CN104483495B - Method for eliminating false positive interference of G test - Google Patents
Method for eliminating false positive interference of G test Download PDFInfo
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- CN104483495B CN104483495B CN201410639046.8A CN201410639046A CN104483495B CN 104483495 B CN104483495 B CN 104483495B CN 201410639046 A CN201410639046 A CN 201410639046A CN 104483495 B CN104483495 B CN 104483495B
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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
Abstract
The invention relates to the G test detection technical field, and particularly discloses a method for eliminating false positive interference of a G test, that is to say, a buffer solution containing a divalent soluble metal salt and Tris is prepared and is mixed with patient plasma or serum containing an interfering substance to prepare a detection sample, and the detection sample is subjected to the G test. With the use of the method, when the patient plasma/serum containing a nonspecific tachypleus amebocyte lysate is detected, the interference effect can be eliminated, the beta-G concentration in the patient plasma/serum is accurately detected, and patient mental and economic burdens caused by clinical misjudgment are greatly reduced.
Description
Technical field
The present invention relates to G Test And Check Technologies field, more particularly, to a kind of side of elimination G experiment false positive interference
Method.
Background technology
Fungi universally present in nature, including the oral cavity of human body, nasopharynx, enteron aisle, female genital tract etc., however these
Its clinical manifestation pole of the deep fungal infection that normal flora causes is not true to type, it is difficult to mutually differentiate with the infection such as other bacteriums, resists true
If bacterium is treated not in time, its prognosis is again bad.Therefore clinically make every effort to find correct, the quick method for judging deep fungal,（1,
3）- β-D glucans（β-G）It is one of component of some fungal cell walls, such as candida albicans and Aspergillus.When thalline metabolism
Or after death, the β-G components in cell membrane can be discharged into environment.Detection candida albicans or the serum of Aspergillus deep infection patient
Or blood plasma, it is found that apparently higher than non-infection population, this shows the detection of-G to candida albicans and the depth of Aspergillus to the content of β-G
Portion's Infect And Diagnose has clinical meaning.
TAL is prepared from by the amoebocyte lysis thing of marine organisms horseshoe crab, and β-G can specific activation TAL
In G clotting factor formed coagulated protein, cause the change of reaction solution light transmittance.Reacted using β-G standard items and TAL,
The standard curve of β-G concentration and light varience relation is set up, quantitatively detects that the experiment of the β-G contents of clinical serum or blood plasma claims
For G is tested.
This experiment is promoted to countries in the world since Japan, is removed as early diagnosis and is combined bacterium（Mucor etc.）, hidden ball
Deep fungal infection beyond bacterium, including the main method of candida albicans, aspergillus etc..Its sensitivity and specificity is about 60~90%.
But some patients, such as 1, injected the patient of Fat Emulsion；2nd, Intravenous immunoglobulin, albumin, blood coagulation
The patient of the factor or blood product；Tried containing nonspecific horseshoe crab in the plasma/serum of the patient for the 3rd, taking sulfa drugs
Agent reactant, can cause the reduction of reaction solution light transmittance in course of reaction.This nonspecific TAL reactant causes
Light varience testing result can be caused false positive results occur, have a strong impact on the clinical diagnosis of deep fungal infection, Er Qietong
Blood sampling is detected again after often needing patient to be discontinued, and affects diagnosis and treatment adversely.
The content of the invention
The technical problem to be solved in the present invention is to overcome the patients serum containing interfering material or blood plasma to be tested with G
The technological deficiency of false positive occurs during detection, there is provided a kind of method of elimination G experiments false positive interference.
Second object of the present invention is to provide a kind of buffer solution of elimination G experiments false positive interference.
The purpose of the present invention is achieved by the following technical programs：
A kind of buffer solution of elimination G experiment false positive interference, the buffer solution be comprising bivalent soluble slaine with
The buffer solution of Tris.
Preferably, described bivalent soluble slaine is magnesium sulfate or calcium chloride.
It is highly preferred that in the buffer solution bivalent soluble slaine final concentration of 0.3~0.8 M, the end of Tris is dense
It is 0.05~0.2 M to spend, and the pH value of buffer solution is 6.0~8.0.
A kind of method of elimination G experiment false positive interference, i.e., with above-mentioned buffer solution and the patient's blood plasma for containing interfering material or
Serum mixes to prepare detection sample, and G testing inspections are carried out to the detection sample.
G testing inspections be fungi cell wall constituent β-G, after the phagocyte phagocytosis fungi of human body, can sustained release
The material, content increases in making blood and body fluid（Mycotic infection of superficial part is without similar phenomenon）.β-G can specific activation horseshoe crab
（Limulus）G-factor in ameboid cell lysate, causes lysate to solidify, and forms coagulated protein, causes reaction solution printing opacity
The change of degree.Can detect whether to infect fungi by the change to reaction solution light transmittance.
