CN109490548A - A kind of cirrhosis detection reagent and its application in cirrhosis detection - Google Patents
A kind of cirrhosis detection reagent and its application in cirrhosis detection Download PDFInfo
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- CN109490548A CN109490548A CN201910038780.1A CN201910038780A CN109490548A CN 109490548 A CN109490548 A CN 109490548A CN 201910038780 A CN201910038780 A CN 201910038780A CN 109490548 A CN109490548 A CN 109490548A
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- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/08—Hepato-biliairy disorders other than hepatitis
- G01N2800/085—Liver diseases, e.g. portal hypertension, fibrosis, cirrhosis, bilirubin
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Abstract
The present invention provides a kind of cirrhosis detection reagents, including C1: being not higher than 2.0%SDS, pH 7.5-8.0；C2: 20mM APTS and the 1M NaCNBH3 of same volume are mixed；The glycosidase of C3: concentration 2 ~ 5U/ μ L and 3 ~ 4% NP-40 1:20 mixed preparing by volume；C4:100mM NH4AC, 1 ~ 2mU/ μ L sialidase and hydrogen peroxide are mixed according to volume 5:1:14；C5: buffer.The present invention also provides a kind of application of composition in cirrhosis detection reagent, the composition is made of NGA2F, NA2, NA3, and the composition passes through NGA2F/(NA2+NA3) ratio detect cirrhosis.The present invention, which uses, has high sensitivity, N- oligonucleotide chain detection method easy to operate, reproducible, stability is good and high-throughput, and accuracy reaches 84.7%, compared with prior art, has higher accuracy.
Description
Technical field
The invention belongs to biomedicine technical fields, and in particular to a kind of cirrhosis detection reagent and its detect in cirrhosis
In application.
Background technique
Cirrhosis (cirrhosis) is clinical common chronic progressive hepatopathy, is since one or more kinds of causes of disease are long
Phase or repeated action and the diffusivity hepatic lesion formed.It is now recognized that causing cirrhosis front three most important the reason is that wine
Essence, hepatitis type B virus (hepatitis B virus, HBV) and infection with hepatitis C virus (hepatitis C virus,
HCV).In global range, 57% cirrhosis be by HBV (30%) and HCV(27%) caused by, in developing country, cirrhosis master
If as caused by HBV.
Needed in cirrhosis clinical diagnostic process consider include clinical manifestation, laboratory inspection, histology, iconography and
The many foundations of histopathology.Liver impedance rheograph can provide the important information of Liver Fibrosis Stages, but there are one for liver impedance rheograph
Determine risk, and the degree of fibrosis of the different surely comprehensive reflection liver entirety of single liver impedance rheograph sample, thus, it sends out in recent years
It has opened up multinomial non-wound diagnostic techniques to be used to assess liver fibrosis, Imaging Technology, serologic marker including measuring liver hardness
With all kinds of points-scoring systems etc..Before serum biochemistry direct indicator mainly includes hyaluronic acid (HA), laminin (LN), type III
Collagen peptide and IV Collagen Type VI etc., indirect indexes mainly include platelet count (PLT), r- globulin, prothrombin time (PT),
Glutamic-pyruvic transaminase, bilirubin and carrier protein A1 etc., but These parameters diagnosis or detection cirrhosis lack specificity.Respectively
Kind common iconography means such as Type B ultrasound, CT, magnetic resonance imaging (MRI) etc. can be found that Glisson's capsule thicken, liver surface profile
Irregularly, the even enhancing of hepatic parenchymal Echo heterogenicity and CT value increase or change in nodositas, each leaf ratio, thickness of spleen increases
And the sign of portal vein and splenic vein diameter broadening equal cirrhosis and portal hypertension.Color Doppler ultrasonography or radioactive nucleus
Element scanning can measure the blood flow and functionality door body tapping condition of liver artery and portal vein.
