WO2024108700A1 - Use of biomarker combination in exhaled breath in novel coronavirus diagnostic reagent - Google Patents
Use of biomarker combination in exhaled breath in novel coronavirus diagnostic reagent Download PDFInfo
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- the present invention belongs to the field of detection and diagnostic reagents related to the new coronavirus, and specifically relates to the application of chromatography-mass spectrometry combined detection, biomarkers in exhaled breath and their combinations in new coronavirus diagnostic reagents.
- Novel coronavirus pneumonia is an acute infectious disease caused by the 2019 novel coronavirus (COVID-19).
- COVID-19 2019 novel coronavirus
- the main symptoms include respiratory symptoms, fever, shortness of breath and difficulty breathing. In more severe cases, the infection can lead to pneumonia, severe acute respiratory syndrome, renal failure, and even death.
- isolation treatment and symptomatic supportive treatment are the main treatments. Therefore, rapid screening of people susceptible to the novel coronavirus and effective isolation are the best ways to defeat the novel coronavirus.
- the existing detection method is mainly nucleic acid detection, but this method is inconvenient to sample, the detection time is long, and the detection effect is still not ideal.
- Detecting COVID-19-related characteristic markers in specific biological samples can provide valuable early COVID-19 diagnosis suggestions and can be used to prepare diagnostic reagents.
- the biological samples include respiratory gases and various pathological tissues, but tissue or body fluid collection is more cumbersome and far less convenient than respiratory gas collection.
- the purpose of the present invention is to overcome the deficiencies of the prior art and to select biomarkers in exhaled breath that have a detection function.
- Another object of the present invention is to provide an application of the biomarker in exhaled breath.
- Another object of the present invention is to provide a method for detecting the biomarkers in the breath.
- a biomarker in exhaled breath wherein the biomarker is derived from a human exhaled breath sample, and the marker is selected from one or more of the following biomarkers A, B, C, D, and E, which respectively correspond to a characteristic mass spectrum and retention time, including:
- the biomarker A has a mass-to-charge ratio of 135.0 ⁇ 0.5amu and a retention time of 23.5 ⁇ 1.0min; and/or,
- the biomarker B has a mass-to-charge ratio of 106.0 ⁇ 0.5amu and a retention time of 26.0 ⁇ 1.0min; and/or,
- the biomarker C has a mass-to-charge ratio of 94.1 ⁇ 0.5amu and a retention time of 29.0 ⁇ 1.0min; and/or,
- the biomarker D has a mass-to-charge ratio of 94.2 ⁇ 0.5amu and a retention time of 19.0 ⁇ 1.0min; and/or,
- the biomarker E has a mass-to-charge ratio of 110.5 ⁇ 0.5amu and a retention time of 39.0 ⁇ 1.0min;
- the mass-to-charge ratio and retention time are measured by a mass spectrometer using an ion trap as a mass analyzer.
- COVID-19 involves lung infection
- COVID-19-related markers in respiratory gases are richer than those in other tissues. Therefore, by collecting respiratory gases from COVID-19 patients and healthy volunteers, and using a combined mass spectrometry-chromatography method to analyze and compare various molecular components, we ultimately screened out some specific biomarkers or their combinations, which can be used in the development and preparation of early diagnostic reagents for COVID-19.
- biomarkers D and E Furthermore, it contains one or both of biomarkers D and E.
- biomarkers A, B, C, D and E are contained.
- the present invention provides the use of the biomarkers in exhaled breath in early diagnostic reagents for COVID-19, including the preparation of diagnostic reagents for COVID-19, the establishment of kits and exhaled breath detection and treatment devices, etc.
- the biomarkers in exhaled breath can be detected by chromatography-mass spectrometry for risk assessment, diagnosis or pathological staging of COVID-19.
- the diagnosis is an early diagnosis, so that early COVID-19 patients detected can receive timely treatment.
