CN104531845A - Kit for detecting lysozyme - Google Patents
Kit for detecting lysozyme Download PDFInfo
- Publication number
- CN104531845A CN104531845A CN201410750845.2A CN201410750845A CN104531845A CN 104531845 A CN104531845 A CN 104531845A CN 201410750845 A CN201410750845 A CN 201410750845A CN 104531845 A CN104531845 A CN 104531845A
- Authority
- CN
- China
- Prior art keywords
- diacetylmuramidase
- test kit
- kit
- lysozyme
- detection
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Abstract
The invention discloses a kit for detecting lysozyme. The kit comprises the following main content: a method for detecting lysozyme based on a desoxyribonucleic acid circle amplification fluorescent signal, and a kit including a reagent required by the method. The main point of the detection method disclosed by the invention is a 'one-pot' mode; spontaneous desoxyribonucleic acid hybridization and desoxyribonucleic acid target (chain)-substituted polymerization reaction are triggered by aptamer recognition of the lysozyme; and a significant amplification effect is provided for detection of the lysozyme by virtue of a desoxyribonucleic acid circulating technology. The kit disclosed by the invention is composed of two signal probes, a primer, polymerase Klenow and a buffer solution NEBuffer. The detection method and the kit have the advantages of being simple to operate, high in sensitivity, and high in specificity on detection of the lysozyme.
Description
Technical field
The present invention relates to thymus nucleic acid (hereinafter referred to as " DNA ") circulation amplify technology, specifically based on DNA circulation amplifying technique, N,O-Diacetylmuramidase is carried out to test kit and the testing method of immunoassay detection.
Background technology
N,O-Diacetylmuramidase is also called N-acetylmuramide lycanohydrlase or muramidase, is a kind of alkaline enzyme that can be hydrolyzed mucopolysaccharide in pathogenic bacterium.Destroy cell walls mainly through the cell wall lipopolysaccharides be hydrolyzed in the cell walls of gram-positive microorganism, cause cell wall rupture content overflow and make bacterolysis.Further, N,O-Diacetylmuramidase directly can be combined with electronegative viral protein, forms double salt with DNA apoprotein, makes virally inactivated.Therefore, N,O-Diacetylmuramidase has antibacterial, anti-inflammatory, the effect such as antiviral.This enzyme is extensively present in the secretory product such as the egg white of birds and poultry, mammiferous saliva, tear, tracheae.N,O-Diacetylmuramidase is the important component part of the endogenous immune system of many animals tissue, and also different in its normal contents of different tissues place.The change of the lysozyme content in a lot of tissue can join with some disease-related usually.Such as, the abnormal disease meaning blood or kidney aspect of lysozyme concentration in the urine of blood; In neonate, N,O-Diacetylmuramidase lacks and may cause bronchial lung development obstacle; In recipe, N,O-Diacetylmuramidase lacks the generation that may increase and suffer from dysentery.Therefore, in a lot of medical diagnosis on disease, very important effect is played to the detection of N,O-Diacetylmuramidase in body.
DNA circulation amplifying technique is one of method often used in a lot of immunodetection, generally includes isothermal duplication, rolling-circle replication, hybridization chain reaction.Because its own signal amplifies, DNA circulation amplifying technique has and consumes few, quick, the sensitive advantage of sample size, is widely used in the detection of DNA, cell, biomolecules and embryo in recent years.Multiple reaction designing being become can the process of automatic circulating, thus signal repeatedly can be amplified, and this effectively will improve and reduce consumption to sample to the detectability of target compound simultaneously.Based on this, the suitable signal probe of design, primer, polysaccharase is selected to be crucial.Since Fleming in 1992 finds N,O-Diacetylmuramidase, the detection of N,O-Diacetylmuramidase in body is constantly carried out always, and has made some progress.But, simple to operate, there is high specific, the N,O-Diacetylmuramidase detection method of high sensitivity remains investigator pays close attention to and constantly to pursue.
Test kit of the present invention is designed to cause with the fit identification of N,O-Diacetylmuramidase, is replaced, polyreaction circulates the amplification realized signal, and then realize detection that is highly sensitive to N,O-Diacetylmuramidase and high specific selectivity by DNA hybridization, DNA chain (target).
