CN105506085A - Treating method for viscous fluid specimen - Google Patents
Treating method for viscous fluid specimen Download PDFInfo
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- CN105506085A CN105506085A CN201511001467.9A CN201511001467A CN105506085A CN 105506085 A CN105506085 A CN 105506085A CN 201511001467 A CN201511001467 A CN 201511001467A CN 105506085 A CN105506085 A CN 105506085A
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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Abstract
The invention discloses a treating method for a viscous fluid specimen, and relates to the technical field of biochemistry. The method includes the steps that the viscous fluid specimen is added into pre-prepared treating agents with the volume equal to that of the viscous fluid specimen, and repetition is carried out to obtain experimental groups; the experimental groups are treated according to the detected type of the viscous fluid specimen to obtain viscous fluid specimen treating fluids, and then the viscous fluid specimen is treated. By means of the method, the liquefaction time of the specimen is significantly shortened, the viscosity of the specimen is reduced, and the diagnosis precision is not affected; the method can be flexibly applied according to diagnosis purposes; meanwhile, medical cost is not increased.
Description
Technical field
The present invention relates to technical field of biochemistry, particularly relate to a kind for the treatment of process of viscous liquid sample.
Background technology
In Clinical Laboratory, often encounter dense phlegm, pus, hydrothorax, gastric juice, TF, pharyngeal mucus, guide the thickness samples such as urethral secretions liquid, these samples carry out microorganism culturing after generally needing the initial stage to process again, biochemical analysis, the check analyses such as immunologic test, and thickness sample is not easy to apply or smear uneven, easily cause omitting pathogenic micro-organism to cultivate, along with biochemical diagnosis technology, the development of immune diagnostic technique and be that the molecular diagnostic techniques of Main Means becomes means indispensable in clinical diagnosis gradually with round pcr, higher to the processing requirements of thickness sample.And the pollutent such as albumen, polysaccharide in humoral specimen easily disturb the activity of biological enzyme or too thickness affect the sensitivity of biochemical immunity diagnostic techniques and specially spend.
Current process thickness sample technology mainly contains boiling method, sodium-hydroxide treatment method, N-acetyl L-Cys-sodium hydroxide (NALC-NaOH) method, trypsinization etc., but these methods or affect the structure of the materials to be checked such as albumen in sample or affect nucleic acid extraction efficiency, affect the sensitivity of diagnostic reagent; Or change the materialization feature of sample to be checked, affect specificity, the main viscous ingredients of viscous liquid sample is the Multiple components such as polysaccharide, glycoprotein and immunocyte, necrotic tissue, fat is main, and Specimen origin is different, and its composition is also not quite similar.Thick substances as main in dense phlegm is glycoprotein MUC5AC, MUC5B etc. of tracheal epithelial cell secretion, and dense phlegm also mixes a small amount of blood and swill etc., causes the viscosity between sample not identical.
These impurity not only hinder nucleic acid amplification to react, also severe jamming nucleic acid extraction and refining.Thickness sample also affects immunization inspection, as the antibody antigen in the antigen in ELISA inspection in sample or antibody and reagent can not abundant contact reacts, Gold standard check in sample be not easy to flow to golden labeling antibody side by capillary phenomenon.In biochemical investigation, thickness sample easily causes capillary pipet to block, and not only affects diagnostic accuracy, also affects machine handing.In order to address these problems, need a kind for the treatment of process for thick liquid sample badly.
Summary of the invention
The object of the present invention is to provide a kind for the treatment of process of viscous liquid sample, thus solve the foregoing problems existed in prior art.
To achieve these goals, the treatment process of viscous liquid sample of the present invention, the method comprises: joined by thick liquid sample in previously prepared treatment agent, described thick liquid sample and described previously prepared treatment agent equal-volume, and do repetition, as experimental group; The type detected according to described thick liquid sample processes experimental group, obtains viscous liquid sample disposal liquid, completes the process to viscous liquid sample.
