Exhaled gas VOCs (volatile organic compounds) marker for early diagnosis of echinococcosis granulosa and application of exhaled gas VOCs marker

Abstract

The invention discloses an exhaled gas VOCs (Volatile Organic Compounds) marker for early diagnosis of echinococcosis granulosa and application of the exhaled gas VOCs marker, and belongs to the technical field of detection. The ROC curve AUC of the biomarker A is equal to 1, the sensitivity and the specificity can reach 100%, the ROC curve AUC of the biomarker B is equal to 0.967, the sensitivity and the specificity can reach 96.4% and 94.9% respectively, and the accuracy reaches 95.0%. The two gas levels have statistical significance, and can be used as a reference basis for diagnosing the CE. The detection system disclosed by the invention is low in cost and simple to operate, and can be used for early screening of echinococcosis granulosus diagnosis methods.

Description

Technical Field

[0001] The invention relates to an exhaled gas VOCs marker for early diagnosis of echinococcosis granulosus and application thereof, belonging to the technical field of detection. Background Art

[0002] Hydatid disease is a common parasitic disease in pastoral areas, also known as echinococcosis. Human echinococcosis (HE) is a zoonotic parasitic disease caused by the larvae of Echinococcus spp. parasitizing humans or animals. It is mainly prevalent in Qinghai, Tibet and other provinces with developed animal husbandry in China. Depending on the type of pathogen, echinococcosis is divided into two types: one is cystic echinococcosis (CE) caused by infection with the larvae of Echinococcus granulosus, and the other is alveolar echinococcosis (AE) caused by infection with the larvae of Echinococcus multilocularis.

[0003] The main reason for human infection with echinococcosis is contact with animals, or accidentally ingesting worm eggs when handling animal carcasses and feces. Echinococcus eggs mainly hatch in the human upper digestive tract, and the formed larvae travel through the portal vein to the liver and then parasitize in the liver. Most patients have a long history of living in pastoral areas and have close contact with animals such as cattle, sheep, and dogs. Humans are the terminal intermediate hosts, so people are not infected from person to person. After people are infected with echinococcosis, there are usually no obvious symptoms in the early stages, so this type of disease is often ignored. Most people are already in the late stages after being diagnosed, and treatment is relatively complicated. When the condition worsens, it may cause death due to liver failure, biliary infection, and metastasis to organs such as the lungs and brain.

[0004] The diagnosis of CE is usually done through ultrasound examination, supplemented by serological tests, computed tomography or magnetic resonance imaging scans. The diagnosis and treatment of CE are both expensive and complicated, and in some cases, require long-term surgery and drug treatment. However, before the examination, most patients have obvious symptoms, and after diagnosis, they have entered the middle and late stages, which is also the shortcoming of these auxiliary examinations and laboratory tests. Therefore, it is an urgent problem to design a simple, accurate and universal early diagnosis method to promote the screening of hepatic echinococcosis cases in nomadic areas, so that patients can be detected and treated early, thereby minimizing the mortality rate of CE. Summary of the invention

[0005] To solve the above problems, the present invention provides an exhaled gas VOCs marker for early diagnosis of echinococcosis and its application, analyzes volatile organic compounds (VOCs) in human exhaled breath by gas chromatography-mass spectrometry (GC-MS) technology, screens out effective biomarkers, and further provides a non-invasive, rapid, easy-to-operate and relatively inexpensive detection system for early diagnosis of echinococcosis.

[0006] The first object of the present invention is to provide an exhaled breath VOCs marker for early diagnosis of echinococcosis granulosus, which is one or more combinations of the following exhaled breath VOCs: nonanoic acid and octanoic acid.

[0007] A second object of the present invention is to provide a detection system for early diagnosis of echinococcosis, the detection system comprising:

[0008] An exhaled gas collection device for collecting volatile organic compounds in the exhaled gas of a subject; and

[0009] An exhaled gas separation device for separating and extracting volatile organic compounds from exhaled gas; and

[0010] Exhaled gas detection device, used to detect the composition and content of markers in organic volatiles;

[0011] Among them, the marker is one or more combinations of the following exhaled gas VOCs: nonanoic acid and octanoic acid.