- the present invention also provides a method for detecting the biomarkers in the exhaled breath, comprising:
- the response value threshold is set at 3 ⁇ 10 4 , and by detecting the types and contents of the biomarkers in the exhaled breath of the subjects, when at least one of the biomarkers A, B, and C is detected in the exhaled breath, and the biomarker D that reaches the response threshold exists and E is absent, it is confirmed that the subject is not infected with the new coronavirus; when at least one of the biomarkers A, B, and C is detected in the exhaled breath, and both D and E exist, that is, both reach the response threshold, it is confirmed that the subject has been infected with the new coronavirus.
- the method for determining the content of each biomarker in the biomarker composition in the subject sample is a method combining liquid chromatography and mass spectrometry.
- sampling steps are as follows:
- the principle of the present invention is that the lung tissue infected with the new coronavirus will produce some specific compounds, which will be discharged through the alveoli by exhalation. Therefore, by comparing this type of new coronavirus biomarkers in the exhaled air, the new coronavirus can be directly diagnosed, which is conducive to timely and accurate clinical diagnosis and treatment of patients.
- the present invention has the following advantages:
- Chromatography and mass spectrometry are combined to accurately locate different COVID-19-related exhaled breath markers through retention time and mass spectrometry characteristics, and finally five biomarkers that can be used for COVID-19 diagnosis are screened out; these five markers can be further analyzed through characteristic mass spectra to determine the molecular structure formula;
- biomarkers can be used for early and rapid diagnosis of COVID-19, and can also be used in the preparation of COVID-19 diagnostic reagents, and have broad prospects.
- Figure 2 is the characteristic mass spectra of the five biomarkers screened out.
- Embodiment 1 is a diagrammatic representation of Embodiment 1:
- Chromatographic feature analysis rapid screening and detection of COVID-19 is performed by determining the types and concentrations of markers corresponding to different retention times in exhaled breath in the chromatogram;
- Chromatography-mass spectrometry was used to accurately locate different biomarkers through retention time and mass spectrometry characteristics, and finally five biomarkers A, B, C, D and E that can distinguish COVID-19 from healthy people were screened out. They have characteristic retention times and charge-to-mass ratios, as shown in Figure 2. They correspond to a characteristic mass spectrum and retention time respectively:
- the biomarker A has a mass-to-charge ratio of 135.0 ⁇ 0.5amu and a retention time of 23.5 ⁇ 1.0min;
- the biomarker B has a mass-to-charge ratio of 106.0 ⁇ 0.5amu and a retention time of 26.0 ⁇ 1.0min;
- the biomarker C has a mass-to-charge ratio of 94.1 ⁇ 0.5amu and a retention time of 29.0 ⁇ 1.0min;
- the biomarker D has a mass-to-charge ratio of 94.2 ⁇ 0.5amu and a retention time of 19.0 ⁇ 1.0min;
- the biomarker E has a mass-to-charge ratio of 110.5 ⁇ 0.5amu and a retention time of 39.0 ⁇ 1.0min;
- the mass-to-charge ratio and retention time are measured by a mass spectrometer using an ion trap as a mass analyzer.
- the five biomarkers A, B, C, D and E screened are used to develop and prepare reagents for early prediction and diagnosis of COVID-19:
- the response value threshold is 3 ⁇ 10 4
- D is present but E is absent
- the response value threshold is 3 ⁇ 10 4
- the present invention analyzed and predicted 20 cases of breath data used for testing. The results showed that there were 9 gas samples from healthy people and 11 gas samples from new coronavirus, which was consistent with the actual situation (10 healthy people and 10 new coronavirus), and the prediction accuracy was as high as 90%.
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Abstract
A biomarker combination in exhaled breath and a use thereof in a novel coronavirus diagnostic reagent. The marker combination comprises some or all of biomarkers A, B, C, D, E, each of which separately corresponds to a characteristic mass spectrogram and chromatographic retention time. The five biomarkers existing in exhaled breath are screened out by detecting the types and measuring relative contents of the biomarkers in breathing gases of a subject and the combined use of the five markers allows the preparation of a reagent for novel coronavirus diagnosis. The plurality of screened novel coronavirus-related biomarkers existing in exhaled breath and a combination thereof can be widely applied to the field of novel coronavirus diagnosis, and have good application prospects.
