CN117512104B - 一种用于ad诊断的标志物组合和试剂盒 - Google Patents
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Abstract
本发明提供了一种用于AD诊断的标志物组合和试剂盒,标志物组合,包括:hsa‑let‑7i‑5p、hsa‑miR‑181c‑5p、hsa‑miR‑21‑5p、hsa‑miR‑29c‑3p、hsa‑miR‑92a‑3p、hsa‑let‑7f‑5p、hsa‑miR‑1285‑5p、hsa‑miR‑193b‑3p、hsa‑miR‑143‑3p。本发明使用血清或血浆样本将AD患者和健康人相关miRNA表达量差异来进行区分,从而快速、准确、无创且低廉地诊断AD。
Description
技术领域
本发明属于阿尔兹海默病(AD)诊断技术领域,涉及一种用于AD诊断的标志物组合和试剂盒。
背景技术
阿尔兹海默病(AD)是一种神经退行性疾病:AD疾病特征表现为一系列症状,包括记忆力减退和认知障碍(即语言、思维和行为方面的困难)。AD大脑中受损的脑细胞(即神经元)首先从海马体开始,海马体是大脑负责认知功能的部分;然而,研究认为需要 20 年或更长时间才可能会出现疾病症状,并且这些症状会随着时间的推移而恶化。严重的AD可能干扰大多数日常活动,患者可能需要全天候护理。AD的确切发病机制尚不明确,通常认为是老化、遗传和环境多种因素的共同结果,目前有多种学说,其中影响较广的是β类淀粉样蛋白级联假说。
AD起病隐匿,临床主要变现为认知障碍、精神行为异常、社会生活功能减退,是一种病情进行性发展的神经退行性疾病。根据患者病情,AD通常分为临床前期、轻度认知障碍(mild cognitive impairment,MCI)、轻度、中度、重度AD,临床确诊时患者通常处于后3个阶段。在临床前期和MCI阶段,虽然患者症状不明显,但生物标志物指标已有异常。
目前尚无足够准确的方法能早期筛查和识别阿尔茨海默症,主要诊断方法为联合诊断。对于拟行痴呆诊断的患者,首选进行认知功能评估,同时常规行CT和MRI等影像学检查,当常规方式不能明确时,考虑使用PET或脑脊液/血液生物标志物检测。对于有痴呆家族史或者快速进展型痴呆,应考虑添加基因检测。
目前AD早期诊断仍存在一定难度,如标志物的检测缺乏一定的规范和标准、患者缺乏对AD早期的认识和接受度等。
发明内容
本发明的目的在于提供一种用于AD诊断的标志物组合和试剂盒,使用血清或血浆样本将AD患者和健康人相关miRNA表达量差异来进行区分,从而快速、准确、无创且低廉地诊断AD。
本发明为实现上述目的采用的技术方案为:
本发明提供了一种用于AD诊断的标志物组合,包括:hsa-let-7i-5p、hsa-miR-181c-5p、hsa-miR-21-5p、hsa-miR-29c-3p、hsa-miR-92a-3p、hsa-let-7f-5p、hsa-miR-1285-5p、hsa-miR-193b-3p、hsa-miR-143-3p。
本发明还提供了一种用于AD诊断的试剂盒,包括上述标志物组合。
优选地,所述试剂盒通过Logit回归方程计算风险评估值,Logit回归方程为:
Logit=-34.6131+1.4038*ΔCthsa-let-7i-5p-1.443*ΔCthsa-miR-181c-5p-4.2333*ΔCthsa-miR-21-5p+5.3845*ΔCthsa-miR-29c-3p-1.9443*ΔCthsa-miR-92a-3p-0.9056*ΔCthsa-let-7f-5p+4.9506*ΔCthsa-miR-1285-5p-2.7654*ΔCthsa-miR-193b-3p+0.9884*ΔCthsa-miR-143-3p
根据靶点和内参的Ct值,将9个靶点的Ct值分别减去内参的Ct值,求得9个靶点相对内参的ΔCt,然后代入Logit回归方程,计算出Logit值。
更优选地,采用U6作为内参。
更优选地,9个靶点若NoCt,应将Ct值赋值为40(最大循环数)后,再进行计算。
优选地,cutoff值:0.00,若logit(P)≥0.00,诊断为AD患者;若logit(P)<0.00,诊断为健康人群。
本发明的有益效果在于:
本发明经过多重筛选,最终选择hsa-let-7i-5p、hsa-miR-181c-5p、hsa-miR-21-5p、hsa-miR-29c-3p、hsa-miR-92a-3p、hsa-let-7f-5p、hsa-miR-1285-5p、hsa-miR-193b-3p、hsa-miR-143-3p这9个位点组合作为分子标记物的miRNA,U6作为内参进行临床样本验证实验,具有很高的灵敏度及特异性。该AD血清miRNA检测试剂盒运用实时荧光定量方法,可以实现区分AD患者和健康人群,从而进行临床辅助诊断,具有较高的临床应用价值。
