CN117757918A - Urine miRNA biomarker combination for BK virus nephropathy and application thereof - Google Patents

Abstract

The invention discloses a urine miRNA biomarker combination for BK virus nephropathy and application thereof, comprising the following urine miRNA: miRNA-b1-5p, miRNA-b1-3p and miRNA-155-5p, and by using a fluorescent real-time quantitative polymerase chain reaction technology, a specific primer and a probe for amplifying miRNA are designed, and the expression condition of miRNA markers in urine of a human to be detected is detected, so that screening and early clinical diagnosis of BK virus nephropathy can be realized, and the method has the characteristics of high accuracy, good specificity, strong stability and the like. The diagnosis score and the expression level of the biomarker in urine of the patient are combined, so that the virus strain type of BK viral nephropathy of the patient can be simply and rapidly judged, and the time is saved for treatment of the patient.

Description

Urine miRNA biomarker combination for BK virus nephropathy and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a urine miRNA biomarker combination for BK virus nephropathy and application thereof.

Background

BK viral nephropathy (BKVAN) is a kidney disease caused by BK virus (BKVAN) infection, which seriously jeopardizes human health. BK virus belongs to the family of human herpesviruses, a DNA virus, which is widely found in the urinary system of humans, and is often detected in the kidneys in particular. BK viral nephropathy occurs primarily in recipients in an immunosuppressive state following organ transplantation. Early after implantation, BK virus can enter organs such as the kidney of the recipient and cause infection and proliferation in epithelial cells and tubular cells due to the limited immune function of the recipient due to the use of immunosuppressants. Inflammatory reactions and cellular damage from such infections can lead to accelerated apoptosis necrosis of tubular epithelial cells, ultimately leading to loss of kidney function. There is usually no obvious clinical symptoms in normal conditions of the human body, and BK virus can be reactivated when the immune function of the patient is reduced or inhibited, probably because the immune function of the patient is reduced due to the fact that the anti-BK virus specific T cells are inhibited after long-term administration of immunosuppressant after transplantation. It has been reported that after a large number of BK viruses are activated, 6-10% of kidney transplant patients may have BK viral kidney disease due to BK virus infection, and that the transplanted kidney function may be compromised, with about 50-70% of BK viral kidney disease recipients eventually potentially losing transplanted kidney.

Traditional detection methods include urocytology, BKV-DNA copy number detection, and biopsy tissue monkey vacuolated virus (SV 40) staining, but these methods have certain limitations. Urine cytology generally detects cell morphology and nuclear changes in urine, with limitations on the detection of BK virus specificity. The urocytology examination requires a medical expert with a high level of experience and expertise, and the reliability of the test results can be compromised for untrained medical workers. Urine cytology examination cannot reflect the replication status and virus quantity of BK virus, and cannot effectively evaluate the disease degree and cure effect of patients. SV40 staining is an invasive examination requiring a kidney puncture biopsy; once the number of SV40 in the biopsy tissue is low, or SV 40-containing cells are present only at specific locations, a stained false positive may occur. Since BK viral nephropathy is manifested in early stages as BK viruria and BK viremia, BKV-DNA copy number detection of viral load in urine and peripheral blood of kidney transplant recipients is an important method for monitoring disease changes in early stages in clinic. BK viral nephropathy has close relation with BKV DNA load in urine and blood, but BKV DNA load only can reflect the content of viruses in body fluid, and cannot reflect the real-time activation degree of BK viruses. The detection method of the blood virus DNA has high specificity and low sensitivity, and is not suitable for screening test; the detection method of the urine virus DNA has high sensitivity, low specificity, high false positive rate and poor diagnosis precision. DNA recombination and mutation of BK virus can also change genome rapidly, so that PCR detection results are misaligned, and adverse effects are caused on clinical treatment.

