CN112626198A

CN112626198A - Molecular marker for liver disease severe treatment and application thereof

Info

Publication number: CN112626198A
Application number: CN202011564730.6A
Authority: CN
Inventors: 杨劲; 施军平
Original assignee: AFFILIATED HOSPITAL OF HANGZHOU NORMAL UNIVERSITY
Current assignee: AFFILIATED HOSPITAL OF HANGZHOU NORMAL UNIVERSITY
Priority date: 2020-12-25
Filing date: 2020-12-25
Publication date: 2021-04-09

Abstract

A liver disease severe biochemical marker and application. The liver disease severity biomarker comprises at least one of three genes of EFEMP1, THBS2 and LUM. The biomarker has obvious correlation with hepatic fibrosis progression, and can be used for preparing auxiliary diagnosis or prognosis preparations for hepatic fibrosis. The biomarker preparation comprises quantitative PCR primers and reagents and a using method thereof, can screen and judge the liver disease fibrosis degree, and provides an effective basis for clinical individualized intervention and treatment.

Description

Molecular marker for liver disease severe treatment and application thereof

Technical Field

The invention relates to the technical field of liver disease diagnosis, and relates to a biomarker for hepatic fibrosis progression and application thereof. In particular, the invention relates to EFEMP1, THBS2 and LUM genes and application of the genes in auxiliary diagnosis or prognosis of liver disease fibrosis.

Background

Various causes of the current liver disease spectrum comprise viral hepatitis, alcoholic liver disease, metabolic liver disease, drug-induced liver disease, hereditary liver disease and the like, and hepatic fibrosis is a key node for the progress of serious diseases in the natural history of a plurality of liver diseases. If liver fibrosis continues to develop to the middle and late stage, liver cirrhosis appears, and the morphology has the manifestations of fibrosis diffusion, liver lobular structure destruction with abnormal nodule formation, portal hypertension and liver function failure caused clinically. In the course of the disease, early hepatic fibrosis can be blocked or reversed by timely intervention, while the late hepatic fibrosis is irreversible, which seriously affects the quality of life and the life of the patient.

With the progress of socio-economic, non-alcoholic fatty liver disease (fatty liver, NAFLD for short) has become one of the main constituents of contemporary liver disease. The prevalence rate of NAFLD of common western people is 20-30%, and the prevalence rate of Asian people is 5-18%. Although most simple fatty liver is benign, 25% of patients can progress to steatohepatitis (NASH) and to end-stage liver diseases such as cirrhosis, liver failure and liver cancer in severe cases.

As a pathological diagnosis of gold criteria, NAFLD pathology grading is usually used such as NAFLD activity score scoring system (NAS), with emphasis on the scoring of ballooning, adiposity, and lobular inflammation; quantitative assessment of GS is often used to assess inflammation and fibrosis of liver diseases such as NAFLD, but the assessment of both is not always consistent. In clinical phenotype, although most NAFLD patients do not develop clinically significant liver disease, some patients may rapidly progress to cirrhosis, liver cancer. For NASH confirmed patients, the severity of NASH is often associated with the stage of fibrosis, with 40-50% of NASH patients expected to progress to cirrhosis, liver cancer. When severe fibrosis occurs, NASH no longer has prognostic significance. In contrast, liver biopsy with progression of fibrosis stage, especially with significant (F ≧ 2) fibrosis, is an independent predictor of liver disease-related mortality. Therefore, early warning of patients with a tendency to become critically ill from a fibrosis perspective is a strong indicator of the management of many liver diseases such as NAFLD.

Currently, the differentiation of hepatic fibrosis depends on: 1) histopathological examination; 2) serological diagnosis of liver fibrosis; 3) and (5) imaging diagnosis. Liver biopsy has its obvious limitations of invasiveness, such as: inhomogeneity of the lesion may cause sampling errors; is difficult to be repeatedly carried out in the same patient, and is inconvenient for observing the dynamic change of the hepatic fibrosis. Four items of hepatic fibrosis, such as serum type III procollagen amino-terminal peptide (PIIINP), type IV collagen (IV-Col), Laminin (LN), Hyaluronic Acid (HA), are commonly used in serological diagnosis. Four liver fibrosis items are greatly affected by liver inflammation, and are also abnormal in other organ diseases such as partial malignant tumor, connective tissue disease, hyperthyroidism, diabetes, etc., and thus are not highly specific. Various commonly used imaging modalities such as B-mode ultrasound, CT, magnetic resonance imaging, etc. have clear characterization of late stage cirrhosis and blurred progression of early stage fibrosis and are machine-limited. Therefore, there is an urgent need for a novel liver fibrosis indication that is simple, convenient, and highly accurate.

