗少的优点,是非常适合的利用差异表达的circRNA诊断活动性肺结核病的方法。
本发明利用circRNA测序,筛选到两条在活动性肺结核病患者PBMC中显著高表达的circRNA,即hsa_circ_0002371和hsa_circ_0007460,hsa_circ_0002371转录区域位于10号染色体,由CCAR1基因的外显子20和21的反向剪接形成,全长230bp,hsa_circ_0007460转录区域位于1号染色体,由GOSR1基因的外显子6~9的反向剪接形成,全长363bp。本发明的技术方案具体如下:
本发明提供一种circRNA标志物在制备结核诊断试剂盒中的应用,circRNA标志物为hsa_circ_0002371或者hsa_circ_0007460中的一种或两种;所述hsa_circ_0002371长230bp,位于人类的第10条染色体上,所述hsa_circ_0002371的碱基序列如SEQ ID NO:5所示;所述hsa_circ_0007460长363bp,位于人类的第1条染色体上,所述hsa_circ_0007460的碱基序列如SEQ ID NO:6所示。
本发明中,hsa_circ_0002371序列为SEQ ID NO:5,具体如下:ATAGGGATGAGGAAGAAATGACCAAACGAGATGACAAAAGAGATATCAACAGATACTGCAAGGAGAGGCCCTCTAAAGATAAGGAAAAAGAAAAGACTCAAATGATCACAATTAACAGAGATCTGTTAATGGCTTTTGTTTATTTTGATCAAAGTCATTGTGGTTACCTTCTTGAAAAGGATTTGGAAGAAATACTTTATACTCTTGGACTACATCTTTCTCGGGCTCAG。
本发明中,hsa_circ_0007460序列为SEQ ID NO:6,具体如下:TTCTGATACAACACCCCTTTTAAATGGATCAAGCCAAGACAGAATGTTTGAGACAATGGCGATTGAGATTGAACAACTTTTGGCAAGGCTTACAGGGGTAAATGATAAAATGGCAGAATATACCAACAGTGCAGGTGTCCCCTCCTTGAATGCAGCCCTGATGCATACATTACAGCGGCATAGAGACATATTGCAGGATTATACACATGAATTCCATAAAACCAAAGCAAACTTTATGGCAATACGGGAAAGGGAGAATCTTATGGGATCAGTACGAAAAGATATTGAGTCATATAAAAGTGGGTCTGGAGTAAACAACAGAAGAACTGAGCTATTTTTGAAAGAACATGACCACCTTCGAAA。
本发明中,结核诊断试剂盒为用于诊断活动性肺结核的活动性肺结核诊断试剂盒。
本发明中,结核诊断试剂盒检测被检测对象的样品中的hsa_circ_0002371、hsa_circ_0007460中一种或多种含量,其中所述hsa_circ_0002371、hsa_circ_0007460的量与所述被检测对象患有肺结核病的概率成正相关关系。
本发明中,hsa_circ_0002371和hsa_circ_0007460的含量是从被检测对象全血样品中的外周血单个核细胞PBMC确定的。
本发明还提供一种引物在制备结核诊断试剂盒中的应用,结核诊断circRNA标志物为hsa_circ_0002371或者hsa_circ_0007460中的一种或两种;所述hsa_circ_0002371长230bp,位于人类的第10条染色体上,所述hsa_circ_0002371的碱基序列如SEQ ID NO:5所示;所述hsa_circ_0007460长363bp,位于人类的第1条染色体上,所述hsa_circ_0007460的碱基序列如SEQ ID NO:6所示;引物用于获得被检测对象的外周血单个核细胞PBMC中的hsa_circ_0002371和hsa_circ_0007460的表达量;引物由以下序列构成:hsa_circ_0002371正向引物序列(SEQ ID NO:1):5'-AGTCATTGTGGTTACCTTCTTGA-3';hsa_circ_0002371反向引物序列(SEQ ID NO:2)5'-GGGCCTCTCCTTGCAGTATC-3’;hsa_circ_0007460正向引物序列(SEQ ID NO:3):5'-GTGGGTCTGGAGTAAACAACAG-3';hsa_circ_0007460反向引物序列(SEQ ID NO:4):5'-CCCTGTAAGCCTTGCCAAAA-3'。
