CN116536412A - Biomarker PGLYRP2 and application of kit thereof - Google Patents

Abstract

The invention relates to the technical field of biomedicine, in particular to a biomarker PGLYRP2 and application of a kit thereof in diagnosing systemic lupus erythematosus activity. The invention provides a biomarker PGLYRP2 for diagnosing systemic lupus erythematosus activity, which is initiated and easy to clinically operate, and a kit thereof, wherein the biomarker can sensitively and specifically diagnose systemic lupus erythematosus disease activity and lipid metabolism abnormality; the invention firstly makes the variable site into the kit and discovers that the variable site is efficiently expressed in the systemic lupus erythematosus patient, so the kit can be used for evaluating the disease activity of the systemic lupus erythematosus disease.

Description

Application of biomarker PGLYRP2 and its kit

technical field

The invention relates to the field of biomedical technology, in particular to the application of biomarker PGLYRP2 in the preparation of products for diagnosing systemic lupus erythematosus or evaluating disease activity.

Background technique

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with multi-organ involvement, affecting multiple end-organs, including the kidney, brain, and heart. Kidney involvement is the main cause of systemic lupus erythematosus morbidity and mortality, affecting about 60% of patients, and is one of the main factors affecting the prognosis of systemic lupus erythematosus. In addition to traditional evaluation indicators of lupus disease activity, such as anti-double-stranded DNA, complement C3, C4, etc., and traditional renal injury markers such as creatinine (SCr), blood urea nitrogen (BUN) and proteinuria, more are still needed Biomarkers that are highly sensitive to systemic lupus erythematosus disease activity and can predict more organ and tissue damage or poor prognosis appear.

Studies have found that systemic lupus erythematosus disease activity is associated with cardiometabolic risk status. Autoantibodies and immune complexes can damage vascular endothelial cells and affect lipoprotein metabolism, making the protective mechanism of endothelial damage and atherosclerosis out of balance, eventually leading to Atherosclerosis, the incidence of atherosclerosis, myocardial infarction, and heart failure in patients with systemic lupus erythematosus is twice as high as that in healthy people. Dyslipidemia plays a key role in atherosclerosis in patients with systemic lupus erythematosus, but the exact mechanism remains unclear. So far, little progress has been made on the relationship between disease activity and dyslipidemia in systemic lupus erythematosus.

Peptidoglycan recognition protein 2 (PGLYRP2), an innate immune pattern recognition receptor, is specifically expressed in liver tissue and participates in the regulation of inflammation. PGLYRP2 can separate cross-linked bacterial peptidoglycan (PGN), digesting it into fragments by hydrolyzing the amide bond between N-acetylcarbamate and L-alanine. There are very few studies on the relationship between PGLYRP2 and autoimmune diseases. Some studies have shown that in arthritis-induced models, PGLYRP2 is necessary for the induction of cytokines, chemokines and some of their receptors; A novel marker for rheumatoid arthritis; PGLYRP2 is a reverse cholesterol transport (RCT) and dyslipidemia-related protein. PGLYRP2 is important in maintaining innate immunity and chronic inflammation. High levels of PGLYRP2 induce long-term inflammation and promote the formation of atherosclerotic lesions , can lead to impaired RCT and chronic inflammation leading to atherosclerosis and myocardial infarction. It has also been reported that PGLYRP2 is also involved in immune responses in the tumor microenvironment, infectious diseases, inflammatory responses, and brain diseases. So far, no studies have described the expression of PGLYRP2 in systemic lupus erythematosus and its correlation with the disease. Therefore, this study aimed to detect the serum PGLYRP2 level in patients with systemic lupus erythematosus and explore its relationship with disease activity and organ involvement.

