CN110702821B - Typing detection kit for chronic obstructive pulmonary disease - Google Patents
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Abstract
The invention belongs to the field of chronic obstructive pulmonary disease metabolome. The invention provides application of a reagent for detecting all-trans-heptaphenyl diphosphate (all-trans-heptaphenyl diphosphate) in serum in preparing a chronic obstructive pulmonary disease typing kit; the typing refers to distinguishing chronic bronchitis type or emphysema type chronic obstructive pulmonary diseases. The invention also provides a corresponding chronic obstructive pulmonary disease typing detection kit. The invention can realize the effective typing of the chronic obstructive pulmonary disease by detecting the content of the all-trans heptaphenyl diphosphate in serum.
Description
Technical Field
The invention belongs to the field of chronic obstructive pulmonary disease metabolome, and particularly relates to a typing detection kit for chronic obstructive pulmonary disease.
Background
Chronic Obstructive Pulmonary Disease (COPD) is a chronic bronchitis and/or emphysema characterized by airflow obstruction that can further progress to the common chronic diseases of pulmonary heart disease and respiratory failure.
The exact cause of chronic obstructive pulmonary disease is not clear, and it is thought that factors involved in the development of both chronic bronchitis and obstructive emphysema may be involved in the onset of chronic obstructive pulmonary disease. Risk factors that have been found can be broadly divided into two categories, external (i.e., environmental factors) and internal (i.e., individual predisposition factors). Extrinsic factors include smoking, inhalation of dust and chemicals, air pollution, respiratory infections, and socioeconomic low (may be related to indoor and outdoor air pollution, crowded rooms, poor nutrition, and other factors associated with socioeconomic low status). Endogenous factors include genetic factors, increased airway responsiveness, individuals with lung development or poor growth during pregnancy, neonatal, infant or childhood due to a variety of causes.
The disability rate and the fatality rate of COPD are high, and the worldwide incidence rate of COPD over 40 years old is up to 9% -10%.
Currently, the diagnosis of COPD relies mainly on pulmonary function examination, chest X-ray examination, chest CT examination, etc., but pulmonary function examination (spirometry), which is a time-consuming and expensive procedure, can only be performed by specialized lung physicians. Furthermore, COPD is a disease with both complexity and heterogeneity, and the characteristics of COPD cannot be objectively reflected by the mere dependence on lung function. COPD is therefore further divided into 2 phenotypes of chronic bronchitis (chronic bronchitis) and emphysema, the former manifesting as long-term productive cough, severe airway obstruction, thickening of the tracheal wall; the latter is mainly manifested as lung hyperinflation. Inexperienced clinicians are subjective in typing patients for COPD and are difficult to accurately type COPD with atypical symptoms, resulting in inaccurate drug administration.
Therefore, there is an urgent need for a tool capable of accurately typing COPD.
Disclosure of Invention
The invention aims to provide a COPD typing marker and a kit, wherein the typing refers to the differentiation of chronic bronchitis type or emphysema type chronic obstructive pulmonary disease.
The technical scheme of the invention is as follows:
the invention provides application of a reagent for detecting all-trans heptaphenyl diphosphate in serum in preparation of a chronic obstructive pulmonary disease parting kit.
The reagent for detecting the all-trans heptaphenyl diphosphate in the serum is used as a reagent for a liquid chromatography-mass spectrometry (LC-MS) method.
As the application, the reagent for detecting the content of the all-trans heptaphenyl diphosphate in the serum is a reagent for a liquid chromatography-mass spectrometry combined metabonomics method.
Further, the reagent for detecting the content of all-trans heptaphenyl diphosphate in serum is a reagent for a liquid chromatography-mass spectrometry non-targeted metabonomics method.
As the application, the reagent for detecting the content of the all-trans heptaphenyl diphosphate in the serum is a reagent for a liquid chromatography method.
The invention also provides a chronic obstructive pulmonary disease parting kit, which comprises a reagent for detecting the content of all-trans heptaphenyl diphosphate in serum.
According to the typing kit, the reagent for detecting the content of the all-trans heptaphenyl diphosphate in the serum is a reagent for a liquid chromatography-mass spectrometry method.
As the typing kit, the reagent for detecting the content of the all-trans heptaphenyl diphosphate in serum is a reagent for a liquid chromatography-mass spectrometry combined metabonomics method.
Further, the reagent for detecting the content of all-trans heptaphenyl diphosphate in serum is a reagent for a liquid chromatography-mass spectrometry non-targeted metabonomics method.