As described in background technology, some patients such as 1, the patient of Fat Emulsion was injected；2nd, venoclysis is immunized
The patient of globulin, albumin, clotting factor or blood product；In the plasma/serum of the patient for the 3rd, taking sulfa drugs
Containing nonspecific TAL reactant, these interfering materials can influence G reagent reactings to produce the change of color or turbidity, from
And produce interference effect.
The present invention will contain patient's blood plasma or serum of interfering material by numerous studies discovery, use special formulation of the present invention
1~3 times of the buffer solution dilution treatment of composition, it is possible to eliminate the interference effect of above disturbing factor；Eliminate false positive.
It is highly preferred that above-mentioned buffer solution is 1 with patient's blood plasma or the mixed volume ratio of serum containing interfering material：1.
Preferably, patient's blood plasma or serum containing interfering material be injected Fat Emulsion patient's blood plasma or serum,
Patient's blood plasma or serum of Intravenous immunoglobulin, albumin, clotting factor or blood product took sulfonamides
Patient's blood plasma or serum of thing.
Preferably, patient's blood plasma or serum containing interfering material first passed through following treatment before mixing with buffer solution：With
Baterial endotoxin test water will contain interfering material patient's plasma/serum dilute 10 times, 70 ~ 75 DEG C heat 10 ~ 15 minutes, so
After be cooled to room temperature.
As a kind of perferred technical scheme, the method for above-mentioned elimination G experiments false positive interference is comprised the following steps：
S1. magnesium sulfate or calcium chloride and Tris without pyrogen are taken, with baterial endotoxin test water dissolves, is configured to
The buffer solution of magnesium sulfate or calcium chloride and Tris, and it is 6.0~8.0 to adjust pH value；
S2. with patient blood plasma or serum of the baterial endotoxin test dilute with water containing interfering material, 70~75 DEG C of heating 10
~15min, is subsequently cooled to room temperature, and patient's blood plasma or serum dilution containing interfering material is obtained；
S3. patient's blood plasma containing interfering material that the buffer solution for being obtained with S1 prepares S2 or serum dilution are again
1~3 times of dilution, you can carry out G experiments.
Further, in above-mentioned optimal technical scheme, the final concentration of 0.65M of magnesium sulfate described in S1 or calcium chloride,
The final concentration of 0.1M of Tris, the pH value of final regulation buffer solution is 7.0.
Further, in above-mentioned optimal technical scheme, in S3 with S1 obtain buffer solution by S2 prepare containing interference
Patient's blood plasma or serum dilution of material dilute 1 times again.
Compared with prior art, the invention has the advantages that：
Present invention firstly provides a kind of buffer solution that can eliminate G experiment false positive interference being specially formulated, then carry again
For eliminate G experiment false positive interference method, that is, prepare the buffer solution containing bivalent soluble slaine Yu Tris, with through process
Rear human plasma containing interfering material or serum mixing prepare detection sample, and G experiments are carried out to the detection sample.Using upper
Method is stated when detection contains patient's plasma/serum of non-specific TAL reactant, interference effect can be eliminated, accurate inspection
β-G the concentration surveyed in patient's plasma/serum.
Brief description of the drawings
Fig. 1 is human normal plasma/serum G experiment kinetic curves, and in smooth anti-s shapes, two lines represent parallel two in figure
Pipe, the kinetic curve is obtained by this area routine G method for testing and detecting；
Fig. 2 is the human plasma containing non-specific TAL reactant/serum routine G experiment kinetic curves, two in figure
Bar line represents parallel two pipe, and the kinetic curve is obtained by this area routine G method for testing and detecting；
Fig. 3 is that the G after the human plasma/serum application the inventive method containing non-specific TAL reactant tests power
Curve is learned, two lines represent parallel two pipe in figure.
Specific embodiment
Present disclosure is further illustrated with reference to Figure of description and specific embodiment, but be should not be construed as to this
The limitation of invention.Without departing from the spirit and substance of the case in the present invention, that the inventive method, step or condition are made is simple
Modification is replaced, and belongs to the scope of the present invention.If not specializing, technological means used is art technology in embodiment
Conventional meanses known to personnel.
Embodiment 1
Blood sample to be measured prepares：
Blood sample 1：Normal G experiments positive human plasma/serum；
Blood sample 2：Injected the plasma/serum of the patient of Fat Emulsion；
The present embodiment test material is as follows：Magnesium sulfate（MgSO4）；Trishydroxymethylaminomethane（C4H11NO3）；Hydrochloric acid
（HCL）；Baterial endotoxin test water（W）；Testing sample is test sample（S）.
