CN102866255B - Kit for detecting fungus (1-3)-beta-D glucan in human body fluid and application method of kit - Google Patents
Kit for detecting fungus (1-3)-beta-D glucan in human body fluid and application method of kit Download PDFInfo
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- CN102866255B CN102866255B CN201210338802.4A CN201210338802A CN102866255B CN 102866255 B CN102866255 B CN 102866255B CN 201210338802 A CN201210338802 A CN 201210338802A CN 102866255 B CN102866255 B CN 102866255B
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a kit for detecting a fungus (1-3)-beta-D glucan in human body fluid. The kit can be industrially produced, and is accurate and high in speed and specificity. The kit is characterized by comprising a main reactant, a body fluid treating agent, an alkaline diluent, an acidic diluent, sterile water, lyophilized serum and a (1-3)-beta-D glucan quality control substance, wherein the main reactant contains a tachypleus tridentatus blood cell G factor, coagulating zymogen and an activator. A detection method is convenient, rapid, safe and high in specificity, the (1-3)-beta-D glucan in body fluid samples of 10 persons can be detected within 80 minutes, a discrimination coincidence rate reaches 100 percent, and the fungus (1-3)-beta-D glucan and bacterial endotoxin can be discriminated within the same reaction time.
Description
Technical field
The present invention relates to a kind of human body fluid fungi (1,3)-β-D glucosan detection kit and application process thereof.
Background technology
In recent years, the morbidity rate of invasive infections with fungi (invasive fungal infections, IFI) obviously rises clinically.IFI also becomes one of the severe complication and important death cause that cause marrow and organ transplant recipients, the malignant hematologic disease accepting chemotherapy and malignant tumor patient, AIDS and other critical patients day by day.Early diagnosis is to the key of Therapy of Invasive Fungal Infections success or failure, after fungi enters blood of human body or deep tissue, through cytophagously to engulf, after digestion and metabolism, (1,3)-β-D glucosan can discharge from cell wall, thus the β-D beta-dextran content in blood or other body fluid is increased, its existence in body fluid can be considered as the mark of IFI (deep fungal infection) to a great extent.Clinical conventional detection method is that microorganism differentiates matrix cultivation, and it is low that this method cultivates positive rate, and easy mistaken diagnosis, and length consuming time (3-7 days), can not provide diagnosis and treatment foundation in time for clinical.
The patent No. is the United States Patent (USP) <reagents for determing peptidoglycan and β-1 of 4970152,3-glucan> (" peptide glycan and β-1, the diagnostic reagent of 3-glucosan ") in, propose a kind of by using composition, measure peptide glycan or β-1, the method of 3-glucosan, described composition comprises the part of silkworm larva blood plasma, its can with β-1,3-glucosan or peptide glycan specific reaction, but do not react with endotoxin.In that patent, the composition that can react with β-1,3-glucosan specificity is by remove by affinity chromatography and the material that reacts of peptide glycan and obtaining.The response composite of the method, be from silkworm larva extract blood plasma be raw material, production will realize industrialization, there is larger limitation.
The day for announcing is on June 15th, 2005, notification number is that the Chinese invention patent of CN1206365C is " for detecting (1, 3) composition of-callose, its preparation method and detection (1, 3) your kit of-callose " propose a kind of for detecting (1, 3) composition of-callose, utilization the sequestrant of chelating calcium ion or damping fluid slightly can extract composition from the plasma cell of Carabidae or Scarabaeidae insect, use calcium ion again, the resin chromatography post of glucosan or vinyl is separated and obtains under calcium ion exists by (1 from thick extraction composition, 3) part of-callose display phenoloxidase activity.The method extracts needed raw material from insect plasma, measures less and limited source, is difficult to industrialization and produces; It is by column chromatography separating effective ingredient, easily by microbial contamination, because this biological reagent can not high-temperature sterilization or sterilizing, and its product standard poor controllability.The method utilizes the activity of phenol oxidase quantitatively to calculate β-1,3-glucosan, and because the material such as lipopolysaccharides, peptide glycan also can activate phenol oxidase, its specificity is not strong, affects measurement result.
