CN116698820A - Airway active oxygen detection method and detection kit - Google Patents
Airway active oxygen detection method and detection kit Download PDFInfo
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- G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
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Abstract
The invention provides a detection method and a kit for airway active oxygen. The method comprises the steps of adding a sample releasing agent into an airway mucus sample for mixing after gargling and deep cough sampling, and then carrying out suction filtration on active oxygen released by the sample by using a test strip fixed with an indicator, wherein a detection area shows a tan as a positive result, and the content of the active oxygen in the sample is indicated to reach or exceed a detection threshold. The method can finish detection within 5 minutes, and the operation process and the used reagents are conventional, safe, noninvasive, convenient and easy to use. The kit based on the method has simple production process, can be stored for a long time, is convenient to carry and use, can be sealed after being used to protect the environment, and is suitable for the point of care testing (POCT) of the airway active oxygen.
Description
Technical Field
The invention belongs to the technical field of biochemistry, and relates to a simple, easy-to-use and rapid airway active oxygen detection method and a detection kit.
Background
Reactive Oxygen Species (ROS), also known as oxygen radicals, are a class of oxygen-containing species with high reactivity and strong oxidizing properties, usually produced in human cells along with processes of oxidative phosphorylation by respiration, including hydrogen peroxide (H) 2 O 2 ) Superoxide radical (O) 2 - ) Hydroxyl radical (OH) - ) Lipid peroxide (ROOH) and peroxynitrite (ONOO) - ) Etc. Nitric Oxide (NO) can also be regarded as an active oxygen species, but its synthetic and metabolic pathways andthe physiological functions are different from those of general active oxygen. Under physiological conditions, the ROS level in the cells is regulated to a lower level by mechanisms such as superoxide dismutase (SOD), catalase (CAT) and glutathione-ascorbic acid reduction system, plays a role in messengers in the normal cell signal transduction process, and participates in various physiological processes such as cell growth proliferation, development differentiation, aging, apoptosis and the like. Various chronic diseases such as smoking, environmental pollutants, radiation, infection, allergy and the like, including cancers, can stimulate phagocytes such as macrophages and neutrophils to excessively produce ROS by NADPH oxidase, and then activate various transcription factors such as NF-kappa B, AP-1, HIF-1 alpha, STAT3 and the like through the ROS to induce and regulate the expression of proteins for inflammation, cell transformation, tumor cell survival, tumor cell proliferation and invasion, angiogenesis and metastasis. ROS also regulate the expression of various tumor suppressor genes such as p53, rb, PTEN, etc. Excessive accumulation of ROS can damage the normal structure of proteins, lipids, and DNA, thereby causing cell senescence death and tissue damage. Recent studies have found that ROS are involved in up to 100 more human diseases, such as: cardiovascular diseases such as hypertension, atherosclerosis, diabetes, chronic inflammation, neurodegenerative diseases and cancer pathogenesis and immune and aging processes.
Chronic obstructive pulmonary disease (chronic obstructive pulmonary disease, COPD) is a group of progressive airway diseases that are not fully reversible by airflow limitation, essentially chronic airway inflammatory diseases characterized by neutrophil infiltration. Inflammatory cells such as macrophages and T lymphocytes are also involved in the pathogenesis of chronic obstructive pulmonary disease. Alveolar macrophages are widely distributed in alveoli and on the inner surface of bronchi, and under the stimulation of factors such as heavy smoking or infection, ROS and various inflammatory factors are generated and released, and the aggregation activation of T lymphocytes and neutrophils is induced, so that the alveolar macrophages are the main mechanism for early starting of chronic obstructive pneumonia. Neutrophils accumulate and activate in the slow-blocking lung airways, continuously generating and releasing ROS, and are an important mechanism for irreversible damage of lung tissue in the course of chronic-blocking pneumonitis. T lymphocytes secrete Interleukin (IL) -13 to activate Aldose Reductase (AR) which causes airway epithelial cells to produce excessive ROS, and excessive increases in intracellular ROS lead to mitochondrial dysfunction which in turn reacts to the respiratory chain to produce more ROS, forming a vicious circle exacerbating airway damage. Abnormal sustained elevation of airway ROS levels is a central mechanism for slow resistance to pulmonary disease and progression of disease. The lung function examination is a standard method for diagnosing the chronic obstructive pulmonary disease, but the method has strong specialization, low efficiency and difficult popularization. By establishing a rapid and simple airway ROS detection method, the development is convenient, the physical examination screening and monitoring method and the product which are suitable for popularization are easy to use, and the prevention, control and diagnosis of the slow-resistance lung can be greatly improved and promoted.
The existing ROS detection method mainly comprises a chemiluminescence method, a fluorescent probe, a spectrophotometry method, an electron paramagnetic resonance method and the like. The methods have the limitation of strong specialization and incapability of being used for instant rapid testing (POCT), and are not beneficial to popularization and application such as physical examination screening. The absorbance method has the lowest requirements on instruments and equipment, but special instruments and equipment such as a spectrophotometer, a liquid shifter and the like are also needed, for example, patent application No. 202010705059.6, a method for detecting the active oxygen level of Morchella cells, and patent application No. 201611056540.7, a method for detecting the influence of electronic smoking products on the secretion of active oxygen of cells and the like all use special equipment and complex special operation, and no detection scheme for ROS in human airways is provided.
Disclosure of Invention
In summary, there is a lack of an airway ROS detection method and product that can be used in non-professional settings such as home or community. Therefore, the invention provides the airway ROS detection method and the kit which are safe, noninvasive, convenient, easy to use, reliable in performance and capable of being used for instant detection.