Although many research discovery liver ultrasound sxemiquantitative marking have good correlation with hepatic tissue classification, right
Early-phase hepatocirrhosis is not sensitive enough, is difficult to quantification for the diagnosis of liver fibrosis.The measurement of liver elastic force mainly includes instantaneous elasticity
Wave scans (Fibroscanh or Fibrotouch).The advantages of such technology is can to measure bigger range even entire liver
Elastic situation, compensate for sampling error existing for liver biopsy to a certain extent；The disadvantage is that Liver fatty deposition,
Situations such as ascites, peritoneal inflammation, can influence testing result to a certain degree.Such technology still cannot distinguish between adjacent liver fiber by stages, but
There is certain Clinical significance of MG for diagnosis Compensated cirrhosis.Currently on the market without the reagent for being exclusively used in detection cirrhosis
Box is clinically diagnosed using some indexs of detection liver function and in conjunction with iconography mostly.
Summary of the invention
The ingredient of glycoprotein glycan is by complicated mutual between hundreds of enzyme, transcription factor, ion channel and albumen
Effect and formed, glycan participate in different kinds of molecules process, such as protein folding, cell adherence, molecule transfer, signal transduction, adjusting
Receptor active etc.；Therefore the change of glycan is closely related with disease.
Find by long-term research, the serum of liver cirrhosis patient and normal person in comparison, N- oligosaccharide NGA2F(a
Galactosylated, core-a-1,6-fucosylated biantennary glycan), NA2(a
Bigalactosylated, core-a-1,6-fucosylated biantennary) and NA3(a
Trigalactosylated, tri-antennary) content change significantly, so as to the mark for detecting cirrhosis
Will object.
Aiming at the problem that in the presence of the detection of existing cirrhosis, such as blood test and iconography detection specificity and standard
Exactness is not high, pathologic finding have it is traumatic, sampling have error, patient's interdependence difference etc.；The present invention provides a kind of apply in liver
Cirrhosis detection reagent in hardening detection.
The technical solution adopted by the invention is as follows:
A kind of cirrhosis detection reagent of the invention, including C1: 2.0% SDS, pH 7.5-8.0 are not higher than；C2: mixing is androgynous
The NaCNBH3 of the APTS and 1M of long-pending 20mM；The glycosidase of C3: concentration 2 ~ 5U/ μ L and 3 ~ 4% NP-40 1:20 by volume
Mixed preparing；NH4AC, 1 ~ 2mU/ μ L sialidase and the hydrogen peroxide of C4:100mM is mixed according to volume 5:1:14；C5: buffering
Liquid.
It is 2 μ L, C3 volumes be 2 μ L, C4 volumes be 2 μ L, C5 volumes is 200 μ that the volume of the C1, which is the volume of 2 μ L, C2,
L。
Reagent preparation:
C1: it is configured to containing the solution for being 7.5 ~ 8.0 not higher than 2% SDS, pH.
C2: the NaCNBH of the APTS and 1M of the 20mM of same volume are mixed3。
The glycosidase of C3: concentration 2 ~ 5U/ μ L and 3 ~ 4% NP-40 1:20 mixed preparing by volume.
The NH of C4:100mM4AC, sialidase (neuraminidase, 1 ~ 2mU/ μ L) and hydrogen peroxide are according to volume 5:
1:14 mixing.
C5: buffer.
The present invention also provides a kind of application of composition in cirrhosis detection reagent, and the composition is by NGA2F, NA2
It is formed with NA3, the composition detects cirrhosis by NGA2F/(NA2+NA3) ratio.
The detection of N- sugar group map:
1. preparation and the label of oligonucleotide chain: taking the 2 μ L of serum inactivated at 90 ~ 95 DEG C, the C1 of 2 μ L is added, mix and place
5min, the C2 that 2 μ L are added are marked, are dried after 37 DEG C of reaction 3h.
2. the label of oligonucleotide chain post-processes: the C3 of 2 μ L is added in sample after the drying, not less than 65 DEG C at react 3h
Afterwards, the C5 that 200 μ L are added terminates reaction.
3. subsequent processing: taking above-mentioned 2 μ L of sample, the C4 of 2 μ L is added, reacts 3h at 45 DEG C, finally adds 200 μ L's
C5 terminates reaction.
4. oligosaccharides chain separation is analyzed: taking 10 μ L of fluid sample 3., N- oligonucleotide chain point is carried out under ABI3500dx instrument
From to obtain N- sugar group map.