Description
本发明属于涉及新冠的检测与诊断试剂领域，具体涉及色谱与质谱联用检测、呼气中的生物标志物及其组合在新冠诊断试剂中的应用。The present invention belongs to the field of detection and diagnostic reagents related to the new coronavirus, and specifically relates to the application of chromatography-mass spectrometry combined detection, biomarkers in exhaled breath and their combinations in new coronavirus diagnostic reagents.
新型冠状病毒肺炎，简称“新冠肺炎”，是一类急性传染性疾病，由2019新型冠状病毒(COVID-19)感染引起。人感染了新型冠状病毒以后，其主要症状包括呼吸道症状、发热、气促和呼吸困难等，在较严重病例中，感染可导致肺炎、严重急性呼吸综合征、肾衰竭，甚至死亡。目前，针对新型冠状病毒引起的疾病，缺乏针对病原体的有效抗病毒药物，以隔离治疗、对症支持治疗为主。因此，对新型冠状病毒易感人群进行快速筛查并进行有效隔离，是战胜新型冠状病毒的最佳方式。现有的检测方法主要是核酸检测，但此方法取样不方便，检测时间长，且检测效果仍不理想。Novel coronavirus pneumonia, referred to as "new coronavirus pneumonia", is an acute infectious disease caused by the 2019 novel coronavirus (COVID-19). After a person is infected with the novel coronavirus, the main symptoms include respiratory symptoms, fever, shortness of breath and difficulty breathing. In more severe cases, the infection can lead to pneumonia, severe acute respiratory syndrome, renal failure, and even death. At present, there is a lack of effective antiviral drugs against pathogens for diseases caused by the novel coronavirus, and isolation treatment and symptomatic supportive treatment are the main treatments. Therefore, rapid screening of people susceptible to the novel coronavirus and effective isolation are the best ways to defeat the novel coronavirus. The existing detection method is mainly nucleic acid detection, but this method is inconvenient to sample, the detection time is long, and the detection effect is still not ideal.
因此，如何实现快速检测具有极为重要的现实意义和社会意义。Therefore, how to achieve rapid detection has extremely important practical and social significance.
研究发现，新型冠状病毒在体内大量繁殖时，会产生挥发性代谢产物并随着人体呼出气体排出体外，因此，通过人体呼出气体中新型冠状病毒挥发性标志物可以用于新型冠状病毒的诊断。另外，与现有核酸检测相比，以呼气为样本具有样品采集迅速、风险低等优点。Studies have found that when the new coronavirus reproduces in large numbers in the body, it will produce volatile metabolites that are excreted from the body with the exhaled air. Therefore, the volatile markers of the new coronavirus in the exhaled air of the human body can be used for the diagnosis of the new coronavirus. In addition, compared with existing nucleic acid tests, using exhaled air as a sample has the advantages of rapid sample collection and low risk.
通过检测特定生物样品中的新冠相关特征标志物可以提供有价值的早期新冠诊断建议，可用于制备诊断试剂。所述生物样品包括呼吸气体和各种病变组织，而组织或者体液采集比较繁琐，远没有呼吸气体采集方便。Detecting COVID-19-related characteristic markers in specific biological samples can provide valuable early COVID-19 diagnosis suggestions and can be used to prepare diagnostic reagents. The biological samples include respiratory gases and various pathological tissues, but tissue or body fluid collection is more cumbersome and far less convenient than respiratory gas collection.
发明内容Summary of the invention
本发明的目的在于克服上述现有技术存在的不足，本发明目的在于：选出具有检测作用的呼气中的生物标志物。The purpose of the present invention is to overcome the deficiencies of the prior art and to select biomarkers in exhaled breath that have a detection function.
本发明的再一目的在于：提供所述呼气中的生物标志物的应用。Another object of the present invention is to provide an application of the biomarker in exhaled breath.
本发明的又一目的在于：提供所述呼气中的生物标志物的检测方法。Another object of the present invention is to provide a method for detecting the biomarkers in the breath.