The technical scheme of the invention is as follows:
the invention provides a detection method of airway ROS, which comprises the following steps: after a user washes the mouth, the user beats the chest or back and performs deep respiration and forceful cough to enable the lung airway to accelerate movement, liquid accumulated in the oral cavity is spitted into a collecting pipe, a sample releasing agent containing sodium dodecyl sulfate, sodium ferrocyanide, sodium chloride and sodium phosphate buffer solution is added for mixing, a test strip with a proper amount of starch, acetic acid, chitosan quaternary ammonium salt, potassium iodide and cysteine fixed in a detection area is used for suction filtration detection, the color change of the detection area on the test strip is observed, tan is displayed as a positive result, the abnormal rise of ROS content in the sample is indicated, and a subject should go to a professional institution for slow pulmonary resistance related examination and diagnosis. The design of the invention is shown in figure 1.
Specifically, the composition of the sample releasing agent provided by the invention is 5-30mM sodium ferrocyanide, 0.5% -2.5% (w/v) sodium dodecyl sulfate, 50-200mM sodium chloride and 5-50mM sodium phosphate buffer water solution with pH value of 5.8-7.4.
Specifically, the preparation process of the test strip provided by the invention comprises the following steps: a detection area is marked at a proper position at the lower end of a filter paper strip with proper size, 1-2 mu l/mM 2% -8% (w/v) starch, 100-500mM acetic acid, 0.5% -2% (w/v) chitosan quaternary ammonium salt, 2.0-7.0M potassium iodide, 0.2-1.0mM cysteine and 10-80mM potassium ferrocyanide mixed aqueous solution are dripped, and then the mixture is dried at 60 ℃ for 30 minutes. The preferable scheme is as follows: 4% (w/v) starch, 350mM acetic acid, 1% (w/v) chitosan quaternary ammonium salt, 2.5M potassium iodide, 0.5mM cysteine, 40mM potassium ferrocyanide in aqueous mixture.
Further, a quality control area can be added at the position 7-20mM above the detection area of the test strip, 1-2 μl/mM of 40-80mM copper sulfate solution is dripped, and then the test strip is dried at 60 ℃ for 30 minutes. The preferable scheme is as follows: 60mM copper sulfate solution.
Specifically, the operation method provided by the invention comprises the following steps: after rinsing, the chest or back is beaten, deep respiration and forceful cough are carried out to accelerate the movement of the lung airway, liquid accumulated in the oral cavity is spitted into a collecting pipe, 4ml of sample releasing agent is added, shaking and vibrating are carried out for about 10 seconds to mix, the lower end of the test strip is immersed in the mixed liquid to approach but not exceed the detection area indication line at the lower end of the test strip, standing is carried out for 10s-90s, an observation result shows a tan color, and the ROS of the airway is indicated to reach or exceed the detection threshold value.
Based on the method, the main components of the airway active oxygen detection kit provided by the invention comprise: the device comprises a sampling tube integrated with an indication groove, a test strip fixed with an indicator, a tube cover, a sample releasing agent and a label. Wherein, the test strip is fixed in the instruction groove, and the sample release agent seals in the tube cover.
Specifically, the composition of the kit sample release agent provided by the invention is as follows: 20mM sodium ferrocyanide, 2% (v/v) sodium dodecyl sulfate, 140mM sodium chloride, 40mM sodium phosphate buffer aqueous solution pH 7.0.
Specifically, the kit test strip provided by the invention consists of a 52mm multiplied by 5mm (length multiplied by width) filter paper strip and a 44mm multiplied by 5mm (length multiplied by width) back support plastic strip, wherein the upper end of the plastic strip is level with the upper end of the filter paper strip; the lower end of the filter paper strip is marked as a detection area 29mM away from the bottom, 3 μl of 4% (w/v) starch, 350mM acetic acid, 1% (w/v) chitosan quaternary ammonium salt, 2.5M potassium iodide, 0.5mM cysteine, 40mM potassium ferrocyanide mixed aqueous solution are added dropwise and dried; a quality control region was marked 12mM above the detection region, and 3. Mu.l of 60mM copper sulfate solution was added dropwise and dried.
Further, the test strip is stuck and fixed in the indication groove of the sampling tube, the lower end of the filter paper of the test strip is close to the bottom of the sampling tube, and the detection area and the quality control area on the test strip correspond to the detection window (D) and the quality control window (Q) on the external label of the indication groove respectively.
Specifically, the kit provided by the invention comprises the following operation steps: 1. after rinsing with clear water, the chest is beaten for 10 times, the chest is deeply breathed for 5 times, and forceful cough is caused for 6 times; 2. spit the liquid accumulated in the oral cavity into the sampling tube; 3. covering the tube cover, screwing, and shaking for about 10s to uniformly mix the liquid in the tube; 4. standing for about 2-3 minutes, and observing and comparing the results. The Q window is brown, and indicates that the operation is effective; the D window is brown, positive (+) is indicated, the ROS content in the sample is abnormally increased, and the subjects should be sent to a professional for examination and diagnosis related to slow lung obstruction.
Further, the detection limit of the above-mentioned kit was 1.0mM (in terms of an equivalent amount of hydrogen peroxide). The value is at least 50% higher than the upper limit of the reference range of the airway active oxygen normal value measured on 250 healthy volunteers airway viscous (sticky) liquid samples by adopting the traditional absorbance method, so that the false positive rate of the detection on healthy volunteers is less than or equal to 0.4%.