Test method and step are as follows：
S1. a small amount of magnesium sulfate without pyrogen is taken（MgSO4）And trishydroxymethylaminomethane（Tris, C4H11NO3）, with thin
Bacterium tiny electrolytic cell water dissolves, are configured to MgSO containing 0.3M4With the buffer solution of 0.05M Tris, then adjusting pH value is
6.0；
S2. with baterial endotoxin test water by above-mentioned 10 times of the 2 kinds of human plasma/serum-dilutions of the present embodiment, 70 DEG C are heated
10min, is subsequently cooled to room temperature, is prepared into 10 times of dilutions of human plasma/serum；
10 times of dilutions of human plasma/serum described in S2 are diluted 2 times by the buffer solution for S3. being obtained with S1 again, are prepared
Human plasma/serum test liquid；
S4. β-the G in G testing inspections human plasma/serum are carried out respectively to 2 kinds of human plasmas/serum test liquid prepared by S3
Concentration.
Diluted without buffer solution of the present invention using this area routine G test method detection blood sample 2, i.e. blood samples 2 simultaneously,
Directly carry out conventional G test methods detection.
Shown in testing result Fig. 1~3, be can be seen that by Fig. 1~3：The present invention can eliminate false positive.
Embodiment 2
Blood sample to be measured prepares：
Blood sample 1：Normal G experiments positive human plasma/serum；
Blood sample 2：The plasma/serum of the patient of Intravenous immunoglobulin；
The present embodiment test material is as follows：Magnesium sulfate（MgSO4）；Trishydroxymethylaminomethane（C4H11NO3）；Hydrochloric acid
（HCL）；Baterial endotoxin test water（W）；Testing sample is test sample（S）.
Test method and step are as follows：
S1. a small amount of magnesium sulfate without pyrogen is taken（MgSO4）And trishydroxymethylaminomethane（Tris, C4H11NO3）, with thin
Bacterium tiny electrolytic cell water dissolves, are configured to MgSO containing 0.5M4With the buffer solution of 0.15M Tris, then adjusting pH value is
7.0；
S2. above-mentioned 2 kinds of human plasma/serum is diluted into 10 times, 73 DEG C of heating respectively with baterial endotoxin test water
12min, is subsequently cooled to room temperature, is prepared into 10 times of dilutions of human plasma/serum；
10 times of dilutions of human plasma/serum described in S2 are diluted 2.5 times by the buffer solution for S3. being obtained with S1 again, are prepared into
To human plasma/serum test liquid；
S4. β-the G for being carried out in G testing inspections human plasma/serum to 2 kinds of human plasmas/serum test liquid prepared by S3 are dense
Degree.
Diluted without buffer solution of the present invention using this area routine G test method detection blood sample 2, i.e. blood samples 2 simultaneously,
Directly carry out conventional G test methods detection.
Shown in testing result Fig. 1~3, the curve of Fig. 1 represents feminine gender, and the curve of Fig. 2 represents the positive, and the curve of Fig. 3 is represented
Feminine gender, this explanation present invention can eliminate false positive.
Embodiment 3
Blood sample to be measured prepares：
Blood sample 1：Normal G experiments positive human plasma/serum；
Blood sample 2：Took the plasma/serum of the patient of sulfa drugs；
The present embodiment test material is as follows：Magnesium sulfate（MgSO4）；Trishydroxymethylaminomethane（C4H11NO3）；Hydrochloric acid
（HCL）；Baterial endotoxin test water（W）；Testing sample is test sample（S）.
Test method and step are as follows：
S1. a small amount of magnesium sulfate without pyrogen is taken（MgSO4）And trishydroxymethylaminomethane（Tris, C4H11NO3）, with thin
Bacterium tiny electrolytic cell water dissolves, are configured to MgSO containing 0.8M4With the buffer solution of 0.2M Tris, it is 8.0 then to adjust pH value；
S2. with baterial endotoxin test water by above-mentioned 10 times of 2 kinds of human plasma/serum-dilutions, 75 DEG C are heated 15min, so
After be cooled to room temperature, be prepared into 10 times of dilutions of human plasma/serum；
10 times of dilutions of human plasma/serum described in S2 are diluted 3 times by the buffer solution for S3. being obtained with S1 again, are prepared
Human plasma/serum test liquid；
S4. β-G the concentration in G testing inspections human plasma/serum is carried out to human plasma/serum test liquid prepared by S3.