For the expression detection of biomarkers, real-time quantitative pcr (qpcr) is the mainstream of nucleic acid detection at present. Wherein the chimeric dye is as

Green 1 or

Is a more economical detection signal. The chimeric dyes such as SYBR have the characteristic of non-sequence-dependent combination, and are not suitable for multiple detection. Sequence-specific hybridization probes such as hydrolysis probes (TaqMan), Molecular beacons (Molecular Beacon), etc. have been developed in succession. All of these probes need to be designed and optimized for each target of detection. Few sequence-specific hybridization probes are currently reported that are more than 2-fold reactive.

Nucleases have been discovered for more than a decade to date, including dnases or rnases. The sequence construct of the nuclease comprises an enzymatically active domain and a substrate recognition domain. Nucleases, in the presence of their substrates, catalyze reactions such as cleavage, ligation, phosphorylation, deglycosylation of RNA or DNA substrates. It is known that the number of naturally occurring ribozymes is growing and that by means of directed molecular evolution in vitro, there is the potential to generate nuclease molecules of specific functional characteristics. For example, an artificial DNase having catalytic function is isolated from a library containing random sequences. Meanwhile, the discovery of the division of the DNase functional domain expands the specific application of the DNase. For example, E6 DNAzymes, as tested by Kolpashchikov, contain non-conserved stem-loop structures and assemble into nuclease structures only when hybridized to a template. Mokany et al used nuclease structures for MNAzyme quantitative PCR reactions. This concept can facilitate the design of generic reporter probes, and then assemble simple and efficient multiplex qPCR reaction schemes.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a biomarker for liver disease intensification, namely liver fibrosis progression and application thereof.

A kind of biological marker is provided, which is a biological marker,

(1) the 3 genes selected from the following group include at least one of the following: LUM (Genbank accession NM-002345); THBS2(NM — 003247); EFEMP1 (NM-001039349).

(2) The products of said at least 3 genes. Preferably said 3 genes. The biomarker can be applied to preparation of liver disease fibrosis detection reagents.

The reagent for detecting the expression levels of the EFEMP1, the THBS2 and the LUM genes comprises a reagent for detecting the expression levels of the EFEMP1, the THBS2, the LUM genes or proteins. The reagent comprises a nucleic acid (e.g. primers and probes) capable of binding to EFEMP1, THBS2, the LUM gene or a substance (e.g. antibody) capable of binding to EFEMP1, THBS2, the LUM protein. The nucleic acid can detect the expression level of EFEMP1, THBS2 and LUM genes; the substance can detect the expression level of EFEMP1, THBS2 and LUM protein.

Detection of the expression of EFEMP1, THBS2, LUM genes can exert their function based on known methods using nucleic acid molecules: such as Polymerase Chain Reaction (PCR), isothermal amplification, nucleic acid hybridization, DNA chips, sequencing, and the like.

Further, the PCR method is a known method such as RT-PCR, multiplex PCR, quantitative PCR and the like. The amplification product can be detected by electrophoresis or fluorescent probe.

The invention provides a quantitative PCR detection method based on a dye method, wherein the sequence of a primer is shown as SEQ ID NO. 1-6. The primer can be prepared by chemical synthesis, appropriately designed and prepared by referring to known information using a method known to those skilled in the art.

Quantitative PCR amplification using primers shown in SEQ ID NO.1-6 enabled significant differentiation between insignificant (F <2) and significant (F ≧ 2) fibrosis based on the expression levels of EFEMP1, THBS2, and LUM genes. According to the findings of the present invention, primers for amplifying EFEMP1, THBS2, LUM genes are not limited to SEQ ID nos. 1 to 6, however, the inventors found that the primers of SEQ ID nos. 1 to 6 shown by EFEMP1, THBS2, LUM have a remarkably excellent effect on distinguishing insignificant from significant fibrosis, and thus are preferable primer pairs.

The invention also provides a universal report probe detection method based on the MNAzyme nuclease principle, which is used for detecting EFEMP1, THBS2 and LUM, and comprises a cooperative probe and a report probe in a combined probe mode, wherein the report probe can be universal and can be used for quickly, simply, highly sensitively and specifically quantitatively detecting nucleic acid.