The basic principle of the invention is that by utilizing the characteristic that the ROS in the airway of a patient with chronic obstructive pulmonary disease is remarkably and abnormally increased, the strong oxidability of airway mucus samples is detected by collecting and synchronously, and the weak acidity with the pH value more than 4.0 is adoptedThe condition of complexing with metal ions excludes the interference of substances with stronger oxidability such as nitric oxide (in the form of nitrite derivatives thereof), and specifically indicates whether the airway ROS level is remarkably abnormally increased. Airway mucus is a body fluid and gel-like protective layer on the inner surface of the airway, and mainly consists of airway mucin, moisture, inorganic salt and other components. In healthy state, the active oxygen in the air passage is mainly O 2 - Is produced in airway epithelial cells and is rapidly converted to hydrogen peroxide by superoxide dismutase (SOD), and is rapidly cleared by highly active Catalase (CAT), with lower airway ROS levels. When chronic pulmonary inflammation occurs, macrophages and neutrophils are aggregated and activated in the airway to generate and release a large amount of ROS, so that the ROS in the airway can be several times higher than that in a healthy state, cell and tissue damage is caused, the obvious abnormal elevation of the ROS in the airway can be stably reflected in airway mucus samples in the forms of exfoliated cells, lipid peroxide and the like, the sampling detection method can be used for detecting the airway mucus before the symptoms are obvious or expectoration is formed, the process is safe, convenient and harmless, an invasive means is not needed, and therefore, the airway mucus active oxygen detection is expected to achieve the purpose of chronic pulmonary injury and noninvasive early screening when the lower respiratory tract symptoms are slight or very slight, and the method has great application value.
The specific detection reaction principle of the invention is as follows: 1) Sample releasing agents mixed with sodium dodecyl sulfate, sodium phosphate buffer, sodium ferrocyanide and sodium chloride are used for releasing and dissolving ROS from airway mucus samples, and meanwhile, interference of nitrite and metal ions is eliminated. Sodium dodecyl sulfate is a commonly used nonionic surfactant that breaks cells, promotes solubilization of lipid materials, including lipid peroxides, and does not interfere with the pH, ionic strength, and redox reactions of the system. The sodium phosphate buffer provides suitable weakly acidic pH conditions so that nitrite does not have strong oxidizing properties and can clear nitrite interference. Sodium ferrocyanide is a good metal ion complexing agent, and can complex and shield various metal ions which can actively participate in redox reaction, such as: cu (Cu) 2+ 、Fe 3+ 、Fe 2+ 、Mn 2+ Etc. to make the sampleThe ROS released in the method can exist stably in a solution system. Sodium chloride supplementation provides ionic strength. 2) The test strip detection zone contains KI, starch, cysteine, chitosan quaternary ammonium salt, potassium ferrocyanide and acetic acid used as a solvent. The combination uses the reducibility of cysteine sulfhydryl reagent to stabilize mother liquor and I of detection zone - And eliminate the interference of dissolved oxygen, so that if the sample does not contain ROS with sufficient concentration, I which can discolor starch is not generated 2 . This is important to eliminate background and ensure specificity of detection. The chitosan quaternary ammonium salt and the potassium ferrocyanide can assist in enhancing I - Stability on mother liquor and test strip, and enhanced color development effect. 3) Under the system condition provided by the invention, ROS released in a sample can be filtered up along with the solution and quickly oxidize sulfhydryl and I in a test strip detection area - Generate I 2 ，I 2 The binding starch then develops a color, and the reaction is very rapid and sensitive, indicating the presence of excessive ROS in the sample. Meanwhile, in the detection operation process, sodium ferrocyanide ascends to the quality control area and Cu along with the liquid 2+ The reaction produced a tan precipitate indicating that the procedure was effective.
Compared with the prior art, the invention has the following advantages:
(1) The invention establishes an internationally initiated suction filtration chromogenic reaction system and an integrated design, integrates sampling, quality control and detection, and can qualitatively judge whether the ROS level of the airway is remarkably abnormal or not within a few minutes through simple and continuous operations of gargling, deep cough sampling, pipe cover screwing, pipe body shaking, standing observation and the like.
(2) Compared to conventional exhaled breath detection. The ROS in the airway related to the slow-release lung mainly exist in the cell in the form of lipid peroxide, a small part of ROS is dissolved in airway mucus in the form of hydrogen peroxide, only a trace part of ROS can be distributed in exhaled breath, and compared with the conventional exhaled breath sampling, the airway mucus sampling can eliminate the air flow interference, so that the airway mucus sampling device has better anti-interference performance, is more sensitive, more specific and easier to use.
(3) Compared with conventional colorimetric or fluorescent methods. These conventional methods require specialized pipetting operations that require drawing a standard curve and diluting the sample to a linear range for re-measurement. The invention has the advantages that quantitative integrated operation is not needed in the use process, extra equipment and facilities are not needed, and background interference is eliminated in the color development threshold of the detection area, so that the invention is favorable for qualitative and visual judgment, is simpler, faster and is more suitable for popularization.
(4) Compared to various blood detection methods. The blood detection related to the slow-resistance lung mainly comprises the inflammatory cell count of neutrophils and the like, can help judge the inflammatory condition, but is not specific to the airway, and the early-stage inflammatory condition of the slow-resistance lung is lighter and probably not reflected in the blood. The invention does not need blood drawing, has excellent safe noninvasive property, and can directly indicate the abnormal condition of the ROS in the air passage more sensitively than blood detection.
