CN113567665B - Lysate for chlamydia trachomatis antigen detection and detection method - Google Patents
Lysate for chlamydia trachomatis antigen detection and detection method Download PDFInfo
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Abstract
The invention provides a lysate for detecting chlamydia trachomatis antigen and a detection method, and relates to the technical field of biological detection. The invention provides a lysate for chlamydia trachomatis antigen detection, which comprises an antigen extraction solution and a sample buffer solution; the antigen extraction solution comprises a solvent, a surfactant, and a lipid extraction agent, and the sample buffer solution comprises a buffer, a surfactant, metal ions, and macromolecular compounds, and a solvent. The cracking liquid is safe and efficient, is convenient to transport and store, and reduces the use difficulty of operators; in addition, the detection method of the lysate improves the sensitivity of the detection of the chlamydia trachomatis antigen.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a lysate for detecting chlamydia trachomatis antigen and a detection method.
Background
Chlamydia trachomatis is a microorganism which is parasitic in cells and is often parasitic in the eyes and genital tract, causing infections of the genitourinary tract. Determining the type of infectious agent is of clinical value for diagnosis and treatment of related diseases.
Currently, of clinical patients, 70% -80% of female patients and 50% of male patients often show no clinical symptoms, so that the antigen typing clinical test has important diagnosis auxiliary effect. Common laboratory examination methods are: 1) Smear microscopy; 2) Performing PCR nucleic acid amplification detection; 3) Antigen detection; 4) Serological tests, and the like. By using the chromatography method based on the nitrocellulose membrane, reliable auxiliary diagnosis basis can be provided in a short time, and meanwhile, the technical requirements on instruments and experimental staff are reduced, so that great convenience is brought to patients and clinical laboratory staff. For the gold-labeled antigen detection method, the lysate formula plays a main role in correctly obtaining target antigens and improving detection sensitivity.
In the prior art, the lysate for detecting the chlamydia trachomatis antigen has more types and different formulas. The cleavage process goes from an early one-step process to a later two-step process. In many two-step schemes on the market, acid-base neutralization schemes are adopted in most cases. In the acid-base neutralization scheme, a dilute solution of strong acid and strong base is used, and the dosage control is inaccurate, so that antigen damage can be caused, and the sensitivity is affected. Meanwhile, the method has a certain potential hazard to related personnel in transportation and detection operations. Thus, there is a need to develop a new lysate formulation and a new lysis method.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide a lysate for chlamydia trachomatis antigen detection and a detection method, and the lysate is mild in formula, reduces the storage, transportation and operation difficulties of the lysate, ensures the sensitivity of colleagues, improves the stability of results and saves the detection cost.
The technical scheme provided by the invention is as follows:
In one aspect, the invention provides a lysate for chlamydia trachomatis antigen detection, the lysate comprising an antigen extract and a sample buffer solution; the antigen extraction solution comprises a solvent, a surfactant, and a lipid extraction agent, and the sample buffer solution comprises a buffer, a surfactant, metal ions, and macromolecular compounds, and a solvent.
The invention provides an improved pyrolysis liquid with a mild formula, which avoids using strong acid and strong alkali for extraction, reduces the damage to chlamydia trachomatis, is convenient to store and transport, and is convenient for operators to use. In addition, the lysate of the invention is used for extracting and detecting chlamydia trachomatis antigen, thereby improving the sensitivity and stability of detection.
In one embodiment, the weight part ratio of the solvent, the surfactant and the lipid extractant in the antigen extracting solution is 60-120: 0.1 to 15:0.1 to 15.
In one embodiment, in the sample buffer solution, the weight part ratio of the buffer solution, the surfactant, the metal ion, the macromolecular compound and the solvent is 0.1-20: 0.1 to 15:0.1 to 5: 1-20: 90 to 110.
The components in the lysate are reasonably designed, and have synergistic interaction, so that the extracted chlamydia trachomatis has high purity and little damage and influence on antigen; the pyrolysis liquid has stable quality and is convenient for storage and transportation.
In one embodiment, the solvent comprises one or more of water, dimethyl sulfoxide, methanol, glycerol.