本发明目的通过以下方案实现：一种呼气中的生物标志物，其中，生物标志物来源于人的呼气样本，所述标志物选择以下生物标志物A、B、C、D、E中的一种或几种，它们分别对应一种特征质谱图和保留时间，包括：The object of the present invention is achieved by the following scheme: a biomarker in exhaled breath, wherein the biomarker is derived from a human exhaled breath sample, and the marker is selected from one or more of the following biomarkers A, B, C, D, and E, which respectively correspond to a characteristic mass spectrum and retention time, including:
所述的生物标志物A，其质荷比为135.0±0.5amu，保留时间为23.5±1.0min；和/或，The biomarker A has a mass-to-charge ratio of 135.0±0.5amu and a retention time of 23.5±1.0min; and/or,
所述的生物标志物B，其质荷比为106.0±0.5amu，保留时间为26.0±1.0min；和/或，The biomarker B has a mass-to-charge ratio of 106.0±0.5amu and a retention time of 26.0±1.0min; and/or,
所述的生物标志物C，其质荷比为94.1±0.5amu，保留时间为29.0±1.0min；和/或，The biomarker C has a mass-to-charge ratio of 94.1±0.5amu and a retention time of 29.0±1.0min; and/or,
所述的生物标志物D，其质荷比为94.2±0.5amu，保留时间为19.0±1.0min；和/或，The biomarker D has a mass-to-charge ratio of 94.2±0.5amu and a retention time of 19.0±1.0min; and/or,
所述的生物标志物E，其质荷比为110.5±0.5amu，保留时间为39.0±1.0min；The biomarker E has a mass-to-charge ratio of 110.5±0.5amu and a retention time of 39.0±1.0min;
所述的质荷比和保留时间由以离子阱为质量分析器的质谱仪测得。The mass-to-charge ratio and retention time are measured by a mass spectrometer using an ion trap as a mass analyzer.
由于新冠涉及肺部感染，呼吸气体中的新冠相关标志物相较于其他组织的新冠相关标志物更为丰富，所以通过采集新冠患者和健康志愿者的呼吸气体，采用质谱-色谱联合的方法分析比较各种分子成分，最终筛选得到一些特异性的生物标志物或其组合物，可应用于新冠的早期诊断试剂研发和制备。Since COVID-19 involves lung infection, COVID-19-related markers in respiratory gases are richer than those in other tissues. Therefore, by collecting respiratory gases from COVID-19 patients and healthy volunteers, and using a combined mass spectrometry-chromatography method to analyze and compare various molecular components, we ultimately screened out some specific biomarkers or their combinations, which can be used in the development and preparation of early diagnostic reagents for COVID-19.
进一步的，含有生物标志物A、B、C中的一种或数种。Furthermore, it contains one or more of biomarkers A, B, and C.
进一步的，含有生物标志物D、E中的一种或两种。Furthermore, it contains one or both of biomarkers D and E.
进一步的，含有生物标志物A、B、C、D和E。Further, biomarkers A, B, C, D and E are contained.
本发明提供了所述的呼气中的生物标志物在新冠的早期诊断试剂中应用，包括新冠诊断试剂的制备，试剂盒及呼气检测诊疗装置的建立等等。呼气中的生物标志物可通过色谱-质谱联合检测，用于进行新冠的患病风险评估、诊断或病理分期。The present invention provides the use of the biomarkers in exhaled breath in early diagnostic reagents for COVID-19, including the preparation of diagnostic reagents for COVID-19, the establishment of kits and exhaled breath detection and treatment devices, etc. The biomarkers in exhaled breath can be detected by chromatography-mass spectrometry for risk assessment, diagnosis or pathological staging of COVID-19.
所述的诊断为早期诊断，使得被检出的早期新冠患者可以得到及时治疗。The diagnosis is an early diagnosis, so that early COVID-19 patients detected can receive timely treatment.