[0012] In one embodiment of the present invention, the detection thresholds are: nonanoic acid threshold is 0.5 ppbv, and / or octanoic acid threshold is 2 ppbv.

[0013] In the present invention, when the group of biomarkers reaches a certain concentration (ie, a threshold value), it can be preliminarily determined that the patient has a risk of Echinococcosis infection.

[0014] In one embodiment of the present invention, the exhaled gas collection device includes an air collection bag, a mouthpiece arranged on the air collection bag, and a sampling cap connected to the mouthpiece.

[0015] In one embodiment of the present invention, the gas separation device is a solid phase microextraction device.

[0016] In one embodiment of the present invention, the solid phase microextraction device comprises a solid phase microextraction handle and an automatic extraction head coated with polydimethylsiloxane.

[0017] In one embodiment of the present invention, the exhaled gas detection device is a gas chromatograph-mass spectrometer.

[0018] In one embodiment of the present invention, the detection parameters of the gas chromatography-mass spectrometer are set as follows: the carrier gas is He, the flow rate is 1 ml / min, the injection temperature is 250°C, and the injection time is 2 minutes; the column box temperature is increased from 40°C to 250°C at a rate of 5°C / min; the chromatographic and mass spectrometric interface temperature is 250°C, the MS ion source temperature is 200°C, and the scanning range is 40-450amu.

[0019] In one embodiment of the present invention, the chromatographic column used in the gas chromatography-mass spectrometer is a DB-1 gas chromatography column.

[0020] The third object of the present invention is to provide the use of the exhaled gas VOCs marker for early diagnosis of echinococcosis granulosus in the preparation of a product for early diagnosis of echinococcosis granulosus.

[0021] Beneficial Effects

[0022] The present invention discloses an exhaled gas VOCs marker for early diagnosis of echinococcosis and its application. In the present invention, the ROC curve AUC of biomarker A is 1, and the sensitivity and specificity can reach 100%; the ROC curve AUC of biomarker B is 0.967, and the sensitivity and specificity can reach 96.4% and 94.9% respectively, and the accuracy reaches 95.0%. Both gas levels are statistically significant and can be used as a reference for diagnosing CE. The detection system of the present invention has low cost and simple operation, and can be used for the diagnosis method of echinococcosis for early screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The sampling device: A is the air collecting bag, mouthpiece and sampling cap; B is the valve open; C is the valve closed.

[0024] Figure 2 This is a comparison of the difference in expression of Nonanoic acid (pelargonic acid) levels between the two groups.

[0025] Figure 3 This is a comparison of the difference in the expression of Octanoic acid levels between the two groups.

[0026] Figure 4 This is the ROC curve of Nonanoic acid expression.

[0027] Figure 5 This is the ROC curve of Octanoic acid expression.

[0028] Figure 6 This is the ROC curve for the clinical validation of Nonanoic acid.

[0029] Figure 7This is the ROC curve for the clinical validation of Octanoic acid. DETAILED DESCRIPTION

[0030] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0031] Materials involved in the examples:

[0032] Teflon FEP film air collection bag was purchased from Dalian Haide Technology Co., Ltd.

[0033] Experimental methods:

[0034] (1) Breath collection

[0035] Medical staff informed the subjects and distributed gas collection bags. Each subject received a Teflon EFP film gas collection bag, a mouthpiece, and a sampling cap ( Figure 1 In the A), the subjects fasted and stopped smoking for 12 hours before sampling, and did not eat foods with strong pungent smells (such as garlic, stinky tofu, leeks, etc.) within 24 hours. They brushed their teeth and fasted in the morning on the day of sampling. The subjects ensured indoor air circulation and exhaled gas into the sampling bag.

[0036] The subject remains at rest and maintains normal breathing. Connect a disposable Teflon mouthpiece to the air outlet of the sampling bag, hold the mouthpiece in the subject's mouth, and slowly open the airtight valve of the sampling bag, which is a plug-through valve ( Figure 1 The subject breathes calmly and blows air slowly into the air bag. After collecting 100 mL of gas, the airtight valve is quickly closed and the sampling cap is tightly closed ( Figure 1 C), store in a sealed container, making sure the air collecting bag is not damaged to prevent air leakage.