Date of publication is on January 4th, 2012, publication No. be 102305787 Chinese invention patent application " preparation of fungi (1-3)-β-D glucosan colorimetric detection reagent box and using method thereof " disclose and a kind of activate king crab blood G-factor by utilization (1-3)-β-D glucosan, after further hydrolysis chromogenic substrate, carry out absorbance detection to reach the object of detection (1-3)-β-D glucosan by spectrophotometer.The problem of the method is that required configuration chromogenic reagent is more, complex operation, and easily has extraneous bacterial contamination to get involved in pilot process, affects testing result.
Summary of the invention
The object of the invention is to provide a kind of can industrialization produce, accurately, fast, human body fluid fungi (1,3)-β-D glucosan detection kit that specificity is high and application process thereof.
For achieving the above object, the present invention is by the following technical solutions:
A kind of human body fluid fungi (1,3)-callose detection kit, it is characterized in that comprising: reaction host, body fluid treating agent, alkaline dilution, acid dilution, sterilized water, freeze-dry blood serum and (1,3)-callose quality-control product, described reaction host contains east limulus blood cell G-factor, proclotting enzyme and promoting agent.
Further, described reaction host is by the preparation of the method that comprises the following steps: live body king crab is disinfected in alcohol ostium blood sampling and adds collected by centrifugation limulus blood cell after anti-coagulants by (1), and the volume ratio of described anti-coagulants and king crab blood is 1:6 ~ 1:10; (2) add the limulus blood cell collected by lysate cracking, obtain limulus blood cell lysate; (3) isolated cell wall, with organic solvent chloroform (CHCl
3) extract limulus blood cell lysate, add chloroform and the PEG 8000 of 1:0.8 ~ 1:1.2 by volume, make every milliliter containing polyglycol 1.5 ~ 3mg, mix 45 ~ 60 minutes immediately, then centrifugal 30 ~ 40 minutes of 1500r/min-3000r/min, obtained reaction host G-factor, proclotting enzyme active component; (4) add promoting agent, the ratio being 1:0.3 ~ 1:0.5 in limulus blood cell extract volume and promoting agent volume ratio adds, and stirs, and obtain reaction host semi-manufacture, described promoting agent adds KCl, MgSO in purified water
4solution or KCl, CaCl
2solution, makes every milliliter of promoting agent containing KCl0.15 ~ 0.30M, containing MgSO
40.15 ~ 0.30M or containing CaCl
20.20 ~ 0.40M, filtered packing, with flowing steam sterilization 30 ~ 40 minutes; (5) 20 ~ 25 DEG C of sealings after-45 DEG C ~-55 DEG C vacuum freeze dryings that the reaction host semi-manufacture of preparation stirred, obtained reaction host finished product.
Further, body fluid treating agent contains sodium chloride, N-tris hydroxymethyl aminomethane and watery hydrochloric acid, and the pH value of described body fluid treating agent is between 5.5 ~ 7.5.Sodium chloride concentration was 8.5mg/ml ~ 15mg/ml, is 0.25mg/ml ~ 1mg/ml containing N-tris hydroxymethyl aminomethane concentration, after mixing, filters packing, with flowing steam sterilization 30 ~ 40 minutes.
Further, the pH value of alkaline dilution is between 10.5 ~ 13.5.Take N-tris hydroxymethyl aminomethane chemical reagent respectively by prescription consumption, put and feed intake in container, add and add water to amount of preparation, make every ml soln containing N-tris hydroxymethyl aminomethane 0.5-1.0mg, after mixing, regulate pH value to be 10.5 ~ 13.5 with watery hydrochloric acid, filter packing, with flowing steam sterilization 30 ~ 40 minutes.
Further, the pH value of acid dilution is between 2.0 ~ 4.0.By prescription consumption, with watery hydrochloric acid or glacial acetic acid solution, the pH value of water recently distilled is adjusted to 2.0 ~ 4.0, after mixing, filters packing, with flowing steam sterilization 30 ~ 40 minutes.
Further, sterilized water is the double distilled water of water for injection.Not containing bacterial endotoxin or (1,3)-callose.
Further, freeze-dry blood serum is human serum albumin or calf serum.