Diluted without buffer solution of the present invention using this area routine G test method detection blood sample 2, i.e. blood samples 2 simultaneously,
Directly carry out conventional G test methods detection.
Shown in testing result Fig. 1~3, the curve of Fig. 1 represents feminine gender, and the curve of Fig. 2 represents the positive, and the curve of Fig. 3 is represented
Feminine gender, this explanation present invention can eliminate false positive.
Embodiment 4
Blood sample to be measured prepares：
Blood sample 1：Normal G experiments positive human plasma/serum；
Blood sample 2：Injected patient's plasma/serum of Fat Emulsion；
The present embodiment test material is as follows：Calcium chloride（CaCl2）；Trishydroxymethylaminomethane（C4H11NO3）；Hydrochloric acid
（HCL）；Baterial endotoxin test water（W）；Testing sample is test sample（S）.
Test method and step are as follows：
S1. a small amount of calcium chloride without pyrogen is taken（CaCl2）And trishydroxymethylaminomethane（Tris, C4H11NO3）, with thin
Bacterium tiny electrolytic cell water dissolves, are configured to CaCl containing 0.4M2With the buffer solution of 0.1M Tris, it is 6.0 then to adjust pH value；
S2. with baterial endotoxin test water by above-mentioned 10 times of 2 kinds of human plasma/serum-dilutions, 70 DEG C are heated 10min, so
After be cooled to room temperature, be prepared into 10 times of dilutions of human plasma/serum；
10 times of dilutions of human plasma/serum described in S2 are diluted 2 times by the buffer solution for S3. being obtained with S1 again, are prepared
Human plasma/serum test liquid；
S4. β-G the concentration in G testing inspections human plasma/serum is carried out to human plasma/serum test liquid prepared by S3.
Diluted without buffer solution of the present invention using this area routine G test method detection blood sample 2, i.e. blood samples 2 simultaneously,
Directly carry out conventional G test methods detection.
Shown in testing result Fig. 1~3, the curve of Fig. 1 represents feminine gender, and the curve of Fig. 2 represents the positive, and the curve of Fig. 3 is represented
Feminine gender, this explanation present invention can eliminate false positive.
Embodiment 5
Blood sample to be measured prepares：
Blood sample 1：Normal G experiments positive human plasma/serum；
Blood sample 2：The plasma/serum of the patient of venoclysis albumin；
The present embodiment test material is as follows：Calcium chloride（CaCl2）；Trishydroxymethylaminomethane（C4H11NO3）；Hydrochloric acid
（HCL）；Baterial endotoxin test water（W）；Testing sample is test sample（S）.
Test method and step are as follows：
S1. a small amount of calcium chloride without pyrogen is taken（CaCl2）And trishydroxymethylaminomethane（Tris, C4H11NO3）, with thin
Bacterium tiny electrolytic cell water dissolves, are configured to CaCl containing 0.6M2With the buffer solution of 0.1M Tris, it is 7.0 then to adjust pH value；
S2. with baterial endotoxin test water by above-mentioned 10 times of 2 kinds of human plasma/serum-dilutions, 73 DEG C are heated 12min, so
After be cooled to room temperature, be prepared into 10 times of dilutions of human plasma/serum；
10 times of dilutions of human plasma/serum described in S2 are diluted 1 times by the buffer solution for S3. being obtained with S1 again, are prepared
Human plasma/serum test liquid；
S4. β-G the concentration in G testing inspections human plasma/serum is carried out to human plasma/serum test liquid prepared by S3.
Diluted without buffer solution of the present invention using this area routine G test method detection blood sample 2, i.e. blood samples 2 simultaneously,
Directly carry out conventional G test methods detection.
Shown in testing result Fig. 1~3, the curve of Fig. 1 represents feminine gender, and the curve of Fig. 2 represents the positive, and the curve of Fig. 3 is represented
Feminine gender, this explanation present invention can eliminate false positive.
Embodiment 6
Blood sample to be measured prepares：Blood sample 1：Normal G experiments positive human plasma/serum；
Blood sample 2：The plasma/serum of the patient of venoclysis clotting factor；
The present embodiment test material is as follows：Calcium chloride（CaCl2）；Trishydroxymethylaminomethane（C4H11NO3）；Hydrochloric acid
（HCL）；Baterial endotoxin test water（W）；Testing sample is test sample（S）.