本发明还提供了所述呼气中的生物标志物的检测方法，包括：The present invention also provides a method for detecting the biomarkers in the exhaled breath, comprising:
取样：取被试者呼出气体；Sampling: Take the exhaled gas of the subject;
设定响应值阈值为3×10
4，通过检测被试者呼出气体中所述生物标志物的种类及含量，当呼出气体中检测到生物标志物A、B、C的至少1种，且存在达响应阈值的生物标志物D、不存在E时，确认被试者未感染新型冠状病毒；当 在呼出气体中检测到生物标志物A、B、C的至少1种，且D、E均存在，即均达到响应阈值时，确认被试者已感染新型冠状病毒。
The response value threshold is set at 3×10 4 , and by detecting the types and contents of the biomarkers in the exhaled breath of the subjects, when at least one of the biomarkers A, B, and C is detected in the exhaled breath, and the biomarker D that reaches the response threshold exists and E is absent, it is confirmed that the subject is not infected with the new coronavirus; when at least one of the biomarkers A, B, and C is detected in the exhaled breath, and both D and E exist, that is, both reach the response threshold, it is confirmed that the subject has been infected with the new coronavirus.
其中，测定受试者样本中所述的生物标志组合物中各生物标志物的含量的方法为液相色谱与质谱联用的方法。Wherein, the method for determining the content of each biomarker in the biomarker composition in the subject sample is a method combining liquid chromatography and mass spectrometry.
取样步骤如下：The sampling steps are as follows:
1)样本的采集：采集健康志愿者及新冠患者的呼吸气体，气体储存在专用呼气袋中；1) Sample collection: Breathing gases from healthy volunteers and COVID-19 patients are collected and stored in special exhalation bags;
2)将呼气袋通过导管连入QITVenture便携式气相色谱-质谱联用仪中，进行气体生物标志物的检测；2) Connect the exhalation bag to the QITVenture portable gas chromatography-mass spectrometer through a catheter to detect gas biomarkers;
3)收集健康志愿者及新冠患者呼吸气体的色谱和标志物质谱图数据。3) Collect chromatographic and marker mass spectrometry data of respiratory gases from healthy volunteers and COVID-19 patients.
本发明的原理在于：新冠感染的肺部组织会产生一些具有特异性的化合物，此类化合物会通过肺泡经由呼气排出，因此，通过对比这类在呼气中的新冠生物标志物可以直接进行新冠的诊断，有利于对患者进行及时准确的临床诊疗。The principle of the present invention is that the lung tissue infected with the new coronavirus will produce some specific compounds, which will be discharged through the alveoli by exhalation. Therefore, by comparing this type of new coronavirus biomarkers in the exhaled air, the new coronavirus can be directly diagnosed, which is conducive to timely and accurate clinical diagnosis and treatment of patients.
与现有技术相比，本发明具有如下优势：Compared with the prior art, the present invention has the following advantages:
1.呼气中的生物标志物采集分析过程方便快捷，易于在医院等地点快速应用；1. The collection and analysis process of biomarkers in exhaled breath is convenient and quick, and can be quickly applied in hospitals and other places;
2.色谱和质谱联用，通过保留时间、质谱特征准确定位不同新冠呼气相关标志物，最终筛选出可以用于新冠诊断的五种生物标志物；后续可以通过特征质谱图对这五种标志物进一步分析，确定分子结构式；2. Chromatography and mass spectrometry are combined to accurately locate different COVID-19-related exhaled breath markers through retention time and mass spectrometry characteristics, and finally five biomarkers that can be used for COVID-19 diagnosis are screened out; these five markers can be further analyzed through characteristic mass spectra to determine the molecular structure formula;
3.通过上述生物标志物可以应用于新冠早期快速诊断，也可以用于新冠诊断试剂的制备，前景广阔。3. The above-mentioned biomarkers can be used for early and rapid diagnosis of COVID-19, and can also be used in the preparation of COVID-19 diagnostic reagents, and have broad prospects.
附图1，健康志愿者和新冠患者呼气色谱图对比；Figure 1, comparison of breath chromatograms of healthy volunteers and COVID-19 patients;
附图2，筛选出的五种生物标志物的特征质谱图。Figure 2 is the characteristic mass spectra of the five biomarkers screened out.
本发明通过下面的实施例进行详细解释，但不仅限于此。The present invention is explained in detail by the following examples, but is not limited thereto.