The universal fluorescent combined Probe comprises 1 Report Probe (RP) and 2 Cooperative Probes (CP). The two ends of the cooperative probe are respectively provided with a target molecule binding sequence and a substrate molecule binding sequence, and the middle part of the cooperative probe contains a nuclease catalysis functional domain so as to realize the site-specific shearing of a specific DNA or RNA basic group. The two ends of the report probe are provided with cooperative probe combination sequences, and the middle of the report probe is provided with a specific DNA enzyme cutting basic group. Two ends of the report probe are respectively marked with a fluorescence emission group or a quenching group. Because the reporter probe is independent of the target molecule, the reporter probe can be designed universally.

In the nuclease-mediated real-time PCR reaction, the partial sequences at the two ends of the 2 cooperative probes are specifically and complementarily combined with the corresponding sequences of the reporter probes. When partial sequences of the 2 cooperative probes are specifically combined with corresponding sequences of target molecules, the intermediate sequences of the cooperative probes form a DNA enzyme structure, the specific sites of the report probes are cut, and fluorescence emission groups or quenching groups are separated to obtain fluorescence detection signals. In the PCR reaction process, the reaction is continuously promoted to occur and circulate along with the formation of a new chain, and the reaction can be monitored in real time through the accumulation of fluorescent signals, so that the target molecules are qualitatively or quantitatively detected.

In particular, the method comprises a detection method based on a universal fluorescence combined probe. The target molecule defines an F3 region, an F2 region, a B2 region and a B3 region in this order from the 5 'end to the 3' end, and defines an F3c region, an F2c region, a B2c region and a B3c region in this order from the 3 'end to the 5' end. The region broadly refers to an oligonucleotide fragment. The regions F3 and F3c, F2 and F2c, B2 and B2c, or B3 and B3c, etc. refer to the two segments with reverse complementarity. This region of the oligonucleotide fragment is synonymous with the primer.

In the method for designing the cooperative probe, the cooperative probe 1(CP1) has a F1 region, a C1 region and an S1 region defined in this order from the 5 'end to the 3' end. The cooperative probe 2(CP2) was defined by a region S2, a region C2 and a region B2 in this order from the 5 'end to the 3' end. The method for designing the report probe is characterized in that when the S1 fragment of CP1 is complementarily combined with the S1C region of RP, the F2 fragment of CP1 is complementarily combined with the F2C region of a target molecule, the B2 fragment of CP2 is complementarily combined with the B2C region of the target molecule, and the S2 fragment of CP2 is complementarily combined with the S2C region of RP, the C1 fragment of CP1 and the C2 region of CP2 form a nuclease structure, and the substrate base in the RP is cut.

The method has the advantages that the report probe is fixed and generalized, and only different primers and cooperative probes need to be designed for nucleic acid detection of different genes without adjusting the report probe, so that the quantitative PCR by the probe method is more economical and practical. Meanwhile, different report probes are designed to enable single-tube multiplex detection to be possible, and the design of a universal report probe enables the optimization of multiplex reaction to be simple and convenient.

An agent for detecting liver fibrosis progression, monitoring treatment progress, or producing or screening a medicament for detecting said biomarker.

The invention has the beneficial effects that: the hepatic fibrosis gene marker (biomarker) according to the invention can be used for producing diagnostic kits, therapeutic drugs and the like; can effectively monitor the liver disease patients with the susceptibility to severe progression and evaluate the treatment effect of the liver fibrosis.

Drawings

FIG. 1 relative quantitative determination of EFEMP1, THBS2, LUM mRNA in clinical patients.

FIG. 2 nuclease-mediated probe method quantitative PCR principle; as shown, the cooperative probes CP1 and CP2 both contained a target template binding region (F2 or B2 sequences), a partial catalytic core (C1 or C2 sequences), and a reporter probe substrate (S1 or S2 sequences) binding region. When not assembled, the catalytic domain structure on the cooperative probe CP is inactive; when PCR is carried out, CP1 and CP2 are hybridized with a template and a substrate to assemble a catalytic domain structure with complete activity. Cleavage of the reporter probe RP and separation of the fluorophore and quencher can generate a signal in real time.

