A miRNA combination, kit and method of use for assisting liver fibrosis staging screening

Abstract

The present invention relates to the field of molecular diagnostic technology, and discloses a miRNA combination, a kit, and a method for use for assisting in the staging screening of liver fibrosis. The miRNA combination includes miR-216b-5p, miR-2681-5p, miR-1178-3p, miR-1323, and miR-4418. The kit is used for ddPCR detection of the content of miRNA markers in a sample, and the kit includes miRNA marker reverse transcription primers and miRNA marker amplification primers. The miRNA combination provided by the present invention is an ideal and reliable miRNA marker for assisting in the staging screening of liver fibrosis. The reverse transcription primers and amplification primers provided by the detection kit have good specificity, high sensitivity, and strong specificity. Based on the ddPCR technology combined with the kit of the present invention, more accurate and reliable miRNA marker concentration values can be obtained.

Description

Technical Field

[0001] The present invention relates to the field of molecular diagnostic technology, and in particular to a miRNA combination, a kit and a method of use for assisting in staging screening of liver fibrosis. Background Art

[0002] Liver fibrosis is a pathological process characterized by excessive deposition of the extracellular matrix (ECM) after the liver is stimulated by various pathogenic factors. It is a repair response of the liver to injury. When this repair response is excessive and uncontrolled, it leads to the occurrence and progression of liver fibrosis. Long-term liver fibrosis can lead to severe damage to the liver's structure and function, and then progress to cirrhosis, liver failure, and even liver cancer, seriously affecting the patient's quality of life and prognosis. Studies have shown that common causes of liver fibrosis include viral hepatitis, alcoholic liver disease, non-alcoholic fatty liver disease, autoimmune liver disease, and drug-induced liver injury. According to statistics, approximately 20%-30% of patients with chronic hepatitis B will develop liver fibrosis within 5 years.

[0003] The METAVIR scoring system is commonly used in clinical liver fibrosis staging. It categorizes liver fibrosis into five stages, F0-F4, based on severity. F0 indicates no fibrosis; F1 indicates portal fibrosis without septal formation; F2 indicates portal fibrosis with a few septal formation; F3 indicates extensive septal formation without cirrhosis; and F4 indicates cirrhosis. The prognosis for patients in different stages is relatively good, as patients in stages F0-F2 have a relatively good prognosis due to the potential for reversal of fibrosis with active treatment of the underlying cause and appropriate interventions. Although patients in stage F3 have a moderate degree of disease progression, timely and effective treatment can still control the disease and slow the progression of cirrhosis, resulting in an intermediate prognosis. However, patients in stage F4, especially those with severe complications, have a poorer prognosis, with a higher risk of serious adverse events such as liver failure and liver cancer, which can significantly impact their quality of life and survival.

[0004] Currently, liver biopsy is the gold standard for diagnosing liver fibrosis. Under local anesthesia, a liver tissue sample is obtained using a needle. Pathological biopsy then examines the tissue under a microscope for pathological changes, accurately determining the extent and stage of fibrosis, as well as the presence of inflammation. However, this is an invasive procedure that can be painful and risky for the patient. It is also subject to sampling errors and may not fully reflect the extent of fibrosis throughout the liver. Conventional serum markers, such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST), as well as hyaluronic acid and laminin, lack specificity, and elevated levels do not necessarily indicate liver fibrosis. These markers may also be elevated in certain inflammatory diseases and tumors. Traditional imaging studies, however, rely heavily on the examiner's experience and technique and cannot accurately assess the extent of fibrosis. Some innovative non-invasive diagnostic technologies, such as FibroScan, can assess the extent of liver fibrosis by measuring liver stiffness. While simple, rapid, and non-invasive, these technologies are subject to patient and disease factors. For example, obesity or a large amount of ascites can interfere with the ultrasound transmission path and signal, making the measurement data inaccurate and potentially overestimating or underestimating the extent of liver fibrosis. Furthermore, they are affected by various technical factors, such as the placement, angle, and pressure of the ultrasound probe, resulting in different results between operators.