Preferably, described previously prepared treatment agent comprises treatment agent A, treatment agent B and treatment agent C;
Described in 1L, treatment agent A comprises: the biological enzyme of the sodium-chlor of the potassium primary phosphate of 0.135g ~ 0.405g, the Sodium phosphate dibasic of 0.71g ~ 2.13g, 4g ~ 12g, the Repone K of 0.1g ~ 0.3g, the polysorbas20 of 2.5mL ~ 7.5mL and 12.5g ~ 37.5g, and the pH of described treatment agent A is 6.5 ~ 7.5;
Described in 1L, treatment agent B comprises: the dithiothreitol (DTT) of the sodium-chlor of the potassium primary phosphate of 0.135g ~ 0.405g, the Sodium phosphate dibasic of 0.71g ~ 2.13g, 4g ~ 12g, the Repone K of 0.1g ~ 0.3g, the sodium lauryl sulphate of 5mL ~ 15ml and 5g ~ 15g, and the pH of described treatment agent B is 6.5 ~ 7.5;
Described in 1L, treatment agent C comprises: the urea of 108g and the dithiothreitol (DTT) of 20g, and the pH of described treatment agent C is 6.5 ~ 7.5.
More preferably, described biological enzyme comprises any proportioning of one or more in trypsinase, Chymotrypsin and deoxyribonuclease.
More preferably, described biological enzyme comprises: the trypsinase of 10g, 10g Chymotrypsin and 5g deoxyribonuclease.
More preferably, when described treatment agent is treatment agent A or treatment agent B, experimental group is shaken 0.8h ~ 1.2h under the condition of 36 DEG C ~ 37.5 DEG C, obtains viscous liquid sample disposal liquid, complete the process to viscous liquid sample.
More preferably, when described treatment agent is treatment agent C, be, after heating 30min ~ 60min under the condition of 95 DEG C, obtain viscous liquid sample disposal liquid, complete the process to viscous liquid sample in temperature by experimental group.
More preferably, described in 1L, treatment agent A comprises: the biological enzyme of the sodium-chlor of the potassium primary phosphate of 0.27g, the Sodium phosphate dibasic of 1.42g, 8g, the Repone K of 0.2g, the polysorbas20 of 5mL and 25g, the pH of described treatment agent A is 6.5 ~ 7.5.
More preferably, described in 1L, treatment agent B comprises: the dithiothreitol (DTT) of the sodium-chlor of the potassium primary phosphate of 0.27g, the Sodium phosphate dibasic of 1.42g, 8g, the Repone K of 0.2g, the sodium lauryl sulphate of 10ml and 10g, the pH of described treatment agent B is 6.5 ~ 7.5.
The invention has the beneficial effects as follows:
The present invention adds trypsinase and tensio-active agent in sample, and reductive agent, can significantly shorten sample liquefying time, reduce sample viscosity, and do not affect diagnostic accuracy, and the treatment agent invented based on this for viscous liquid sample and treatment process, this treatment agent is with the treatment agent A of microorganism culturing for main application, treatment agent C that the treatment agent B of main application and molecular diagnosis are purposes is diagnosed as to meet various clinical demand with biochemical immunity, this additive can be preloaded onto specimen collection container, realize single container process, facilitate sample disposal, reduce schedule of operation, avoid biological pollution to greatest extent.This treatment process for diagnostic purpose flexible Application, should not affect medical treatment cost simultaneously.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearly understand, the present invention is further elaborated.Should be appreciated that embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
Embodiment 1
Viscous liquid sample disposal is become the method for the treatment solution of microorganism culturing: get qualified clinical sputum sample 20 parts, every part arranges control group and experimental group respectively, be specially: getting volume is that the thick liquid sample I of XmL joins in the previously prepared treatment agent A of XmL, do repetition, as experimental group; Getting volume is that the thick liquid sample I of XmL joins in XmL physiological saline, does repetition, as a control group; Control group and experimental group are shaken 0.8 ~ 1.2h under the condition of 36 DEG C ~ 37.5 DEG C, obtains viscous liquid sample disposal liquid, complete the process to viscous liquid sample.
Described in 1L, treatment agent A comprises: the biological enzyme of the sodium-chlor of the potassium primary phosphate of 0.27g, the Sodium phosphate dibasic of 1.42g, 8g, the Repone K of 0.2g, the polysorbas20 of 5mL and 25g, and the pH of described treatment agent A is 6.5 ~ 7.5; Described biological enzyme comprises: the trypsinase of 10g, 10g Chymotrypsin and 5g deoxyribonuclease.
