CN115236220A - 一种诊断新型冠状病毒的挥发性标志物及其应用 - Google Patents
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Abstract
本发明涉及一种诊断新型冠状病毒的挥发性标志物及其应用,所述的挥发性标志物包括以下挥发性有机物:环己酮、乙苯、1‑辛烯、2‑甲基环己酮、异丙基苯、苯丙烯、癸烷、1‑癸烯中的多种或全部。与现有技术相比,本发明具有准确性高、快速、成本低、能够进行大面积新型冠状病毒病例筛查等优点。
Description
技术领域
本发明涉及新型冠状病毒检测技术领域,具体涉及一种诊断新型冠状病毒的挥发性标志物及其应用。
背景技术
COVID-19是一种由新型冠状病毒引起的急性传染病。COVID-19感染的主要症状包括发热、咳嗽、气短、呼吸困难等呼吸道症状新型冠状病毒的传播途径多,主要是经呼吸道飞沫传播,密切接触传播以及气溶胶传播新型冠状病毒肺炎患者缺乏特异性的临床特征,部分患者缺乏确切的流行病学暴露史,为疾病的早期识别带来困难
目前,主要的诊断技术是基于COVID-19病毒核酸和抗体,但仍存在样本采集困难、检测时间长、检测结果假阳性或阴性等缺点。
发明内容
本发明的目的是提供一种诊断新型冠状病毒的挥发性标志物及其应用。
本发明的目的可以通过以下技术方案来实现:一种诊断新型冠状病毒的挥发性标志物,包括以下呼气挥发性有机物(VOCs):环己酮(108-94-1)、乙苯(100-41-4)、 1-辛烯(111-66-0)、2-甲基环己酮(583-60-8)、异丙基苯(98-82-8)、苯丙烯 (300-57-2)、癸烷(124-18-5)、1-癸烯(872-05-9)中的多种或全部的组合物。
一种如上述诊断新型冠状病毒的挥发性标志物的应用,通过检测被试者呼气中所述的挥发性标志物的含量,进而判断是否感染新型冠状病毒。
优选地,通过气相色谱质谱仪检测被试者呼气中所述的挥发性标志物的含量。
进一步优选地,所述的气相色谱质谱仪内置Tenax-TA富集管,通过精密气泵抽取气袋内的呼气样本,并将VOC成分富集到Tenax-TA富集管中。
进一步优选地,所述的气相色谱质谱仪为便携式气相色谱质谱仪。
更进一步优选地,所述的气袋通过TPFE管路连接至气相色谱质谱仪,管路外部配备加热封层,在进样过程中保持55~65摄氏度。优选地,在进样过程中保持 60摄氏度,减少呼气样本中的VOC成分在管道内壁的吸附。
优选地,所述的气相色谱质谱仪内部的精密气泵控制气袋内呼气样本预浓缩在富集管中,预浓缩完成后,富集管中的VOC成分在270~280摄氏度下脱附,高纯氦气作为载气将VOC成分传输至气相色谱质谱系统中进行检测。优选地,富集管中的VOC成分在275摄氏度下脱附。
优选地,气相色谱质谱仪内部的精密气泵控制气袋内呼气样本以5毫升/分钟的流速通过富集管,进样时间为20分钟,即将1升气体预浓缩在富集管中;预浓缩完成后,富集管中的VOC成分在275摄氏度下脱附。
优选地,高纯氦气(99.99%)作为载气将VOC成分传输至气相色谱质谱系统中时,流速为1毫升/分钟,升温程序:起始温度40度,保持时间10分钟,2.5度 /分升温至200度,保持5分钟。
优选地,在样本检测前,不进样的条件下对仪器进行老化,去除富集管、气体管路以及各个连接部位的杂质。
优选地,指示呼气样本来自阳性个体的VOC含量指标(峰面积)包括:环己酮>0.017、乙苯>0.067、1-辛烯>0.037、2-甲基环己酮>0.046、异丙基苯>0.057、苯丙烯>0.074、癸烷>0.028、1-癸烯>0.021,当上述8个条件至少满足5个时,被试者判断为感染新型冠状病毒,反之,被试者判断为未新型冠状病毒。
一种用于检测新冠感染标志物的检测系统,包括气袋预处理装置、呼气收集装置和气相色谱-质谱仪。
所述的气袋预处理装置用于保证气袋的洁净干燥,呼气收集装置用于收集被试者的呼出气体,气相色谱-质谱仪用于检测呼气中挥发性有机物(VOCs):环己酮、乙苯、1-辛烯、2-甲基环己酮、异丙基苯、苯丙烯、癸烷、1-癸烯的含量。
所述的检测系统使用气泵将收集到的呼气样本富集到Tenax-TA富集管中、经过高温脱附装置将样品中的VOC成分递送至气相色谱-质谱仪,检测新冠相关标志物的含量。
与现有技术相比,本发明具有以下优点:
1.本发明基于呼气样本得到检测方法具有采集迅速、被试者无不适感、成本低廉、易于储存和运输等优点;
2.本发明所筛选的呼气中的新冠相关VOC标志物种类多,通过检测多个标志物组合的指标,确保诊断结果准确性高;
3.本发明所筛选的呼气中的新冠标志物在感染新冠病毒的患者组和健康对照度之间存在显著性差异,保证诊断效果准确可靠;
4.本发明准确性高、快速、成本低、能够进行大面积新型冠状病毒病例筛查;
5.本发明提供了呼气中多种挥发性有机物作为标志物用于新冠感染的非侵入式诊断,在新冠筛查与疫情防控中应用前景广阔;
6.本发明标志物直接来自于人体呼气,对比了新冠病毒阳性和阴性患者呼气中挥发性标志物的差异,使用了呼气成分的差异进行新冠诊断,样本均来自于真实的个体,更能代表新冠病毒感染造成的人体代谢情况的变化,对于新冠筛查更具价值。
附图说明
图1为新冠阳性与阴性志愿者的气相色谱-质谱图及相应的VOC标志物;
图2为新冠相关VOC标志物在新冠阳性组与阴性组中含量对比图。
具体实施方式
下面结合附图和具体实施例对本发明进行详细说明。以下实施例在以本发明技术方案为前提下进行实施,给出了详细的实施方式和具体的操作过程,但本发明的保护范围不限于下述的实施例。
实施例1
被试者募集:
40名提供呼气的志愿者(20名感染新冠病毒患者和20名正常人),在前期被试者筛选中,所有被试者经过间隔24小时的两次核酸检测以及血清中的IgG与IgM 抗体检测,所有的检测结果显示为阳性的被试者,被选入新冠感染阳性组;所有检测结果显示为阴性的被试者,被选入新冠感染阴性组,即对照组。对每个受试者的呼气样本进行分析,使用共计40个呼气样品。样本选取标准,排除具有过量饮酒史(折合乙醇量:男性≥30g/d,女性≥20g/d)、心、肝、肾功能不全、患有恶性肿瘤或血液疾病或代谢系统疾病如糖尿病等的志愿者。所有被试者在样本采集前被告知项目背景及内容,并签署之情同意书。记录每个志愿者年龄、性别、核酸检测结果、抽烟饮酒史、当前服用药物情况等信息。
实施例2
气袋预处理与呼气样本采集:
项目中使用带密封条的3L泰德拉气袋,在采样前进行彻底清洗,在洁净的条件下将气袋翻折,使气袋内壁朝外;将气袋平整地放入干净的烘箱中在80摄氏度的条件下烘烤4-5天;将气袋内壁翻折到内侧,使用密封条将气袋密封;用高纯氮气(纯度为99.99%)清洗气袋,重复5次,得到纯净的采样袋。样本收集前,每个志愿者要在48小时内保持自然平和的情绪,24小时内,不得抽烟和喝酒。样本收集前1小时内,所有志愿者禁止摄入任何食物以保持口腔清洁。采样开始前,先用纯水漱口,5分钟后开始收集。吹气前先深吸气,憋气3秒,前2-3秒的呼气不收集,通过聚四氟乙烯管吹满3L泰德拉气袋的80%左右。
实施例3
呼气样本的检测:
检测仪为便携式气相色谱质谱仪,仪器内置Tenax-TA富集管,通过精密气泵抽取气袋内的呼气样本,并将VOC成分富集到Tenax-TA富集管中。在样本检测前,不进样的条件下对仪器进行老化,去除富集管、气体管路以及各个连接部位的杂质。气袋通过TPFE管路连接至仪器,管路外部配备加热封层,在进样过程中保持60摄氏度,减少呼气样本中的VOC成分在管道内壁的吸附;仪器内部的高精度气泵控制气袋内呼吸气以5毫升/分钟的流速通过富集管,进样时间为20分钟,即将1升气体预浓缩在富集管中;预浓缩完成后,富集管中的VOC成分在275摄氏度下脱附;高纯氦气(纯度为99.99%)作为载气将VOC成分传输至气相色谱质谱系统中,流速为1毫升/分钟。升温程序:起始温度40度,保持时间10分钟, 2.5度/分升温至200度,保持5分钟。
实施例4
呼气样本中标志物筛选:
经过气相色谱质谱仪检测出的VOC成分,通过与NIST谱库对比对VOC成分进行定性,在对色谱图进行平滑和去除基线后,对色谱峰进行积分实现得到每种 VOC成分的含量。VOC含量在新冠阳性组和阴性组之间具有显著性差异的成分作为新冠相关的VOC标志物。新冠感染阳性患者与阴性志愿者的色谱图对比曲线如图1所示,相应的VOC成分如表1所示。图2为新冠阳性组与阴性组中8种VOC 的含量对比图。
表1:新冠感染阳性与阴性志愿者呼气中的VOC标志物
峰号 | 化合物名称 | CAS号 |
1 | 环己酮 | 108-94-1 |
2 | 乙苯 | 100-41-4 |
3 | 1-辛烯 | 111-66-0 |
4 | 2-甲基环己酮 | 583-60-8 |
5 | 异丙基苯 | 98-82-8 |