In recent years, research on the pathogenesis of systemic lupus erythematosus has continued to deepen, but there is still no biological marker that can specifically evaluate systemic lupus erythematosus disease activity, organ and tissue involvement, and abnormal blood lipid metabolism at the same time, which has been widely recognized and accepted. Finding a biomarker that can effectively predict early renal involvement in the early stages of systemic lupus erythematosus is very important to improve the treatment and prognosis of lupus nephritis. Our study found a biomarker that is convenient for clinical operation. It is significantly correlated with the activity index of systemic lupus erythematosus, and can be used as one of the indicators for judging the clinical disease activity of systemic lupus erythematosus. At the same time, it is related to lipid metabolism indicators, which can provide predictive value for clinical synchronous prediction of systemic lupus erythematosus disease activity and abnormal lipid metabolism-related diseases.

Contents of the invention

The present invention finds for the first time that the serum expression level of PGLYRP2 in patients with systemic lupus erythematosus is increased, and it is a good indicator of systemic disease activity in patients with systemic lupus erythematosus, and can be used as a predictor of longitudinal deterioration of renal function, and is also a risk of poor prognosis of lupus nephritis Factors; and for the first time found that the expression level of PGLYRP2 is associated with abnormal lipid metabolism in systemic lupus erythematosus, thereby predicting the risk of cardiovascular disease in systemic lupus erythematosus.

In view of this, the present invention provides the application of the biomarker PGLYRP2 in the preparation of products for diagnosing systemic lupus erythematosus or evaluating disease activity. The invention overcomes the defects of the prior art in diagnosing and evaluating the disease activity of systemic lupus erythematosus, and provides a novel biomarker for diagnosing systemic lupus erythematosus and evaluating the disease activity.

In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

The invention provides a marker for auxiliary diagnosis of systemic lupus erythematosus, which can more accurately measure systemic lupus erythematosus, diagnose the formed systemic lupus erythematosus and evaluate its activity.

A gene expression profile detection kit for detecting systemic lupus erythematosus, the kit contains a substance specifically binding to detect PGLYRP2 protein or its protein fragment; and/or, a substance specifically binding to PGLYRP2 gene or its gene fragment.

The substances specifically combined with the PGLYRP2 gene or its gene fragments include one or more of primer pairs, probes, antisense oligonucleotides and aptamers.

The primer pair includes: a primer pair for amplifying the PGLYRP2 gene, as follows:

The detection sites of the PGLYRP2 gene are rs34440547, rs892145, rs74688727, rs3813135, and the upstream and downstream primers of the above sites are as follows:

The amplification primers are used for the gene expression level of PGLYRP2.

A SNP site related to the detection of systemic lupus erythematosus, the SNP site is located in the coding region of the PGLYRP2 gene, which is the 3407th position of the entire gene sequence, and its base is A/G; the coding region of the PGLYRP2 gene is the entire The 7216th position of the gene sequence, its base is T; the PGLYRP2 gene coding region is the 7692nd position of the entire gene sequence, its base is T; the PGLYRP2 gene coding region is the 7889th position of the entire gene sequence, its base Any one or a combination of two or more of A.

A test kit for detecting systemic lupus erythematosus, comprising the following reagents:

(1) Reagent 1: the reagent for the first long fragment PCR amplification;

(2) Reagent 2: a reagent for the second high-content PCR amplification;

(3) primers and probes: the reagents for PCR amplification include primer sets and DNA probes of the present invention;

(4) Internal standard: housekeeping gene primer pair.

The nucleotide sequence of the DNA probe is as follows:

Nucleotide sequence

Probe 1CGCCTTATCTGGGCCGCCCGAAGCTGCTGCAGCTGCCGCTGGG

Probe 2CGCCTTATCCGGGCCGCCCGAAGCTGCTGCAGCTGCCGCTGGG

Probe 3TTAACCAAGGCCTTCCTCAATGGCGCCCTGGATGG

Probe 4TGGTACTGGCACCTGATGGCTCGACCGTGGCTGTGGA

Probe 5AAAGTGCCAGCTGCCAAGACCAGACACACAGCTTCT

Both ends of the DNA probe are marked with a fluorescent group and a quenching group respectively, the fluorescent group is FAM, and the quenching group is Eclipse.