实施例1：Embodiment 1:
1.呼气样本数据的采集：1. Collection of breath sample data:
1)通过呼气的方式采集健康志愿者及新冠患者的呼吸气体，气体储存在专用呼气袋中；1) Collect respiratory gases from healthy volunteers and COVID-19 patients by exhaling, and store the gases in special exhalation bags;
2)将呼气袋通过导管连入QITVenture便携式气相色谱-质谱联用仪中，进行气体标志物的检测；2) Connect the exhalation bag to the QITVenture portable gas chromatography-mass spectrometer through a catheter to detect gas markers;
3)收集健康志愿者及新冠患者呼吸气体的色谱和标志物质谱图数据，见附图1和附图2。3) Collect chromatographic and marker mass spectrometry data of respiratory gases from healthy volunteers and COVID-19 patients, see Figures 1 and 2.
2.呼气样本数据分析：2. Breath sample data analysis:
1)色谱特征分析，通过判断色谱图中呼气中的不同保留时间对应的标志物种类及其浓度来进行新冠的快速筛查与检测；1) Chromatographic feature analysis: rapid screening and detection of COVID-19 is performed by determining the types and concentrations of markers corresponding to different retention times in exhaled breath in the chromatogram;
2)对不同保留时间出峰的标志物进行质谱分析，双重定位特征标志物类型：2) Perform mass spectrometry analysis on markers with different retention times, and double locate the characteristic marker types:
色谱质谱联用，通过保留时间，质谱特征准确定位不同的生物标志物，最终筛选出可以区别新冠和健康者的五种生物标志物A、B、C、D和E，它们具有特征的保留时间和荷质比，见附图2，它们分别对应一种特征质谱图和保留时间为：Chromatography-mass spectrometry was used to accurately locate different biomarkers through retention time and mass spectrometry characteristics, and finally five biomarkers A, B, C, D and E that can distinguish COVID-19 from healthy people were screened out. They have characteristic retention times and charge-to-mass ratios, as shown in Figure 2. They correspond to a characteristic mass spectrum and retention time respectively:
所述的生物标志物A，其质荷比为135.0±0.5amu，保留时间为23.5±1.0min；The biomarker A has a mass-to-charge ratio of 135.0±0.5amu and a retention time of 23.5±1.0min;
所述的生物标志物B，其质荷比为106.0±0.5amu，保留时间为26.0±1.0min；The biomarker B has a mass-to-charge ratio of 106.0±0.5amu and a retention time of 26.0±1.0min;
所述的生物标志物C，其质荷比为94.1±0.5amu，保留时间为29.0±1.0min；The biomarker C has a mass-to-charge ratio of 94.1±0.5amu and a retention time of 29.0±1.0min;
所述的生物标志物D，其质荷比为94.2±0.5amu，保留时间为19.0±1.0min；The biomarker D has a mass-to-charge ratio of 94.2±0.5amu and a retention time of 19.0±1.0min;
所述的生物标志物E，其质荷比为110.5±0.5amu，保留时间为39.0±1.0min；The biomarker E has a mass-to-charge ratio of 110.5±0.5amu and a retention time of 39.0±1.0min;
所述的质荷比和保留时间由以离子阱为质量分析器的质谱仪测得。The mass-to-charge ratio and retention time are measured by a mass spectrometer using an ion trap as a mass analyzer.
3.所筛选的五种生物标志物A、B、C、D和E用于研发和制备新冠早期预测与诊断的试剂：3. The five biomarkers A, B, C, D and E screened are used to develop and prepare reagents for early prediction and diagnosis of COVID-19:
当呼出气体中检测到A、B、C的至少1种(响应值阈值为3×10
4)， 且存在D、不存在E时，确认被试者未感染新型冠状病毒；
When at least one of A, B, and C is detected in the exhaled breath (the response value threshold is 3×10 4 ), and D is present but E is absent, it is confirmed that the subject is not infected with the new coronavirus;
当在呼出气体中检测到A、B、C的至少1种，且D、E均存在(响应值阈值为3×10
4)时，确认被试者已感染新型冠状病毒。
When at least one of A, B, and C is detected in the exhaled breath, and both D and E are present (the response value threshold is 3×10 4 ), it is confirmed that the subject has been infected with the new coronavirus.