5. Data Management Analysis: obtained N- sugar group map being carried out peak value quantization, passes through function NGA2F/(NA2+
NA3 further statistical analysis is carried out) to detect cirrhosis.
Beneficial effects of the present invention:
(1) present invention is using with high sensitivity, N- oligonucleotide chain easy to operate, reproducible, stability is good and high-throughput
Detection method obtains glycan NGA2F, the NA2 in experimenter's serum according to the N- oligonucleotide chain map of the experimenter's serum of foundation,
NA3 peak value, by function NGA2F/(NA2+NA3) to 959 samples (wherein 480, cirrhosis sample, non-cirrhosis sample
479) it is detected.The results show that the accuracy of this detection method reaches 84.7%, compared with prior art, there is higher standard
Exactness.
(2) this detection method is used, numerous liver cirrhosis patients can be allowed to receive conventional, Non-invasive detection, to help doctor
And patient detects the generation of cirrhosis and the progress of the state of an illness in time, is expected to promote the use of in clinic.
Detailed description of the invention
Fig. 1 is the N- oligonucleotide chain map of cirrhosis serum sample；
Fig. 2 is the N- oligonucleotide chain map of healthy control group serum sample；
Fig. 3 be detect sample pass through NGA2F/(NA2+NA3) made by ROC indicatrix；Detecting total sample number is 959,
Wherein 480, cirrhosis sample, 479, healthy control group sample；Area under the curve AUC=0.847.
Specific embodiment
This kit is described in further detail combined with specific embodiments below.It should be noted that the following example is only used for
Illustrate rather than limits the scope of the invention.Test method without specific conditions in following embodiment, usually according to normal
Condition test, or the condition suggested according to manufacturer are advised, reagent is all that cell culture is dedicated.
Embodiment one:
(1) experimental facilities:
ABI3500dx analyzer (Applied Biosystems), PCR, centrifuge
(2) prepared by reagent:
C1: it is configured to containing the solution for being 7.5 ~ 8.0 not higher than 2% SDS, pH.
C2: the NaCNBH of the APTS and 1M of the 20mM of same volume are mixed3。
The glycosidase of C3: concentration 2 ~ 5U/ μ L and 3 ~ 4% NP-40 1:20 mixed preparing by volume.
The NH of C4:100mM4AC, sialidase (neuraminidase, 1 ~ 2mU/ μ L) and hydrogen peroxide
It is mixed according to volume 5:1:14.
C5: buffer.
(3) N- sugar group map detects
1. preparation and the label of oligonucleotide chain: taking the 2 μ L of serum inactivated at 90 ~ 95 DEG C, the C1 of 2 μ L is added, mix and place
5min, the C2 that 2 μ L are added are marked, are dried after 37 DEG C of reaction 3h.
2. the label of oligonucleotide chain post-processes: the C3 of 2 μ L is added in sample after the drying, not less than 65 DEG C at react 3h
Afterwards, the C5 that 200 μ L are added terminates reaction.
3. subsequent processing: taking above-mentioned 2 μ L of sample, the C4 of 2 μ L is added, reacts 3h at 45 DEG C, finally adds 200 μ L's
C5 terminates reaction.
4. oligosaccharides chain separation is analyzed: taking 10 μ L of fluid sample 3., N- oligonucleotide chain point is carried out under ABI3500dx instrument
From to obtain N- sugar group map.
Detection sample pass through NGA2F/(NA2+NA3) made by ROC indicatrix；Detecting total sample number is 959,
480, middle cirrhosis sample, 479, healthy control group sample；Area under the curve AUC=0.847.
The results show that the accuracy of this detection method reaches 84.7%, compared with prior art, there is higher accuracy.It adopts
With detection method, numerous liver cirrhosis patients can be allowed to receive conventional, Non-invasive detection, thus help doctor and patient and
When detection cirrhosis generation and the state of an illness progress, be expected to promote the use of in clinic.
The specific embodiment in conjunction with described in attached drawing above carries out the purpose of the present invention, technical scheme and beneficial effects
It is further described, it should be understood that the above is only a specific embodiment of the present invention, but not to the present invention
The limitation of protection scope, those skilled in the art should understand that, all within the spirits and principles of the present invention, do not need to pay
Any modification, equivalent substitution, improvement and etc. that creative work can be made, should all be included in the protection scope of the present invention.