本发明中,circRNA作为活动性肺结核病特异性标志物;本发明通过提取活动性肺结核病患者和健康对照者全血样本中PBMC的总RNA,设计RT-qPCR特异性引物,并通过RT-qPCR验证活动性肺结核病特异性circRNA的表达情况,并通过ROC曲线评价其诊断表现;本发明提供了鉴别可能患有活动性肺结核对象的方法及其引物序列;本发明中,circRNA相对表达量分析采用GraphPad Prism 9软件,采用软件中单样本T检验和独立样本T检验,P<0.05时,认为结果在统计学上具有显著性差异。
本发明发现:circRNA测序结果显示hsa_circ_0002371和hsa_circ_0007460在活动性肺结核病人里面显著上调,进一步扩大样本,用RT-qPCR检测活动性肺结核和健康人的PBMC,相对健康人样本,活动性肺结核病人hsa_circ_0002371和hsa_circ_0007460的表达量发生了显著性上调表达,在统计学上具有显著性差异,且ROC曲线分析显示AUC(hsa_circ_0002371)=0.8063,AUC(hsa_circ_0007460)=0.7474,AUC(combined)=0.8177,两者单独诊断具有较高的特异性和敏感性,且联合诊断可以进一步提高两者的诊断效能。可以说明hsa_circ_0002371、hsa_circ_0007460中的一种或者多种可以作为活动性肺结核病特异性标志物用于诊断。
附图说明
图1为circRNA芯片检测结果图。
图2为hsa_circ_0002371在活动性肺结核病人和正常健康人之间的差异表达图。
图3为hsa_circ_0007460在活动性肺结核病人和正常健康人之间的差异表达图。
图4hsa_circ_0002371、hsa_circ_0007460以及两者联合诊断表现评估图。
具体实施方式
下面结合附图和实施例对本发明的技术方案进行详细阐述。
实施例中,人淋巴细胞分离液购自深圳市达科为生物工程有限公司;总RNA提取试剂购自广州美基生物科技有限公司;Epicentre Ribo-Zero Gold Kit购自因美纳公司;RNA-Seq样品制备试剂盒购自因美纳公司;RT-qPCR反转录试剂盒购自南京诺唯赞生物科技股份有限公司;Taq Pro Universal SYBR qPCR Master Mix购自南京诺唯赞生物科技股份有限公司;96孔板及膜购自Axygen公司。
实施例1活动性肺结核患者与健康志愿者全血中PBMC的circRNA芯片表达分析
A1:收集3例临床确诊的活动性肺结核患者和3例健康对照者。所有参加者血样为晨起空腹抽取,采用一次性真空EDTA抗凝采血管,采集外周血5.0mL,用等体积无菌PBS稀释全血。在离心管中加入一定体积的人淋巴细胞分离液,将稀释后的血样平铺到分离液液面上方,保持两液面界面清晰,分离液、抗凝未经稀释全血、无菌PBS体积为1:1:1。室温,水平转子800g离心25min。离心结束后,管底是红细胞,中间层是分离液,最上层是血浆/组织匀浆层,血浆层与分离液层之间是一层薄较致密的白膜,即:单个核细胞(包括淋巴细胞和单核细胞)层。小心吸取白膜层到另一离心管中。用无菌PBS稀释到一定体积,颠倒混匀。室温,水平转子250g,离心10min弃上清。重复洗涤1~2次。用1mL的TRIzol轻轻悬浮,-80℃冰箱保存。
A2:将活动性肺结核病患者和健康对照者含有TRIzol的PBMC样本于-80℃冰箱中取出,待4℃自然溶解。加入0.2mL氯仿,剧烈振荡15s,室温放置3min后4℃12000g离心15min。样品分为三层:底层为黄色有机相,上层为无色水相和一个中间层(DNA),RNA主要在水相中。把水相转移到新管中,加0.5mL异丙醇沉淀水相中的RNA,混匀,冰箱中放置30min后4℃12000g离心10min,弃上清。用1mL 75%乙醇洗涤RNA沉淀,旋涡振荡30s后4℃12000g离心5min,弃上清,在超净台中鼓风静置3~5min。加入20μL的DEPC。管中的液体即为提取的总RNA,可放置在-80℃冰箱保存。
A3:提取总RNA后,构建大于200nt的去rRNA链特异性文库。根据Epicentre Ribo-Zero Gold Kit试剂盒操步骤,对10μg总RNA进行核糖体RNA的去除。随后,在高温下使用二价阳离子将poly(A)-或poly(A)+RNA片段分割成小段。然后按照RNA-Seq样品制备试剂盒(Illumina,San Diego,USA)的方案,对裂解的RNA片段进行反转录,建立最终的cDNA文库,文库的平均插入长度为300bp(±50bp)。然后,我们按照供应商推荐的方案,在IlluminaHiseq 4000上进行配对末端测序。提取circRNA芯片原始数据,用R软件包对数据进行归一化及后续数据处理,寻找2组样本间差异表达的circRNA,通过倍数变化、circRNA长度、物种保守性和P值等条件进行筛选,差异circRNA的标准为ATB组与HC组表达比值≥2倍且P<0.05。