In recent years, more and more researches indicate that the development of BK virus nephropathy is closely related to the abnormal expression of miRNAs. mirnas are a class of small molecule RNAs 20-25nt in length that regulate gene expression by binding to the 3' utr of a particular mRNA at post-transcriptional levels. There are techniques for detecting BK viral nephropathy using miRNA as a marker, but single miRNA detection effect is limited. BK virus has two states of latent infection and activating infection, the two states possibly cause different miRNA expression modes, and the two states cannot be distinguished by simple miRNA copy number detection, so that accurate judgment on the BK virus infection state is limited. In addition, BK viruses exist in multiple strains in the east Asia population, wherein the most common BK virus strains are BK virus strain-I and BK virus strain-IV, the prognosis of different BK virus strains is also different, and only the virus expression quantity can be reflected by taking a single miRNA as a marker, so that the specific BK virus strain cannot be identified. The current method is to identify BK virus strain by gene sequencing technology. Firstly, collecting urine samples in patients, then, extracting BK virus DNA by using a kit, synthesizing a specific primer for PCR amplification of the virus DNA, sequencing the amplified DNA to obtain sequence information, finally, comparing the obtained BK virus sequence with known I type and IV type reference sequences by using sequence comparison software, and judging whether the BK virus is the strain type I or the strain type IV according to the sequence comparison result. However, this technique has problems such as high cost and long time.

Disclosure of Invention

In order to solve the technical problems, the invention provides a urine miRNA marker combination for BK virus nephropathy. The biomarker combination consisting of miRNA-b1-5p, miRNA-b1-3p and miRNA-155-5p is used, so that the detection accuracy is improved compared with that of single miRNA or double miRNA. Meanwhile, three miRNAs are used as expression levels of biomarkers, so that the BK virus strain type can be judged.

It is a first object of the present invention to provide a miRNA biomarker combination for BK viral kidney disease, including miRNA-b1-5p, miRNA-b1-3p and miRNA-155-5p.

Further, the nucleotide sequence of the miRNA-b1-5p is shown as SEQ ID NO.1, the nucleotide sequence of the miRNA-b1-3p is shown as SEQ ID NO.2, and the nucleotide sequence of the miRNA-155-5p is shown as SEQ ID NO. 3.

Further, the miRNA is derived from urine.

A second object of the present invention is to provide a primer composition for amplifying the above miRNA biomarker, comprising:

a forward primer for amplifying miRNA-b1-5p shown in SEQ ID NO.4 and a reverse primer shown in SEQ ID NO. 5;

a forward primer for amplifying miRNA-b1-3p shown in SEQ ID NO.6 and a reverse primer shown in SEQ ID NO. 7;

the forward primer for amplifying miRNA-155-5p shown as SEQ ID NO.8 and the reverse primer shown as SEQ ID NO. 9.

A third object of the present invention is to provide a BK virus kidney disease detection kit for detecting BK virus kidney disease by detecting the above miRNA biomarker combination.

Further, the kit comprises the primer composition.

Further, the kit further comprises:

(1) The reverse transcription primer for synthesizing cDNA, wherein the reverse transcription primer of miRNA-b1-5p is shown as SEQ ID NO.10, the reverse transcription primer of miRNA-b1-3p is shown as SEQ ID NO.11, and the reverse transcription primer of miRNA-155-5p is shown as SEQ ID NO. 12;

(2) The Taqman probe, wherein the probe sequence of the miRNA-b1-5P is shown as SEQ ID NO.13, the probe sequence of the miRNA-b1-3P is shown as SEQ ID NO.14, and the probe sequence of the miRNA-155-5P is shown as SEQ ID NO. 15.

Further, the reverse transcription primer used for cDNA synthesis is a specific stem-loop primer.

Further, the method for detecting by adopting the kit comprises the following steps:

(1) Collecting a urine sample to be tested;

(2) Extracting total RNA in urine, and performing reverse transcription to obtain cDNA;

(3) Using cDNA as a template, and detecting the expression level of the miRNA marker in urine by using a fluorescent real-time quantitative PCR technology based on a Taqman probe;

(4) Calculate a composite diagnostic score Y, where y=33.55+β _miRNA-b1-5p ×(-0.3190)+β _miRNA-b1-3p ×(-0.4529)+β _miRNA-155-5p X (-0.2054), β is an argument in the formula; this value is a presentation of the result of fluorescent quantitative PCR for calculating the gene expression difference or gene copy number.

When the comprehensive diagnosis score is smaller than 37, the detection result is considered positive, and the risk of BK virus nephropathy of the person to be detected is high; when the comprehensive diagnosis score is greater than 37, the detection result is considered negative, and the risk of BK virus nephropathy of the human to be detected is low.