(5) Compared with the traditional sputum detection method. Sputum detection has the advantages of non-invasiveness and low cost. However, although the secretion level of the mucus of the lung airway is increased, expectoration can be formed, the sputum is mostly generated in the early morning or late night, and is not beneficial to sample collection and detection, especially symptoms of the early stage of the lung with slow obstruction are not obvious, and obvious expectoration symptoms are not generated, so that the traditional sputum detection is not beneficial to early screening and monitoring of the lung with slow obstruction. The invention can collect and detect airway mucus samples in a regular time period in a specified mode, and has better sensitivity and universality.
(6) Compared to lung function examination. The lung function examination is a standard method for diagnosing the chronic obstructive pulmonary disease, but has strong professional property, low efficiency and difficult popularization, the popularization rate is in a very low level for a long time, and the early stage of the chronic obstructive pulmonary disease, in which irreversible damage to the lung function does not occur, cannot be detected. The method is beneficial to the lung function examination, and can discover and prompt patients with chronic obstructive pulmonary disease and high risk groups to pay attention to disease prevention and treatment and timely participate in necessary lung function examination. In addition, based on the characteristic of limited ROS half-life, the invention has good dynamic property, is suitable for timely indicating dynamic changes of chronic obstructive pulmonary inflammation, and is also suitable for home monitoring of patients.
In a word, the invention pointedly solves the problem of insufficient convenience and usability of airway ROS detection, has the advantages of safety, no wound, convenience, easiness in use, reliable performance, moderate price and the like, is favorable for popularization and application, is suitable for slow-resistance lung early screening and dynamic monitoring of slow-resistance lung patients and high-risk groups, and is greatly favorable for improving and promoting prevention, control and diagnosis of slow-resistance lung.
Drawings
The invention is further described below with reference to the accompanying drawings:
FIG. 1 is a schematic diagram of test strips and test results.
FIG. 2 is a photograph of the finished test strip and the detected fruit.
Fig. 3 is a top view of the tube cap.
FIG. 4 is a schematic diagram of an integrated design.
FIG. 5 is a photograph of the finished product of the kit and the detected fruit.
1: a sampling tube; 2: a tube cover; 3: an indication groove; 4: a base; 6: a sharp protrusion; 32: detecting test paper; 33: a result indication window (D window); 34: a quality control indication window (Q window); 321: a detection zone; 322: and a quality control area.
Detailed Description
The present invention will be described in further detail with reference to the following examples. It should be understood that the detailed description is intended to illustrate the invention more clearly, and is not intended to limit the invention. The embodiments described herein are only some, but not all, embodiments of the invention.
The means used in the examples, unless otherwise specified, are all conventional in the art.
The reagents used in the examples were all commercially available unless otherwise specified.
FIG. 1 is a schematic diagram of a test strip. The test strip 32 of this example was set as a detection zone 321 at 29mM from the bottom, and was added dropwise with 4% (w/v) starch, 350mM acetic acid, 1% (w/v) chitosan quaternary ammonium salt, 2.5M potassium iodide, 0.5mM cysteine, 40mM potassium ferrocyanide mixed aqueous solution; above it, 12mM, is a quality control zone 322, which is dried with 60mM copper sulfate in water. After drying treatment at 60 ℃ for 30 minutes, the test strip can be directly used for sample detection, and can also be sealed, dried and stored at normal temperature for standby. The detection area 321 is brown, and the quality control area 322 is brown, so that the sample is positive; before the test strip is used, the quality control area 322 is brown, which indicates that the test strip has deteriorated and is not recommended to continue to be used. If the strip is not tan after use, this area will not be used, indicating improper operation and unreliable results.
Fig. 2 is a schematic diagram of a test strip finished product and a detection result, 321: a detection zone; 322: and a quality control area.
The invention discloses a detection kit for airway active oxygen, which is established according to the method of the invention, and is shown in fig. 3-5, and mainly comprises a sampling tube 1 (comprising a base 4), a tube cover 2 and an indication groove 3 comprising detection test paper.
In the embodiment of the invention, the sampling tube 1 is a frosted ABS tube, and has the caliber: 25mm height: 90mm, can stand, have tube cap 2, base 4. After the detection of the sample is completed, the whole kit and the sample solution are in a tightly covered and sealed state, so that the environmental pollution and cross infection are avoided.
The length of the sampling tube 1 is matched with that of the indication groove 3, the detection test paper 32 is fixedly arranged in the indication groove 3, the size of the detection test paper 32 is determined according to the size of the indication groove, the sampling tube 1 is shown in fig. 4, and the base 4 contains a sample releasing agent and an airway mucus mixture.
A cylindrical sealing tube is fixed in the tube cover 2, so that a sample releasing agent can be fixed, and the sealing film can be pierced by a sharp protrusion 6 on the upper edge of the sampling tube 1 to enable liquid to fall into the tube bottom in the process of screwing in the tube cover. In this example, the sample releasing agent was 4ml in volume and consisted of 20mM sodium ferrocyanide, 2% (v/v) sodium dodecyl sulfate, 140mM sodium chloride, 40mM sodium phosphate buffer aqueous solution at pH 7.0.
In the embodiment, the test strip 32 placed in the indicator tank 3 is composed of a test strip having dimensions (length×width) of 52mm×5mm and a back support plastic strip having dimensions of 44mm×5 mm. To the bottom end of the test paper was added dropwise a mixed aqueous solution of 4% (w/v) starch, 350mM acetic acid, 1% (w/v) chitosan quaternary ammonium salt, 2.5M potassium iodide, 0.5mM cysteine, 40mM potassium ferrocyanide in an amount of 3. Mu.L, and at 12mM above it, a mixed aqueous solution of 60mM copper sulfate was added dropwise in an amount of 3. Mu.L. The indication groove (D) 33 was provided with a result of about 2mM diameter at about 29mM at the upper end of the bottom of the indication groove 3, and a clear tan band was observed when the ROS content was more than 1.0mM. The upper end of the bottom of the finger groove 3 is provided with a quality control indication groove (Q) 34 with the diameter of about 2mm, and if the window is brown before use, the kit is not recommended to continue use. If the window does not appear brown after detection, the sample or operation is not qualified, and the result is unreliable.