[0037] After collecting the gas, the subject should store it in a cool, dry, dark place until it is recovered. To ensure accuracy, the gas collection bag should be recovered in the afternoon of the same day.

[0038] (2) Solid phase microextraction and GC-MS detection of exhaled VOCs

[0039] The VOCs in the samples were separated and enriched by solid phase microextraction. According to the properties of the test substances, the solid phase microextraction (SPME) handle of Supelco and the automatic extraction head coated with 100um polydimethylsiloxane (PDMS) were used. The needle was inserted into the gas collection bag through the sampling cap septum; the handle piston was pushed to place the fiber head in the gas bag, and the extraction was carried out for 1 hour at a constant temperature of 60℃; the fiber head was retracted and the needle was pulled out, and the samples were transferred to GC-MS for analysis. Qualitative analysis was performed by searching the NIST mass spectrometry database, and the peak area was calculated by calculus for quantitative analysis.

[0040] The gas chromatograph-mass spectrometer was a Shimadzu GC-MS-QP2020, the capillary column was a DB-1 gas chromatograph column (30 m × 0.25 mm × 0.25 um) produced by Agilent, and the instrument parameters were set as follows: the carrier gas was He, the flow rate was 1 ml / min, the injection temperature was 250 °C, and the injection time was 2 min; the column box temperature was increased from 40 °C to 250 °C at a rate of 5 °C / min; the chromatographic and mass spectrometric interface temperature was 250 °C, the MS ion source temperature was 200 °C, and the scanning range was 40-450 amu.

[0041] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0042] Example 1: Differential analysis of biomarker levels in exhaled breath

[0043] A total of 60 CE patients and 60 control group were selected.

[0044] (1) CE group: patients from all parts of Qinghai Province who were diagnosed with CE within the past year, regardless of gender or age, with a gender ratio of 1:1.

[0045] (2) CCE group: 60 patients without echinococcosis infection and without Echinococcus granulosus infection, matched 1:1 by age and sex.

[0046] (3) Exclusion criteria:

[0047] ① Suffering from hepatitis A, hepatitis B, hepatitis C, hepatitis D, or hepatitis E;

[0048] ② History of alcoholism and drug allergy;

[0049] ③ Patients with liver and kidney dysfunction or abnormality caused by other reasons;

[0050] ④ Those who cannot cooperate with the collection of exhaled gas.

[0051] The exhaled air collection method of the present invention was used to collect exhaled VOCs, and the exhaled VOCs solid phase microextraction and GC-MS detection methods were used to detect the collected gas. SPSS23.0 was used to complete the statistical analysis. The difference in the VOCs peak area between the CE group and the CCE group was compared.

[0052] The GC-MS results showed that there were more than 400 compounds in each breath sample, and there were statistical differences in the concentrations of multiple VOCs in the exhaled VOCs of the CE group and the control group (CCE group, non-echinococcosis), and the concentrations of two biomarkers increased (Nonanoic acid, Octanoic acid). According to the GC-MS analysis, it can be found that both biomarkers are statistically different compared with healthy people.

[0053] Among them, the difference analysis of Nonanoic acid level is as follows Figure 2 As shown, the average peak area of ​​the CE group is 4.5×10 8 The average peak area of ​​the CCE group was 3.1×10 7 The peak areas of the two groups were statistically significant by non-parametric test (P<0.0001).

[0054] Differential analysis of octanoic acid levels Figure 3 As shown, the average peak area of ​​the CE group is 2.4×10 8 The average peak area of ​​the CCE group was 1.9×10 7 The peak areas of the two groups were statistically significant by non-parametric test (P<0.0001).

[0055] Example 2: Reliability of biomarkers in CE diagnosis

[0056] After statistical analysis, the ROC curve AUC of biomarker A was 1, and both sensitivity and specificity could reach 100%. Figure 4 ; The ROC curve of biomarker B has an AUC of 0.974, with a sensitivity and specificity of 98.2% and 97.4% respectively, and an accuracy of 97.5%. Figure 5 The levels of both gases were statistically significant and could be used as a reference for diagnosing CE.