The fourth object of the invention is to provide the application of the miRNA biomarker combination in the preparation of BK virus strain identification products.

A fifth object of the invention is to provide the application of the kit in preparing BK virus strain identification products.

Further, when BK virus strain detection is carried out by adopting the kit, under the condition that the comprehensive diagnosis score is smaller than 37, the initial copy number is higher than 7.15log copies/mL, the detection sample is BK virus strain I, the initial copy number is lower than 7.15log copies/mL, and the detection sample is BK virus strain IV.

The invention has the beneficial effects that:

(1) Diagnosis is carried out by taking miRNA-b1-3p, miRNA-b1-5p and miRNA-155-5p as biomarkers, and the accuracy is higher than that of single miRNA or any two of the miRNAs, and the accuracy is highest in all three miRNA combinations obtained by screening.

(2) The expression amounts of the biomarkers of miRNA-b1-3p, miRNA-b1-5p and miRNA-155-5p are obviously different in different strain types of BK viruses, and the specific strain type of BK viruses of a patient can be simply, conveniently and rapidly judged by combining the expression amounts of the biomarkers with diagnostic scores.

Drawings

FIG. 1 is a standard graph of miRNA-b1-5 p;

FIG. 2 is a standard graph of miRNA-b1-3 p;

FIG. 3 is a standard graph of miRNA-155-5p;

FIG. 4 is a ROC graph of miRNAs-b 1-5 p;

FIG. 5 is a ROC graph of miRNAs-b 1-3 p;

FIG. 6 is a ROC graph of miRNA-155-5p;

FIG. 7 is a fitting chart of a Hosmer-Lemeshow test model;

FIG. 8 is a ROC graph of a triple variable miRNA versus a duplex and a simplex variable miRNA;

FIG. 9 is a graph of BK virus strain ROC curve analysis;

fig. 10 is a ROC graph of trivariate miRNA cross-validation.

Detailed Description

The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.

The specific experimental procedures or conditions are not noted in the examples and may be followed by the operations or conditions of conventional experimental procedures described in the literature in this field. The reagents or apparatus used were conventional reagent products commercially available without the manufacturer's knowledge.

Example 1: fluorescent quantitative PCR (polymerase chain reaction) quantitative miRNA-b1-5p, miRNA-b1-3p and miRNA-155-5p

(1) Urine collection and preservation

8-10mL of morning urine from BKV kidney disease patients and healthy people are collected by a sterile centrifuge tube, 1-2 tubes of each patient are put into an ice chest or stored at 4 ℃ and transported back to a laboratory for treatment.

(2) Urine pretreatment

The collected morning urine was placed in a centrifuge at room temperature at 3000rpm for 10 minutes, and then the supernatant was transferred to a new RNase-free centrifuge tube, centrifuged again at 10000rpm for 5 minutes at room temperature, and transferred to a new tube.

(3) Extraction of total RNA from urine supernatant

Total RNA was extracted using a Kaijie miRcute miRNA extraction and separation kit (centrifugation column). Adding an equal volume of lysate at a temperature of 4 ℃ into 200 mu L of supernatant, shaking and mixing for 30 seconds, standing for 5 minutes at room temperature, centrifuging at 12000rpm for 10 minutes at room temperature, transferring the supernatant into a new 1.5mL EP tube without RNase, adding 200 mu L of chloroform (chloroform), shaking for 15 seconds rapidly and vigorously until the chloroform is completely mixed, and standing for 5 minutes at room temperature. Centrifuge was centrifuged at 12000rpm for 15min at room temperature and the liquid in the EP tube was found to separate into 3 layers (upper clear aqueous phase for RNA, middle white for protein, lower yellow organic phase for DNA). The upper clear aqueous layer was carefully transferred to a fresh RNase-free 1.5mL EP tube, avoiding as much as possible the uptake of the middle layer protein and the lower layer DNA. Measuring the volume of the transfer solution, slowly adding absolute ethyl alcohol with the volume being 1.5 times that of the transfer solution, and shaking and uniformly mixing. The solution and the precipitate were transferred to an adsorption column, centrifuged at 12000rpm for 30 seconds at room temperature, and the effluent was discarded after centrifugation, leaving the column. 500. Mu.L deproteinized solution was added thereto, allowed to stand at room temperature for 2 minutes, centrifuged at 12000rpm at room temperature for 30 seconds, and the waste liquid was discarded. 500. Mu.L of the rinse solution was added, allowed to stand at room temperature for 2 minutes, centrifuged at 12000rpm at room temperature for 30 seconds, and the waste solution was discarded, and the rinsing step was repeated. The column was placed in a 2mL collection tube and centrifuged at 12000rpm for 1 min at room temperature to remove residual liquid. The column was transferred to a fresh RNase-free 1.5mL EP tube, 30-100. Mu.L of DEPC water was added, and the mixture was allowed to stand at room temperature for 2 minutes and centrifuged at 12000rpm for 2 minutes. 1. Mu.L of the total RNA solution was taken, and the concentration was measured and recorded by an ultra-micro spectrophotometer.