A method for using a kit for detecting systemic lupus erythematosus, the detection method of the kit comprises the following steps:

1) Nucleic acid extraction: extract the DNA of the sample to be tested;

Prepare reaction system: Prepare 15 μL quantitative PCR reaction system, including template DNA, dNTP mixture, DNA polymerase, primers, probes, 10×PCR Buffer and water;

2) The first PCR reaction: including pre-denaturation at 96°C for 5 minutes; denaturation at 95°C for 15 seconds, annealing and extension at 59°C for 90 seconds, 30-38 cycles;

3) The second PCR reaction: including pre-denaturation at 96°C for 5 minutes; denaturation at 95°C for 15 seconds, annealing and extension at 59°C for 90 seconds, 30-38 cycles;

4) Result detection: adding DNA probes for real-time fluorescence signal interpretation and drawing a standard curve.

In the specific embodiments provided by the present invention, the methods for detecting the presence or absence of the PGLYRP2 gene, or the expression level include but are not limited to: PCR-based detection methods, Southern hybridization, Northern hybridization, dot hybridization, fluorescence in situ hybridization, One or more of DNA microarrays and high-throughput sequencing platforms.

In specific embodiments provided by the present invention, PCR-based detection methods include but are not limited to reverse transcription PCR and real-time quantitative PCR.

In the embodiment provided by the present invention, the test samples of the product include one or more of serum, skin, joint, serosa, placenta, skeletal muscle, kidney, aortic endothelial cells or liver.

In the embodiments provided by the present invention, the kits include but are not limited to qPCR kits, Western blot detection kits, immunochromatography detection kits, flow cytometry analysis kits, immunohistochemical detection kits, ELISA kits and Electrochemiluminescence detection kit.

In the embodiment provided by the present invention, the method for diagnosis or auxiliary diagnosis of systemic lupus erythematosus is as follows: if the serum PGLYRP2 level of the sample is detected to be 5299.94±570.87 pg/ml, it is diagnosed as highly active systemic lupus erythematosus.

In the embodiments provided by the present invention, in assessing or assisting in assessing the disease activity of systemic lupus erythematosus: for patients with stable systemic lupus erythematosus, the level of serum PGLYRP2 in the test sample is 4468.99±457.02pg/ml; for systemic lupus erythematosus For those with active LN, the serum PGLYRP2 level was 5152.93±446.13pg/ml.

In a specific embodiment provided by the present invention, in the diagnosis or auxiliary diagnosis of systemic lupus erythematosus, the expression level of serum PGLYRP2 in the hyperlipidemia species sample of systemic lupus erythematosus is correlated with C3, C4 and renal function parameters eGFR, IgA, 24-hour urine protein Correlation.

In a specific embodiment provided by the present invention, the serum PGLYRP2 level in active systemic lupus erythematosus for diagnosis or auxiliary diagnosis is correlated with lipid metabolism indexes HDL-c, Apo-A1, and Apo B/A1 ratio.

In the embodiment provided by the present invention, the main detection samples are serum and morning urine.

Another aspect of the present invention provides a method for screening candidate drugs for the prevention and/or treatment of systemic lupus erythematosus, comprising the following steps:

(1) contacting the substance to be tested with a system containing or expressing PGLYRP2 protein;

(2) detecting the expression level of PGLYRP2 protein in the system;

(3) Detection of HDL-c, Apo-A1, Apo-B/A1 ratio;

(4) Selecting substances that can reduce the expression level of PGLYRP2 protein as candidate drugs for preventing and/or treating systemic lupus erythematosus.

Compared with prior art, the beneficial effect that the present invention has is:

The present invention provides an original biomarker PGLYRP2 for diagnosing the activity of systemic lupus erythematosus that is easy to operate clinically. The biomarker can sensitively and specifically diagnose the activity of systemic lupus erythematosus with high specificity and sensitivity , through experimental verification, the coincidence rate of the biomarker PGLYRP2 is above 85%.