In one embodiment, the surfactant in the antigen extract comprises one or more of polyvinylpyrrolidone, polyethylene glycol, tween 80, sodium dodecyl sulfate, triton-100; the surfactant of the sample buffer solution includes one or more of polyvinylpyrrolidone, polyethylene glycol, tween 80, sodium dodecyl sulfate, triton-100, and brisedge (brij).
In one embodiment, the lipid extractant comprises one or more of cholesterol, phenol, glycerol, high density lipoproteins.
In one embodiment, the buffer comprises one or more of a tris buffer, a boric acid-borax buffer, or a citric acid buffer; preferably, the pH of the buffer is 7-10.
In one embodiment, the metal ion salt comprises one or more of magnesium chloride, sodium chloride, trisodium citrate, ferrous sulfate.
In one embodiment, the macromolecular compound comprises one or more of polyethylene glycol, polyvinylpyrrolidone, sodium caseinate, bovine serum albumin.
The invention also encompasses a chlamydia antigen extraction kit comprising the lysate and sample buffer solution mentioned in the previous schemes.
The preparation method of the lysate for chlamydia trachomatis antigen detection comprises the steps of sequentially adding the surfactant, the lipid extractant and the solvent, and stirring and uniformly mixing at normal temperature to obtain the antigen extract; sequentially adding buffer solution, surfactant, macromolecular compound and metal ion salt, and stirring and mixing uniformly at normal temperature to obtain the sample buffer solution.
In another aspect, the present invention provides a method for detecting chlamydia trachomatis antigen for non-disease diagnosis purposes, using the lysate to detect it, comprising: and (3) performing lysis and extraction on the sample by adopting the antigen extracting solution, then adding the sample buffer solution, and then performing antigen detection.
In a specific embodiment, the sample to be tested is lysed for a period of 1-2 min.
In one embodiment, the cleaved sample to be detected is detected using an immunochromatographic test strip.
In a specific embodiment, the sample to be tested is a human urinary tract secretion or a female cervical secretion withdrawal, for example, with a sterile swab withdrawal.
The beneficial effects are that:
(1) The lysate provided by the invention is safer in reagent. In the prior art strong acid-base hydrolysis neutralization method, sodium hydroxide is used to break up pathogens and hydrochloric acid is used for neutralization. In the invention, the system of the components of the tris (hydroxymethyl) aminomethane and the citric acid is used for eliminating the interference of acidic pH in the sample, and simultaneously reducing the possible corrosion injury of the test solution to the skin of the human body, thereby being safer and more friendly to experimental personnel and related transportation and storage personnel.
(2) The invention has high detection sensitivity. In the strong acid-alkali cracking method, sodium hydroxide in alkali liquor is used as a membrane breaker to obtain corresponding antigens. Since sodium hydroxide is a strong base, its irreversible destruction requires precise control of the reaction time, otherwise it is liable to cause destruction of the LPS antigen and thus affect the sensitivity. The invention combines the surfactant with the lipid extractant, can mildly and completely obtain LPS antigen, and greatly improves the sensitivity.
(3) The operation difficulty of antigen detection by using the lysate of the invention is low. In the strong acid alkaline hydrolysis neutralization method, the reaction time of the alkaline solution needs to be precisely controlled to ensure the output of effective antigen. If the test is performed inadvertently, the antigen in the sample may be lost, resulting in erroneous test results. In the invention, the influence of the addition time of the second component on the test result is not required to be considered, the operation difficulty is greatly reduced, and the possibility of error operation is reduced.
Detailed Description
The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
A lysate for chlamydia trachomatis antigen detection comprises antigen extract (solution A) and sample buffer solution (solution B).
In the antigen extracting solution, the solvent is glycerol, and the adding amount is 80g; the surfactant is Tween 80 and polyvinylpyrrolidone with the addition amount of 1g and 2g respectively, and the lipid extractant is cholesterol with the addition amount of 5g. The surfactant, the lipid extractant and the solvent are added in sequence and stirred and mixed uniformly at normal temperature.
In the sample buffer solution, the tris buffer solution in the buffer system comprises 1.2g of tris, and the citric acid buffer solution comprises 0.96g of citric acid with pH of 7.5; surfactants include sodium lauryl sulfate: 5g, triton-100: 5g; the metal ion salt is sodium chloride 1.5g; the macromolecular compound comprises 2.2g of polyethylene glycol, 1.5g of sodium caseinate and 0.5g of bovine serum albumin; purified water 100g (100 mL). The buffer solution, the surfactant, the macromolecular compound, the metal ion salt and the purified water are added in sequence and stirred and mixed uniformly at normal temperature.