(4) cDNA synthesis by reverse transcription

The extracted total RNA is reversely transcribed into cDNA by using the reagent of the cDNA first strand synthesis kit, the HyperScript III Enzyme Mix frozen at-20 ℃ is put on ice for standby, and the frozen 5 xRT Mix is thawed and is gently inverted and mixed uniformly after being thawed. 1 μg of each sample was subjected to reverse transcription. Reverse transcription of miRNAs has its specificity, and first, for each miRNA, its specific reverse transcription primer is designed. Compared with a tailing method and a dye method, the stem-loop primer does not need complicated temperature circulation steps, has higher sensitivity, and can be amplified at very low nucleic acid concentration. The use of stem-loop primers improves the specificity of amplification and reduces the occurrence of false positive results.

i. Primer synthesis

The sequences in this example are shown in Table 1:

table 1 sequence listing

ii. configuring the reaction System in a PCR tube for the DeRNase

TABLE 2 reaction system

Mixing the above solutions, and standing at 25deg.C for 5min;

iii.50 ℃ for 15min;

iriiii the mixture was left at 85 ℃ for 5s.

(5) Real-time fluorescent quantitative PCR

Detecting the expression levels of miRNA-b1-5p, miRNA-b1-3p and miRNA-155-5p in the synthesized cDNA based on a probe method fluorescence quantitative PCR amplification technology, wherein the kit is a miRNA fluorescence quantitative detection kit, and melting 2X Taqman Master Mix and Reverse Primer at room temperature; and the melted 2X Taqman Master Mix is mixed up and down, and is slightly centrifuged for standby. Placing the synthesized cDNA, probe and other reactants on ice; each sample was provided with 2 multiplex wells (i.e., all conditions were unchanged, 2 replicates were performed). The initial copy number of the target miRNA in the sample was quantified by an absolute quantification standard curve.

i. Primer design

TABLE 3qPCR primer set and Probe set

The purification modes of the primer set and the probe set can be selected from the following modes: HAP, PAGE and HPLC purification formats. The primer set and the probe set were synthesized by general biosystems (Anhui) Inc.

ii, configuring a reaction system

TABLE 4 reaction system

Reaction conditions

TABLE 5 reaction conditions

iii real-time fluorescent quantitative PCR result analysis

The detection result of miRNA can be obtained through absolute quantitative calculation. Firstly, diluting positive standard substances with known concentrations into a series of substances with concentration gradients according to the same multiple, then measuring a threshold Cycle number (Ct value) by using a qPCR instrument, and drawing a standard curve by taking the Ct value as an ordinate and the logarithm of the copy number of the initial template as an abscissa. Substituting the Ct value of the sample into a standard curve equation to obtain the copy number of the initial template to quantify miRNA.

(6) Statistical treatment

Statistical analysis was performed with SPSS13.0 software. Data conforming to normal distribution, wherein metering data are represented by mean ± standard deviation, and two independent sample t-test is adopted for comparison between two groups; data which do not accord with normal distribution are compared between two groups by rank sum test; the comparison of two by two in the set was performed using the Kruskal-Wallis test. Data conforming to normal distribution, wherein the linear correlation adopts pearson correlation analysis; data not meeting normal distribution, using the spin scale correlation analysis, P <0.05 considered the difference statistically significant.