本发明用于辅助诊断AD的试剂盒,可以简单、有效、无创地检测AD。与常规脑脊液样本在取样难度方面有明显优势,相比蛋白检测在有很大的成本优势。
附图说明
图1为采用本发明试剂盒进行检测的操作流程图。
图2为hsa-let-7i-5p灵敏度检测结果。
图3为hsa-miR-181c-5p灵敏度检测结果。
图4为hsa-miR-21-5p灵敏度检测结果。
图5为hsa-miR-29c-3p灵敏度检测结果。
图6为hsa-miR-92a-3p灵敏度检测结果。
图7为hsa-let-7f-5p灵敏度检测结果。
图8为hsa-miR-1285-5p灵敏度检测结果。
图9为hsa-miR-193b-3p灵敏度检测结果。
图10为hsa-miR-135a-5p灵敏度检测结果。
图11为hsa-miR-223-3p灵敏度检测结果。
图12为hsa-miR-660-5p灵敏度检测结果。
图13为hsa-miR-143-3p灵敏度检测结果。
图14为6个标志物组合绘制的ROC曲线。
图15为7个标志物组合绘制的ROC曲线。
图16为8个标志物组合绘制的ROC曲线。
图17为9个标志物组合绘制的ROC曲线。
图18为10个标志物组合绘制的ROC曲线。
图19为11个标志物组合绘制的ROC曲线。
图20为12个标志物组合绘制的ROC曲线。
图21为不同数量miRNA组合性能测试结果。
具体实施方式
为了更清楚地说明本发明,下面结合实施例并对照附图对本发明作进一步详细说明。本领域技术人员应当理解,下面所具体描述的内容是说明性的而非限制性的,不应以此限制本发明的保护范围。
实施例
一、血清miRNA标志物的初步筛选
首先使用NGS方法筛选出若干个差异表达明显的AD相关miRNA,并且经qPCR验证,最终确认AD诊断相关的生物标志物。标志物包括但不限于如下miRNA:
表1. miRNA序列和miRBase数据库登录号
二、血清miRNA标志物组合的优化
结合图1,使用以上12个miRNA位点组合引物探针,进行血清及血浆样本进行miRNA提取、反转录及qPCR实验。
1、miRNA的提取
使用血清/血浆miRNA提取试剂盒(柱纯化)(麦锐克,中国苏州,货号:MRE00101)提取血清/血浆样本的总RNA,包括非编码小RNA。
a. 取200 μL血清或血浆等液体样本转移至1.5 mL DNase/RNase-free离心管中;
b. 分别加入200 μL裂解液和10 μL蛋白酶K,充分涡旋振荡混匀后短暂离心;
c. 65℃ 静置10 min后,冷却至室温;
d. 将洗脱柱1套入收集管中,备用;
e. 加入600 μL无水乙醇至步骤a.中的1.5 mL DNase/RNase-free管中,涡旋混匀后,将混合液加入到上述准备好的洗脱柱1中,12000 rpm离心1 min,弃滤液;
f. 将洗脱柱1放回收集管,加入500 μL洗液1,12000 rpm离心30 sec,弃滤液;
g. 将洗脱柱1放回收集管,加入500 µL洗液2,12000 rpm离心30 sec,弃滤液;
h. 重复上步操作;
i. 将洗脱柱1放回收集管,12000 rpm离心2 min,以除去残留的液体;
j. 将洗脱柱1放入新的1.5 mL DNase/RNase-free离心管中,向洗脱柱1中央加入30-100 µL RNase-free H2O,室温放置2 min;
k. 12000 rpm离心1 min。收集滤液,即为总RNA溶液,长期保存应置于-80℃。
2、反转录反应
表2. 特异性茎环反转录引物名称和序列
表3. 反转录反应体系
表4. 反转录反应条件
逆转录产物立即进行qPCR反应或置于-20℃ 保存。
3、qPCR反应
qPCR试剂:2×miRNA stem-loop qPCR Master Mix(麦锐克,苏州)
表5. 特异性上游引物和通用下游引物的名称及序列
表6. 特异性探针名称及序列
表7. qPCR反应体系
表8. qPCR反应条件
在60℃ 时进行荧光信号采集。
4、灵敏度检测
基于步骤2和3,对合成RNA模板进行稀释后检测,灵敏度检测结果如图2~13以及表6所示,曲线从左往右表示合成模板起始浓度依次为10 fg/μL、0.1 fg/μL、0.01 fg/μL、0.001 fg/μL、0.0001 fg/μL、0.00001 fg/μL。由此可见,该检测方法可检测到至少0.001fg/μL浓度下的miRNA。
表9. 不同位点灵敏度检测结果
5、组合优化
对从筛选中获得的不同数量miRNA标志物组合来检测灵敏度、特异性。根据AUC,当组合个数为9个时AUC为0.995,当组合个数增加到12个时AUC依旧为0.995,当组合个数减少为8个、7个、6个时,AUC均小于0.995,因此最终选择miRNA个数为9个的组合。具体数据如表10,ROC曲线如图14~21。