Summary of the invention
The object of this invention is to provide a kind of test kit detecting N,O-Diacetylmuramidase, this test kit schedule of operation is simple, highly sensitive, specific selectivity is good, is applicable to the rapid detection of N,O-Diacetylmuramidase in urine, human serum.
The consisting of of test kit in the present invention: two bars probes, primer, polysaccharase Klenow, a damping fluid NEBuffer.
For achieving the above object, the technical solution used in the present invention is:
1, synthesising probing needle
1: design three oligodeoxynucleotide sequences (S1, S2, S3), S1 is for containing the fit sequence of N,O-Diacetylmuramidase and modifying tetramethylrhodamine fluorophor; S2 is a terminal modified amino, is convenient to be combined with magnetic bead, and its Sequence can be complementary with S1 and S3; S3 is then the sequence with S2 partial complementarity.Article three, Oligoribonucleotides sequence is combined by hybridization, and to be combined with the magnetic bead of carboxyl modified by the amino on S2 thus to define signal probe
1.
2, synthesising probing needle
2: design two oligodeoxynucleotide sequences (S4, S5), S4 is the hairpin structure of a terminal modified amino, is convenient to be combined with magnetic bead; S5 be a terminal modified tetramethylrhodamine fluorophor and its sequence can with the stem complementary of S4.Article two, Oligoribonucleotides sequence is combined by hybridization, and to be combined with the magnetic bead of carboxyl modified by the amino on S4 thus to define signal probe
2.
3, N,O-Diacetylmuramidase detects: N,O-Diacetylmuramidase, primer (S6) joins in the probe of above-mentioned synthesis, and first N,O-Diacetylmuramidase is caused by Complementary hybridization fit with it; Probe
1middle S1 and S2 hybridization is destroyed, and partial sequence fragment is exposed; By primer, under polysaccharase and dNTP effect, cause DNA polyreaction, the complex body of N,O-Diacetylmuramidase and S1 and S3 are replaced by the new chain that reaction extends, and are discharged in solution; Under polysaccharase and dNTP effect, cause second polyreaction, now N,O-Diacetylmuramidase is released, and can enter next reaction formation one circulation.The S3 replaced runs into signal probe
2, combined by hybridization; Initiated polymerization under primer, polysaccharase and dNTP effect, S3 is released in solution again, enters next reaction and forms another circulation.
4, fluorescence intensity test: carry out fluorometric investigation after getting above-mentioned reacted supernatant liquor dilution.
Principal innovative of the present invention and superiority are:
1, the present invention adopts the method for " treating different things alike ", and experimental implementation is simple.
2, the present invention causes with the fit identification of N,O-Diacetylmuramidase, can the detection N,O-Diacetylmuramidase of high specific selectivity.
3, fluorescent signal is amplified by two DNA circulation, can high-sensitive detection N,O-Diacetylmuramidase.
Accompanying drawing explanation
Fig. 1 is the schematic diagram of the present invention for fluoroscopic examination N,O-Diacetylmuramidase.
Fig. 2 A is fluorescence response curve; B is working curve.
Embodiment
Below for implementing concrete example of the present invention, its role is to illustrate content of the present invention further, reader is easier to understand, but the restriction do not formed the protection domain of application claims or restriction.
Experiment condition:
F-4500 Fluorescence spectrophotometer (Hitachi, Ltd), oligodeoxynucleotide sequence used (match Parkson, Beijing biotechnology company limited), (happy chromatographic technique development centre PSC-3412 is doubly thought in Tianjin to carboxyl modified magnetic microsphere, particle diameter 0.5-1.0 μm, stoste magnetic bead content 50 mg mL
-1), ethyl-(3-dimethyl propyl) carbodiimide hydrochloride (EDAC, Tianjin BASF Chemical Co., Ltd.), imidazoles (Tianjin Bo Di Chemical Co., Ltd.), the mixture (dNTP) (Dalian precious biotechnology company limited) of polysaccharase Klenow fragment, damping fluid and four kinds of deoxynucleotide triphosphoric acids, the composition of 10 × Klenow damping fluid: 100 mM Tris-HCl, 70 mL MgCl
2with 1 mM DTT, pH=7.5.