The present invention found for the first time that the expression of PGLYRP2 is higher in patients with high disease activity and neuropsychiatric lupus patients, so this biomarker can also be used to evaluate the disease activity; the present invention found for the first time that the expression of plasma PGLYRP2 in patients with systemic lupus erythematosus increased, It can be used as a predictor for the worsening of systemic lupus erythematosus nephritis; for the first time, the present invention found that plasma PGLYRP2 was significantly negatively correlated with lipid metabolism indexes HDL-c and Apo-A1, and was significantly negatively correlated with the ratio of Apo-B/A1, indicating that PGLYRP2 can be used To predict the risk and severity of cardiovascular disease in systemic lupus erythematosus;

The present invention also provides a kit for systemic lupus erythematosus diagnosis and detection of disease activity, including: real-time fluorescent quantitative PCR amplification reagent premix, primer pair of PGLYRP2 gene, primer pair of housekeeping gene GAPDH, and Specific primer sequences are given. Through the serum detection of 45 qualified systemic lupus erythematosus patients and 15 healthy volunteers who have been diagnosed, the accuracy of the kit of the present invention is further verified, and it is found through sensitivity and specificity experiments that the coincidence rate of the kit is Reached more than 90%.

The present invention also finds that PGLYRP2 can be used to screen candidate drugs for preventing and/or treating systemic lupus erythematosus, so as to further develop effective therapeutic drugs for the disease.

Description of drawings

Figure 1 Serum PGLYRP2 levels in healthy volunteers, patients with SLE in stable condition, patients with active LN and NP-SLE. The results in Figure 1 show that compared with healthy volunteers (3938.56±576.07pg/ml), the stable phase of systemic lupus erythematosus (4468.99±457.02pg/ml), the active phase of LN (5152.93±446.13pg/mL) and the NP-system Serum PGLYRP2 levels were significantly elevated in patients with lupus erythematosus (5141.52±579.61pg/mL). Serum PGLYRP2 levels in active LN patients were higher than those in stable SLE patients, but there was no significant difference between active LN patients and NP-SLE patients (P>0.05).

Figure 2 Relative expression of PGLYRP2 in serum in healthy volunteers, patients with stable systemic lupus erythematosus and patients with active LN. The results showed that the expression level of serum PGLYRP2 in patients with active LN or NP-systemic lupus erythematosus was higher than that in healthy volunteers (P<0.01).

Figure 3 The ROC curve of PGLYRP2 in serum that can distinguish active and stable patients with systemic lupus erythematosus. The diagnostic potential of PGLYRP2 in systemic lupus erythematosus was tested by plotting the ROC curve, as shown in Figure 3, AUC=0.841, this result indicates that the prediction accuracy is good, proving that PGLYRP2 can distinguish active and stable systemic lupus erythematosus patients Sensitivity and specificity.

Figure 4 Serum PGLYRP2 levels in healthy volunteers, low activity LN patients (0-9), moderate activity LN patients (10-14) and high activity LN patients (≥15). Serum PGLYRP2 in the high activity group (5299.94±570.87pg/mL) was significantly higher than that in the low activity group (4610.64±533.59pg/mL) (P<0.01). There was no significant difference between the moderate activity group (4970.85±402.61pg/mL) and the high activity group (5299.94±570.87pg/mL) (P>0.05). However, the content of PGLYRP2 in the serum of all patients with systemic lupus erythematosus was higher than that of healthy volunteers.

Fig. 5 Correlation curve between serum PGLYRP2 and disease activity of systemic lupus erythematosus. In patients with systemic lupus erythematosus, there is a positive correlation between serum PGLYRP2 content and systemic lupus erythematosus DAI. With the increase of serum PGLYRP2 content, the disease activity of patients with systemic lupus erythematosus increases (R=0.5783, p<0.01 , n=45)

Figure 6 ROC curve of PGLYRP2 kit detecting patients with systemic lupus erythematosus

specific embodiment

Specific embodiments of the present invention have been described in detail so that those skilled in the art will easily understand. However, according to all the disclosed descriptions, various modifications or substitutions can be made to those details, and these changes are all within the protection scope of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Example 1: Primer design and construction

According to the PGLYRP2 gene selected from GenBank, the gene kit of the present invention is used for detection, and the positive rate is as high as 95%. The Primer Premier 5.0 online primer design software is used to design primers and probe compositions. The primers are provided by a Shanghai biotechnology limited company. The specific sequences of the primers synthesized by the company are shown in Table 1 below.