A kind of human body fluid fungi (1, 3) detection method of-callose detection kit, it is characterized in that in the limulus blood cell of east, G-factor is by (1 in sample to be detected, 3)-callose activates, form coagulated protein glue, (1, 3) content of-callose is higher, then form the coagulated protein glue reaction time shorter, by being captured in the transmittance of different time, carrying out regretional analysis (Lgt=a+blgc) realizes in sample to be detected (1, 3) detection of-callose content, specific implementation step comprises standard curve making, body fluid (1, 3) extraction purification of-callose, the preparation of the positive liquid of sample, upper machine testing.
Further, in blood matrix, the linear detection range of (1,3)-callose is 10pg/ml ~ 640pg/ml.
Further, the detection recovery of additional (1, the 3)-callose standard solution of human body fluid is 80% ~ 140%.
A kind of human body fluid fungi (1,3)-callose detection kit, is characterized in that the mensuration being mainly used in fungi (1,3)-callose content in human blood or other body fluid.
Linear coefficient r >=0.99 of the typical curve set up by this human body fluid fungi (1,3)-callose detection kit; 10pg/ml ~ 640pg/ml is reached in blood matrix neutral line sensing range; The qualitative and quantitative analysis to (1,3)-callose can be realized; By at the additional bacterial endotoxin standard solution of body fluid sample and (1,3)-callose standard solution carries out detection recovery test, endotoxin recovery < 5%, prove whole detection experiment process, endotoxin does not affect this Product checking (1,3)-callose, realizes (1 simultaneously, 3) recovery of-callose reaches 80% ~ 140%, more accurate than 50% ~ 200% of international standard requirement.Detection method of the present invention is easy, quick, safety, (1 of the body fluid sample of 10 person-portions can be completed in 80 minutes, 3)-callose detects, Coincidence rate reaches 100%, and specificity is high, fungi (1,3)-β-D glucosan and bacterial endotoxin (Fig. 1) can be differentiated within the same reaction time.
Accompanying drawing explanation
Fig. 1 is bacterial endotoxin and the same time response curve of fungi (1,3)-β-D glucosan, and curve a is bacterial endotoxin response curve, and curve b is (1,3)-β-D glucosan response curve.
Fig. 2 is typical curve
Fig. 3 is typical curve each concentration point reaction optical density curve figure: curve A is concentration is 200pg/ml glucosan reaction optical density curve; Curve B is concentration is 100pg/ml glucosan reaction optical density curve; Curve C is concentration is 50pg/ml glucosan reaction optical density curve; Curve D is concentration is 25pg/ml glucosan reaction optical density curve; Curve E is concentration is 12.5pg/ml glucosan reaction optical density curve; Curve F is negative control optical density response curve.
Fig. 4 is for detecting sample dynamic response curve map: G is blood sample reaction optical density curve figure; H is sample positive liquid reaction optical density curve figure; I is positive control reaction optical density curve figure; J is negative control reaction optical density curve figure.
Specific embodiment
The present invention will be further described to be applied as example below in conjunction with accompanying drawing with the detection of blood (1,3)-β-D glucosan.
The manufacturing of human body fluid fungi (1,3)-callose detection kit:
1. human body fluid fungi (1,3) formation of-callose detection kit comprises: dynamic turbidimetric reaction host, No. I, body fluid treating agent (body fluid treating agent), true No. II dilution (alkaline dilution), true No. III dilution (acid dilution), (1,3)-callose quality-control product, baterial endotoxin test use water, freeze-dry blood serum.
2. the preparation technology of dynamic turbidimetric reaction host, mainly by using anti-coagulants, cell pyrolysis liquid, promoting agent to prepare the extraction of limulus blood cell lysate, specifically comprises the following steps:
2.1 blood samplings: new fresh and alive king crab is disinfected in alcohol ostium blood sampling, the volume ratio of anti-coagulants and king crab blood is be advisable between 1:6 ~ 1:10.
2.2 anti-coagulants were with Purified Water q. s, add pre-assigned auxiliary material solution, make it dilute, and containing caffeine or theophylline 0.015M ~ 0.04M in every 100ml solution, containing NaCl0.25M ~ 0.55M, filter packing, with flowing steam sterilization 30 ~ 40 minutes.
2.3 isolated cells: in the centrifugal bottle suitable by king crab blood dislocation, centrifuging under the speed of 750-850 rev/min, abandons serum.