Can obviously obtain: experimental group viscosity comparatively control group obviously reduces, be inoculated into blood agar according to a conventional method, observe colony number, experimental group colony number is many compared with control group, the suspicious bacterium colony of visual inspection afterwards, picking colony carries out bacteria culture discriminating, isolate pathogenic bacterium for 13 parts in experimental group 20 parts of thick liquid samples, in control group 20 parts of thick liquid samples, isolate pathogenic bacterium for 5 parts, after adding treatment agent A, significantly reduce viscosity, smear more even, pathogenic bacterium separation rate obviously raises.
Treatment agent A: be mainly used in microorganism culturing thick liquid sample disposal; compared with the past sample disposal agent; this treatment agent can liquefy thickness sample completely; inorganic salts ingredients in this treatment agent is as the activity of buffer reagent protection biological enzyme; maintain the physiologically active of microorganism; polysorbas20 is as nonionogenic tenside; reduce agriculture denseness; biological enzyme is fully mixed with sample; improve the effect of biological enzyme; and biological enzyme decomposes the impurity that mucoprotein and host DNA etc. cause thickness, reduce the viscosity of sample, create favorable conditions for microorganism is smearing culture.
Embodiment 2
The difference of the present embodiment and embodiment 1 is: described in 1L, treatment agent A comprises: 0.135g potassium primary phosphate, 0.71g Sodium phosphate dibasic, 4g sodium-chlor, 0.1g Repone K, 2.5mL polysorbas20 and 12.5g biological enzyme, and the pH of described treatment agent A is 7.0.
Embodiment 3
The difference of the present embodiment and embodiment 1 is: described in 1L, treatment agent A comprises: 0.405g potassium primary phosphate, 2.13g Sodium phosphate dibasic, 12g sodium-chlor, 0.3g Repone K, 7.5mL polysorbas20 and 37.5g biological enzyme, and the pH of described treatment agent A is 7.5.
Embodiment 4
Viscous liquid sample disposal is become the method for immunodetection or biochemical diagnosis treatment solution used: get qualified clinical sputum sample 38 parts, every part arranges control group and experimental group respectively, be specially: getting volume is that the thick liquid sample I of YmL joins in the previously prepared treatment agent A of YmL, do repetition, as experimental group; Getting volume is that the thick liquid sample I of YmL joins in YmL physiological saline, does repetition, as a control group; Control group and experimental group are shaken 0.8 ~ 1.2h under the condition of 36 DEG C ~ 37.5 DEG C, obtains viscous liquid sample disposal liquid, complete the process to viscous liquid sample.
Described in 1L, treatment agent B comprises: the dithiothreitol (DTT) of the sodium-chlor of the potassium primary phosphate of 0.27g, the Sodium phosphate dibasic of 1.42g, 8g, the Repone K of 0.2g, the sodium lauryl sulphate of 10ml and 10g, the pH of described treatment agent B is 6.5 ~ 7.5.
Treatment agent B: be mainly used in immunodetection and biochemical diagnosis thick liquid sample disposal used, compared with the past sample disposal agent, this treatment agent not only can liquefy sample completely, sample is easily flowed, inorganic salts ingredients in this treatment agent is as buffer reagent protection and stablize the protein such as antigen-antibody in sample, SDS as macromolecular substance such as discrete dose of dispersed protein to reduce viscosity, and the disulfide bond reduction effect between reduction DNA dimer effect and protein strengthening DTT that cooperates, reduce the viscosity of sample, increase the interaction between protein, improve the susceptibility of immune diagnostic reagent and specially spend, improve the biochemical investigation efficiency of sample.
Can obviously obtain: experimental group viscosity comparatively control group obviously reduces, get the treatment solution of 100uL viscous liquid sample disposal liquid and control group respectively, be added drop-wise to sputum tuberculosis antigen gold-marking immunity diagnostic reagent specimen hole respectively, experimental group flow velocity is obviously fast than control group, experimental group all shows the positive, positive line is clear, nature controlling line is clear, the display positive time comparatively control group shift to an earlier date 2 ~ 3 minutes, control group has 3 sample displays negative, display positive time comparatively slow 2 ~ 3 minutes of experimental group, sample positive rate through treatment agent B process obviously improves than control group detection time.
Embodiment 5
The difference of the present embodiment and embodiment 4 is: described in 1L, treatment agent B comprises: 0.135g potassium primary phosphate, 0.71g Sodium phosphate dibasic, 4g sodium-chlor, 0.1g Repone K, 5ml sodium lauryl sulphate and 5g dithiothreitol (DTT), and the pH of described treatment agent B is 6.5.