Example 1: preferred confirmation of test strip composition
The test strip consisted of a 52mm by 5mm filter strip and a 44mm by 5mm back support plastic strip. The filter paper strip is aligned with the plastic strip at the top end. Marking a detection area (D) at a position 29mm away from the bottom end of the filter paper strip, and uniformly dripping a mixed solution of starch, acetic acid, chitosan quaternary ammonium salt, potassium iodide, cysteine and potassium ferrocyanide into the pipette tip; the upper 12mm of the device is marked as a quality control area (Q), and the copper sulfate solution is evenly dripped into the gun head of the liquid transfer device. The reagent formulations for the detection zone and the quality control zone are shown in Table 3, and the volumes are 3. Mu.L. The composition of the sample release agent is: 20mM sodium ferrocyanide, 2% (v/v) sodium dodecyl sulfate, 140mM sodium chloride, 40mM sodium phosphate buffer aqueous solution pH 7.0. The negative reference substance is purified water, and the positive reference substance is 1.5mM H 2 O 2 . The detection method is carried out according to instructions. After the operation is finished, the Q area can be observed to be obviously blackish brown, and the correct operation is indicated; no obvious color development is observed in the zone D of the negative control product, and the detection result is negative; obvious brown color can be observed in the detection areas of the positive control substances, and the detection results are indicated to be positive, so that the test strips prepared in the concentration range of the tested reagent can reach the expected detection results (table 1).
Table 1 control test and statistics of test strip composition
Example 2: sample Release agent composition test
Test strip specification and preparation are described in the text of the specification and in example 1. The detection zone was 3. Mu.l of 4% (w/v) starch added dropwisePowder, 350mM acetic acid, 1% (w/v) chitosan quaternary ammonium salt, 2.5M potassium iodide, 0.5mM cysteine, 40mM potassium ferrocyanide mixed solution; the mass control zone consisted of 3. Mu.l of 60mM copper sulfate solution added dropwise. The sample release formulations are shown in table 2. The negative reference substance is purified water, and the positive reference substance is 1.5mM H 2 O 2 . The detection method is carried out according to instructions. After the operation is finished, the quality control area can observe obvious brown color, and the correct operation is indicated; no obvious color development is observed in the detection area of the negative control substance, and the detection result is negative; obvious brown color can be observed in the positive control detection areas, and the positive detection result is indicated, so that the sample release agent prepared in the concentration range of the tested reagent can reach the expected detection result.
TABLE 2 composition of sample Release agent
Example 3: the influence of the common beverage and medicine remained in the sample on the judgment of the result
Test strip specification and preparation are described in the text of the specification and in example 1. The detection area is dropwise filled with 3 μl of a mixed aqueous solution of 4% (w/v) starch, 350mM acetic acid, 1% (w/v) chitosan quaternary ammonium salt, 2.5M potassium iodide, 0.5mM cysteine and 40mM potassium ferrocyanide; the quality control area was constituted by dropping 3. Mu.l of copper sulfate solution. The negative reference substance is purified water, and the positive reference substance is 1.5mM H 2 O 2 . The detection method is carried out according to instructions. The interfering substances tested are shown in Table 3. The content of the interfering substance in the sample is unified to be that the conventional dosage of the substance accounts for 2% of the volume of the sample, which is higher than the possible volume ratio of the residual quantity of the substance after the conventional use and the gargling in the sampling. 3 replicates were performed for each sample, for a total of 3 runs. The result observation statistics show that after the operation is finished, the quality control area can observe obvious black brown color to indicate that the operation is correct; no obvious color development is observed in the detection area of the negative control substance, and the detection result is negative; the positive control detection areas can all observe obvious brown color, which indicates that the detection result is positive, thus, the detection result is concentrated in the testWithin the range of the degree, the tested interfering substances do not influence the detection result, i.e. do not interfere with the detection result.
TABLE 3 interfering substances
Example 4: the invention relates to the production of a rapid detection kit for airway active oxygen
In this example 1000 sets of planned kit production were performed. First, a lot number and a production schedule were prepared (table 4), and material preparation was performed (table 5).
Table 4.1000 set of schedule component tables
| Numbering device | Component (A) | Specification of specification | 1000 sets of plan amounts | Remarks |
| KKG121 | Sampling tube | Personal (S) | 1000 | |
| KKG122 | Pipe cover | Personal (S) | 1000 | |
| KKG123 | Sample releasing agent | 4.0mL | 4000mL | |
| KKG124 | Test paper strip | Personal (S) | 1000 | |
| KKG125 | Label (Label) | Personal (S) | 1000 |
TABLE 5 bill of materials
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Key procedure-test strip preparation: taking a pencil mark line as a detection area from the bottom of 270g of common qualitative filter paper cut into a specification of 300mM multiplied by 60mM, taking the pencil mark line as the detection area from 12mM above, taking the pencil mark line as the detection area, and dripping 4% (w/v) starch, 350mM acetic acid, 1% (w/v) chitosan quaternary ammonium salt, 2.5M potassium iodide, 0.5mM cysteine and 40mM potassium ferrocyanide mixed aqueous solution into the detection area by a sample application pen according to the text method of the specification; a12 mM quality control area was streaked with 60mM copper sulfate solution. After drying at 60 ℃ for 30 minutes, the filter paper strip is stuck and fixed on the PVC back plate, the lower end of the filter paper strip is 26mm longer than the lower end of the PVC back plate, and the filter paper strip is vertically cut into strips of 5 mm. After the test strip is prepared, performing spot check of key working procedures to determine that the test strip quality is qualified.
