CN114791459A - 用于检测肺结核的血清代谢标志物及其试剂盒 - Google Patents
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Abstract
本发明公开了一组用于检测肺结核的血清代谢标志物及其试剂盒,所述血清代谢标志物共17种,经验证后所述17种代谢物的水平变化确实与肺结核相关。基于上述17种代谢标志物水平的肺结核检测模型在肺结核检测中ROC曲线下的面积可达到99.5%,敏感性98.2%,特异性95.8%。本发明还提供一种肺结核检测试剂盒,包括上述的17种肺结核病人血清中的代谢物的检测试剂,所述检测试剂用于对所述17种肺结核病人血清中的代谢物进行定量检测。本发明的用于检测肺结核的血清代谢标志物以及试剂盒能够帮助医生准确的进行疾病诊断,对于肺结核的诊断和治疗具有重要意义。
Description
技术领域
本发明属于生物技术领域,具体涉及一种用于检测肺结核的血清代谢标志物及其试剂盒。
背景技术
肺结核病的检测以病原学检测为金标准,由于涉及大量活菌操作,耗时较长,而且对技术人员操作熟练程度及实验室生物安全级别要求较高,在经济欠发达地区受到限制,不利于结核病的早期控制。血清学也可用于结核检测,而且检测操作简单,但是现有的检测方法的敏感性与特异性均有待提高。
近几年发展起来的分子生物学检测技术如聚合酶链反应(PCR)、DNA探针技术、DNA序列测定、基因芯片技术等因操作要求较高、价格昂贵等问题使其使用受到限制。准确、敏感、特异、方便、快速的早期鉴别与检测对控制病菌感染、减少传播机会、抑制结核流行具有非常重要的意义。
代谢组学揭示的小分子代谢变化是一系列生命活动的终端,直接反映生命活动的最终状态。疾病发展过程的各个阶段病理生理的变化都可由代谢变化反映出来,代谢组学可以根据这些变化筛选出相关的生物标志物,还可以在疾病早期就检测到代谢物含量的改变,借以揭示疾病发生发展过程中的代谢变化机制,为进一步探索疾病治疗靶点提供有用的信息。肺结核引起的系统性变化不仅反映在结核杆菌感染的部位,全身代谢也发生了变化。因此使用代谢组方法寻找结核的标志物具有重要的临床价值。
目前,代谢组学已经用于结核研究。有研究者使用液相色谱质谱联用技术(LC-MS)分析血清、尿液、痰液样本,利用统计学方法如主成分分析法(PCA)、正交偏最小二乘判别分析(OPLS-DA)对质谱数据进行分析,寻找结核的生物标志物。但这些研究大多仅针对小量样本,实际临床意义十分有限。
发明内容
针对肺结核现缺少能准确鉴别诊断肺结核的血清标志物,本发明提供了一组用于检测肺结核的血清代谢标志物及其试剂盒。所述血清代谢标志物可有效克服现有技术在肺结核检测方面的缺陷及不足,能够提高肺结核检测的准确率。
本发明采用以下技术方案实现:
一组用于检测肺结核的血清代谢标志物,所述标志物为在肺结核患者血清中的代谢物(17种),见表1。
本发明的代谢标志物对于肺结核具有较高的灵敏性和特异性,对于结核治疗具有重要意义。
本发明的用于检测肺结核的血清代谢标志物的筛选方法,包括以下步骤:
(1)于空腹状态下用BD黄色5.0ml 367955SST II血清分离胶管采血管采集结核病患者以及健康人的血(静脉)1-2ml;
(2)在采集后的两小时内,将血液于4℃,3500rpm离心10min;
(3)分离上层血清0.3mL到1.5mL于新的EP管中,保存于-80℃冰箱;
(4)在LDI MS实验中,铁颗粒以1mg mL-1的浓度分散在水中作为基质。将稀释10倍的血清1μL直接点于抛光板上,室温空气干燥,然后加入基质浆液1μL,干燥后进行LDI MS分析。
(5)采用LDI MS技术对每个分析样本进行分析,得到各血清样本的原始代谢指纹图谱;质谱记录在AutoFlex TOF/TOF质谱仪(Bruker,Germany)上。
(6)利用MATLAB(R2016a,the MathWorks,USA)对原始质谱数据进行基线校正、峰值检测、提取、比对、归一化、标准化等预处理,将从仪器中获得的原始数据转换为可用于进一步统计分析的数据形式。
(7)使用机器学习偏最小二乘法(Partial Least Squares,PLS)算法学习步骤(6)的数据,随机将上述结核及健康血清样本数据的3/4作为训练集,1/4作为测试集进行学习。
(8)应用偏最小二乘法PLS建立检测模型,绘制ROC曲线。
(9)应用MetaboAnalyst 5.0软件进行差异分析和富集分析。
(10)根据模型选出频率大于90%,p值小于0.05,VIP值大于1的作为差异代谢物。
(11)将差异数据与人类代谢组数据库(HMDB)中的谱图信息进行比对,从而对代谢物进行鉴定,即可得到上述17种血清代谢标志物。
基于筛选出的17种血清代谢标志物,使用机器学习偏最小二乘法(Partial LeastSquares,PLS)算法建立肺结核检测模型。将需检测的患者血液经步骤(1)-(6)所述方法处理,并将步骤(6)所得数据输入所建立的肺结核检测模型中,即可检测出患者是否患有肺结核。
本发明还提供一种肺结核检测试剂盒,包括上述的17种肺结核病人血清中的代谢物的检测试剂,所述检测试剂用于对所述17种肺结核病人血清中的代谢物进行定量检测。