Table 1 Primers selected by 4 loci

In order to further optimize the detection program, DNA probes were designed for the conservative sequences of each gene, and the two ends of the DNA probes were respectively marked with a fluorescent group and a quencher group, the fluorescent group was FAM, and the quencher group was The group is Eclipse. The probes were all synthesized by a company in Shanghai, and the specific sequences are shown in Table 2 below.

The sequence and nucleotide of table 2 probe

Nucleotide sequence Probe 1 CGCCTTATCTGGGCCGCCCGAAGCTGCTGCAGCTGCCGCTGGG Probe 2 CGCCTTATCCGGGCCGCCCGAAGCTGCTGCAGCTGCCGCTGGG Probe 3 TTAACCAAGGCCTTCCTCAATGGCGCCCTGGATGG Probe 4 TGGTACTGGCACCTGATGGCTCGACCGTGGCTGTGGA Probe 5 AAAGTGCCAGCTGCCAAGACCAGACACACAGCTTCT

Embodiment 2, systemic lupus erythematosus cell gene extraction

Use a nucleic acid extraction kit and a nucleic acid extractor to extract DNA from systemic lupus erythematosus cells, use guanidine isothiocyanate-containing lysate to disrupt and lyse systemic lupus erythematosus cells, and wash twice with Wash Buffer A and Buffer B to remove protein and other impurities, and finally separate the protein from the nucleic acid, collect the liquid, and dissolve it in TE buffer. The final elution volume is 50 μL, that is, 50 μL of extract containing systemic lupus erythematosus cell DNA is prepared as the sample to be tested.

Example 3, gene amplification and detection of systemic lupus erythematosus cells

The first PCR amplification reaction: During the extraction of systemic lupus erythematosus cell genes, there are many interferences from other DNAs. Using the extracted and standardized DNA in Example 2 as a template, the nested PCR method was used to amplify, and the reaction System (15μl): 1.5μl 10×Taq Buffer (Mg2+Plus), 1μl 2mM dNTP, 0.2μl 10mM Primer, 1μl template DNA (50ng), 0.5UTaq, add 15μl with ddH2O.

Purification of the first PCR product: according to the DNA content of each sample, dilute to 30-50ng/μl with 1×TE, and store at -20°C for future use.

The second PCR amplification reaction: 1 μL of the diluted extract was taken for PCR amplification experiment, and 8 pairs of primers of the present invention were respectively screened for 2 amplifications, and the PCR reaction system was 20ul. Reaction system (15μl): 1.5μl 10×TaqBuffer (Mg2+Plus), 1μl 2mM dNTP, 0.2μl 10mM Primer, 1μl template DNA (50ng), 0.5UTaq, make up 15μl with ddH2O, DNA probe primer (1.5ng/ml ). The sequence result of the first amplification is used as the DNA template for the second amplification result, and finally further amplified through the screened SNPs sites, the specific reaction system and primers are shown in Table 3:

Table 3 reaction system and primers

The first PCR amplification reaction PCR reaction control process is as follows:

Table 4 PCR reaction primers and PCR process of each gene

Second PCR amplification

Table 4 PCR reaction primers and PCR process of each gene

The primers of the second PCR reaction process in table 5

Table 6 reaction system and primers

Result detection: Real-time fluorescence signal interpretation and standard curve drawing.

Embodiment 4: Second annealing extension temperature optimization

Due to the presence of DNA probes in the second PCR amplification reaction, the design software cannot be used as a reference standard. According to the design of annealing temperature and base sequencing, five sets of reaction procedures are designed:

Reaction program 1: Step 1: Pre-denaturation at 95°C for 5 minutes; Step 2: Denaturation at 95°C for 15 seconds, annealing and extension at 59°C for 90 seconds, fluorescence collection after extension; repeat the second step for 38 cycles.

Reaction program 2: Step 1: Pre-denaturation at 95°C for 5 minutes; Step 2: Denaturation at 95°C for 15 seconds, annealing and extension at 55°C for 90 seconds, fluorescence collection after extension; repeat the second step for 38 cycles.