Specific operation process:
1, signal probe
1preparation, with 0.1M imidazole hydrochloride salts solution (pH 6.8) cleaning magnetic bead three times, magnetic bead is re-dispersed in imidazole hydrochloride salts solution and makes magnetic bead content reach 50 mg mL
-1, in the bead suspension after washing to 600 μ L, add 1 mL EDAC solution (0.1 M), priming reaction 60 min.The solution of 200 μ LS2 is joined 200 μ L imidazole solution (0.1 M, pH 6.8) priming reaction 30 min, then it is mixed with the magnetic bead activated, react 12 h, the solution of 200 μ L S1 and S3 is added after dilution, at 37 DEG C, hybridize 1 h, Magneto separate, solution dilution to 500 μ L is for subsequent use.
2, signal probe
2preparation, magnetic bead activation and signal probe
1in identical, the solution of 200 μ L S4 is joined 200 μ L imidazole solution (0.1 M, pH 6.8) priming reaction 30 min, then it is mixed with the magnetic bead activated, react 12 h, add the solution of 200 μ L S5 after dilution, at 37 DEG C, hybridize 1 h, Magneto separate, solution dilution to 500 μ L is for subsequent use.
3, circulating reaction experiment: 20 μ L(2 × 10
-7) signal probe
1suspension, 40 μ L(5 × 10
-7) signal probe
2suspension, 20 μ L S6(5.0 μM), 12 μ L Klenow polymerase buffer (10 ×), 10 μ L dNTP, 1 μ L polysaccharase (5.0 μ/mL), lysozyme soln 10 μ L to be measured, adds deionized water and forms 120 μ L reaction systems, at 37 DEG C of reaction 2 h.
4, fluorometric investigation: above-mentioned reaction is complete after Magneto separate, gets supernatant liquor and is diluted to 600 μ L and carries out fluorometric investigation.
Experimental result:
As shown in Figure 2 A, along with the rising of lysozyme concentration, fluorescence signal intensity increases.Mapped to concentration after the peak fluorescence button at 575 nm places goes blank signal by every bar curve, working curve is shown in shown in Fig. 2 B.When lysozyme concentration is 2.0 × 10
-13to 4.0 × 10
-11scope in, fluorescence response is linear with it, and regression equation is Δ F=3.24c+15.32(Δ F, a.u.; C, 10
-13m), according to 3 σ rules, the detection of this test kit to N,O-Diacetylmuramidase is limited to 9.2 × 10
-14m.
Claims (6)
1. for detecting a test kit for N,O-Diacetylmuramidase, it is characterized in that: the method for employing " treating different things alike " achieves the detection to N,O-Diacetylmuramidase high specific and high sensitivity.
2. according to the test kit for detecting N,O-Diacetylmuramidase according to claim 1, it is characterized in that: this test kit is the testing method based on thymus nucleic acid circulation amplify fluorescent signal.
3. according to the test kit for detecting N,O-Diacetylmuramidase according to claim 1, it is characterized in that: in test kit, comprise two signal probes, primer, polysaccharase and a buffered soln.
4. according to test kit according to claim 3, the composition characteristic of test kit is: two signal probes are made up of five kinds of oligonucleotides, have caused thymus nucleic acid circulating reaction by self.
5. a test kit for high-sensitive detection N,O-Diacetylmuramidase, is characterized in that: replace polyreaction by thymus nucleic acid hybridization, DNA chain, thymus nucleic acid target replacement polyreaction circulates the amplification achieved fluorescent signal.