2.4 cell lysis: by the receiving flask of limulus blood cell dislocation cleaning, add 40% ~ 50% cell pyrolysis liquid by volume, cell lysis, obtain limulus blood cell lysate.
2.5 lysates were with Purified Water q. s, add the tris-HCl solution of 0.05M/ml, adjust pH value to be 6.0 ~ 8.0, with flowing steam sterilization 30 ~ 40 minutes.
2.6 isolated cell walls, with organic solvent chloroform (CHCl
3) extract limulus blood cell lysate, add chloroform and the PEG 8000 of 1:0.8 ~ 1:1.2 by volume, make every milliliter containing polyglycol 1.5 ~ 3mg, mix 45 ~ 60 minutes immediately, then dislocation centrifuge 30-40 minute (2000 ~ 2500 revs/min) separating extractive, obtained reaction host G-factor, proclotting enzyme active component.
2.7 add promoting agent, preparation semi-manufacture: the promoting agent that the ratio being 1:0.3 ~ 1:0.5 in limulus blood cell extract volume and promoting agent volume ratio adds pre-coordination good stirs, and make reaction host semi-manufacture.2.8 promoting agents add KCl, MgSO in appropriate purified water
4or CaCl
2solution, makes every milliliter of heavy manual labour agent containing KCl0.15 ~ 0.30M, containing MgSO
40.15 ~ 0.30M or containing CaCl
20.20 ~ 0.40M, filtered packing, with flowing steam sterilization 30 ~ 40 minutes.
2.9 filling, freeze drying and sealing: the reaction host semi-manufacture of preparation are stirred, carries out packing by production ordering requirement, in subzero 45 DEG C ~ 55 DEG C vacuum freeze dryings, 20 ~ 25 DEG C of sealings, obtained reaction host finished product.
3. the preparation method of No. I, body fluid treating agent: take sodium chloride, N-tris hydroxymethyl aminomethane chemical reagent respectively by prescription consumption, put and feed intake in container, add and add water to amount of preparation, make every ml soln sodium chloride-containing 8.5mg ~ 15mg, containing N-tris hydroxymethyl aminomethane 0.25mg ~ 1mg, regulate pH value 5.5 ~ 7.5, after mixing with watery hydrochloric acid, filter packing, with flowing steam sterilization 30 ~ 40 minutes.
4. the preparation method of true No. II dilution: take N-tris hydroxymethyl aminomethane chemical reagent respectively by prescription consumption, put and feed intake in container, add and add water to amount of preparation, make every ml soln containing N-tris hydroxymethyl aminomethane 0.5-1.0mg, after mixing, regulate pH value to be 10.5 ~ 13.5 with watery hydrochloric acid, filter packing, with flowing steam sterilization 30 ~ 40 minutes.
5. the preparation method of true No. III dilution: by prescription consumption, was adjusted to 2.0 ~ 4.0 with watery hydrochloric acid or glacial acetic acid solution by the pH value of water recently distilled, after mixing, filters packing, with flowing steam sterilization 30 ~ 40 minutes.
6. the preparation method of (1,3)-callose quality-control product: the concentration being diluted to needs with (1,3)-callose national standard, mix rear packing, subzero 45 DEG C ~ 55 DEG C freeze-drying, 35 DEG C gets product with lower sealing.Often prop up quality-control product to use by labelled amount dilution.
7. inspection water: be the double distilled water of water for injection, packing 2ml/ props up, after sealing, sterilizing, detection must not have bacterial endotoxin or (1,3)-callose, qualified, is used as the double solvents of reaction host or freeze-dry blood serum.
8. the preparation method of freeze-dry blood serum: after human serum albumin or calf serum dilution, add certain excipient mixing, packing, subzero 45 DEG C ~ 55 DEG C freeze-drying, 35 DEG C one gets product with lower sealing.Often prop up after dried frozen aquatic products adds the redissolution of inspection water by labelled amount and use.
Body fluid fungi (1,3) the application performing step of-callose detection kit comprises standard curve making, body fluid (1,3) preparation of the positive liquid of the extraction purification of-callose, sample, upper machine testing, with blood (1,3)-β-D glucosan detects and is applied as example, and its concrete steps are as follows:
1. the making of typical curve:
The preparation of 1.1 titers
Get callose standard items 1, after 75% alcohol disinfecting, open, add 1ml serum solution to dissolve, put eddy mixer mixing 10min, then dextran standard serum solution is diluted to 1280pg/ml, all the other dilution process operate with reference to table 1.