Sample release agent preparation and split charging: 4000ml of the sample release agent of the kit shown in the text of the specification is prepared, and the sample release agent is subpackaged into tube covers according to 4 ml/part and sealed by sealing films.
Assembling a sampling tube: sticking double-sided foam rubber on the back of the test strip, placing and sticking the test strip in a sampling tube indication groove, enabling the lower end of the test strip to be just close to the bottom of the tube, and sealing and fixing the sampling tube base on the tube body; and (3) sticking labels of a reserved D window and a reserved Q window outside the indication groove, so that the D window and the Q window respectively correspond to a detection area and a quality control area on the test strip. And after the workshop inspector checks the assembly to be qualified on site, attaching a qualification mark on the top of the indication groove. And (3) placing the tube cover and the sampling tube into a self-sealing bag provided with a drying agent, sealing, temporarily storing at room temperature to obtain a semi-finished product, and carrying out external packaging after the sampling inspection is qualified.
And (3) external packing: and (5) filling the semi-finished products and specifications which are subjected to the sampling inspection into an external packaging box, attaching a qualification certificate, and warehousing for later use.
Example 5: the invention relates to a rapid detection kit for airway active oxygen quality inspection
1. Key procedure spot check
And performing spot check on key procedures after the test strip is prepared, so as to determine that the test strip quality is qualified.
(1) Appearance of
The appearance size of the test strip is in accordance with the setting.
(2) Negative reference compliance
And (5) taking 10 enterprise negative reference products for detection, wherein the detection results are negative.
(3) Compliance rate of positive reference
And (5) taking 10 enterprise positive reference products for detection, wherein the detection results are positive.
(4) Detection limit
And (3) taking the enterprise detection limit reference to carry out repeated detection for 20 times, wherein the detection result of at least 19 times is positive.
2. Inspection of semifinished products
(1) Appearance of
The components are complete, the self-sealing bag and the drying agent are well packaged, no leakage, no damage and no moisture regain are caused; the label writing is clear, the top of the indication groove is stuck with a qualified mark, and the position of the window is matched with the corresponding area on the test strip.
(2) Negative reference compliance
And directly adopting a key procedure spot check result.
(3) Compliance rate of positive reference
And directly adopting a key procedure spot check result.
(4) Detection limit
And directly adopting a key procedure spot check result.
(5) Repeatability of
And (3) taking enterprise repetitive reference, and repeatedly detecting for 10 times, wherein the detection results are positive and the color development is uniform.
(6) Sample processing efficiency
According to the airway mucus sampling method disclosed by the invention, 5 negative samples obtained by mixing and sub-packaging airway mucus samples of 10 healthy volunteers and 5 positive samples obtained by mixing and sub-packaging airway mucus samples of 10 slow-resistance lung patients are respectively detected, wherein the negative sample detection results are negative, and the positive sample detection results are positive.
3. Inspection of finished products
(1) Appearance of
The components are complete, the self-sealing bag and the drying agent are well packaged, no leakage, no damage and no moisture regain are caused; the label writing is clear, the top of the indication groove is stuck with a qualified mark, and the position of the window is matched with the corresponding area on the test strip. The total weight of the finished product should be more than or equal to 24.70g, which indicates that the sealed reagent is sufficient.
(2) Performance of
After the appearance check is correct, the performance indexes of the key process spot check and the semi-finished product check are directly adopted.
Example 6: healthy volunteer test
The finished kit is shown in example 4. 50 healthy volunteers are collected on site in a park, the age is 20-50 years, the occupation is high-tech industry practitioners, the proportion of men and women is about 50%, the patients take comprehensive physical examination in one year, the patients have no abnormal conditions, the patients have no medical history in the severe smoking or severe pollution industry, the patients have no lung diseases such as lung cancer or chronic lung obstruction, and the patients have no family history of related diseases. Some females are pregnant or lactating and feel well on their own. The staff hosts the registration, dispenses the finished product of the kit, and carries out operation explanation demonstration. The test operation was then performed by the volunteer as described in the text of the present specification, and the result was reported to the staff. And after comparison and confirmation, carrying out result statistical analysis on the staff. The results show that 50 cases of detection Q windows all show obvious blackish brown, and the operation accuracy is 100%; the 50 cases of detection D windows have no obvious color development, are all negative, and have low risk of slow lung resistance morbidity of the detected volunteers.
Example 7: slow pulmonary disease patient test
The finished kit is shown in example 4. 30 volunteers of patients with chronic obstructive pulmonary disease are collected, the age is 40-70 years, the occupation is unlimited, the symptoms of chronic obstructive pulmonary disease or self-description of the symptoms of acute respiratory syndrome, asthma aggravation, increased sputum quantity, purulent sputum or mucoid sputum, chest distress and the like appear in one year through the diagnosis of pulmonary function examination, and the symptoms are relieved by oneself, and medicaments such as bronchodilators, glucocorticoids, phosphodiesterase-4 inhibitors and the like are not used. Women are not in gestation or lactation. The staff hosts the registration, dispenses the finished product of the kit, and carries out operation explanation demonstration. The test operation was then performed by the volunteer as described in the text of the present specification, and the result was reported to the staff. And after comparison and confirmation, carrying out result statistical analysis on the staff. The results show that 30 cases of detection Q windows all show obvious blackish brown, and the operation accuracy is 100%; the 30 cases of detection D windows all show obvious tan, are positive, indicate abnormal rise of the level of active oxygen in the airway, prompt the risk of inflammation of the airway related to slow-resistance lung and suggest to go to a hospital to accept deep diagnosis and treatment.