本发明对20例用于测试的呼气数据进行了分析预测，结果显示有9例健康者气体样本，11例为新冠气体样本，和实际情况(10例健康，10例新冠)较为符合，预测准确率高达90％。The present invention analyzed and predicted 20 cases of breath data used for testing. The results showed that there were 9 gas samples from healthy people and 11 gas samples from new coronavirus, which was consistent with the actual situation (10 healthy people and 10 new coronavirus), and the prediction accuracy was as high as 90%.
Claims (8)
- 一种呼气中的生物标志物，其特征在于，生物标志物来源于人的呼气样本，所述标志物选择以下生物标志物A、B、C、D、E中的一种或几种，它们分别对应一种特征质谱图和保留时间，包括：A biomarker in exhaled breath, characterized in that the biomarker is derived from a human exhaled breath sample, and the biomarker is selected from one or more of the following biomarkers A, B, C, D, and E, which respectively correspond to a characteristic mass spectrum and retention time, including:所述的生物标志物A，其质荷比为135.0±0.5amu，保留时间为23.5±1.0min；和/或，The biomarker A has a mass-to-charge ratio of 135.0±0.5amu and a retention time of 23.5±1.0min; and/or,所述的生物标志物B，其质荷比为106.0±0.5amu，保留时间为26.0±1.0min；和/或，The biomarker B has a mass-to-charge ratio of 106.0±0.5amu and a retention time of 26.0±1.0min; and/or,所述的生物标志物C，其质荷比为94.1±0.5amu，保留时间为29.0±1.0min；和/或，The biomarker C has a mass-to-charge ratio of 94.1±0.5amu and a retention time of 29.0±1.0min; and/or,所述的生物标志物D，其质荷比为94.2±0.5amu，保留时间为19.0±1.0min；和/或，The biomarker D has a mass-to-charge ratio of 94.2±0.5amu and a retention time of 19.0±1.0min; and/or,所述的生物标志物E，其质荷比为110.5±0.5amu，保留时间为39.0±1.0min；The biomarker E has a mass-to-charge ratio of 110.5±0.5amu and a retention time of 39.0±1.0min;所述的质荷比和保留时间由以离子阱为质量分析器的质谱仪测得。The mass-to-charge ratio and retention time are measured by a mass spectrometer using an ion trap as a mass analyzer.
- 根据权利要求1所述的呼气中的生物标志物，其特征在于，至少含有生物标志物A、B、C中的一种或数种。The biomarker in exhaled breath according to claim 1, characterized in that it contains at least one or more of biomarkers A, B, and C.
- 根据权利要求1所述的呼气中的生物标志物，其特征在于，至少含有生物标志物D、E中的一种或两种。The exhaled breath biomarker according to claim 1, characterized in that it contains at least one or two of biomarkers D and E.
- 根据权利要求1所述的呼气中的生物标志物及其组合，其特征在于，含有生物标志物A、B、C、D和E。The biomarkers in exhaled breath and the combination thereof according to claim 1, characterized in that they contain biomarkers A, B, C, D and E.
- 一种根据权利要求1至4任一项所述的生物标志物的应用，其特征在于，用于包括新冠诊断试剂的制备，试剂盒及呼气检测诊疗装置的建立；对呼气中的生物标志物通过色谱-质谱联合检测，用于进行新冠的患病风险评估、诊断或病理分期。An application of the biomarker according to any one of claims 1 to 4, characterized in that it is used for the preparation of COVID-19 diagnostic reagents, the establishment of kits and breath detection and treatment devices; the biomarkers in the exhaled breath are detected by combined chromatography-mass spectrometry for COVID-19 risk assessment, diagnosis or pathological staging.