与现有技术相比,本发明的有益效果如下:
本发明首次发现上述17种代谢标志物的水平变化与肺结核相关,采用代谢组学分析发现检测肺结核病人血清中17种代谢物的水平异常。基于所述17种代谢标志物水平,结合代谢组学分析以及机器学习方法,构建肺结核检测模型,并使用受试者工作曲线(ROC)评价其检测肺结核的效能。基于上述17种代谢标志物水平的肺结核检测模型在肺结核检测中ROC曲线下的面积可达到99.5%,敏感性98.2%,特异性95.8%。
本发明采用血清代谢组学技术以及机器学习方法得到适合于肺结核的检测标志物和肺结核检测模型。本发明的肺结核检测模型效果良好,灵敏度高,特异性好,适合于肺结核的检测。本发明仅通过取血检测就能实现肺结核的诊断,检测速度快,且样本仅需50nL,有利于肺结核的早诊早治,具有很好的临床使用和推广价值。
附图说明
图1是原始代谢指纹图谱的质谱图。
图2是使用17种血清代谢标志物构建的肺结核检测模型的分类性能图。
具体实施方式
下面结合具体实施实例,进一步阐释本发明,本发明的实施例仅用于解释本发明,并不意味着限制本发明的保护范围。
1、研究对象
本研究共包含来自于浙江省杭州市红十字会医院118例健康人血清样本以及110例结核样本。其中结核检测标准是经菌培养,涂片,影像学检测,分子检测等综合检测结果判定。
2、血液样本预处理
(1)静脉抽血1-2mL(避免血清溶脂,溶血),注入真空采血管,立刻上下颠倒混匀5-8次,使附着在管壁上的促凝剂能够充分混合于血液中。
(2)在室温(20℃-25℃)下,静置约30min,等血液完全凝固。
(3)待血清充分析出后,在离心力为3500转的条件下,离心10min。
(4)谨慎吸取上层透明液体约0.3mL到1.5mL离心管中,并将离心管压好盖,检查契合度,放入样本盒,置于-80度冰箱。
3、上机检测
通过铁粒子辅助激光解吸/电离质谱检测,其中铁颗粒以1mg/mL-1的浓度分散在水中作为基质。将稀释10倍的血清1μL直接点于抛光板上,室温空气干燥,然后加入基质浆液1μL,干燥后进行LDI MS分析。质谱记录在AutoFlex TOF/TOF质谱仪(Bruker,Germany)上,配备Nd:YAG激光器(2kHz,355nm)。采集采用正反射离子模式,采用延迟提取,重复频率为1,000Hz,加速电压为20kV。本实验的延迟时间被优化为250ns。在所有LDI MS实验中,每次分析的激光射击次数为2000次。在获取原始质谱数据后(如图1),使用MATLAB代码(R2016a,the Mathworks,USA)进行峰提取、比对、归一化和标准化预处理,将原始数据转换为可用于进一步分析的可用形式。
4、机器学习随机森林建模
使用偏最小二乘法(Partial Least Squares)算法建立模型,随机将肺结核与健康人血清样本数据的3/4作为训练集,1/4作为测试集进行学习,即可得到所述模型。绘制ROC曲线,获得的模型的AUC为94.5%。
5、代谢物的筛选
差异代谢产物的鉴定
搜索满足VIP值>1、模型选中频率>90%和P值<0.05的代谢产物,最终鉴定得到17种差异代谢物,如表1所示。
6、使用偏最小二乘法算法根据筛选得到的17个差异代谢物进行建模并绘制ROC曲线。获得的肺结核检测模型的分类性能见图2,AUC为99.5%,敏感性98.2%,特异性95.8%。可知采用筛选得到的17种血清代谢标志物进行肺结核检测的准确性和特异性均较好。
利用所述的肺结核检测模型进行肺结核检测,具体方法为:
采用如上述步骤2的方法进行血液样本预处理;接着采用如步骤3所述的方法进行上机检测,得到原始代谢指纹图谱,并对其进行一系列预处理;然后将前一步得到的数据输入到步骤6中利用17种血清代谢标志物建立的模型中,根据模型输出结果即可得血液样本所属的个体是否患有肺结核。
采用试剂盒对肺结核进行检测,具体方法为:
采用如上述步骤2的方法进行血液样本预处理;接着应用试剂盒检测上述17种血清代谢标志物的水平;分析17种血清标志物的水平,确定是否患有肺结核。
表1差异代谢物列表
Claims (2)
1.用于检测肺结核的血清代谢标志物,其特征在于,包括17种肺结核病人血清中的代谢物,具体为:D-Leucic acid、Leucinic acid、L-Cystine、MG(0:0/14:0/0:0)、S-Lactoylglutathione、Octacosanoic acid、Cer(d18:1/12:0)、Raffinose、LysoPC(20:1(11Z)/0:0)、LysoPC(22:4(7Z,10Z,13Z,16Z)/0:0)、PC(18:1(9Z)e/2:0)、Torvoside G、CE(16:1(9Z))、all-trans-Heptaprenyl diphosphate、PC(15:0/16:0)、NADP、TG(20:0/20:0/20:1(11Z))。
2.一种肺结核检测试剂盒,其特征在于,包括如权利要求1所述的17种肺结核病人血清中的代谢物的检测试剂,所述检测试剂用于对所述的17种肺结核病人血清中的代谢物进行定量检测。
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