[0005] miRNA (microRNA) is a small, non-coding RNA molecule of approximately 22 nucleotides that is widely present in eukaryotes. It is crucial for cell development, differentiation, proliferation, and apoptosis, regulating and influencing the entire life process. Currently, miRNA in blood is recognized as one of the most sensitive biomarkers in the medical field. Extensive research evidence indicates that miRNA can serve as a new generation of biomarkers, with important application prospects and significance in the diagnosis and prognosis of various diseases. Currently, over 2,800 miRNA species have been discovered, and one of the objectives of the present invention is to screen for miRNA markers that can assist in the staging and screening of liver fibrosis. Furthermore, the levels of miRNA molecules in human blood are generally low, and conventional fluorescence quantitative PCR methods cannot guarantee accurate detection. A second objective of the present invention is to provide a method to improve the accuracy of miRNA detection. Summary of the Invention

[0006] In order to solve the above problems, the present invention provides a miRNA combination, kit and method of use for assisting in the staging screening of liver fibrosis. The specific combination of miRNAs provided by the present invention (miR-216b-5p, miR-2681-5p, miR-1178-3p, miR-1323 and miR-4418) can be used to assist in the staging screening of liver fibrosis. The kit of the present invention combined with droplet digital PCR technology can simply, effectively and accurately obtain the concentration of specific miRNA in peripheral blood, with the characteristics of high sensitivity and strong specificity.

[0007] In order to solve the above technical problems, the present invention provides the following technical solutions.

[0008] In a first aspect, the present invention provides a miRNA combination for assisting in the staging screening of liver fibrosis, comprising miR-216b-5p, miR-2681-5p, miR-1178-3p, miR-1323, and miR-4418.

[0009] In a second aspect, the present invention provides a kit for assisting in the staging screening of liver fibrosis, wherein the kit is used for detecting the content of miRNA markers in a sample by dd PCR, wherein the miRNA markers include miR-216b-5p, miR-2681-5p, miR-1178-3p, miR-1323 and miR-4418.

[0010] Furthermore, the kit includes miRNA marker reverse transcription primers and miRNA marker amplification primers.

[0011] Furthermore, the miRNA marker reverse transcription primer includes:

[0012] Primer for reverse transcription of miR-216b-5p: SEQ ID No: 1;

[0013] Primer for reverse transcription of miR-2681-5p: SEQ ID No: 4;

[0014] Primer for reverse transcription of miR-1178-3p: SEQ ID No: 7;

[0015] Primer for reverse transcription of miR-1323: SEQ ID No: 10;

[0016] Primer for reverse transcription of miR-4418: SEQ ID No: 13.

[0017] Furthermore, the miRNA marker amplification primers include:

[0018] Primer pair used to amplify miR-216b-5p: SEQ ID No: 2 and SEQ ID No: 3;

[0019] Primer pair used to amplify miR-2681-5p: SEQ ID No: 5 and SEQ ID No: 6;

[0020] Primer pair used to amplify miR-1178-3p: SEQ ID No: 8 and SEQ ID No: 9;

[0021] Primer pair used to amplify miR-1323: SEQ ID No: 11 and SEQ ID No: 12;

[0022] Primer pair used to amplify miR-4418: SEQ ID No: 14 and SEQ ID No: 15.

[0023] Furthermore, the kit further comprises a quality control product and its reverse transcription primer and amplification primer, and the RNA sequence of the quality control product is shown in SEQ ID No: 16. The addition of the quality control product ensures the reliability of the ddPCR detection results.

[0024] Furthermore, the sequence of the reverse transcription primer of the quality control product is shown as SEQ ID No: 17.

[0025] Furthermore, the sequences of the amplification primers of the quality control product are shown as SEQ ID No: 18 and SEQ ID No: 19.

[0026] In a third aspect, the present invention provides a method for obtaining the concentration of miRNA markers using the above kit, which comprises the following steps:

[0027] Step 1: Obtain a blood sample and centrifuge to obtain a plasma sample. A volume of C μL of the plasma sample is aspirated for total free RNA extraction. After extraction, the total free RNA concentration is adjusted to 20-40 ng / μL to obtain a total free RNA sample volume of A μL.

[0028] Step 2: Configure a reverse transcription reaction system to perform a reverse transcription reaction to obtain a reverse transcription sample; the reverse transcription reaction system includes dNTPs, reverse transcriptase, reverse transcription buffer, RNase inhibitor, reverse transcription primer, total free RNA sample and ultrapure water; further, the reverse transcription reaction system contains the following components per 15 μL: 0.2 μL of dNTPs (with dTTP), 1 μL of Reverse Transcriptase, 1.5 μL of RT Buffer, 0.2 μL of RNase Inhibitor, 3 μL of reverse transcription primer, 3 μL of total free RNA sample, and 6.1 μL of ultrapure water; the reverse transcription primer is a miRNA marker reverse transcription primer, and the reverse transcription sample is a miRNA marker reverse transcription sample; further, the reaction conditions of the reverse transcription reaction are: 16° C. for 30 min; 42° C. for 30 min; 85° C. for 5 min; and storage at 4° C.