6. high specific detects a test kit for N,O-Diacetylmuramidase, it is characterized in that: cause with the fit identification of N,O-Diacetylmuramidase, improve the Selective recognition of test kit to N,O-Diacetylmuramidase.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410750845.2A CN104531845A (en) | 2014-12-10 | 2014-12-10 | Kit for detecting lysozyme |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410750845.2A CN104531845A (en) | 2014-12-10 | 2014-12-10 | Kit for detecting lysozyme |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104531845A true CN104531845A (en) | 2015-04-22 |
Family
ID=52847464
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410750845.2A Pending CN104531845A (en) | 2014-12-10 | 2014-12-10 | Kit for detecting lysozyme |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104531845A (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103364353A (en) * | 2013-07-19 | 2013-10-23 | 广西师范大学 | Aptamer nanogold resonance Rayleigh scattering spectrum method for determination of lysozyme |
| CN103399005A (en) * | 2013-07-12 | 2013-11-20 | 青岛科技大学 | Method for determining lysozyme based on interaction between carboxylation carbon nanoparticles and DNA (Deoxyribose Nucleic Acid) |
-
2014
- 2014-12-10 CN CN201410750845.2A patent/CN104531845A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103399005A (en) * | 2013-07-12 | 2013-11-20 | 青岛科技大学 | Method for determining lysozyme based on interaction between carboxylation carbon nanoparticles and DNA (Deoxyribose Nucleic Acid) |
| CN103364353A (en) * | 2013-07-19 | 2013-10-23 | 广西师范大学 | Aptamer nanogold resonance Rayleigh scattering spectrum method for determination of lysozyme |
Non-Patent Citations (3)
| Title |
|---|
| 于兆涛: "整合适体识别和DNA循环反应放大网络的一体化高灵敏度溶菌酶传感", 《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》, no. 06, 15 June 2013 (2013-06-15) * |
| 张红鸽等: "以适体和金纳米颗粒为探针比色法检测溶菌酶", 《西北大学学报(自然科学版)》, vol. 4, no. 4, 31 August 2011 (2011-08-31), pages 617 - 622 * |
| 黄珊等: "CdSe量子点探针共振光散射法检测溶菌酶", 《高等学校化学学报》, vol. 30, no. 10, 31 October 2009 (2009-10-31), pages 1951 - 1955 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20240052400A1 (en) | Unimolecular aptamer-based sensors for pathogen detection | |
| JP2011511949A5 (en) | ||
| US20240018577A1 (en) | Method for detection of analytes via polymer complexes | |
| KR20210028277A (en) | Specific detection of deoxyribonucleic acid sequences using novel CRISPR enzyme-mediated detection strategies | |
| US20210285032A1 (en) | Compositions and methods for the detection of nucleic acids | |
| KR20220018036A (en) | Methods and reagents for amplification and/or detection of nucleic acids | |
| US9429575B2 (en) | DNA aptamer specifically binding to EN2 (engrailed-2) and use thereof | |
| CN116096884A (en) | Isothermal methods, compositions, kits and systems for detecting nucleic acids | |
| CN104611419B (en) | A kind of detection method based on graphene oxide chip DNA unwindase | |
| CN101215600B (en) | Detection probe and detection method for nucleic acid aim sequence | |
| ES2637489T3 (en) | HEV test | |
| WO2021149829A1 (en) | Method for detecting specific dna in sample | |
| CN104531845A (en) | Kit for detecting lysozyme | |
| Long et al. | Sensitive two-color whole-mount in situ hybridizations using digoxygenin-and dinitrophenol-labeled RNA probes | |
| JP6012085B2 (en) | Nucleic acid molecules that bind to Salmonella and uses thereof | |
| JP6642859B2 (en) | Cortisol analysis sensor, cortisol analysis method, stress evaluation reagent, stress evaluation method, test reagent for cortisol-related disease, and method for testing morbidity of cortisol-related disease | |
| KR101152354B1 (en) | Dna aptamer specifically binding to anthrax toxins and uses thereof | |
| KR20190031705A (en) | DNA aptamer specifically binding to Avian influenza virus and uses thereof | |
| CN103215389A (en) | Porcine reproductive and respiratory syndrome and porcine Japanese B encephalitis dual one-step RT-PCR (Reverse Transcription-Polymerase Chain Reaction) diagnosis kit | |
| WO2017189878A1 (en) | Compositions and methods for the detection of nucleic acids | |
| Wang et al. | Visual and label-free ASFV and PCV2 detection by CRISPR-Cas12a combined with G-quadruplex | |
| KR101971474B1 (en) | Nucleotide Aptamer for Use in Detection of benzylpenicillin | |
| CN104136611B (en) | The detection method of nucleic acid | |
| JP6598315B2 (en) | Nucleic acid molecules that bind to wheat allergens and uses thereof | |
| JP5888470B2 (en) | Method for measuring telomerase activity |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20150422 |
|
| RJ01 | Rejection of invention patent application after publication |