- 一种呼气中的生物标志物的检测方法，其特征在于，包括：A method for detecting biomarkers in exhaled breath, comprising:采样：采集被试者呼出气体进行收集；Sampling: Collect the exhaled gas of the subjects;通过检测被试者呼出气体中所述生物标志物的种类及含量，用于新冠的评估、诊断；By detecting the types and contents of the biomarkers in the exhaled breath of the subjects, it can be used for the evaluation and diagnosis of COVID-19;设定生物标志物的响应阈值3×10 4，当呼出气体中检测到A、B、C的至少1种达到阈值，且生物标志物D达到阈值，而不存在生物标志物E时，确认被试者未感染新型冠状病毒；当在呼出气体中检测到A、B、C的至少1种达到阈值，且D、E均达到响应值阈值时，确认被试者已感染新型冠状病毒。 The response threshold of the biomarker is set at 3×10 4 . When at least one of A, B, and C is detected in the exhaled breath and reaches the threshold, and biomarker D reaches the threshold, but biomarker E is absent, it is confirmed that the subject is not infected with the new coronavirus. When at least one of A, B, and C is detected in the exhaled breath and reaches the threshold, and both D and E reach the response value threshold, it is confirmed that the subject is infected with the new coronavirus.
- 权利要求6所述的生物标志物的检测方法，其特征在于，所述的生物标志组合物的含量采用液相色谱与质谱联用的方法检测。The biomarker detection method according to claim 6 is characterized in that the content of the biomarker composition is detected by a method combining liquid chromatography and mass spectrometry.
- 根据权利要求6或7所述的生物标志组合物的检测方法，其特征在于，按下述步骤：The method for detecting the biomarker composition according to claim 6 or 7, characterized by the following steps:1)呼气数据收集：采集健康志愿者及新冠患者的呼吸气体，气体储存在专用呼气袋中；1) Exhalation data collection: The respiratory gases of healthy volunteers and COVID-19 patients are collected and stored in special exhalation bags;2)将呼气袋通过导管连入QITVenture便携式气相色谱-质谱联用仪中，进行呼气中生物标志物的检测；2) Connect the exhalation bag to the QITVenture portable gas chromatography-mass spectrometer through a catheter to detect biomarkers in the exhaled breath;3)收集健康志愿者及新冠患者呼吸气体的色谱和标志物质谱图数据。3) Collect chromatographic and marker mass spectrometry data of respiratory gases from healthy volunteers and COVID-19 patients.
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| CN112067712A (en) * | 2020-08-18 | 2020-12-11 | 上海纳米技术及应用国家工程研究中心有限公司 | Volatile marker for diagnosing novel coronavirus and application thereof |
| WO2022032039A1 (en) * | 2020-08-04 | 2022-02-10 | Richard Postrel | Rapid screening device for detecting covid19 and other infections diseases |
| WO2022058796A1 (en) * | 2020-09-17 | 2022-03-24 | Universite De Versailles-St Quentin En Yvelines | A method for analysing a breath sample for screening, diagnosis or monitoring of sars-cov-2 carriage or infection (covid-19) on humans |
| WO2022094405A1 (en) * | 2020-10-30 | 2022-05-05 | The Children's Hospital Of Philadelphia | Breath biomarkers of sars-cov-2 in children and methods of use thereof for the diagnosis and treatment of covid-19 |
| CN115236220A (en) * | 2022-06-23 | 2022-10-25 | 上海交通大学 | Volatile marker for diagnosing novel coronavirus and application thereof |
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| WO2022032039A1 (en) * | 2020-08-04 | 2022-02-10 | Richard Postrel | Rapid screening device for detecting covid19 and other infections diseases |
| CN112067712A (en) * | 2020-08-18 | 2020-12-11 | 上海纳米技术及应用国家工程研究中心有限公司 | Volatile marker for diagnosing novel coronavirus and application thereof |
| WO2022058796A1 (en) * | 2020-09-17 | 2022-03-24 | Universite De Versailles-St Quentin En Yvelines | A method for analysing a breath sample for screening, diagnosis or monitoring of sars-cov-2 carriage or infection (covid-19) on humans |
| WO2022094405A1 (en) * | 2020-10-30 | 2022-05-05 | The Children's Hospital Of Philadelphia | Breath biomarkers of sars-cov-2 in children and methods of use thereof for the diagnosis and treatment of covid-19 |
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