FIG. 3 multiplex PCR principle using combination probes; the reporter probe sequence is fixed, and the cooperative probe CP1 is fixed with the catalytic core of CP2 and the substrate (reporter probe) binding region. When facing different targets, only different target template binding regions need to be designed. In multiple reactions, a universal report probe marked with a unique fluorescent group is selected, and a cooperative probe is designed to correctly combine the report probe and a template.

FIG. 4 Absolute quantitative determination of LUM mRNA.

FIG. 5 absolute quantitative detection of THBS2 mRNA.

FIG. 6 Absolute quantitative measurement of EFEMP1 mRNA.

FIG. 7 Absolute quantitative measurements of EFEMP1, THBS2, LUM mRNA in clinical patients.

Detailed Description

The technical solutions of the present invention are described below with specific examples, but the technical solutions of the present invention are not limited thereto. It will be understood by those skilled in the art that the present invention may be modified and equivalents may be made without departing from the spirit and scope of the invention in any way or by partial replacement thereof.

Example 1: screening of biomarkers for liver fibrosis

In GEO and Arrayexpress database, NAFLD liver fibrosis is used as key word to search fatty liver related liver fibrosis gene chip data, and the data requirement is as follows:

1) the selected dataset must be whole genome expression profile data;

2) the selected dataset contains fibrosis versus control expression profiles; all samples were from human liver tissue.

3) The selected data set must comprise more than 4 samples.

The GSE49541 dataset was first selected as the exploration cohort with clear fibrosis staging because it contains the most NAFLD samples. The data of the verification queue is sourced from E-MEXP-3291, GSE48452 and GSE 59045; the other 5 data sets were fatty liver samples, but without clinical information annotation such as fibrosis (GSE17470, GSE24807, GSE37031, GSE46300 and GSE 63067). The positive control used the HBV-associated liver fibrosis GSE84044 dataset, containing 124 samples with different fibrosis stages. Finally, a total of 10 data sets were included in our study, as shown in table 1.

TABLE 1 basic cases of the complete genome data set

A gene co-expression model was constructed using a weighted gene co-expression network analysis (WGCNA) approach. Through the correlation with the fibrosis character, the fibrosis gene module is found in the exploration queue and is repeatedly embodied in all the verification queues.

Gene importance (GS) and intra-module connectivity (k.in) are discriminatory elements of key genes within the fibrosis module. Three core genes (EFEMP1, THBS2, LUM) were identified based on discrimination between k.in and GS. Meanwhile, EFEMP1, THBS2 and LUM in each validation queue have significant differential expression compared with a control group. Therefore, EFEMP1, THBS2 and LUM are important genes in the progress of fibrosis of liver diseases such as NAFLD and HBV.

Example 2 dye-based PCR quantitative detection of EFEMP1, THBS2, LUM

Study subjects: 20 patients with fatty liver disease, fatty liver diagnosis in accordance with Chinese non-alcoholic fatty liver disease diagnosis and treatment guidelines (revised 2010). The tissue specimens after liver puncture were collected in the affiliated hospital of Hangzhou university. Liver penetrating tissues were routinely HE stained and scored for fibrosis stage by a professional pathologist. Wherein the fibrosis stage is 12 cases in 0-1 stage, 8 cases in 2-4 stage.

The experimental method comprises the following steps:

RNA extraction

1) Approximately 50mg of liver tissue was placed in a 1.5ml EP tube containing a small steel ball, 1ml Trizol solution was added, and the mixture was placed in a grinder and quickly ground. Centrifuging at 12000r/min and 4 deg.C for 5min, centrifuging, and standing at room temperature for 5 min.

2) A separation stage: transferring the supernatant to a new EP tube, adding 200ul chloroform into the EP tube, mixing well, standing at room temperature for 10min, and centrifuging at 12000r/min for 15 min.

3) RNA precipitation: sucking the supernatant into a new EP tube, adding 500ul isopropanol, shaking and mixing uniformly, standing at room temperature for 5min, and centrifuging at 12000r/min for 10min again.

4) Elution of RNA: carefully pour off the supernatant without touching the pellet, add 1mL of 75% ethanol which was chilled in situ, wash the tube walls gently upside down, and centrifuge at 12000r/min for 5 min.

5) Re-solubilization of RNA: the supernatant was carefully aspirated off, the pellet air dried, and 500ul of DEPC water was added to the tube to dissolve.

6) Determination of RNA concentration: RNA with OD260/280 values in the range of 1.80-2.00 was acceptable.

2.qPCR

The GAPDH gene serves as an internal control.