Embodiment 6
The difference of the present embodiment and embodiment 4 is: described in 1L, treatment agent B comprises: 0.405g potassium primary phosphate, 2.13g Sodium phosphate dibasic, 12g sodium-chlor, 0.3g Repone K, 15ml sodium lauryl sulphate and 15g dithiothreitol (DTT), and the pH of described treatment agent B is 7.5.
Embodiment 7
Viscous liquid sample disposal is become the method for the treatment solution of molecular diagnosis: get qualified clinical sputum sample 100 parts, every part arranges control group and experimental group respectively, be specially: getting volume is that the thick liquid sample I of ZmL joins in the previously prepared treatment agent A of ZmL, do repetition, as experimental group; Getting volume is that the thick liquid sample I of ZmL joins in ZmL physiological saline, does repetition, as a control group; After control group and experimental group are heated 30min by control group and experimental group under temperature is the condition of 95 DEG C, obtain viscous liquid sample disposal liquid, complete the process to viscous liquid sample.
Described in 1L, treatment agent C comprises: the urea of 108g and the dithiothreitol (DTT) of 20g, and the pH of described treatment agent C is 6.5 ~ 7.5.
Treatment agent C: be mainly used in molecular diagnosis agents, compared with the past sample disposal agent, this treatment agent liquefaction sample is complete, easy extraction nucleic acid, protein denaturation activity is high simultaneously, reduce the interference that the impurity such as albumen react nucleic acid amplification, urea in treatment agent not only plays dispersion agent effect, protein denaturation can be made simultaneously, Action of Surfactant can also be played, the cytolemma such as destroy microorganisms, also promote the disulfide bond reduction effect of DTT, the viscosity of great minimizing sample, DTT can destroy cytolemma more thoroughly through heating, promote the releasing of intracellular nucleic acid, nucleic acid is hindered to form dimer, be conducive to the efficiency improving PCR equimolecular diagnostic means.
Viscous liquid sample disposal liquid conventional nucleic acid extracting method control group and experimental group obtained extracts nucleic acid, and design mycobacteria specific primer, carries out PCR reaction, can obviously obtain: experimental group 97 parts of positives, 3 parts of 1+ samples present feminine gender, and more than 2+ sample all presents the positive, control group 69 parts of PCR positives, 31 parts of PCR feminine genders, 17 parts of positives in 38 parts of 1+ samples, 21 parts present feminine gender, positive 54 parts of more than 2+ sample 62 parts of PCR, negative 10 parts, experimental group positive rate significantly improves.
By adopting technique scheme disclosed by the invention, obtain effect useful as follows: the present invention adds trypsinase and tensio-active agent in sample, and reductive agent, can significantly shorten sample liquefying time, reduce sample viscosity, and do not affect diagnostic accuracy, and the treatment agent invented based on this for viscous liquid sample and treatment process, this treatment agent is with the treatment agent A of microorganism culturing for main application, treatment agent C that the treatment agent B of main application and molecular diagnosis are purposes is diagnosed as to meet various clinical demand with biochemical immunity, this additive can be preloaded onto specimen collection container, realize single container process, facilitate sample disposal, reduce schedule of operation, avoid biological pollution to greatest extent.This treatment process for diagnostic purpose flexible Application, should not affect medical treatment cost simultaneously.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should look protection scope of the present invention.
Claims (8)
1. a treatment process for viscous liquid sample, is characterized in that, the method comprises:
Thick liquid sample is joined in previously prepared treatment agent, described thick liquid sample and described previously prepared treatment agent equal-volume, and do repetition, as experimental group; The type detected according to described thick liquid sample processes experimental group, obtains viscous liquid sample disposal liquid, completes the process to viscous liquid sample.
2. the treatment process of viscous liquid sample according to claim 1, it is characterized in that, described previously prepared treatment agent comprises treatment agent A, treatment agent B and treatment agent C;
Described in 1L, treatment agent A comprises: the biological enzyme of the sodium-chlor of the potassium primary phosphate of 0.135g ~ 0.405g, the Sodium phosphate dibasic of 0.71g ~ 2.13g, 4g ~ 12g, the Repone K of 0.1g ~ 0.3g, the polysorbas20 of 2.5mL ~ 7.5mL and 12.5g ~ 37.5g, and the pH of described treatment agent A is 6.5 ~ 7.5;
Described in 1L, treatment agent B comprises: the dithiothreitol (DTT) of the sodium-chlor of the potassium primary phosphate of 0.135g ~ 0.405g, the Sodium phosphate dibasic of 0.71g ~ 2.13g, 4g ~ 12g, the Repone K of 0.1g ~ 0.3g, the sodium lauryl sulphate of 5mL ~ 15ml and 5g ~ 15g, and the pH of described treatment agent B is 6.5 ~ 7.5;
Described in 1L, treatment agent C comprises: the urea of 108g and the dithiothreitol (DTT) of 20g, and the pH of described treatment agent C is 6.5 ~ 7.5.