Claims (3)
1. a kind of cirrhosis detection reagent, it is characterised in that: including C1: being not higher than 2.0% SDS, pH 7.5-8.0；C2: mixing
The NaCNBH3 of the APTS and 1M of the 20mM of same volume；The glycosidase of C3: concentration 2 ~ 5U/ μ L and 3 ~ 4% NP-40 by volume 1:
20 mixed preparings；NH4AC, 1 ~ 2mU/ μ L sialidase and the hydrogen peroxide of C4:100mM is mixed according to volume 5:1:14；C5: buffering
Liquid.
2. cirrhosis detection reagent according to claim 1, it is characterised in that: the volume of the C1 is the volume of 2 μ L, C2
It is 2 μ L, C4 volumes for 2 μ L, C3 volumes be 2 μ L, C5 volumes is 200 μ L.
3. a kind of application of composition in cirrhosis detection reagent, the composition is made of NGA2F, NA2 and NA3, described
Composition detects cirrhosis by NGA2F/(NA2+NA3) ratio.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2018116332876 | 2018-12-29 | ||
| CN201811633287 | 2018-12-29 |
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| Publication Number | Publication Date |
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| CN109490548A true CN109490548A (en) | 2019-03-19 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114032282A (en) * | 2021-09-15 | 2022-02-11 | 陈翠英 | Prostate cancer detection reagent and application thereof in prostate cancer detection |
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| CN1759320A (en) * | 2003-01-14 | 2006-04-12 | Vib研究所 | A serum marker for measuring liver fibrosis |
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| CN109100507A (en) * | 2017-06-20 | 2018-12-28 | 江苏先思达生物科技有限公司 | The method for building up of the seroglycoid N- sugar group spectrum model of chronic hepatitis hepatic injury |
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2019
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| CN1662817A (en) * | 2002-04-16 | 2005-08-31 | 福拉姆斯大学生物技术研究所 | Marker for measuring liver cirrhosis |
| CN1759320A (en) * | 2003-01-14 | 2006-04-12 | Vib研究所 | A serum marker for measuring liver fibrosis |
| CN109100410A (en) * | 2017-06-20 | 2018-12-28 | 江苏先思达生物科技有限公司 | The method for building up of the seroglycoid N- sugar group spectrum model of cirrhosis |
| CN109100507A (en) * | 2017-06-20 | 2018-12-28 | 江苏先思达生物科技有限公司 | The method for building up of the seroglycoid N- sugar group spectrum model of chronic hepatitis hepatic injury |
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| 房萌: "肝纤维化、肝癌的N-糖组学动态研究。", 《中国博士学位论文全文数据库 医药卫生科技辑》 * |
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| CN114032282A (en) * | 2021-09-15 | 2022-02-11 | 陈翠英 | Prostate cancer detection reagent and application thereof in prostate cancer detection |
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Effective date of registration: 20200206 Address after: 225312 Jiangsu city of Taizhou province China pharmaceutical Road East, Xinyang City Road on the north side of building six on the eastern side of G26 Applicant after: JIANGSU XIANSIDA BIOTECHNOLOGY Co.,Ltd. Applicant after: XIANSIDA NANJING BIOTECHNOLOGY Co.,Ltd. Address before: 225312 Jiangsu city of Taizhou province China pharmaceutical Road East, Xinyang City Road on the north side of building six on the eastern side of G26 Applicant before: JIANGSU XIANSIDA BIOTECHNOLOGY Co.,Ltd. Applicant before: XIANSIDA NANJING BIOTECHNOLOGY Co.,Ltd. Applicant before: CHANGZHOU JITAI BIOLOGY TECH Co.,Ltd. |
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Inventor after: Chen Cuiying Inventor after: Tan Zongnan Inventor after: Zhu Hongzhang Inventor after: Zhang Fanghong Inventor after: Liu Xueen Inventor after: Zhuang Hui Inventor before: Chen Cuiying Inventor before: Tan Zongnan Inventor before: Zhu Hongzhang Inventor before: Zhang Fanghong Inventor before: Liu Xueen Inventor before: Zhuang Hui |
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Application publication date: 20190319 |
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