LUM

A forward primer: 5'-CTCTCTTCTAACTCTCTACTTA-3' (SEQ ID NO: 1)

Reverse primer: 5'-GGGATGACACATTGAAAG-3' (SEQ ID NO: 2)

THBS2

A forward primer: 5'-CAACCTCAATCTGGTCTG-3' (SEQ ID NO: 3)

Reverse primer: 5'-GCAGTTGTCCTTCTCATC-3' (SEQ ID NO: 4)

EFEMP1

A forward primer: 5'-ATGCCAACACCATCAATA-3' (SEQ ID NO: 5)

Reverse primer: 5'-TCCCTATACTGCTGACTG-3' (SEQ ID NO: 6)

GAPDH

A forward primer: 5'-AGAAGGCTGCCGCTCATTTG-3' (SEQ ID NO: 7)

Reverse primer: 5'-AGGGGCCATCCACAGTCTTC-3' (SEQ ID NO: 8)

TABLE 2 qRT-PCR reaction preparation

The reaction conditions are as follows: 30min at 50 ℃; 95 ℃ for 2 min. Then 40 cycles at 95 ℃ for 10sec, 60 ℃ for 30 sec. After the reaction is finished, the expression value in each reaction tube is calculated by a 2-delta-delta-ct method. The metric data are expressed as mean ± sd, the comparisons between groups were performed using t-test, and P <0.05 as the difference is statistically significant and expressed as a x.

Results of the experiment

In 20 liver disease specimens, the expression levels of LUM, EFEMP1, THBS2 in liver tissues were examined. As shown in fig. 1, the expression of liver tissue LUM, EFEMP1, THBS2 was significantly increased in the case of significant fibrosis (F ≧ 2) compared to the case of no significant fibrosis (F <2) (P < 0.01). The expression levels for samples in which there was no significant fibrosis were all set to 1 as shown.

Example 3 Universal Probe expression detection

1. Construction of standards

According to the coding region sequence of human THBS2 gene (Genbank gene accession number is NM-003247), the THBS 2-containing full-length cDNA vector is constructed on pGEM-T vector, and is biosynthesized by Oncomelania. The plasmid standard was named pGEM-THBS 2.

According to the sequence of the coding region of human LUM gene (Genbank gene accession No. NM-002345), the LUM-containing full-length cDNA vector is constructed on pGEM-T vector and biosynthesized by Pomaceae. The plasmid standard was named pGEM-LUM.

According to the coding region sequence of human EFEMP1 gene (Genbank gene accession number is NM-001039349), the full-length cDNA vector containing EFEMP1 is constructed on pGEM-T vector, and is biosynthesized by Oncomelania. The plasmid standard was designated pGEM-EFEMP 1.

Plasmid DNA was linearized by digestion with ApaI and transcribed in vitro with T7 RNA polymerase (T7 RiboMAX)^TMExpress Large Scale RNA Production System, Promega), 1h at 37 ℃, DNase I digestion for 15min, EDTA termination reaction of 0.2mol/L, purification, quantification by a micro-spectrometer and conversion to copies/ml, and subpackaging as molecular standard.

2. Nuclease-mediated universal probe detection method

The same reporter probe is used for detection of different molecules.

2.1 LUM primers and probes:

forward primer 5 LUM: 5'-TAAACCTGACCTTCATCC-3' (SEQ ID NO: 9)

Reverse primer 3 LUM: 5'-TTAGAAGAGAGACAGGGA-3' (SEQ ID NO: 10)

Synergistic probe LUM-CP 1:

5’-CGAATACCTTGACTTGAGCACAACGAGAGGCGTGAT-3’(SEQ ID NO：11)

synergistic probe LUM-CP 2:

5’-CTGGGAGGAAGGCTAGCTTCAATCAGATAGCCAGACT-3’(SEQ ID NO：12)

reporter probe RP 1:

5’-FAM-ATCACGCCTCguTCCTCCCAG-BHQ1-3’(SEQ ID NO：13)

the 3' end of the cooperative probe is labeled with a phosphate group to prevent self-extension. FAM in the reporter Probe means that 6-FAM was used at the 5' end of the probe^TMThe fluorescent reporter group is labeled, BHQ1 indicates that the 3' end is labeled with BHQ1 quencher group, and the inserted RNA bases are in lower case bold.