[0057] Example 3: Clinical Validation of Biomarker Sensitivity, Specificity and Accuracy

[0058] Research subjects: In addition, 30 CE patients and 30 control group people (CCE group) were selected.

[0059] (1) CE group: patients from all parts of Qinghai Province who were diagnosed with AE within the past year, regardless of gender or age, with a gender ratio of 1:1.

[0060] (2) CCE group: 30 patients without echinococcosis infection and without Echinococcus granulosus infection, matched 1:1 by age and sex.

[0061] (3) Exclusion criteria:

[0062] ① Suffering from hepatitis A, hepatitis B, hepatitis C, hepatitis D, or hepatitis E;

[0063] ② History of alcoholism and drug allergy;

[0064] ③ Patients with liver and kidney dysfunction or abnormality caused by other reasons;

[0065] ④ Those who cannot cooperate with the collection of exhaled gas.

[0066] The exhaled breath collection method of the present invention is used to collect exhaled VOCs, and the exhaled VOCs solid phase microextraction and GC-MS detection methods are used to detect the peak areas of the collected gas markers A and B. Standard products are used to perform quantitative concentration detection through the external standard method to determine the threshold, statistical analysis is performed, and the sensitivity, specificity and accuracy are calculated through the ROC curve.

[0067] The pathological reference concentration thresholds recommended for the three VOCs are:

[0068] VOC Normal concentration / ppbv Pathological concentration / ppbv VOC A 0.05-0.5 0.5-5 and above VOC B 0.05-2 2-8 and above

[0069] The ROC curve of biomarker A is AUC = 1, and both sensitivity and specificity can reach 100%. Figure 6 ; The ROC curve of biomarker B has an AUC of 0.967, with a sensitivity and specificity of 96.4% and 94.9% respectively, and an accuracy of 95.0%. Figure 7 The levels of both gases were statistically significant and could be used as a reference for diagnosing CE.

[0070] It should be noted that the specific features, structures, materials or characteristics described in this specification may be combined in any one or more embodiments in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments and features of the different embodiments described in this specification without contradicting each other.

Claims

1. An exhaled gas VOCs marker for early diagnosis of echinococcosis, characterized in that: It is one or more combinations of the following exhaled gas VOCs: nonanoic acid and octanoic acid.

2. A detection system for early diagnosis of echinococcosis, characterized in that: The detection system comprises: An exhaled gas collection device for collecting volatile organic compounds in the exhaled gas of a subject; and An exhaled gas separation device for separating and extracting volatile organic compounds from exhaled gas; and Exhaled gas detection device, used to detect the composition and content of markers in organic volatiles; Among them, the marker is one or more combinations of the following exhaled gas VOCs: nonanoic acid and octanoic acid.

3. The detection system according to claim 2, characterized in that: The detection thresholds are: 0.5 ppbv for nonanoic acid and / or 2 ppbv for octanoic acid.

4. The detection system according to claim 2, characterized in that: The exhaled gas collection device comprises an air collection bag, a mouthpiece arranged on the air collection bag, and a sampling cap sleeved with the mouthpiece.

5. The detection system according to claim 2, characterized in that: The gas separation device is a solid phase microextraction device.

6. The detection system according to claim 5, characterized in that: The solid phase microextraction device comprises a solid phase microextraction handle and an automatic extraction head coated with polydimethylsiloxane.

7. The detection system according to claim 2, characterized in that: The exhaled gas detection device is a gas chromatograph-mass spectrometer.

8. The detection system according to claim 7, characterized in that: The detection parameters of the gas chromatography-mass spectrometer were set as follows: carrier gas was He, flow rate was 0.8-1.2 ml / min, injection temperature was 240-260°C, and injection time was 1-3 minutes; column box temperature was increased from 35-45°C to 240-260°C at a rate of 4-6°C / min; chromatographic and mass spectrometric interface temperature was 240-260°C, MS ion source temperature was 190-210°C, and scanning range was 40-450 amu.

9. The detection system according to claim 7, characterized in that: The chromatographic column used in the gas chromatography-mass spectrometry instrument is a DB-1 gas chromatography column.

10. Use of the exhaled gas VOCs marker for early diagnosis of echinococcosis according to claim 1 in the preparation of a product for early diagnosis of echinococcosis.

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