A4:关于活动性肺结核患者与健康志愿者全血中PBMC的circRNA芯片聚类分析见图1,芯片筛查发现多条表达上调和表达下调的circRNA。其中hsa_circ_0002371和hsa_circ_0007460符合我们的筛选标准,在活动性肺结核患者的PBMC中显著上调,鉴于其可能在活动性肺结核患者的PBMC中存在特异性高表达,本发明通过以下实施例采用大规模样本进行验证。
实施例2活动性肺结核病人hsa_circ_0002371的表达量发生了显著性上调表达A1:收集30例临床确诊的活动性肺结核患者和32例健康对照者。所有参加者血样为晨起空腹抽取,采用一次性真空EDTA抗凝采血管,采集外周血5.0mL,用等体积无菌PBS稀释全血。在离心管中加入一定体积的人淋巴细胞分离液,将稀释后的血样平铺到分离液液面上方,保持两液面界面清晰,分离液、抗凝未经稀释全血、无菌PBS体积为1:1:1。室温,水平转子800g离心25min。离心结束后,管底是红细胞,中间层是分离液,最上层是血浆/组织匀浆层,血浆层与分离液层之间是一层薄较致密的白膜,即:单个核细胞(包括淋巴细胞和单核细胞)层。小心吸取白膜层到另一离心管中。用无菌PBS稀释到一定体积,颠倒混匀。室温,水平转子250g,离心10min弃上清。重复洗涤1~2次。用1mL的TRIzol轻轻悬浮,-80℃冰箱保存。
A2:将活动性肺结核病患者和健康对照者含有TRIzol的PBMC样本于-80℃冰箱中取出,待4℃自然溶解。加入0.2mL氯仿,剧烈振荡15s,室温放置3min后4℃12000g离心15min。样品分为三层:底层为黄色有机相,上层为无色水相和一个中间层(DNA),RNA主要在水相中。把水相转移到新管中,加0.5mL异丙醇沉淀水相中的RNA,混匀,冰箱中放置30min后4℃12000g离心10min,弃上清。用1mL 75%乙醇洗涤RNA沉淀,旋涡振荡30s后4℃12000g离心5min,弃上清,在超净台中鼓风静置3~5min。加入20μL的DEPC。管中的液体即为提取的总RNA,可放置在-80℃冰箱保存。
取活动性肺结核病患者和健康对照者总RNA样本5.0μL,加入0.5μL的10×LoadingBuffer,130V,电泳30min,紫外灯下观察条带分布。经Thermo分析仪器nanodrop 2000检测OD值,在紫外分光光度计上测定A260/A280数值,测定结果总RNA浓度在50~500ng/μL之间,A260/A280数值在1.8~2.2之间,表示总RNA的纯度较好,无蛋白或者其他有机物的污染亦无总RNA降解。
A3:设计circRNA RT-qPCR引物对RT-qPCR引物长度20bp左右,退火温度Tm值在60℃左右,GC含量约40%~60%;引物对的序列为SEQ ID NO:1、SEQ ID NO:2或者SEQ ID NO:3、SEQ ID NO:4,参见表1。
以内参GAPDH在活动性肺结核病患者全血样本分离出来的PBMC中扩增为例,观察扩增情况,扩增曲线平滑且随着循环数的增加,荧光信号具有明显的对数期,而空白对照无连续曲线生成,说明扩增情况良好。对扩增的circRNA产物做溶解曲线,有且只有一个明显的单峰,而无模板对照无明显波峰出现,说明经扩增后产物是特异的circRNA,即此定量无污染,准确,可靠。
表1 RT-qPCR引物
Primers | Sequence(5'-3') | nt | |
hsa_circ_0002371-F | AGTCATTGTGGTTACCTTCTTGA | 23 | SEQ ID NO:1 |
hsa_circ_0002371-R | GGGCCTCTCCTTGCAGTATC | 20 | SEQ ID NO:2 |
hsa_circ_0007460-F | GTGGGTCTGGAGTAAACAACAG | 22 | SEQ ID NO:3 |
hsa_circ_0007460-R | CCCTGTAAGCCTTGCCAAAA | 20 | SEQ ID NO:4 |
GAPDH-F | TCTCTGCTCCTCCTGTTCGA | 20 | |
GAPDH-R | GCGCCCAATACGACCAAATC | 20 |
A4:活动性肺结核病诊断试剂盒RT-qPCR活动性肺结核病相关的特异性circRNA
用HiScript III All-in-one RT SuperMix Perfect for qPCR反转录试剂盒进行反转录,反转录反应体系:5×All-in-one qRT SuperMix 4μL,Enzyme Mix 1μL,总RNA 1μg,总体积20μL。反应条件:50℃,15min;85℃,5sec。反应完毕后,保存于-80℃冰箱。
RT-qPCR体系:2×ChamQ Universal SYBR qPCR Master Mix 10μL,模板cDNA2μL,正向/反向引物(10μM)0.4μL,ddH2O 7.2μL,总体积20μL。混匀后3000rpm离心5min,在CFX96荧光定量PCR仪中进行荧光定量PCR反应,反应参数设置为:95℃30sec,然后95℃10sec,60℃30sec,共40个循环,95℃15sec,60℃60sec,95℃15sec。