TABLE 3 preparation of reaction mixtures

In Bio-Rad CFX96^TMReacting in a PCR amplification instrument at 50 ℃ for 30min and at 95 ℃ for 5 min; 5 cycles comprised 95 ℃ for 15s and 60 ℃ for 30 s. The subsequent 40 cycles comprised 95 ℃ for 10s and 52 ℃ for 60 s.

The results are shown in fig. 4, which shows a good linear relationship between Ct value and log value of the starting concentration of the template (Slope-3.148, r-1.00); the lowest detection limit is 100 copies/mL; in the accuracy experiment, the molecular standard is set to be 1.0 multiplied by 10⁵copies/mL、1.0×10²Two copies/mL gradient, each gradient sample completes 3 repeated detection, the absolute deviation of the detection result does not exceed plus or minus 0.5. In the precision experiment, the reference product is set to be 5.0 multiplied by 10⁴copies/mL、5.0×10³copies/mL、5.0×10²Three copies/mL gradients, 8 repeated detections are completed for each gradient sample, and the CV value is 5.27% -13.68%.

2.2 THBS2 primer and Probe:

forward primer 5THBS 2: 5'-CAGGAAGACTTTGACAAG-3' (SEQ ID NO: 14)

Reverse primer 3THBS 2: 5'-AACCTCATCCTTGTCATA-3' (SEQ ID NO: 15)

Cooperative probe THBS2-CP 1:

5’-GTGACCGATGAGAAGGACACAACGAGAGGCGTGAT-3’(SEQ ID NO：16)

cooperative probe THBS2-CP 2:

5’-CTGGGAGGAAGGCTAGCTCCAGCTCCTCTTCAATCC-3’(SEQ ID NO：17)

reporter probe RP 1:

5’-FAM-ATCACGCCTCguTCCTCCCAG-BHQ1-3’(SEQ ID NO：13)

the reaction configuration was as shown in 2.1, using the above-described THBS2 primer and probe. The reaction set-up is shown in 2.1. The results obtained with the molecular standard reaction are shown in fig. 5, and the standard curve shows a good linear relationship between Ct value and template concentration value (Slope-2.567, r-0.9918).

2.3EFEMP1 primers and probes:

forward primer 5EFEMP 1: 5'-CAAAGATGCGTGAATACA-3' (SEQ ID NO: 18)

Reverse primer 3EFEMP 1: 5'-AACCAAGAATGTTGTAGC-3' (SEQ ID NO: 19)

Synergistic probe EFEMP1-CP 1:

5’-ACAACTATACCTGCGTAGATATACAACGAGAGGCGTGAT-3’(SEQ ID NO：20)

synergistic probe EFEMP1-CP 2:

5’-CTGGGAGGAAGGCTAGCTAATGAATGTGATGCCAGC-3’(SEQ ID NO：21)

reporter probe RP 1:

5’-FAM-ATCACGCCTCguTCCTCCCAG-BHQ1-3’(SEQ ID NO：13)

the reaction configuration was as shown in 2.1, using the EFEMP1 primer and probe described above. The reaction set-up is shown in 2.1. The results obtained with the molecular standard reaction are shown in fig. 6, and the standard curve shows a good linear relationship between Ct value and template concentration value (Slope-2.983, r-0.9829).

Example 4 Triplex monotube expression assays of LUM, THBS2 and EFEMP1 in clinical samples

3 different report probes are set for establishing a single-tube 3-fold reaction system.

LUM primer:

forward primer 5 LUM: 5'-TAAACCTGACCTTCATCC-3' (SEQ ID NO: 9)

Reverse primer 3 LUM: 5'-TTAGAAGAGAGACAGGGA-3' (SEQ ID NO: 10)

Synergistic probe LUM-CP 1:

5’-CGAATACCTTGACTTGAGCACAACGAGAGGCGTGAT-3’(SEQ ID NO：11)

synergistic probe LUM-CP 2:

5’-CTGGGAGGAAGGCTAGCTTCAATCAGATAGCCAGACT-3’(SEQ ID NO：12)

reporter probe RP 1:

5’-FAM-ATCACGCCTCguTCCTCCCAG-BHQ1-3’(SEQ ID NO：13)

THBS2 primers and probes:

forward primer 5THBS 2: 5'-CAGGAAGACTTTGACAAG-3' (SEQ ID NO: 14)

Reverse primer 3THBS 2: 5'-AACCTCATCCTTGTCATA-3' (SEQ ID NO: 15)