Reaction program 3: Step 1: Pre-denaturation at 95°C for 5 minutes; Step 2: Denaturation at 95°C for 15 seconds, annealing and extension at 56°C for 90 seconds, fluorescence collection after extension; repeat the second step for 38 cycles.

Reaction program 4: Step 1: Pre-denaturation at 95°C for 5 minutes; Step 2: Denaturation at 95°C for 15 seconds, annealing and extension at 57°C for 90 seconds, fluorescence collection after extension; repeat the second step for 38 cycles.

Reaction program 5: Step 1: Pre-denaturation at 95°C for 5 minutes; Step 2: Denaturation at 95°C for 15 seconds, annealing and extension at 58°C for 90 seconds, fluorescence collection after extension; repeat the second step for 38 cycles.

Table 7 Comparison test of optimization of annealing extension temperature

Reaction procedure 1 Reaction procedure 2 Reaction program 3 Reaction Program 4 Reaction program 5 rs34440547 N/A -- -- N/A N/A rs892145 N/A N/A N/A N/A N/A rs74688727 N/A N/A N/A N/A -- rs3813135 N/A -- N/A -- N/A

The difference between the above 5 groups of reaction procedures is that the annealing extension temperature is different, and the amplification results are shown in the table, N/A

Indicates that there is no amplification, and the test result is negative, and the value is the detection Ct value, indicating that there is amplification, and the test result is positive (reaction program 2: 21.5, 35.2; reaction program 3: 30.5; reaction program 4: 24.7; Reaction program 5 is: 23.1). It can be seen from the amplification results that when the annealing temperature is 59°C, the 4 sets of primer pairs have the best specificity, and there is no false positive amplification result; however, when the annealing temperature is 56-58°C, some non-specific The opposite sex is significant, that is, a false positive test result occurs.

Example 5: Gene purification and detection of systemic lupus erythematosus

After the amplification was completed, 5 μl was taken from each sample, and positive individuals were detected on a 1% DNA gel. The results showed that the fragment length was 105bp for rs34440547, 135bp for rs892145, 114bp for rs74688727, and 109bp for rs3813135. It can be seen that the target fragment can be clearly seen through 2 times of PCR amplification, which shows that the effect of PCR is good, and other conditions of the present invention meet the detection requirements.

Example 6: A kit for detecting the activity of systemic lupus erythematosus

Including systemic lupus erythematosus cell extraction kit, primers and DNA probes for 6 genes, housekeeping gene GAPDH (primer alignment 5′-TCAGTGGTGGACCTGACCTG-3′ and 5′-TGCTGTAGCCAAATTCGTTG-3′).

Example 7: Research materials and specimens

From January 1, 2019 to November 1, 2020, 45 eligible patients with systemic lupus erythematosus (15 patients with stable systemic lupus erythematosus, 15 patients with active LN and 15 patients with active NP-systemic lupus erythematosus patients and 15 healthy volunteers. Among the 45 systemic lupus erythematosus patients, 39 were females (86.6%), 6 were males (13.4%), and the average age was 36.53±13.54 years old.

Classification criteria:

Systemic lupus erythematosus was diagnosed and classified according to the systemic lupus erythematosus classification criteria revised by the American College of Rheumatology in 1982, and the selected patients with systemic lupus erythematosus were divided into low disease activity group (systemic lupus erythematosus DAI≦9, n= 19), moderate disease activity group (10<systemic lupus erythematosus DAI<14, n=12) and high disease activity group (systemic lupus erythematosus DAI≧15, n=14).

PGLYRP2 detection method in serum:

①Morning venous blood was collected from 45 patients with systemic lupus erythematosus and 15 volunteers after fasting for 8 hours.

②The blood collected from each participant was centrifuged at 1000×G for 10 minutes, aliquoted and stored at -80°C.

③Take out the sample and let it thaw at room temperature. Dilute the serum sample 1:10. Note that each sample is only cycled and thawed once to prevent protein degradation.

④Detect the serum level of PGLYRP2 with PGLYRP2 ELISA kit.