[0029] Step 3: Configure a PCR reaction system to prepare PCR reaction droplets, transfer the PCR reaction droplets to a 96-well plate for PCR reaction, and transfer the 96-well plate after the reaction to a dd PCR reader to obtain a result value B; the PCR reaction system includes a Supermix reagent, an amplification primer, a reverse transcription sample, and ultrapure water; further, each 20 μL of the PCR reaction system contains the following components: 10 μL of Supermix reagent, 1 μL of amplification primer, 3 μL of reverse transcription sample, and 6 μL of ultrapure water; the amplification primer is a miRNA marker amplification primer, and the reverse transcription sample is a miRNA marker reverse transcription sample; further, the reaction conditions of the PCR reaction are: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 15 s, annealing and extension at 61°C for 1 min, 40 cycles; extension at 95°C for 10 min; and storage at 10°C.

[0030] Step 4: Calculate the miRNA marker concentration according to formula (I);

[0031] (I),

[0032] Where A is the volume of the total free RNA sample in step 1;

[0033] C is the volume of the plasma sample from which total free RNA was extracted in step 1;

[0034] G is the total volume of the reverse transcription reaction system in step 2;

[0035] F is the volume of the total free RNA sample in the reverse transcription reaction system in step 2;

[0036] B is the reading result of the ddPCR reader in step 3, in copies / μL;

[0037] D is the total volume of the PCR reaction system in step 3;

[0038] E is the volume of the miRNA marker reverse transcription sample in the PCR reaction system in step 3;

[0039] The units used in A, C, D, E, F, and G are consistent.

[0040] Furthermore, in some embodiments of the present invention, the above method further includes quality control of the reverse transcription reaction and the PCR reaction. Specifically, in the above step 1, H μL of quality control material (concentration of 5×10 4 After 100 copies / μL), total free RNA was extracted. In step 2, the reverse transcription primers of the quality control were used to obtain the reverse transcription sample of the quality control. In step 3, the amplification primers and reverse transcription samples were used to obtain the dd PCR result value. The concentration of the quality control was calculated by replacing C with H in formula (I). If the calculated concentration of the quality control was between 4×10 4 ~5×10 4 If the miRNA marker concentration is within the range of 10 copies / μL, the calculated result is valid. If it is outside this range, re-extraction and testing are required. Quality control can ensure the reliability of miRNA marker concentrations, thereby ensuring the accuracy of liver fibrosis staging screening.

[0041] Droplet Digital™ PCR (ddPCR), as a third-generation PCR technology, represents a significant innovation and breakthrough in trace detection technology. Based on the Droplet Digital PCR platform, this invention provides a kit and method for use that ensures the accuracy of miRNA marker content detection results, with high sensitivity and strong specificity.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] The miRNA combination and detection kit for assisting in liver fibrosis staging screening provided by the present invention use the reverse transcription-droplet digital PCR method and have been verified and evaluated in a large number of patients with different liver fibrosis stages. It has high sensitivity and strong specificity. The miRNA combination provided by the present invention is an ideal and reliable miRNA marker for assisting in liver fibrosis staging screening. The reverse transcription primers and amplification primers provided by its detection kit have good specificity. Based on the ddPCR technology combined with the kit of the present invention, more accurate and reliable miRNA marker concentration values can be obtained. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is the ROC curve for distinguishing patients in F0-F2 stage from patients in F3-F4 stage in an embodiment of the present invention;

[0045] Figure 2 This is the ROC curve for distinguishing between F3 stage patients and F4 stage patients in the embodiment of the present invention. DETAILED DESCRIPTION

[0046] In order to make those skilled in the art better understand the technical solution of the present invention, the present invention is further described in detail below in conjunction with specific examples and drawings. The reagents involved in the embodiments of the present invention are all commercially available products. Unless otherwise specified, they can be purchased through commercial channels. In the embodiments of the present invention, the quality control products, reverse transcription primers and PCR amplification primers used were synthesized by a biological company based on the sequences submitted by the applicant of the present invention.