Example 2: fluorescent quantitative PCR standard curve formulation

The system for preparing PCR amplification reactions for the different standards using the kit of example 1 was:

PCR master mix, 12.5. Mu.L; upstream primer F,10uM, 1. Mu.L; downstream primer R, 10. Mu.M, 1. Mu.L; a probe, 10. Mu.M, 1uL; DNA template (standard of diluted concentration), 1. Mu.L; RNase-free water was made up to 25. Mu.L.

The conditions for the PCR amplification reaction were: pre-denaturation at 95℃for 10min; denaturation at 95℃for 5s, annealing at 60℃for 40s, extension at 60℃for 40s,40 cycles. Fluorescence signals were collected at the time of annealing in each cycle.

miRNA-b1-5p, miRNA-b1-3p and miRNA-155-5p were 2X 10, respectively ⁴ -2×10 ⁸ The copies/. Mu.L as a template is prepared according to the system of the PCR amplification reaction, fluorescent quantitative PCR is performed according to the conditions of the PCR amplification reaction, ct values of standard substances with various concentrations are taken as an ordinate, and logarithm of the copy number of the initial template is taken as an abscissa to be taken as a standard curve, so that the PCR amplification reaction can be performedObtaining linear relation expression between copy number logarithm and Ct value, slope (Slope) of miRNA-b1-5p, miRNA-b1-3p and miRNA-155-5p, linear correlation coefficient (R ² ) And the amplification efficiencies (E) are-3.537/-3.928/-3.475, 0.9961/0.9988/0.9985 and 91.7%/93.7%/93.9%, respectively, and the results show that the linear relationship between the Ct value of the BK virus miRNA detected by the Taqman fluorescence quantitative PCR and the logarithm of the copy number of the initial template is good. The recombinant plasmid is qualified as a standard of Taqman fluorescent quantitative PCR detection. According to the standard curve equation, substituting the Ct value of the sample to be detected into the equation to obtain the initial copy number of the sample, thereby achieving the purpose of quantifying the miRNA of the sample (figures 1-3).

Example 3: repeatability verification

Will be 6.57×10 ⁹ copies/μL、6.57×10 ⁸ copies/μL、6.57×10 ⁷ Three miRNA standards with different concentrations of copies/mu L are respectively used as DNA templates for carrying out batch-to-batch repeated experiments, and the following PCR amplification reaction system is prepared:

TABLE 6 reaction system

The conditions for the PCR amplification reaction were: pre-denaturation at 95℃for 10min; denaturation at 95℃for 5s, annealing at 60℃for 40s, extension at 60℃for 40s,40 cycles. Fluorescence signals were collected by annealing at 60℃in each cycle.

In order to verify the repeatability of the invention, the error of Ct values of three BK virus positive samples detected by 3 repeated batches and 3 repeated batches is less than 0.5, and the variation coefficient is less than 5%, which indicates that the method has higher repeatability. In addition, through analysis on the average value, standard deviation and variation coefficient of ct, the p value is 0.4787, no obvious difference (p is more than 0.05) exists between the two groups, and the experimental repeatability of the invention is good.

TABLE 7 fluorescent quantitative PCR in-batch repeat experiment results

TABLE 8 fluorescent quantitative PCR results of batch-to-batch repeat experiments

Table 9 results of the verification

The sample-to-sample and batch-to-batch replicates at three different copy number concentrations were not significantly different from one another by statistical analysis.