Example 2
A lysate for chlamydia trachomatis antigen detection comprises antigen extract (solution A) and sample buffer solution (solution B).
In the antigen extracting solution, the solvent comprises glycerin, the adding amount is 50g, the purified water and the adding amount is 30g; the surfactant is Tween 80,1g, and polyvinylpyrrolidone 2g; the lipid extractant is 3g of high density lipoprotein. The surfactant, the lipid extractant and the solvent are added in sequence and stirred and mixed uniformly at normal temperature.
In the sample buffer solution, the buffer system is a tris buffer solution, comprising 1.2g of tris buffer solution and boric acid-borax buffer solution (1 g of boric acid, 0.25g of borax) with pH of 7; the surfactant comprises 10g of sodium dodecyl sulfate; the metal ion salt is magnesium chloride 0.5g and sodium chloride 1g; the macromolecular compound comprises 2.5g of polyethylene glycol, 1g of sodium caseinate and 1g of bovine serum albumin; purified water 100g (100 mL). The buffer system, the surfactant, the macromolecular compound, the metal ion salt and the purified water are added in sequence and stirred and mixed uniformly at normal temperature.
Example 3
A lysate for chlamydia trachomatis antigen detection comprises antigen extract (solution A) and sample buffer solution (solution B).
In the antigen extracting solution, dimethyl sulfoxide is selected as a solvent, and the adding amount is 75g; the surfactant is polyvinylpyrrolidone 2g, and the lipid extractant is phenol 5g. The surfactant, the lipid extractant and the solvent are added in sequence and stirred and mixed uniformly at normal temperature.
In the sample buffer solution, the buffer system is a tris buffer solution, comprising 1.2g of tris, and a citric buffer solution, comprising 0.96g of citric acid and having a pH of 7.5; the surfactant comprises 5g of sodium dodecyl sulfate, 5g of tween 80; the metal ion salt is ferrous sulfate 1g; the macromolecular compounds comprise 0.5g of polyvinylpyrrolidone, 1.5g of casein sodium and 0.5g of bovine serum albumin; purified water 100g (100 mL). The buffer system, the macromolecular compound, the surfactant, the metal ion salt and the purified water are added in sequence and stirred and mixed uniformly at normal temperature.
Example 4
A lysate for chlamydia trachomatis antigen detection comprises antigen extract (solution A) and sample buffer solution (solution B).
In the extracting solution, 75g of dimethyl sulfoxide and 5g of methanol are selected as solvents, 0.5g of sodium dodecyl sulfate, 0.5g of polyethylene glycol and 2g of polyvinylpyrrolidone are selected as surfactants, and 5g of glycerol is selected as lipid extracting agents. The surfactant, the lipid extractant and the solvent are added in sequence and stirred and mixed uniformly at normal temperature.
In the sample buffer solution, the buffer system is a tris buffer solution, comprising 1.2g of tris buffer solution and boric acid-borax buffer solution (1 g of boric acid, 0.25g of borax) with pH of 7; the surfactant comprises 5g of sodium dodecyl sulfate and 5g of brijie; the metal ion salt comprises 1g of sodium chloride and 0.8g of trisodium citrate; the macromolecular compound comprises 2.2g of polyethylene glycol and 1.5g of casein sodium; purified water 100g (100 mL). The buffer system, the surfactant, the macromolecular compound, the metal ion salt and the purified water are added in sequence and stirred and mixed uniformly at normal temperature.
The prepared diluent is canned in a polyethylene plastic bottle and stored in a dark place.
Comparative example 1
A lysate for chlamydia trachomatis antigen detection comprises antigen extract (solution A) and sample buffer solution (solution B).
In the antigen extracting solution, the solvent is glycerol, and the adding amount is 80g; the surfactant is Tween 80 and polyvinylpyrrolidone, and the addition amounts are 1g and 2g respectively. The surfactant and the solvent are added in sequence and stirred and mixed uniformly at normal temperature.