表10. 不同数量miRNA标志物组合性能检测结果
三、AD患者诊断模型的构建
通过Logit回归方程拟合各miRNA指标构建AD患者诊断模型。通过数据分析,得到该诊断模型公式,及回归方程为:
Logit=-34.6131+1.4038*ΔCthsa-let-7i-5p-1.443*ΔCthsa-miR-181c-5p-4.2333*ΔCthsa-miR-21-5p+5.3845*ΔCthsa-miR-29c-3p-1.9443*ΔCthsa-miR-92a-3p-0.9056*ΔCthsa-let-7f-5p+4.9506*ΔCthsa-miR-1285-5p-2.7654*ΔCthsa-miR-193b-3p+0.9884*ΔCthsa-miR-143-3p
注意:ΔCt为靶标Ct-内参Ct的差值。
根据靶点和内参(U6)的Ct值,将9个靶点的Ct值分别减去内参(U6)的Ct值,求得9个靶点相对内参的ΔCt,然后代入以下公式,计算出Logit值。
9个靶点若NoCt,应将Ct值赋值为40(最大循环数)后,再进行计算。
cutoff值:0.00,若logit(P)≥0.00,诊断为AD患者;若logit(P)<0.00,诊断为健康人群。
对检测结果,采用MedCalc 20.015进行统计分析,9个靶点联合构建模型的ROC曲线如图17所示。用这9个miRNA标志物组合灵敏度、特异性分别为:95.83、100.00。可以很好区分AD患者和健康对照人群。
显然,本发明的上述实施例仅仅是为更清楚地说明本发明所作的举例,而并非是对本发明的实施方式的限定,对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其他不同形式的变化或变动,这里无法对所有的实施方法予以穷举,凡是属于本发明的技术方案所引申出的显而易见的变化或变动仍处于本发明的保护范围之列。
Claims (6)
1.一种用于AD诊断的标志物组合,包括:hsa-let-7i-5p、hsa-miR-181c-5p、hsa-miR-21-5p、hsa-miR-29c-3p、hsa-miR-92a-3p、hsa-let-7f-5p、hsa-miR-1285-5p、hsa-miR-193b-3p、hsa-miR-143-3p。
2.一种用于AD诊断的试剂盒,包括权利要求1所述的标志物组合。
3.根据权利要求2所述的试剂盒,其特征在于,所述试剂盒通过Logit回归方程计算风险评估值,Logit回归方程为:
Logit=-34.6131+1.4038*ΔCthsa-let-7i-5p-1.443*ΔCthsa-miR-181c-5p-4.2333*ΔCthsa-miR-21-5p+5.3845*ΔCthsa-miR-29c-3p-1.9443*ΔCthsa-miR-92a-3p-0.9056*ΔCthsa-let-7f-5p+4.9506*ΔCthsa-miR-1285-5p-2.7654*ΔCthsa-miR-193b-3p+0.9884*ΔCthsa-miR-143-3p
根据靶点和内参的Ct值,将9个靶点的Ct值分别减去内参的Ct值,求得9个靶点相对内参的ΔCt,然后代入Logit回归方程,计算出Logit值。
4.根据权利要求3所述的试剂盒,其特征在于,采用U6作为内参。
5.根据权利要求3所述的试剂盒,其特征在于,9个靶点若NoCt,应将Ct值赋值为40后,再进行计算。
6.根据权利要求3~5任意一项所述的试剂盒,其特征在于,cutoff值:0.00,若logit(P)≥0.00,诊断为AD患者;若logit(P)<0.00,诊断为健康人群。
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Dolores Siedlecki-Wullich.Altered microRNAs related to synaptic function as potential plasma biomarkers for Alzheimer’s disease.Alzheimer's Research & Therapy.2019,第11卷(第46期),全文. * |
Fengjun Cao.Diagnostic value of miR-193a-3p in Alzheimer's disease and miR-193a-3p attenuates amyloid-β induced neurotoxicity by targeting PTEN.Experimental Gerontology.2019,全文. * |
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