Example 4: combined prediction BK virus replication risk assessment model

The data set containing the target variable and the characteristic variable is collected, then the data is preprocessed, including characteristic selection, characteristic scaling and the like, then a proper logistic regression model is selected, and model fitting is performed on the data. An optimization algorithm (e.g., gradient descent) is used to minimize the loss function to update the model parameters. Model parameters are optimized through multiple iterations until the model converges, after which coefficients in the logistic regression model can be obtained that represent the extent of contribution of each feature to the target variable. BKV-miRNA difference analysis is carried out on BK virus urine samples by adopting Wilcoxon signed rank sum test (P is less than or equal to 0.001), and the average difference is more than 0.2. The same test was performed 100 times to determine the difference in urine samples ct for suspected BKVN patients and for confirmed BKVN patients, three-fourths of the total samples were randomly drawn each time. A minimum absolute shrinkage and selection operator (Least absolute shrinkage and selection operator, LASSO) regression analysis and random forest selection variables in the R-packets were used and a diagnostic model was built using urine samples from BKVN patients in the training cohort. We used sequencing data to identify BKV-mirnas and performed LASSO regression analysis to select variables using Lambda determined by 10-fold cross validation. And (3) establishing a random forest model, removing the variable with the minimum feature importance, and using the rest variables for model construction to remove useless variables by using the same method. This process is iterated until the optimal variable is identified based on the highest classification accuracy. We also calculated the area under the curve (AUC) to compare the identification performance of the model to urine miRNA-b1-3p, miRNA-b1-5p and miRNA-155-5p levels, as shown in fig. 4-6. Coefficients of 3 markers in the Logistic regression calculation model are adopted, and the calculation formula of the comprehensive diagnosis Score (miR-Score) is as follows:

Y＝33.55+β _miRNA-b1-5p ×(-0.3190)+β _miRNA-b1-3p ×(-0.4529)+β _miRNA-155-5p ×(-0.2054)；

where β is an argument in the formula; this value is a presentation of the result of fluorescent quantitative PCR for calculating the gene expression difference or gene copy number.

miR-Score <37 is positive, namely miR-Score >37 is negative.

Table 10miRNA signature data

The embodiment adopts the goodness of fit of a Hosmer-Lemeshow test (HL test) model, and predicts X after substituting data ² Is 10.685, and p value is 0.2202 (p>0.05 It was demonstrated that the model fits well by HL test, i.e. there is no very significant difference between the predicted and the actual values (fig. 7).

The embodiment also provides the comparison of the accuracy of the single, double and triple miRNA markers for detecting the replication activity of the BK virus, and as shown in figure 8, the accuracy of the single miRNA markers is higher than that of the single miRNA markers or any two miRNAs when the miRNA-b1-3p, the miRNA-b1-5p and the miRNA-155-5p are used as the biological markers.

Example 5: strain differentiation of BK Virus nephropathy in the east Asia population

BK virus exists in multiple strains in the east Asia population, with BK virus strain-I and strain-IV being the most common. Research has shown that BK virus strain-I/c is more common among eastern Asian populations in China, japan, korea, etc. Its relatively high replication capacity during infection is more likely to lead to BK virus-related diseases such as BK viral nephropathy. On the other hand, BK virus strains-IV/a-1, IV/a-2, IV/b-1, IV/b-2, IV/c-1 are also distributed to a certain extent in the northeast Asia population. Strain IV/c-2 is less common in patients in east Asia, and is common in Western, indian subcontinent, africa, etc.

In this example, specific regions of target strains were amplified by designing specific primers for different strains, and PCR amplification was performed on the viral RNA extracted using the primers. And carrying out sequencing analysis on the amplified product, and judging whether the strain-I and the strain-IV exist in the sample according to the sequencing result.

TABLE 11BK Virus Strain-I and Strain-IV detection Table

In this example, 3 miRNAs of BK virus type-I and type-IV were subjected to ROC curve analysis, and the AUC area of type-I and the AUC area of type-IV were subjected to variance analysis, the p value was 0.004 (p < 0.05), and the 3 miRNAs were significantly different between type-I and type-IV (FIG. 9). According to the risk assessment model established in example 4, in BKV kidney disease positive diagnosis patients, when the initial copy number of miRNA-score is less than 37 and is greater than 7.15log copies/mL, the detection sample is considered to be BK virus strain I, and when the initial copy number is less than 7.15log copies/mL, the detection sample is considered to be BK virus strain IV. In BK viral nephropathy patients, the initial copy number of strain I ranges from 7.15 to 8.55log, and that of strain IV ranges from 6.50 to 7.10log. In BK virus-carrying patients, the initial copy number of strain I ranges from 6.23 to 7.35log, and that of strain IV ranges from 4.69 to 6.45log.

Example 6: detection result of kit

Urine samples from 15 kidney transplant patients with known pathological outcomes were tested using example 1 and the results are shown in the following table:

table 12 clinical sample verification

Note that: "+" indicates positive detection and "-" indicates negative detection.