⑤Use a microplate reader to detect the optical density at 450nm, and measure each sample twice. The expression level of serum PGLYRP2 is PG/ml.

Test results and analysis

Serum PGLYRP2 levels in healthy volunteers, patients with stable systemic lupus erythematosus, active LN patients and NP-systemic lupus erythematosus patients; Relative expression in patients with chronic LN; PGLYRP2 in serum can distinguish active and stable patients with systemic lupus erythematosus ROC curve; in healthy volunteers, low activity LN patients, moderate activity LN patients and high activity LN patients The content of serum PGLYRP2 in the blood; the correlation curve between serum PGLYRP2 and the disease activity of systemic lupus erythematosus.

After testing, it was found that serum PGLYRP2 in the high activity group (5299.94±570.87pg/mL) was significantly higher than that in the low activity group (4610.64±533.59pg/mL) (P<0.01). There was no significant difference between the moderate activity group (4970.85±402.61pg/mL) and the high activity group (5299.94±570.87pg/mL) (P>0.05). However, the content of PGLYRP2 in the serum of all patients with systemic lupus erythematosus was higher than that of healthy volunteers.

Example 8: The coincidence rate of PGLYRP2 kit used to detect the activity of systemic lupus erythematosus

In order to further verify the accuracy of the kit of the present invention, 3 types of 6 genes were classified and combined, specifically as shown in the combination in Table 7. The theory of the combination is to reduce the cost as much as possible and improve the experimental efficiency.

The kit is selected from the kit in Example 6, and the operation is as follows:

The first PCR amplification reaction: During the extraction of systemic lupus erythematosus cell genes, there are many interferences from other DNAs. Using the extracted and standardized DNA in Example 2 as a template, the nested PCR method was used to amplify, and the reaction System (15μl): 1.5μl 10×Taq Buffer (Mg2+Plus), 1μl 2mM dNTP, 0.2μl 10mM Primer, 1μl template DNA (50ng), 0.5UTaq, add 15μl with ddH2O.

Purification of the first PCR product: according to the DNA content of each sample, dilute to 30-50ng/μl with 1×TE, and store at -20°C for future use.

The second PCR amplification reaction: 1 μL of the diluted extract was taken for PCR amplification experiment, and 6 pairs of primers of the present invention were respectively screened for 2 amplifications, and the PCR reaction system was 20ul. Reaction system (15μl): 1.5μl 10×TaqBuffer (Mg2+Plus), 1μl 2mM dNTP, 0.2μl 10mM Primer, 1μl template DNA (50ng), 0.5UTaq, make up 15μl with ddH2O, DNA probe primer (1.5ng/ml ). The sequence result of the first amplification is used as the DNA template of the second amplification result, which is finally further amplified and displayed through the screened SNPs sites.

According to the existing theory, it can be seen from the combination that the detection of combination 3 should be the most efficient, but the combination of primers and other reagents used in combination 3 is more time-consuming and costly. The specific test results are shown in Table 8 and Table 9.

Combination

Table 8 Probe combinations

Combination site ① Probe 1+Probe 3 ② Probe 1+Probe 3+Probe 4 ③ Probe 1+Probe 3+Probe 4+Probe 5 ④ Probe 1+Probe 2+Probe 3+Probe 4+Probe 5

Table 9 The results of three different combinations of gene detection

Combination positive number True Positives number of false positives number of false negatives Positive rate (%) ① 31 14 17 31 68.9 ② 30 twenty two 8 12 66.7 ③ 37 33 4 7 82.2 ④ 43 42 1 2 95.6

It can be seen from Table 8 that the detection result of combination ④ is more reliable, with an accuracy rate of over 95%, and nearly half of the patients in the detection results of combination ①-③ were not detected. Therefore, it is not recommended to use the detection method of combination ①-③.

Example 9: Using PGLYRP2 kit to detect the degree of different activities of systemic lupus erythematosus

Kit: PGLYRP2 kit, using the probes in the ④ combination method in Example 8.