Example 8: upper respiratory tract inflammation patient test
The finished kit is shown in example 4. The patients suffering from common cold, cough, rhinitis and sphagitis are respectively 20 cases, the ages are 20-40 years, the occupation is unlimited, the proportion of men and women is about 50%, the patients take comprehensive physical examination within one year, no other abnormal conditions, no medical history in heavy smoking or severe pollution industries, no pulmonary disease history such as lung cancer or chronic obstructive pulmonary disease, and no family history of related diseases. Women are not in gestation or lactation. The staff hosts the registration, dispenses the finished product of the kit, and carries out operation explanation demonstration. After volunteers cleaned the nasal discharge, and were lightly couched to clear the throat and eliminate the expectoration, the test procedure was performed as described in the text of this specification and the results reported to the staff. And after comparison and confirmation, carrying out result statistical analysis on the staff. The results show that 60 cases of detection Q windows all show obvious blackish brown, and the operation accuracy is 100%; the D windows of 60 cases are not obvious in color development, are negative, and the risk of slow pulmonary disease of the tested volunteers is low.
Example 9: screening application of slow-blocking lung examination
The finished kit is shown in example 4. A staff is arranged in the physical examination center and is responsible for registering forms and issuing and recycling the reagent kit, necessary answering guidance is carried out when a subject is in question, and propaganda explanation videos are rolled and played. The using method of the kit is described in the text of the specification. Physical examination subjects can follow up, taking an average of about 5 minutes. Multiple persons can be self-tested in parallel when the persons arrive at the scene, and 30 persons can only take about 10 minutes for detection. From this estimate, detection was completed about 1440 times for 1 day (8 hours). While lung function examination requires professional 1 to 1 professional instruction and quality control, physical examination subjects usually need to wait in line, 30 times of examination need about 40 minutes, and 8 hours can only complete the examination about 120 times. The invention can improve the screening efficiency of physical examination by about 12 times.
Example 10: middle-aged and elderly people screening application
The finished kit is shown in example 4. 30 middle-aged and elderly people who have symptoms of acute breath, severe wheezing, increased sputum volume, purulent sputum or mucopurulent sputum, chest distress and the like and can release the symptoms by themselves are collected within one year, the age is 40-70 years, the proportion of men and women is about 50%, and atomized inhalation or other treatment is not carried out. The staff hosts the registration, dispenses the finished product of the kit, and carries out operation explanation demonstration. If the throat has foreign body sensation, the throat is lightly cough and clear, and after expectoration is eliminated, the detection operation is carried out according to the method described in the text of the specification, and the result is reported to staff. And after comparison and confirmation, carrying out result statistical analysis on the staff. The results show that 30 cases of detection Q windows all show obvious blackish brown, and the operation accuracy is 100%; the 30 cases of detection D windows all show obvious tan, are positive, indicate abnormal rise of airway ROS level, prompt the risk of chronic obstructive pulmonary-related airway inflammation and suggest to go to a hospital to accept deep diagnosis and treatment. Early intervention and sustained management of slow lung resistance is beneficial to alleviating symptoms and reducing the frequency and severity of acute exacerbations. However, currently, auxiliary tests, such as lung function measurement, chest X-ray test, etc., which are helpful for diagnosis of chronic obstructive pulmonary disease are limited by equipment conditions and are difficult to screen in large scale in middle aged and elderly people. The invention has the advantages of convenient and easy-to-understand sampling and testing process, and is beneficial to the early diagnosis and early treatment of the slow-blocking lung and early screening of middle-aged and elderly people.
Example 11: community application
The finished kit is shown in example 4. Putting 50 sets of finished products of the kit in a test point community supermarket, pasting propaganda posters with two-dimension codes, enabling a user to scan the codes by a mobile phone to watch video explanation, acquiring the kit on site to automatically detect after online registration, performing detection operation according to the method disclosed by the specification of the invention, and updating the detection result online. Summarizing and counting detection results, counting positive according to the reported results of users, and counting negative according to the reported results of users and not reported results; the logical basis is positive, so that the user's communication desire can be stimulated, the report missing rate is very low, and negative can reduce the user's communication desire, and the report missing rate is high. Statistics show that 6 cases of positive cases are total, the positive rate is 12%, the rate of chronic lung resistance of adults aged 20 years and older is obviously higher than 8.6%, and the rate of chronic lung resistance of 40 persons aged 40 years and older in 50 users is 13.7%, so that the total positive rate is 12% more reasonable. The medical and health institutions of the basic level lack of slow-resistance lung diagnosis professional facilities such as lung function examination and the like, can not directly diagnose the slow-resistance lung, and need to go to a second-level hospital or more for examination, thus being unfavorable for residents to participate in the slow-resistance lung screening. The kit is convenient for residents to participate in the slow pulmonary resistance screening in basic medical and health institutions, and is hopeful to develop into powerful supplement for the health construction of the whole population.