In the sample buffer solution, the tris buffer solution in the buffer system comprises 1.2g of tris, and the citric acid buffer solution comprises 0.96g of citric acid with pH of 7.5; surfactants include sodium lauryl sulfate: 5g, triton-100: 5g; the metal ion salt is sodium chloride 1.5g; the macromolecular compound comprises 2.2g of polyethylene glycol, 1.5g of sodium caseinate and 0.5g of bovine serum albumin; purified water 100g (100 mL). The buffer solution, the surfactant, the macromolecular compound, the metal ion salt and the purified water are added in sequence and stirred and mixed uniformly at normal temperature.
Comparative example 2
A lysate for chlamydia trachomatis antigen detection comprises antigen extract (solution A) and sample buffer solution (solution B).
In the antigen extracting solution, the solvent is glycerol, and the adding amount is 80g; the surfactant is Tween 80 and polyvinylpyrrolidone, and the addition amounts are 1g and 2g respectively; the lipid extractant is diethyl ether with an addition amount of 5g. The surfactant, the lipid extractant and the solvent are added in sequence and stirred and mixed uniformly at normal temperature.
In the sample buffer solution, the tris buffer solution in the buffer system comprises 1.2g of tris, and the citric acid buffer solution comprises 0.96g of citric acid with pH of 7.5; surfactants include sodium lauryl sulfate: 5g, triton-100: 5g; the metal ion salt is sodium chloride 1.5g; the macromolecular compound comprises 2.2g of polyethylene glycol, 1.5g of sodium caseinate and 0.5g of bovine serum albumin; purified water 100g (100 mL). The buffer solution, the surfactant, the macromolecular compound, the metal ion salt and the purified water are added in sequence and stirred and mixed uniformly at normal temperature.
Comparative example 3
The same as in example 1, except that the antigen-extracting solution was glycerol as the solvent, and 50g of the solvent was added; the surfactant is Tween 80 and polyvinylpyrrolidone with the addition amount of 10g and 20g respectively, and the lipid extractant is cholesterol with the addition amount of 5g. The surfactant, the lipid extractant and the solvent are added in sequence and stirred and mixed uniformly at normal temperature.
Comparative example 4
In the acid-base pyrolysis neutralization method, in 100ml of antigen extracting solution, water is selected as a solvent, sodium hydroxide is used as an alkaline substance, and the addition amount is 0.8g; the surfactant is triton 100, and the addition amount is 2g. Adding alkaline substances and surfactant in sequence, and stirring and mixing at normal temperature.
In 100ml of the acidic neutralization solution, the solvent was a 0.2M dilute hydrochloric acid solution.
Detection method and detection result:
Antigen-antibody detection (colloidal gold immunochromatography)
The detection step comprises:
1. obtaining samples to be tested (taking cervical or urethral secretion samples with sterile swabs)
Cervical swab: inserting the cervical swab into the cervix until the end of the cervical swab completely enters, rotating the swab on the inner wall of the cervix for 15-20 seconds, and taking out;
Urethral swab: the urethral swab is inserted into the male urethra for 2-4 cm, and the urethral swab is rotated for 3-5 seconds and then taken out.
2. Splitting to obtain chlamydia trachomatis antigen:
Into the lysis tube, 150. Mu.l of the antigen-extracting solution (solution A) was dropped, and a cervical swab or a urethral swab was inserted. The swab was pressed while being rotated at least 10 times. After squeezing out excess liquid from the swab cotton head, the swab is discarded. Timing for 1 minute and standing.
Mu.l of the sample buffer solution (solution B) was dropped into the lysis tube. After mixing upside down, 3 drops of sample solution were added to the test wells of the flat-laid test card. The time was 15 minutes, and the results were read after 15 minutes.
Effects of antigen extraction by lysis:
the cleavage effect was examined by colloidal gold immunochromatography.
The antigen extraction using the extracts of examples 1-4 showed a visible test band, whereas comparative example 1 (without the addition of lipid extractant) showed only a faint band or a band that could not be seen visually, with poor extraction.
Obvious test strips were seen using the specific lipid extractants of examples 1-4, and the use of the remaining components of the extractant (the extract of comparative example 2) resulted in failure of the colloidal gold test strip to function properly, resulting in failure of the test.
Obvious test strips were visible with the correct component ratios of the extract, and no effective results could be obtained with either insufficient or exceeded ratios (extract of comparative example 3).