[0047] A miRNA combination for assisting in the staging and screening of liver fibrosis includes miR-216b-5p, miR-2681-5p, miR-1178-3p, miR-1323, and miR-4418. The sequences of miR-216b-5p, miR-2681-5p, miR-1178-3p, miR-1323, and miR-4418 involved in the present invention are shown in Table 1 below.

[0048] Table 1. Public database information of the five miRNA sequences provided by the present invention

[0049]

[0050] A kit for assisting in the staging screening of liver fibrosis, the kit is used for ddPCR detection of the content of miRNA markers in a sample, the miRNA markers including miR-216b-5p, miR-2681-5p, miR-1178-3p, miR-1323, and miR-4418. The kit includes miRNA marker reverse transcription primers, miRNA marker amplification primers, reverse transcription primers and amplification primers for quality control products. The quality control product is 5×10 4 copies / μL of synthetic single-stranded miRNA (cel-miRNA-39). The sequences of quality control products and each reverse transcription primer and amplification primer are shown in Table 2 below.

[0051] Table 2. Sequence list of quality control products, reverse transcription primers, and amplification primers

[0052]

[0053] The specific steps of the embodiment of the present invention for assisting liver fibrosis staging screening are as follows:

[0054] 1. Draw approximately 3 ml of venous blood and centrifuge at 2000 g for 10 minutes at 4°C to separate the upper plasma layer. Centrifuge again at 12000 g for 20 minutes at 4°C to collect the upper plasma sample. Aspirate approximately 200 μL of plasma and extract total free RNA using the QIAGEN miRNeasy Serum / Plasma Kit (note that 10 μL of a quality control sample should be added before RNA extraction). After extraction, determine the concentration and adjust it to 20-40 ng / μL to obtain the total free RNA sample.

[0055] 2. Prepare the reverse transcription reaction system of miRNA markers and quality control products, carry out the reaction, and obtain reverse transcription samples of miRNA markers and quality control products.

[0056] The reverse transcription reaction system for miRNA markers and quality control products is shown in Table 3 below, and the reverse transcription reaction conditions are shown in Table 4 below (Note: Each miRNA and quality control product is tested in a separate tube).

[0057] Table 3. Reverse transcription reaction system

[0058]

[0059] Table 4. Reverse transcription reaction conditions

[0060]

[0061] 3. Configure the digital PCR reaction system, prepare digital PCR reaction droplets, and then transfer them to a 96-well plate for PCR reaction. After the reaction, transfer the 96-well plate to the QX200™ Digital PCR Reader to read the results.

[0062] The digital PCR reaction system is shown in Table 5 below, and the reaction conditions are shown in Table 6 below (Note: Each miRNA and quality control product is tested in a separate tube).

[0063] Table 5. Digital PCR detection reaction system

[0064]

[0065] Table 6. Digital PCR reaction conditions

[0066]

[0067] 4. Calculation of results:

[0068] (1) Calculation of quality control products: Assuming that the total volume of the total free RNA sample is A (μL), and the result value read by the QX200™ digital PCR reader is B (copies / μL), the concentration of the quality control product X (copies / μL) is calculated according to the following formula (II):

[0069] (II).

[0070] (2) The test results of quality control products should meet the requirements of 4×10 4 ~5×10 4 copies / μL, if the sample meets the requirements, the miRNA detection results of the sample are valid; otherwise, the experiment is invalid and needs to be re-extracted for detection.

[0071] (3) If the quality control product results are qualified, the concentration Y (copies / μL) of the miRNA marker in the plasma sample (i.e., the content of the miRNA marker in the sample to be tested) is calculated according to the following formula (III):

[0072] (III).

[0073] 5. Risk Assessment

[0074] First, calculate the P1 value according to formula (IV): (IV);

[0075] Wherein, S1=2.125+0.02×m1+0.014×m2-0.033×m3+0.052×m4+0.017×m5, where m1 is the miR-216b-5p content in the test sample; m2 is the miR-2681-5p content in the test sample; m3 is the miR-1178-3p content in the test sample; m4 is the miR-1323 content in the test sample; and m5 is the miR-4418 content in the test sample. The unit of m1, m2, m3, m4, and m5 is copies / μL.

[0076] When P1≤0.39, it means that the subject has no liver fibrosis or only mild liver fibrosis (F0-F2).

[0077] When P1>0.39, it means that the subject has significant liver fibrosis or cirrhosis (F3-F4).