3. the treatment process of viscous liquid sample according to claim 2, it is characterized in that, described biological enzyme comprises any proportioning of one or more in trypsinase, Chymotrypsin and deoxyribonuclease.
4. the treatment process of viscous liquid sample according to claim 3, it is characterized in that, described biological enzyme comprises: the trypsinase of 10g, 10g Chymotrypsin and 5g deoxyribonuclease.
5. the treatment process of viscous liquid sample according to claim 2, it is characterized in that, when described treatment agent is treatment agent A or treatment agent B, experimental group is shaken 0.8h ~ 1.2h under the condition of 36 DEG C ~ 37.5 DEG C, obtain viscous liquid sample disposal liquid, complete the process to viscous liquid sample.
6. the treatment process of viscous liquid sample according to claim 2, it is characterized in that, when described treatment agent is treatment agent C, be after heating 30min ~ 60min under the condition of 95 DEG C in temperature by experimental group, obtain viscous liquid sample disposal liquid, complete the process to viscous liquid sample.
7. the treatment process of viscous liquid sample according to claim 2, is characterized in that,
Described in 1L, treatment agent A comprises: the biological enzyme of the sodium-chlor of the potassium primary phosphate of 0.27g, the Sodium phosphate dibasic of 1.42g, 8g, the Repone K of 0.2g, the polysorbas20 of 5mL and 25g, the pH of described treatment agent A is 6.5 ~ 7.5.
8. the treatment process of viscous liquid sample according to claim 2, it is characterized in that, described in 1L, treatment agent B comprises: the dithiothreitol (DTT) of the sodium-chlor of the potassium primary phosphate of 0.27g, the Sodium phosphate dibasic of 1.42g, 8g, the Repone K of 0.2g, the sodium lauryl sulphate of 10ml and 10g, the pH of described treatment agent B is 6.5 ~ 7.5.
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| CN201511001467.9A CN105506085A (en) | 2015-12-28 | 2015-12-28 | Treating method for viscous fluid specimen |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110964842A (en) * | 2019-12-31 | 2020-04-07 | 广州金域医学检验集团股份有限公司 | Sample processing reagent and kit for detecting mycobacterium tuberculosis nucleic acid and nucleic acid amplification method of mycobacterium tuberculosis |
| CN112345319A (en) * | 2020-09-29 | 2021-02-09 | 郑州安图生物工程股份有限公司 | Digestion method of sputum sample |
| CN112345318A (en) * | 2020-09-29 | 2021-02-09 | 郑州安图生物工程股份有限公司 | Compound sputum digestive juice |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110964842A (en) * | 2019-12-31 | 2020-04-07 | 广州金域医学检验集团股份有限公司 | Sample processing reagent and kit for detecting mycobacterium tuberculosis nucleic acid and nucleic acid amplification method of mycobacterium tuberculosis |
| CN110964842B (en) * | 2019-12-31 | 2023-11-03 | 广州金域医学检验集团股份有限公司 | Sample processing reagent for detecting mycobacterium tuberculosis nucleic acid, kit and mycobacterium tuberculosis nucleic acid amplification method |
| CN112345319A (en) * | 2020-09-29 | 2021-02-09 | 郑州安图生物工程股份有限公司 | Digestion method of sputum sample |
| CN112345318A (en) * | 2020-09-29 | 2021-02-09 | 郑州安图生物工程股份有限公司 | Compound sputum digestive juice |
| CN112345319B (en) * | 2020-09-29 | 2024-05-17 | 郑州安图生物工程股份有限公司 | Digestion method of sputum sample |
| CN112345318B (en) * | 2020-09-29 | 2024-05-17 | 郑州安图生物工程股份有限公司 | Compound phlegm digestive juice |
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Application publication date: 20160420 |