Table 1: the preparation of standard solution
| Test tube is numbered | ① | ② | ③ | ④ | ⑤ |
| Standard items initial concentration (pg/ml) | 1200 | 200 | 100 | 50 | 25 |
| Standard solution (ml) | 0.4 | 0.9 | 0.9 | 0.9 | 0.9 |
| Serum solution (ml) | 2.0 | 0.9 | 0.9 | 0.9 | 0.9 |
| Standard items final concentration (pg/ml) | 200 | 100 | 50 | 25 | 12.5 |
1.2 application of sample
Take out reaction host 12, flick at the bottom of a bottle wall makes white powder fall into bottle, with 75% alcohol disinfecting after, unlatching, is inserted in test tube rack by reagent, respectively by table 2 application of sample.
Table 2: prepared by standard items
1.3 detect and drawing standard curve
Application of sample is complete, namely sealed membrane closed test tube mouth is used, test tube is inserted successively in the detecting instrument hole of constant temperature (37 DEG C ± 1 DEG C), and press instrument requirements input relevant information, the logarithm value (x) of measured reaction density and the logarithm value (y) in reaction time are carried out regretional analysis, are calculated as follows the linear coefficient (r) of typical curve:
Table 3: standard items test data
Obtain r=0.9979 according to above-mentioned formula, typical curve: LgT=4.3334-0.5793LgC (r=0.9979), meets experimental standard requirement, standard curve making effectively (Fig. 2, Fig. 3).
2. detect the preparation of sample
The preparation of 2.1 blood samples (A liquid)
2.1.1 get blood sample 1ml, add body fluid treating agent No. I 1ml, mix after 15 seconds, get the centrifugal layering of 1ml mixed liquor, obtain 2 times of supernatants.Centrifugal condition: centrifugation time 5-10min, rotating speed 2000r/min ~ 4500r/min).
2.1.2 getting 0.3ml2 times of supernatant and add isopyknic true No. II dilution, in vitro mixing 15 seconds, sealing orifice, putting water bath incubation 5-10min (water temperature 70-90 DEG C).
2.1.3 incubation is complete, adds true No. III dilution of 0.6ml, sealing orifice, mixes after 15 seconds, centrifugal, centrifugation time 5-10min (rotating speed 2000r/min ~ 4500r/min), and obtained test sample liquid A liquid, gets A liquid supernatant for subsequent use.
The preparation of 2.2 positive control solutions (B liquid): (1,3)-β-D dextran standard serum solution is diluted to 100pg/ml and obtains B liquid, for subsequent use.
The preparation of 2.3 Sample Positive solution (C liquid): the preparation method of Sample Positive liquid, operates by table 4, obtained C liquid.
Table 4: Sample Positive solution
3. application of sample
Host 8 is answered in negate, after 75% alcohol disinfecting, opens, is inserted on test tube rack, by table 5 application of sample.
Table 5: pattern detection system
4. go up machine testing and result discriminatory analysis
Application of sample is complete, with sealed membrane closed test tube mouth, mix 3 seconds (bubble must not be produced) gently, test tube is inserted successively in detecting instrument (the BET-24A type bacteria endotoxin detector) hole of constant temperature (37 DEG C ± 1 DEG C), and by instrument operation instructions requirement input relevant information, (Sample Dilution multiple is set as 8 times, and other is set as 1 times; The typical curve that selected step (1) makes is this lot number reaction host typical curve; OD value is set as 0.02), reaction terminates, and by data analysis, calculates the recovery:
When the recovery is in 50% ~ 200% scope, test sample testing result is effective.
5. test result analysis (Fig. 4)
By data analysis, calculate the recovery:
Visible, the recovery is 101%, and in 50% ~ 200% scope, and negative control detected value is not more than the minimum of typical curve, and this testing inspection result is effective.