From the above results, in the results of the test performed on urine samples of 15 kidney transplant patients in example 1 of the present invention, the accuracy of the fluorescent quantitative PCR test results was 86.7%, the false negative rate was 6.7%, and the false positive rate was 6.7%. The invention has the advantage of high accuracy in detecting the replication activity of BK virus by using a fluorescent quantitative PCR method.

Comparative example 1: two-class Logistic regression assessment of the effect of mirnas on disease and normal groups

The effect of miRNA-b1-5p, miRNA-b1-3p, miRNA-155-5p, miRNA-331-3p, miRNA-221-3p, miRNA-31-5p, miRNA-17-5p, and miRNA-205-5p (sequences shown in Table 13) on the disease group (label-1) and the normal group (label-0) was evaluated using two-class Logistic regression in this comparative example. Wherein the training set sample size: test set sample size 68: 12. and the data are randomly split into a training set and a testing set, wherein the testing set proportion is as follows: 15%, the random factor is: 1. the training set AUC is 0.965, the test set AUC is 0.97, and the model prediction effect is good. Of the variables incorporated into the model, the miRNA-b1-5p had a coefficient of-0.382 and a p value of 0.033, meaning that an influence relationship was to be exerted on the predicted result. The factor of miRNA-b1-3p is-0.584 and the p value is 0.007, which means that the influence relationship on the prediction result is generated. The factor for miRNA-155-5p was-0.399 and the p value was 0.018, meaning that the effect on the predicted outcome would be relevant. The miRNA-331-3p has a coefficient of-0.109 and a p value of 0.238, without significant difference. The miRNA-221-3p had a coefficient of-0.142 and a p value of 0.295 without significant differences. The miRNA-31-5p had a coefficient of-0.012 and a p value of 0.909, and there was no significant difference, meaning that the results were not affected. The miRNA-17-5p had a coefficient of 0.068 and a p value of 0.546 without significant difference. The miRNA-205-5p has a coefficient of-0.199 and a p value of 0.129, and has no significant difference and no effect on the results.

Table 13 five miRNA sequence listing

Comparative example 2: triple miRNA (micro ribonucleic acid) as biomarker detection accuracy contrast

The method of example 1 was used to detect the AUC value of the multivariate combination of 1-or 3-miRNA based on the non-variable miRNA for 8 miRNAs (miRNA-b 1-5p, miRNA-b1-3p, miRNA-155-5p, miRNA-331-3p, miRNA-221-3p, miRNA-31-5p, miRNA-17-5p and miRNA-205-5 p) obtained by screening, and the result is shown in Table 14, from which it can be seen that the combined effect of the triple miRNA is higher than that of the single miRNA as the biomarker.

Table 14 mean AUC values for miRNA combinations

This comparative example also provides experimental studies of the correlation of miRNA-b1-5p, miRNA-b1-3p, miR-155-5p, miRNA-331-3p, miRNA-221-3p, miRNA-31-5p, miRNA-17-5p and miRNA-205-5p with BKVA viral nephrosis events, in which 8 miRNAs were analyzed for ROC curves in a large sample queue by comparing the miRNA expression profiles of BKVA viral nephrosis and suspected BKVA viral nephrosis, and compared with the ROC curves of the tri-variable miRNAs, the accuracy of detecting BK viral replication activity by the triple miRNAs (miRNA-b 1-5p, miRNA-b1-3p and miRNA-155-5 p) is better than other combinations of miRNAs as shown in FIG. 10. Fluorescent quantitative PCR (polymerase chain reaction) of triple combinations of miRNA-b1-5p, miRNA-b1-3p and miRNA-155-5p is the optimal combination for detecting the replication activity of BK viruses.

Comparative example 3: comparison of SV40 and miRNA detection results

The comparative example provides comparison of BK virus SV40 and miRNA qPCR detection results, and three-way miRNAs of miRNA-b1-5p, miRNA-b1-3p and miRNA-155-5p are used as biomarker combinations, urine samples of 30 kidney biopsy patients are detected, and the clinical manifestation is as follows:

table 15BK Virus SV40 vs. miRNA qPCR detection results

In the comparative example, the SV40 staining method is used as a gold standard for detecting the replication activity of BK virus, the specificity is almost 100%, and the accuracy of the triple miRNA qPCR method reaches 96.6%.