For the 45 systemic lupus erythematosus patients and 15 healthy volunteers in Example 7, in order to evaluate the test effect of the PGLYRP2 kit, the above-mentioned systemic lupus erythematosus patients and 15 healthy volunteers were detected. The diagnostic results were divided into four grades: healthy volunteers, patients with low activity LN, patients with moderate activity LN, and patients with high activity LN, and the results were represented by 1-4, as shown in Table 10.

Table 10 Test scoring results of PGLYRP2 kit

The specific test results are shown in Figure 6. The test results show that the area under the ROC curve of the kit is 0.936, which is significantly different from 0.5 (P<0.001), indicating that the kit has better detection ability.

Claims (10)

1. A gene expression profile detection kit for detecting the activity of systemic lupus erythematosus, which is characterized in that the kit contains a substance for detecting the specific binding of PGLYRP2 protein or protein fragments thereof; and/or a substance that specifically binds to the PGLYRP2 gene or a gene fragment thereof.

2. The kit for detecting gene expression profile according to claim 1, wherein the substance specifically binding to PGLYRP2 gene or gene fragment thereof comprises one or more of primer pair, probe, antisense oligonucleotide, and aptamer.

3. The gene expression profiling kit of claim 2, wherein the primer pair comprises: primer pairs for amplifying the PGLYRP2 gene were as follows:

4. the gene expression profile test kit of claim 2, wherein the single nucleotide polymorphism sites of the PGLYRP2 gene are rs34440547, rs892145, rs74688727, rs3813135.

5. The gene expression profile detection kit of claim 4, wherein the upstream and downstream primers of rs34440547, rs892145, rs74688727, rs3813135 are as follows:

6. the kit for detecting gene expression profile according to claim 1, wherein the amplification primer is used for the expression level of PGLYRP2 gene.

7. The SNP locus related to the activity of detecting the systemic lupus erythematosus is characterized in that the SNP locus is positioned in a PGLYRP2 gene coding region and is 3407 th site of the whole sequence, and the base is A or G; the PGLYRP2 gene coding region is 7216 th position of the whole sequence, and the base is T; the coding region of PGLYRP2 gene is 7692 th position of the whole sequence, and the base is T; the PGLYRP2 gene coding region is 7889 th site of the whole sequence, and the base is one or more than two of A.

8. A kit for detecting systemic lupus erythematosus activity comprising the following reagents:

(1) Reagent 1: reagents for the first long fragment PCR amplification;

(2) Reagent 2: a second high-content PCR amplification reagent;

(3) Primer and probe: the reagent for PCR amplification comprises the primer set according to any one of claims 1 to 2 and a DNA probe.

9. The kit of claim 8, wherein the DNA probe has the nucleotide sequence as follows:

probe 1CGCCTTATCTGGGCCGCCCGAAGCTGCTGCAGCTGCCGCTGGG

Probe 2CGCCTTATCCGGGCCGCCCGAAGCTGCTGCAGCTGCCGCTGGG

Probe 3TTAACCAAGGCCTTCCTCAATGGCGCCCTGGATGG

Probe 4TGGTACTGGCACCTGATGGCTCGACCGTGGCTGTGGA

Probe 5AAAGTGCCAGCTGCCAAGACCAGACACACAGCTTCT

The two ends of the DNA probe are respectively marked with a fluorescent group and a quenching group, the fluorescent group is FAM, and the quenching group is Eclipse.

10. The kit according to claim 9, wherein the method of detection of the kit comprises the steps of:

1) Nucleic acid extraction: extracting DNA of a sample to be detected;

preparing a reaction system: preparing a 15 mu L quantitative PCR reaction system comprising template DNA, dNTP mixture, DNA polymerase, primer, probe, 10 XPCR Buffer and water;

2) First PCR reaction: comprises pre-denaturation at 96 ℃ for 5 minutes; denaturation at 95℃for 15 seconds, annealing at 59℃for 90 seconds, 30-38 cycles;

3) Second PCR reaction: comprises pre-denaturation at 96 ℃ for 5 minutes; denaturation at 95℃for 15 seconds, annealing at 59℃for 90 seconds, 30-38 cycles;

4) And (3) result detection: and adding a DNA probe for real-time fluorescent signal interpretation and drawing a standard curve.