Example 12: household self-test application
The finished kit is shown in example 4. 30 volunteers of patients with chronic obstructive pulmonary disease who routinely use one or more of bronchodilators, glucocorticoids, phosphodiesterase-4 inhibitors were collected and tested by home self using the kit of the present invention, 1 time per week for a total of 4 times. The sampling test period is selected prior to inhalation administration. Volunteers age 40-60 years, sex ratio about 50%, and administration course not more than 2 years and not shorter than 1 month. The method of using the kit is carried out according to the method of the specification. The user returns the detection result through the network or collects the use condition by the phone call or WeChat follow-up visit of the staff and registers the detection result. The statistics of the results show that 4 of 10 patients were negative and that at least 1 of 7 patients appeared negative. The remaining 13 patients were positive for 4 tests. The decrease or disappearance of the positive frequency of detection during the course of patient medication may be correlated with the control and improvement of airway inflammatory conditions. The specific diagnosis also needs to go to the second grade and above hospitals for diagnosis confirmation. The medication maintenance treatment of the slow-acting lung is a long-term process, and the medication is stopped after the illness state of many patients is improved, so that the illness state is aggravated, and hidden danger is caused to the control of the slow-acting lung. The invention is convenient for home self-test tracking of patients and is beneficial to promoting the patients to adhere to the medication.
Example 13: kit preservation and stability test
The finished kit is shown in example 4. The sampling tube (containing test strip) is sealed, dried and assembled in the finished product package of the kit. The preservation condition of the kit is that the room temperature is 25+/-5 ℃ or the refrigeration temperature is low, and the humidity is less than or equal to 60%. The negative reference is prepared by mixing airway mucus samples of healthy volunteers and sub-packaging with 1 ml/part, and H is shown by standard absorbance method 2 O 2 The content is less than 1.2mM. The positive reference isMixing airway mucus samples of volunteers of patients with chronic obstructive pulmonary disease, subpackaging at 1 ml/part, and measuring by standard absorbance method to show H 2 O 2 The content is more than or equal to 2.0mM. The repetitive reference was 2.0mM H 2 O 2 The detection limit reference is 1.0mM H 2 O 2 . The detection method is carried out according to instructions. Each batch was tested 3 times each, 5 times at the limit of detection, and 2 batches per month. After 4 times of detection in the 2 nd month, preliminary statistics show that the coincidence rate of the negative/positive/repeatability detection limit reaches 100%, and meets the quality requirement.
Example 14: portability and transport stability
The finished kit is shown in example 4. Kit storage conditions and references were the same as in example 14. Taking 3 batches of kits, storing at a high temperature of 40 ℃ for 3 days, and then, falling the kits from 1.5m height, repeating the three times for 3 times, performing shock drop tests, observing whether the external package, the internal package and the like of the kits are damaged, and then, testing by using a negative reference, a positive reference and a detection limit reference. Results: only one kit of one batch has damaged outer package part, no damage to inner package, and detection statistics show that the female/male/repeatability detection limit meets 100% and meets the quality requirement.
Claims (6)
1. The airway active oxygen detection method is characterized in that after rinsing mouth with clear water, drinking a mouthful of water, beating the front chest 10 times, deeply breathing 5 times, forcefully cough 6 times, spitting and collecting intraoral liquid and mixing with a sample release agent, and then sucking and filtering active oxygen released by a test sample by using a test strip fixed with an indicator, wherein a detection area shows a tan color as a positive result, and the content of the active oxygen in the sample is indicated to reach or exceed a detection threshold value.
2. The method of claim 1, wherein the sample releasing agent is composed of 5-30mM sodium ferrocyanide, 0.5% -2.5% (w/v) sodium dodecyl sulfate, 50-200mM sodium chloride, 5-50mM sodium phosphate buffer aqueous solution with pH of 5.8-7.4.
3. The method according to claim 1, wherein the test strip with the indicator immobilized thereon is a filter strip of a suitable size, and is dried with 1-2 μl/mM of 2% -8% (w/v) starch, 100-500mM acetic acid, 0.5% -2% (w/v) chitosan quaternary ammonium salt, 2.0-7.0M potassium iodide, 0.2-1.0mM cysteine, 10-80mM potassium ferrocyanide mixed aqueous solution at a suitable position at the lower end, and 40-80mM copper sulfate mixed solution is dried 7-20mM above the region.
4. An airway active oxygen detection kit is characterized by mainly comprising the following components: the device comprises a sampling tube integrating an indication groove, a test strip fixed with an indicator, a tube cover, a sample releasing agent and a label. The test paper strip is a core component and consists of a filter paper strip with the length of 52mm multiplied by 5mm (length multiplied by width) and a back support plastic strip with the length of 44mm multiplied by 5mm (length multiplied by width), wherein the upper ends of the filter paper strip and the plastic strip are level; the lower end of the filter paper strip is 29mM away from the bottom and is a detection zone, and 3 μl of 4% (w/v) starch, 350mM acetic acid, 1% (w/v) chitosan quaternary ammonium salt, 2.5M potassium iodide, 0.5mM cysteine and 40mM potassium ferrocyanide mixed aqueous solution are dried; a quality control zone was located 12mM above the detection zone and dried with 3. Mu.l of 60mM copper sulfate in water.
5. The kit for detecting airway active oxygen according to claim 4, wherein the sample releasing agent is a film which can be pierced by a spike in the sampling tube and is sealed in a tube cover, and the composition of the sample releasing agent is 20mM sodium ferrocyanide, 2% (v/v) sodium dodecyl sulfate, 140mM sodium chloride, 40mM sodium phosphate buffer aqueous solution of pH 7.0.
6. The kit for detecting airway active oxygen according to claim 4, wherein the detection limit of the kit is H 2 O 2 Calculated as 1.0mM.
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| CN202211658151.7A CN116698820A (en) | 2022-12-22 | 2022-12-22 | Airway active oxygen detection method and detection kit |
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| CN202211658151.7A CN116698820A (en) | 2022-12-22 | 2022-12-22 | Airway active oxygen detection method and detection kit |
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