According to the typing kit, the reagent for detecting the content of the all-trans heptaphenyl diphosphate in the serum is a reagent for a liquid chromatography method; when used, the all-trans heptaphenyl diphosphate standard is used as a reference.
The inventor finds that the content of all-trans heptaphenyl diphosphate in the serum of COPD (chronic bronchitis type/emphysema type) patients with different phenotypes is remarkably different, and the content of all-trans heptaphenyl diphosphate in the serum of COPD patients with chronic bronchitis type is far higher than that of COPD patients with emphysema type. The conventional compound detection method, such as a liquid chromatography (such as high performance liquid chromatography, ultra-high performance liquid chromatography), liquid chromatography-mass spectrometry combined use and a liquid chromatography-mass spectrometry combined metabonomics method, can detect all-trans heptaphenyl diphosphate, and COPD typing judgment can be carried out by using the detection data of the existing liquid chromatography, liquid chromatography-mass spectrometry combined use or liquid chromatography-mass spectrometry combined metabonomics (including targeted metabonomics and non-targeted metabonomics) method as a reference.
Further, the liquid chromatography-mass spectrometry combined non-targeted metabonomics method used in example 1 of the present invention detects specifically quantified values of all-trans heptaphenyl diphosphate in serum of COPD (chronic bronchitis/emphysema) patients with different phenotypes, and performs ROC analysis, and the area under the curve (AUC) obtained is 0.690, and when the detection threshold value (cut-off value) is 1.263, the specificity is 0.900, and the sensitivity is 0.500. Therefore, if the kit is used for typing detection of COPD by a liquid chromatography-mass spectrometry non-targeted metabonomics method, the size relation between the detection value of the all-trans heptaphenyl diphosphate and 1.263 can be used as a typing judgment basis, when the detection value is more than 1.263, chronic bronchitis type COPD is judged, and when the detection value is less than 1.263, emphysema type COPD is judged.
The COPD typing detection kit can realize objective, accurate and rapid COPD typing by detecting all-trans heptaphenyl diphosphate in serum.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1 is a ROC plot of all-trans heptaphenyl diphosphate concentration versus COPD typing.
Detailed Description
Example 1 detection of all-trans heptaphenyl diphosphate in the serum of patients with COPD (chronic bronchitis/emphysema) of different phenotypes
1. Object to be inspected
COPD patients with chronic bronchitis (group a) 30 people and COPD patients with emphysema (group B) 30 people. The subject population was informed and consented to prior to the experiment.
2. Method of producing a composite material
30 cases of serum of COPD patients with chronic bronchitis phenotype and emphysema phenotype are respectively subjected to sample pretreatment, metabolite extraction, LC-MS full-scan detection, data pretreatment and statistical analysis. Based on the non-targeted metabonomics function of the UPLC-VION IMS Q-Tof high-resolution mass spectrometer, the method combines the metabonomics data processing software Progenetics QI v2.3 to carry out qualitative and relative quantitative analysis on the original data and carry out standardized pretreatment on the original data.
The specific experimental steps are as follows:
(1) sample pretreatment
1) Samples stored at-80 ℃ were removed, thawed at room temperature, 100. mu.L serum was removed, and internal standard (L-2-chlorophenylalanine, 0.3 mg/mL; c-17, 0.01mg/mL, all in methanol) each 10. mu.L, vortex for 10 s;
2) adding 300 μ L of protein precipitant methanol-acetonitrile (V: V is 2: 1), and vortexing and shaking for 1 min;
3) ultrasonic extracting in ice water bath for 10 min;
4) standing at-20 deg.C for 30 min;
5) centrifuge for 10min (13000rpm, 4 ℃), aspirate 200 μ L of supernatant with syringe, filter using a 0.22 μm organic phase pinhole filter, transfer to LC injection vial, store at-80 ℃ until LC-MS analysis.
6) The quality control sample (QC) is prepared by mixing the extractive solutions of all the samples in equal volume, and the quality control sample (QC) is prepared by mixing the extractive solutions of all the samples in equal volume
The volume is the same as the sample.
Remarking: all extraction reagents were pre-chilled at-20 ℃ prior to use. Quality Control (QC) samples: each set of samples was taken in equal amounts and mixed to QC. One QC was inserted into each 10 samples and used to evaluate system stability throughout the experiment.
(2) Conditions for liquid chromatography-mass spectrometry
The analytical instrument of the experiment is a liquid chromatography-mass spectrometry system consisting of a Voltte I-Class ultra-high performance liquid phase tandem VION IMS Q-Tof high-resolution mass spectrometer.