Claims (7)
1. a human body fluid fungi (1,3)-callose detection kit, it is characterized in that comprising: reaction host, body fluid treating agent, alkaline dilution, acid dilution, sterilized water, freeze-dry blood serum and (1,3)-callose quality-control product, described reaction host contains east limulus blood cell G-factor, proclotting enzyme and promoting agent;
The method preparation of described reaction host by comprising the following steps: (1) disinfects live body king crab in alcohol ostium, takes a blood sample after adding anti-coagulants, collected by centrifugation limulus blood cell, and the volume ratio of described anti-coagulants and king crab blood is 1:6 ~ 1:10; (2) add the limulus blood cell collected by lysate cracking, obtain limulus blood cell lysate; (3) isolated cell wall, with organic solvent chloroform (CHCl
3) extract limulus blood cell lysate, add chloroform and the PEG 8000 of 1:0.8 ~ 1:1.2 by volume, make every milliliter containing polyglycol 1.5 ~ 3mg, mix 45 ~ 60 minutes immediately, then centrifugal 30 ~ 40 minutes of 1500r/min-3000r/min, obtained reaction host G-factor, proclotting enzyme active component; (4) add promoting agent, the ratio being 1:0.3 ~ 1:0.5 in limulus blood cell extract volume and promoting agent volume ratio adds, and stirs, and obtain reaction host semi-manufacture, described promoting agent adds KCl, MgSO in purified water
4solution or KCl, CaCl
2solution, makes every milliliter of promoting agent containing KCl 0.15 ~ 0.30M, containing MgSO
40.15 ~ 0.30M or containing CaCl
20.20 ~ 0.40M, filtered packing, with flowing steam sterilization 30 ~ 40 minutes; (5) 20 ~ 25 DEG C of sealings after-45 DEG C ~-55 DEG C vacuum freeze dryings that the reaction host semi-manufacture of preparation stirred, obtained reaction host finished product.
2. a kind of human body fluid fungi (1,3)-callose detection kit as claimed in claim 1, it is characterized in that body fluid treating agent contains sodium chloride, N-tris hydroxymethyl aminomethane and watery hydrochloric acid, the pH value of described body fluid treating agent is between 5.5 ~ 7.5.
3. a kind of human body fluid fungi (1,3)-callose detection kit as claimed in claim 2, is characterized in that the pH value of alkaline dilution is between 10.5 ~ 13.5.
4. a kind of human body fluid fungi (1,3)-callose detection kit as claimed in claim 3, is characterized in that the pH value of acid dilution is between 2.0 ~ 4.0.
5. a kind of human body fluid fungi (1,3)-callose detection kit as claimed in claim 4, is characterized in that sterilized water is the double distilled water of water for injection.
6. a kind of human body fluid fungi (1,3)-callose detection kit as claimed in claim 5, is characterized in that freeze-dry blood serum is human serum albumin or calf serum.
7. a kind of human body fluid fungi (1,3)-callose detection kit as claimed in claim 1, is characterized in that the mensuration being mainly used in fungi (1,3)-callose content in human blood or other body fluid.
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| CN103214566A (en) * | 2013-03-30 | 2013-07-24 | 福州新北生化工业有限公司 | G factor separating and purifying method |
| CA2932192C (en) * | 2013-12-05 | 2020-10-06 | Biothera, Inc. | .beta.-glucan assay methods |
| 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 |
| CN106974876A (en) * | 2017-03-14 | 2017-07-25 | 天津喜诺生物医药有限公司 | A kind of method that eye care solution is prepared by horseshoe crab cell pyrolysis liquid |
| JP7110360B2 (en) | 2017-10-09 | 2022-08-01 | テルモ ビーシーティー バイオテクノロジーズ，エルエルシー | Freeze-drying method |
| CA3130700A1 (en) | 2019-03-14 | 2020-09-17 | Terumo Bct Biotechnologies, Llc | Lyophilization container fill fixture, system and method of use |
| CN115840038A (en) * | 2023-01-16 | 2023-03-24 | 郑州安图生物工程股份有限公司 | Method for detecting (1-3)-β-D-glucan content and its application |
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Application publication date: 20130109 Assignee: Zhanjiang Bokang ocean Biotechnology Co. Ltd. Assignor: Mo Shuijing|Mo Shiwen|Mo Shijun Contract record no.: 2015440000141 Denomination of invention: Human body liquid fungus (1,3) - beta -D glucan detection kit and application method thereof Granted publication date: 20141224 License type: Exclusive License Record date: 20150519 |
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