Comparative example 4: DNA and miRNA as biomarker detection contrast

The comparative example provides a comparison of BK viral DNA PCR and miRNA qPCR detection results, with triple miRNAs of miRNA-b1-5p, miRNA-b1-3p and miRNA-155-5p as biomarker combinations, from urine samples of 30 kidney biopsy patients. The clinical manifestations are as follows:

table 16BK viral DNA PCR vs miRNA qPCR detection results

In this comparative example, the detection result accuracy of the triple miRNA qPCR was 96.6% compared to the SV40 staining method, whereas the detection accuracy of the BKV DNA copy number method was 90%. The miRNA qPCR method is more accurate in detection.

The above-described embodiments are merely preferred embodiments for fully explaining the present invention, and the scope of the present invention is not limited thereto. Equivalent substitutions and modifications will occur to those skilled in the art based on the present invention, and are intended to be within the scope of the present invention. The protection scope of the invention is subject to the claims.

Claims (10)

1. A miRNA biomarker combination for bk viral nephropathy characterized by comprising miRNA-b1-5p, miRNA-b1-3p and miRNA-155-5p;

   the nucleotide sequence of the miRNA-b1-5p is shown as SEQ ID NO.1, the nucleotide sequence of the miRNA-b1-3p is shown as SEQ ID NO.2, and the nucleotide sequence of the miRNA-155-5p is shown as SEQ ID NO. 3.
2. 2. Use of the miRNA biomarker combination of claim 1 in the preparation of a product for BK virus nephropathy screening, assisting in BK virus nephropathy pathological identification or in BK virus nephropathy early clinical diagnosis.
3. 3. A primer composition for amplifying the miRNA biomarker combination of claim 1, comprising:

   primer pairs for amplifying miRNA-b1-5p as shown in SEQ ID No. 4-5;

   primer pairs for amplifying miRNA-b1-3p as shown in SEQ ID No. 6-7;

   primer pairs for amplifying miRNA-155-5p as shown in SEQ ID No. 8-9.
4. BK virus nephrosis detection kit, its characterized in that: the kit detects BK virus kidney disease by detecting the miRNA biomarker combination of claim 1.
5. 5. The kit of claim 4, wherein: the kit comprising the primer composition of claim 3.
6. 6. The kit of claim 5, further comprising:

   (1) The reverse transcription primer for synthesizing cDNA is shown in SEQ ID NO.10, the reverse transcription primer of miRNA-b1-5p is shown in SEQ ID NO.11, and the reverse transcription primer of miRNA-155-5p is shown in SEQ ID NO. 12.

   (2) The Taqman probe, wherein the probe sequence for detecting miRNA-b1-5p is shown as SEQ ID NO.13, the probe sequence for detecting miRNA-b1-3p is shown as SEQ ID NO.14, and the probe sequence for detecting miRNA-155-5p is shown as SEQ ID NO. 15.
7. 7. The kit of claim 6, wherein the detection is performed by:

   (1) Collecting a urine sample to be tested;

   (2) Extracting total RNA in urine, and performing reverse transcription to obtain cDNA;

   (3) Using cDNA as a template, and detecting the expression level of the miRNA marker in urine by using a fluorescent real-time quantitative PCR technology based on a Taqman probe;

   (4) Calculating a composite diagnostic score Y, wherein Y = beta _miRNA-b1-5p ×(-0.3190)+β _miRNA-b1-3p ×(-0.4529)+β _miRNA-155-5p X (-0.2054) +33.55; when the comprehensive diagnosis score is less than 37, the detection result is considered to be positive; when the integrated diagnosis score is greater than 37, the detection result is considered negative.
8. 8. Use of the miRNA biomarker combination of claim 1 in the preparation of a BK virus strain identification product.
9. 9. Use of the kit of any one of claims 4-7 for the preparation of a BK virus strain identification product.
10. 10. The use according to claim 9, characterized in that: when the kit is used for BK virus strain detection, under the condition that the comprehensive diagnosis score is smaller than 37, the initial copy number is higher than 7.15log copies/mL, the detection sample is BK virus strain I, the initial copy number is lower than 7.15log copies/mL, and the detection sample is BK virus strain IV.