6 | 苯丙烯 | 300-57-2 |
7 | 癸烷 | 124-18-5 |
8 | 1-癸烯 | 872-05-9 |
实施例5
呼气中的VOC标志物用于新冠感染筛查:
收集13例确诊的新冠阳性志愿者与9例新冠阴性志愿者的呼气样品,严格按照实施例1-3的步骤对呼气样本中的VOC成分进行检测。指示呼气样本来自阳性个体的VOC含量指标(峰面积)包括:环己酮>0.017、乙苯>0.067、1-辛烯>0.037、 2-甲基环己酮>0.046、异丙基苯>0.057、苯丙烯>0.074、癸烷>0.028、1-癸烯>0.021。当上述8个条件至少满足5个时,被试者判断为感染新冠病毒,反之,被试者判断为未感染新冠病毒。结果显示,只有一例实际的阴性样本被错误分类为阳性样本,其余样本的预测结果与实际情况相符,即诊断准确率高于95%,敏感度可达100%。
本发明提供了一种利用呼气中的VOC标志物进行新冠筛查的新方法,该标志物组合可用于通过对人的呼气的快速检测快速诊断新冠感染。本发明的原理在于:当人体感染新型冠状病毒后,病毒本身的代谢过程及其引起的相关组织的感染等代谢状态变化,部分挥发性有机代谢物的种类与含量随之改变,通过血液循环到达肺部随呼气排出体外。因此利用呼气中的VOC标志物进行新冠感染的诊断,能够实现新冠的快速筛查,具有非标记、快速、无创、经济有效等优点。
上述的对实施例的描述是为便于该技术领域的普通技术人员能理解和使用发明。熟悉本领域技术的人员显然可以容易地对这些实施例做出各种修改,并把在此说明的一般原理应用到其他实施例中而不必经过创造性的劳动。因此,本发明不限于上述实施例,本领域技术人员根据本发明的揭示,不脱离本发明范畴所做出的改进和修改都应该在本发明的保护范围之内。
Claims (10)
1.一种诊断新型冠状病毒的挥发性标志物,其特征在于,包括以下挥发性有机物:环己酮、乙苯、1-辛烯、2-甲基环己酮、异丙基苯、苯丙烯、癸烷、1-癸烯中的多种或全部。
2.一种如权利要求1所述的诊断新型冠状病毒的挥发性标志物的应用,其特征在于,通过检测被试者呼气中所述的挥发性标志物的含量,进而判断是否感染新型冠状病毒。
3.根据权利要求2所述的诊断新型冠状病毒的挥发性标志物的应用,其特征在于,通过气相色谱质谱仪检测被试者呼气中所述的挥发性标志物的含量。
4.根据权利要求3所述的诊断新型冠状病毒的挥发性标志物的应用,其特征在于,所述的气相色谱质谱仪内置Tenax-TA富集管,通过气泵抽取气袋内的呼气样本,并将VOC成分富集到Tenax-TA富集管中。
5.根据权利要求4所述的诊断新型冠状病毒的挥发性标志物的应用,其特征在于,所述的气袋通过TPFE管路连接至气相色谱质谱仪,管路外部配备加热封层,在进样过程中保持55~65摄氏度。
6.根据权利要求5所述的诊断新型冠状病毒的挥发性标志物的应用,其特征在于,气相色谱质谱仪内部的气泵控制气袋内呼气样本预浓缩在富集管中,预浓缩完成后,富集管中的VOC成分在270~280摄氏度下脱附,高纯氦气作为载气将VOC成分传输至气相色谱质谱系统中进行检测。
7.根据权利要求6所述的诊断新型冠状病毒的挥发性标志物的应用,其特征在于,所述的气相色谱质谱仪内部的气泵控制气袋内呼气样本以5毫升/分钟的流速通过富集管,进样时间为20分钟,即将1升气体预浓缩在富集管中;预浓缩完成后,富集管中的VOC成分在275摄氏度下脱附。
8.根据权利要求6所述的诊断新型冠状病毒的挥发性标志物的应用,其特征在于,高纯氦气作为载气将VOC成分传输至气相色谱质谱系统中时,流速为1毫升/分钟,升温程序:起始温度40度,保持时间10分钟,2.5度/分升温至200度,保持5分钟。
9.根据权利要求3所述的诊断新型冠状病毒的挥发性标志物的应用,其特征在于,在样本检测前,不进样的条件下对仪器进行老化,去除富集管、气体管路以及各个连接部位的杂质。
10.根据权利要求3所述的诊断新型冠状病毒的挥发性标志物的应用,其特征在于,以峰面积计,以下8个条件:环己酮>0.017、乙苯>0.067、1-辛烯>0.037、2-甲基环己酮>0.046、异丙基苯>0.057、苯丙烯>0.074、癸烷>0.028、1-癸烯>0.021,当上述8个条件至少满足5个时,被试者判断为感染新型冠状病毒,反之,被试者判断为未新型冠状病毒。
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