Test method and step are as follows：
S1. a small amount of calcium chloride without pyrogen is taken（CaCl2）And trishydroxymethylaminomethane（Tris, C4H11NO3）, with thin
Bacterium tiny electrolytic cell water dissolves, are configured to CaCl containing 0.7M2With the buffer solution of 0.2M Tris, it is 8.0 then to adjust pH value；
S2. with baterial endotoxin test water by above-mentioned 10 times of 2 kinds of human plasma/serum-dilutions, 75 DEG C are heated 15min, so
After be cooled to room temperature, be prepared into 10 times of dilutions of human plasma/serum；
10 times of dilutions of human plasma/serum described in S2 are diluted 3 times by the buffer solution for S3. being obtained with S1 again, are prepared
Human plasma/serum test liquid；
S4. β-G the concentration in G testing inspections human plasma/serum is carried out to human plasma/serum test liquid prepared by S3.
Diluted without buffer solution of the present invention using this area routine G test method detection blood sample 2, i.e. blood samples 2 simultaneously,
Directly carry out conventional G test methods detection.
Shown in testing result Fig. 1~3, the curve of Fig. 1 represents feminine gender, and the curve of Fig. 2 represents the positive, and the curve of Fig. 3 is represented
Feminine gender, this explanation present invention can eliminate false positive.
Claims (7)
1. application of the buffer solution in G experiment false positive interference is eliminated, it is characterised in that the buffer solution is by bivalent soluble gold
Category salt and Tris buffer solutions composition.
2. application according to claim 1, it is characterised in that described bivalent soluble slaine is magnesium sulfate or chlorination
Calcium.
3. application according to claim 2, it is characterised in that the final concentration of bivalent soluble slaine in the buffer solution
It is 0.3~0.8 M, final concentration of 0.05~0.2 M of Tris, the pH value of buffer solution is 6.0~8.0.
4. a kind of method that elimination G experiments false positive is disturbed, it is characterised in that with the buffering described in any one of claims 1 to 3
Liquid mixes to prepare detection sample with patient's blood plasma or serum containing interfering material, and G testing inspections are carried out to the detection sample.
5. the method for eliminating G experiment false positive interference according to claim 4, it is characterised in that the buffer solution with containing dry
The mixed volume ratio of the patient's blood plasma or serum of disturbing material is 1~3：1.
6. the method for eliminating G experiment false positive interference according to claim 4, it is characterised in that described containing interfering material
Patient's blood plasma or serum be injected patient's blood plasma or serum of Fat Emulsion, Intravenous immunoglobulin, albumin, blood coagulation because
Patient's blood plasma or serum of son or blood product took patient's blood plasma or serum of sulfa drugs.
7. the method for eliminating G experiment false positive interference according to claim 4, it is characterised in that the patient containing interfering material
Blood plasma or serum first passed through following treatment before mixing with buffer solution：To contain interfering material with baterial endotoxin test water
Patient's plasma/serum dilutes 10 times, and 70 ~ 75 DEG C are heated 10 ~ 15 minutes, are subsequently cooled to room temperature.
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| CN106290890B (en) * | 2016-07-15 | 2018-10-23 | 科赫生物科技（北京）有限公司 | A kind of the human body fluid development process fungi 1,3- calloses detection kit and its application process of improvement |
| CN111521830B (en) * | 2020-04-27 | 2024-02-23 | 四川沃文特生物技术有限公司 | BNP detection kit, buffer solution, enzyme working solution and application |
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|---|---|---|---|---|
| CN1105757A (en) * | 1993-02-26 | 1995-07-26 | 生化学工业株式会社 | Reagent for endotoxin assay and method for endotoxin assay using the same |
| CN102147415A (en) * | 2010-12-30 | 2011-08-10 | 上海市肿瘤研究所 | Rapid magnetic separation method dominated detection kit for liver cancer alpha fetoprotein variant |
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| WO2012037719A2 (en) * | 2010-09-20 | 2012-03-29 | 天津市一瑞生物工程有限公司 | PREPARATION AND USING METHOD OF KIT FOR COLORIMETRIC DETECTING FUNGAL (1-3)-β -D-GLUCAN |
| CN105021817B (en) * | 2014-04-24 | 2017-02-15 | 天津汇滨生物科技有限公司 | Developing-process fungus 1,3-beta-D-glucan detection kit for human body fluid |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1105757A (en) * | 1993-02-26 | 1995-07-26 | 生化学工业株式会社 | Reagent for endotoxin assay and method for endotoxin assay using the same |
| CN102147415A (en) * | 2010-12-30 | 2011-08-10 | 上海市肿瘤研究所 | Rapid magnetic separation method dominated detection kit for liver cancer alpha fetoprotein variant |
Non-Patent Citations (1)
| Title |
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| 偶联分子簇功能基内毒素吸附剂的合成及应用;屈亚辉;《中国优秀硕士学位论文全文数据库》;20110915(第9期);第I、10-11、28、31页 * |
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