circRNA荧光定量数据分析以GAPDH为内参基因,对目标基因进行归一化处理,已确保相等数量的样品中比较目标基因的量。circRNA表达倍数的变化公式为RE=2-△△Ct,其中△△Ct=(CtcircRNA-CtGAPDH)ATB-(CtcircRNA-CtGAPDH)Mean HC。RE代表相对表达变化量,CtcircRNA和CtGAPDH分别代表同个样本中目标circRNA和内参基因GAPDH的Ct值,ATB代表活动性肺结核病患者,HC代表健康对照者,Mean HC代表所有正常对照组中的平均值。实验设立阴性对照和重复实验,阴性对照为反应体系中不加cDNA模板,以ddH2O代替,所有RT-qPCR均做3次重复。circRNA相对表达量分析采用GraphPad Prism 9软件,采用软件中单样本T检验和独立样本T检验。P<0.05时,认为结果在统计学上具有显著性差异,差异表达的circRNA数据处理结果以平均值±标准误表示,绘制含误差线的散点图。
分别在30例肺结核病人和32例正常健康人中检测hsa_circ_0002371和hsa_circ_0007460的表达量,结果显示在肺结核病人中hsa_circ_0002371和hsa_circ_0007460发生了显著性上调表达,hsa_circ_0002371表达量差异见图2,hsa_circ_0007460表达量差异见图3,提示两者皆可作为诊断标志物。受试者工作特征曲线(ROC)下面积AUC(hsa_circ_0002371)=0.8063,AUC(hsa_circ_0007460)=0.7474,AUC(combined)=0.8177,表明hsa_circ_0002371和hsa_circ_0007460对于诊断活动性肺结核准确性较好,且两者联合诊断可以进一步提高诊断的准确性,hsa_circ_0002371、hsa_circ_0007460及两者联合诊断表现评估图见图4。
上述为本发明较佳的具体实施方式,本发明的保护范围不限于此,任何熟悉本技术领域的技术人员在本发明披露的技术范围内,可显而易见地得到的技术方案的简单变化或等效替换均落入本发明的保护范围内。
Claims (5)
1.一种circRNA标志物在制备结核诊断试剂盒中的应用,其特征在于, circRNA标志物
为hsa_circ_0002371或者hsa_circ_0007460中的一种或两种;所述hsa_circ_0002371长230 bp,位于人类的第10条染色体上,所述hsa_circ_0002371的碱基序列如SEQ ID NO:5所示;所述hsa_circ_0007460长363 bp,位于人类的第1条染色体上,所述hsa_circ_0007460的碱基序列如SEQ ID NO:6所示。
2.根据权利要求1所述的应用,其特征在于:结核诊断试剂盒为用于诊断活动性肺结核
的活动性肺结核诊断试剂盒。
3.如权利要求1或2所述的应用,其特征在于,结核诊断试剂盒检测被检测对象的样品
中的hsa_circ_0002371、hsa_circ_0007460中一种或多种含量,其中所述hsa_circ_0002371、hsa_circ_0007460的量与所述被检测对象患有肺结核病的概率成正相关关系。
4.如权利要求3所述的应用,其特征在于,hsa_circ_0002371和hsa_circ_0007460的含量
是从被检测对象全血样品中的外周血单个核细胞PBMC确定的。
5.一种引物在制备结核诊断试剂盒中的应用,其特征在于,结核诊断circRNA标志物为hsa_circ_0002371或者hsa_circ_0007460中的一种或两种;所述hsa_circ_0002371长230bp,位于人类的第10条染色体上,所述hsa_circ_0002371的碱基序列如SEQ ID NO:5所示;所述hsa_circ_0007460长363 bp,位于人类的第1条染色体上,所述hsa_circ_0007460的碱基序列如SEQ ID NO:6所示;引物用于获得被检测对象的外周血单个核细胞PBMC中的hsa_circ_0002371和hsa_circ_0007460的表达量;引物由以下序列构成:
hsa_circ_0002371正向引物序列:5'-AGTCATTGTGGTTACCTTCTTGA- 3';hsa_circ_0002371反向引物序列5'-GGGCCTCTCCTTGCAGTATC-3’;hsa_circ_0007460正向引物序列:5'-GTGGGTCTGGAGTAAACAACAG-3';hsa_circ_0007460反向引物序列:5'-CCCTGTAAGCCTTGCCAAAA-3'。
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