[0078] For patients who were assessed as having significant liver fibrosis or cirrhosis (F3-F4), the P2 value was calculated according to formula (V): (V);

[0079] Wherein, S2 = 6.114 + 0.034 × m1 + 0.047 × m2 - 0.061 × m3 + 0.034 × m4 + 0.022 × m5, where m1 represents the miR-216b-5p content in the sample to be tested; m2 represents the miR-2681-5p content in the sample to be tested; m3 represents the miR-1178-3p content in the sample to be tested; m4 represents the miR-1323 content in the sample to be tested; and m5 represents the miR-4418 content in the sample to be tested. The unit of m1, m2, m3, m4, and m5 is copies / μL.

[0080] When P2≤0.44, it means that the subject has a large number of septa formed in liver fibrosis but no cirrhosis (F3).

[0081] When P2>0.44, it means the subject has liver cirrhosis (F4).

[0082] 46 patients with liver fibrosis in stages F0-F2 with a relatively good expected prognosis, 42 patients with liver fibrosis in stage F3 with a moderate expected prognosis, and 45 patients with liver cirrhosis in stage F4 with a poor expected prognosis were included. Results were calculated and risk assessed according to the above-mentioned steps for assisting liver fibrosis staging screening (plasma collection, total miRNA extraction from plasma, digital PCR reaction, and calculation of P1 or P2 values according to the above steps after the reaction). Data analysis was then performed. GraphPad Prism software was used for data analysis, and the ROC curve for the test results was obtained. Figure 1 and Figure 2 shown. Figure 1 The ROC curve for distinguishing patients in stage F0-F2 from those in stage F3-F4 showed an AUC of 0.91, a sensitivity of 89.5%, and a specificity of 78.2%; Figure 2 The ROC curve for distinguishing patients in stage F3 from patients in stage F4 had an AUC of 0.85, a sensitivity of 84.4%, and a specificity of 73.8%.

[0083] In summary, the miRNA combination, kit, and method of use provided by the present invention can assist in the staging screening of liver fibrosis. They have high sensitivity and strong specificity, and are simple, rapid, and non-invasive to operate. They are less affected by external factors and have good prospects in clinical applications.

[0084] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be construed as limiting the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. Persons skilled in the art will appreciate that improvements and modifications may be made without departing from the spirit and scope of the present invention, and such improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Use of a primer for detecting miRNA markers in the preparation of a kit for assisting in the staging screening of liver fibrosis, characterized in that: The kit is used for ddPCR detection of the content of miRNA markers in a sample, and the miRNA markers consist of miR-216b-5p, miR-2681-5p, miR-1178-3p, miR-1323 and miR-4418.

2. The use according to claim 1, characterized in that: The kit includes a miRNA marker reverse transcription primer and a miRNA marker amplification primer.

3. The use according to claim 2, characterized in that: The miRNA marker reverse transcription primer includes: Primer for reverse transcription of miR-216b-5p: SEQ ID No: 1; Primer for reverse transcription of miR-2681-5p: SEQ ID No: 4; Primer for reverse transcription of miR-1178-3p: SEQ ID No: 7; Primer for reverse transcription of miR-1323: SEQ ID No: 10; Primer for reverse transcription of miR-4418: SEQ ID No:

13.

4. The use according to claim 2, characterized in that: The miRNA marker amplification primers include: Primer pair used to amplify miR-216b-5p: SEQ ID No: 2 and SEQ ID No: 3; Primer pair used to amplify miR-2681-5p: SEQ ID No: 5 and SEQ ID No: 6; Primer pair used to amplify miR-1178-3p: SEQ ID No: 8 and SEQ ID No: 9; Primer pair used to amplify miR-1323: SEQ ID No: 11 and SEQ ID No: 12; Primer pair used to amplify miR-4418: SEQ ID No: 14 and SEQ ID No:

15.

5. The use according to claim 2, characterized in that: The kit further includes a quality control product and reverse transcription primers and amplification primers for the quality control product. The RNA sequence of the quality control product is shown in SEQ ID No:

16.

6. The use according to claim 5, characterized in that: The sequence of the reverse transcription primer of the quality control product is shown in SEQ ID No:

17.

7. The use according to claim 5, characterized in that: The sequences of the amplification primers of the quality control product are shown in SEQ ID No: 18 and SEQ ID No: 19.

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