Ten minutes after the samples of the same samples were extracted using the antigen extracts of examples 1-4, a sample dilution was added and mixed well, and then a visible band was observed. Whereas comparative example 4 failed to obtain a distinct band after more than 2 minutes due to the cleavage of the extracted LPS molecules caused by prolonged exposure to alkaline substances. Therefore, the control difficulty of the cracking time is reduced, and the possibility of false negative of the test result caused by uncontrollable time in the test is reduced.
Compared with comparative example 4, the same positive sample is used, the detection activity of the antigen is stronger, the detection line intensity reflected on the detection result is improved by about 150%, and the sensitivity is improved by 2-3 times.
The antigen has better stability in the extracting solution, and can still have detection activity after being kept for 30min.
In the nonpolar antigen extracting solution, the specificity of the test is improved, and the possibility of false positive of the test result is reduced.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (2)
1. A lysate for chlamydia trachomatis antigen detection, characterized in that the lysate consists of an antigen extraction solution and a sample buffer solution; the antigen extracting solution consists of a solvent, a surfactant and an extracting agent, and the sample buffer solution consists of a buffer solution, a surfactant, metal ions, a macromolecular compound and a solvent;
In the antigen extracting solution, the weight part of the solvent is 60-120 parts, the weight part of the surfactant is 0.1-15 parts, and the weight part of the extracting agent is 0.1-15 parts;
In the sample buffer solution, the weight part of the buffer solution is 0.1-20 parts, the weight part of the surfactant is 0.1-15 parts, the weight part of the metal ion is 0.1-5 parts, the weight part of the macromolecular compound is 1-20 parts, and the weight part of the solvent is 90-110 parts;
the pH of the buffer solution is 7-10;
the solvent of the antigen extracting solution and the sample buffer solution comprises one or more of water, dimethyl sulfoxide, methanol and glycerol;
The surfactant in the antigen extracting solution comprises one or more of polyvinylpyrrolidone, polyethylene glycol, tween 80, sodium dodecyl sulfate and triton-100; the surfactant of the sample buffer solution comprises one or more of polyvinylpyrrolidone, polyethylene glycol, tween 80, sodium dodecyl sulfate, triton-100 and brijie;
The extractant comprises one or more of cholesterol, phenol, glycerol and high-density lipoprotein;
the buffer solution comprises one or more of a tris buffer solution, a boric acid-borax buffer solution or a citric acid buffer solution;
The metal ions comprise one or more of magnesium chloride, sodium chloride, trisodium citrate and ferrous sulfate;
The macromolecular compound comprises one or more of polyethylene glycol, polyvinylpyrrolidone, sodium caseinate and bovine serum albumin.
2. Use of the lysate of claim 1 for the preparation of a reagent for the detection of chlamydia trachomatis antigens.
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| GB0130947D0 (en) * | 2001-12-24 | 2002-02-13 | Lee Helen | Improved sample preparation for the detection of infectious agents |
| US20100255002A1 (en) * | 2003-06-26 | 2010-10-07 | Chiron Corporation | Immunogenic compositions for chlamydia trachomatis |
| WO2011112670A2 (en) * | 2010-03-09 | 2011-09-15 | Board Of Regents, University Of Texas System | Methods and compositions for chlamydial antigens for diagnosis and treatment of chlamydial infection and disease |
| CN102885029B (en) * | 2012-10-23 | 2013-11-27 | 安徽信灵检验医学科技有限公司 | Non-water leucorrhea specimen storing liquid and preparation method and pH value test collection tube |
| CN103820471A (en) * | 2014-03-17 | 2014-05-28 | 英诺特（唐山）生物技术有限公司 | Recombined chlamydia trachomatis protein and application thereof |
| CN108802368B (en) * | 2018-03-27 | 2021-05-04 | 中国农业大学 | An enzyme-linked immunosorbent assay kit for the detection of bovine abortion chlamydia |
| CN110218802B (en) * | 2019-04-30 | 2023-05-16 | 广州普世利华科技有限公司 | Method for detecting respiratory pathogen nucleic acid |
| CN110308145A (en) * | 2019-07-10 | 2019-10-08 | 迪瑞医疗科技股份有限公司 | A kind of chlamydia trachomatis glycogen detection reagent, chlamydia trachomatis glycogen test strip and preparation method thereof |
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