Chromatographic conditions are as follows:
a chromatographic column: ACQUITY UPLC BEH C18(100 mm. times.2.1 mm, 1.7 um); column temperature: 45 ℃; mobile phase: a-water (with 0.1% formic acid), B-acetonitrile/methanol (2/3) (v/v) (with 0.1% formic acid); flow rate: 0.4 mL/min;
sample introduction volume: 1 μ L.
Mass spectrum conditions: an ion source: ESI; and the sample mass spectrum signal acquisition respectively adopts a positive and negative ion scanning mode.
3. Results
A. The results of the detection of all-trans heptaphenyl diphosphate in group B sera are shown in the following table:
remarking: FC (fold change) refers to the amount of this protein in serum of the chronic bronchitis phenotype/the emphysema phenotype.
As can be seen, the content of all-trans heptaphenyl diphosphate in the serum of chronic bronchitis type COPD patients is obviously higher than that of emphysema type COPD patients.
The ROC analysis of the content of all-trans heptaphenyl diphosphate in A, B sera showed that the area under the curve (AUC) is 0.690, as shown in FIG. 1; when the cut-off value (cut-off value) was 1.263, the specificity was 0.9 and the sensitivity was 0.5.
Based on the detection of the content of all-trans heptaphenyl diphosphate in the serum of a COPD patient, a COPD typing kit can be developed. If the kit is used for typing detection of COPD by an LC-MS non-targeted metabonomics method (as in the embodiment), the size relation between the detection value of the all-trans heptaphenyl diphosphate and 1.263 can be used as a typing judgment basis, when the detection value is more than 1.263, chronic bronchitis type COPD is judged, and when the detection value is less than 1.263, emphysema type COPD is judged.
Of course, the LC-MS non-targeted metabonomics method is only a conventional means for detecting compounds, and theoretically, various kits (such as a liquid chromatography method kit and a liquid chromatography-mass spectrometry combined method kit) capable of detecting the content of all-trans heptaphenyl diphosphate in the serum of a COPD patient can realize COPD typing. Taking a liquid chromatography method kit as an example, the method only needs to detect the content of all-trans heptaphenyl diphosphate in the serum of each known chronic bronchitis type and emphysema type COPD patient in advance as a reference standard; the same kit is used for detecting the serum of an unknown COPD patient to obtain the content value of all-trans-heptaphenyl diphosphate, and the content value is compared with a reference standard, so that the COPD typing judgment can be carried out.
Example 2 kit of the invention
1. Compositions of the kits of the invention
Methanol, formic acid, acetonitrile and L-2-chlorophenylalanine.
2. Kit using method
The experimental procedure was as in example 1.
When the detection value of all-trans heptaphenyl diphosphate in serum is higher than 1.263, chronic bronchitis type COPD can be distinguished; otherwise, emphysema type COPD can be distinguished.
In conclusion, the kit can realize effective typing of chronic bronchitis type or emphysema type COPD through quantitative detection of all-trans heptaphenyl diphosphate, can simply and quickly provide valuable reference information for clinicians, is convenient for symptomatic medication and has good application prospect.
Claims (3)
1. The application of a reagent for detecting the content of all-trans-heptaprenyl diphosphate in serum in preparing a chronic obstructive pulmonary disease parting kit; the typing refers to distinguishing chronic bronchitis type or emphysema type chronic obstructive pulmonary diseases.
2. The use according to claim 1, wherein the reagent for detecting the content of all-trans-heptaprenyl diphosphates in serum is a reagent for a liquid chromatography-mass spectrometry method.
3. The use according to claim 1, wherein the reagent for measuring the amount of all-trans-heptaprenyl diphosphates in serum is a reagent for liquid chromatography.
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| CN112285232B (en) * | 2020-10-20 | 2022-06-10 | 四川大学华西医院 | Application of Ulexin C detection reagent in preparation of COPD (chronic obstructive pulmonary disease) diagnostic kit |
| CN112285355B (en) * | 2020-10-20 | 2023-01-13 | 四川大学华西医院 | Application of Viridiflorin detection reagent in preparation of COPD (chronic obstructive pulmonary disease) diagnostic kit |
| CN115616227B (en) * | 2022-11-18 | 2023-05-16 | 四川大学华西医院 | Use of indole-3-acryloylglycine detection reagent, and kit and system for diagnosis or auxiliary diagnosis of chronic obstructive disease |
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