Cooperative probe THBS2-CP 1:

5’-GTGACCGATGAGAAGGACACAACGAGGTTGTGCTG-3’(SEQ ID NO：22)

cooperative probe THBS2-CP 2:

5’-CGGTTGGTGAGGCTAGCTCCAGCTCCTCTTCAATCC-3’(SEQ ID NO：23)

reporter probe RP 2:

5’-HEX-CAGCACAACCguCACCAACCG-BHQ2-3’(SEQ ID NO：24)

EFEMP1 primers and probes:

forward primer 5EFEMP 1: 5'-CAAAGATGCGTGAATACA-3' (SEQ ID NO: 18)

Reverse primer 3EFEMP 1: 5'-AACCAAGAATGTTGTAGC-3' (SEQ ID NO: 19)

Synergistic probe EFEMP1-CP 1:

5’-ACAACTATACCTGCGTAGATATACAACGAGAGGAAACCTT-3’(SEQ ID NO：25)

synergistic probe EFEMP1-CP 2:

5’-TGCCCAGGGAGGCTAGCTAATGAATGTGATGCCAGC-3’(SEQ ID NO：26)

reporter probe RP 3:

5’-ROX-AAGGTTTCCTCguCCCTGGGCA-BHQ2-3’(SEQ ID NO：27)

table 4: report Probe mixture (3plex Probe mix)

Table 5: primer and cooperative probe mixture (3plex Oligo mix)

Table 6: reaction mixture

As a result, as shown in fig. 7, the expression levels of LUM, EFEMP1, and THBS2 genes in patients with significant fibrosis (F ≧ 2) were significantly increased (P <0.01) as compared to those without significant fibrosis (F < 2).

It should be understood that the detailed description of the present invention is only for illustrating the present invention and is not limited by the technical solutions described in the embodiments of the present invention, and those skilled in the art should understand that the present invention can be modified or substituted equally to achieve the same technical effects; as long as the use requirements are met, the method is within the protection scope of the invention.

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<213> Unknown (Unknown)

<400> 23

cggttggtga ggctagctcc agctcctctt caatcc 36

<210> 24

<211> 19

<212> DNA

<213> Unknown (Unknown)

<400> 24

cagcacaacc caccaaccg 19

<210> 25

<211> 40

<212> DNA

<213> Unknown (Unknown)

<400> 25

acaactatac ctgcgtagat atacaacgag aggaaacctt 40

<210> 26

<211> 3

<212> DNA

<213> Unknown (Unknown)

<400> 26

<210> 27

<211> 20

<212> DNA

<213> Unknown (Unknown)

<400> 27

aaggtttcct cccctgggca 20

Claims

1. A biomarker for detecting the severity of liver disease, wherein the biomarker is at least one of the following genes: EFEMP1, THBS2, LUM.

2. The biomarker of claim 1, wherein the DNA or RNA or gene expression protein is selected from EFEMP1, THBS2, LUM gene.

3. A reagent for detecting the severity of liver disease, which comprises a reagent for detecting the expression level of nucleic acid or protein of at least one of EFEMP1, THBS2 and LUM genes.

4. The reagent of claim 3, wherein the GAPDH gene serves as an internal control.

5. The application of the reagent according to claim 4 in a fluorescence quantitative PCR method comprises the following specific steps: extracting RNA from a sample; and detecting the expression of the marker by adopting a fluorescent quantitative PCR method.

6. The reagent of claim 5, which is used in a fluorescent quantitative PCR method, wherein the reagent is used in dye-based relative quantitative PCR, and comprises at least one of EFEMP1, THBS2 and LUM genes, and a forward primer and a reverse primer of the genes; the sequences of the forward primer and the reverse primer of the gene are shown as follows:

LUM

a forward primer: 5'-CTCTCTTCTAACTCTCTACTTA-3'

Reverse primer: 5'-GGGATGACACATTGAAAG-3'

THBS2

A forward primer: 5'-CAACCTCAATCTGGTCTG-3'

Reverse primer: 5'-GCAGTTGTCCTTCTCATC-3'

EFEMP1

A forward primer: 5'-ATGCCAACACCATCAATA-3'

Reverse primer: 5'-TCCCTATACTGCTGACTG-3' are provided.

7. The use of the reagent according to claim 4 for quantitative PCR by probe method, wherein detection of the amount of molecular expression is carried out by nuclease-mediated universal probe method.