MiRNA combination and kit for assisting stage screening of hepatic fibrosis and use method of miRNA combination and kit

Abstract

The invention relates to the technical field of molecular diagnosis, and discloses a miRNA combination for assisting hepatic fibrosis staged screening, a kit and a use method, the miRNA combination comprises 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 comprises a miRNA marker reverse transcription primer and a miRNA marker amplification primer. The miRNA composition provided by the invention is an ideal and reliable miRNA marker used for assisting in hepatic fibrosis staged screening, the reverse transcription primer and the amplification primer provided by the detection kit have good specificity, high sensitivity and strong specificity, and a more accurate and reliable miRNA marker concentration value can be obtained based on the ddPCR technology in combination with the kit provided by the invention.

Description

Technical Field

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

[0002] Liver fibrosis is a pathological process mainly characterized by excessive deposition of 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 out of control, the occurrence and development of liver fibrosis will be caused. Long-term liver fibrosis can lead to severe damage to the structure and function of the liver, and then develop into cirrhosis, liver failure, and even liver cancer, seriously affecting the quality of life and prognosis of patients. Research shows 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, etc. According to statistics, among patients with chronic hepatitis B, about 20%-30% will develop liver fibrosis within 5 years.

[0003] The METAVIR scoring system is commonly used for clinical liver fibrosis staging. According to the severity, liver fibrosis is divided into 5 stages from F0 to F4. F0 indicates no fibrosis; F1 is fibrosis in the portal area without septum formation; F2 is fibrosis in the portal area with a few septum formations; F3 is a large number of septum formations without cirrhosis; F4 is cirrhosis. From the perspective of the prognosis of patients in different stages, for patients in the F0 - F2 stages, after actively treating the cause and taking appropriate intervention measures, liver fibrosis may be reversed and the prognosis is relatively good. For patients in the F3 stage, although the condition has progressed to a certain extent, if effective treatment can be carried out in a timely manner, it is still possible to control the condition and delay the process of cirrhosis, and the prognosis is at a medium level. For patients with F4-stage cirrhosis, especially those with severe complications, the prognosis is poor, and the risk of serious adverse events such as liver failure and liver cancer is relatively high, and the quality of life and survival period may be greatly affected.

[0004] Currently, liver biopsy is the gold standard for diagnosing liver fibrosis. By using a puncture needle to obtain a liver tissue sample under local anesthesia, followed by pathological section examination, the pathological changes of the liver tissue are observed under a microscope to accurately judge the degree, stage of liver fibrosis, and the presence of inflammation, etc. However, it is an invasive examination, which may bring certain pain and risks to patients, and there is sampling error. Sometimes it may not fully reflect the fibrosis situation of the entire liver. Conventional serological indicators such as alanine aminotransferase (ALT), aspartate aminotransferase (AST) and other conventional liver biochemical indicators, or hyaluronic acid, laminin, etc., all lack specificity, and their elevated levels are not necessarily entirely caused by liver fibrosis. In some cases of inflammatory diseases, tumors, etc., these indicators may also increase. Traditional imaging examinations rely on the experience and operation techniques of the examiners and cannot accurately judge the degree of fibrosis. Some innovative non-invasive diagnostic techniques, such as FibroScan, can evaluate the degree of liver fibrosis by measuring the liver stiffness value. It is simple, rapid, and non-invasive, but it is affected by patient or disease factors. For example, in patients with obesity or a large amount of ascites, it will interfere with the propagation path and signal of ultrasound, making the measured data inaccurate, and may overestimate or underestimate the degree of liver fibrosis. Moreover, it is also affected by various technical factors, such as the placement position, angle, pressure of the ultrasound probe, and different operators may obtain different measurement results.

[0005] miRNA (microRNA) is a non-coding small RNA molecule about 22 nucleotides in length widely present in eukaryotes. It is crucial for cell development, differentiation, proliferation, and apoptosis, regulating and influencing the entire process of life activities. Currently, miRNA in blood is one of the most sensitive biomarkers recognized in the current medical field. A large amount of research evidence shows that miRNA can be used as a new generation of biomarker, having important application prospects and significance in the diagnosis and prognosis of various diseases. Currently, about more than 2,800 types of miRNA have been discovered. One of the purposes of the present invention is to screen out miRNA markers that can assist in the screening of liver fibrosis staging. And the content of miRNA molecules in human blood is generally low, and the conventional fluorescence quantitative PCR method cannot guarantee the accuracy of detection. The second purpose of the present invention is to provide a method for improving the accuracy of miRNA content detection. Summary of the Invention

[0006] To solve the above problems, the present invention provides a miRNA combination, a kit and a usage method for assisting in the screening of liver fibrosis staging. 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 screening of liver fibrosis staging. By combining the kit of the present invention with droplet digital PCR technology, the concentration of specific miRNAs in peripheral blood can be simply, effectively and accurately obtained, with the characteristics of high sensitivity and strong specificity.

[0007] To solve the above technical problems, the present invention provides the following technical solutions.

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

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

[0010] Further, the kit includes reverse transcription primers for miRNA markers and amplification primers for miRNA markers.

[0011] Further, the reverse transcription primers for miRNA markers include: Primer for reverse transcribing miR-216b-5p: SEQ ID No: 1; Primer for reverse transcribing miR-2681-5p: SEQ ID No: 2; Primer for reverse transcribing miR-1178-3p: SEQ ID No: 3; Primer for reverse transcribing miR-1323: SEQ ID No: 4; Primer for reverse transcribing miR-4418: SEQ ID No: 5.

[0012] Further, the amplification primers for miRNA markers include: Primer pair for amplifying miR-216b-5p: SEQ ID No: 6 and SEQ ID No: 7; Primer pair for amplifying miR-2681-5p: SEQ ID No: 8 and SEQ ID No: 9; Primer pairs for amplifying miR-1178-3p: SEQ ID No: 10 and SEQ ID No: 11; Primer pairs for amplifying miR-1323: SEQ ID No: 12 and SEQ ID No: 13; Primer pairs for amplifying miR-4418: SEQ ID No: 14 and SEQ ID No: 15.

[0013] Furthermore, the kit further includes a quality control product, its reverse transcription primer and amplification primer, and the RNA sequence of the quality control product is as shown in SEQ ID No: 16. The addition of the quality control product ensures the reliability of the ddPCR detection result.

[0014] Furthermore, the sequence of the reverse transcription primer of the quality control product is as shown in SEQ ID No: 17.

[0015] Furthermore, the sequences of the amplification primers of the quality control product are as shown in SEQ ID No: 18 and SEQ ID No: 19.

[0016] In a third aspect, the present invention provides a method for obtaining the concentration of miRNA markers using the above kit, which includes the following steps: Step 1: Obtain a plasma sample by centrifuging a blood sample, aspirate a plasma sample with a volume of C μL for extraction of total free RNA, and adjust the concentration of the total free RNA to 20 - 40 ng / μL after extraction to obtain a total free RNA sample with a volume of A μL; 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; furthermore, each 15 μL of the reverse transcription reaction system contains the following components: 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, 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; furthermore, the reaction conditions of the reverse transcription reaction are: incubation at 16°C for 30 min; incubation at 42°C for 30 min; incubation at 85°C for 5 min; storage at 4°C; Step 3: Configure the PCR reaction system to prepare PCR reaction droplets, transfer the PCR reaction droplets to a 96-well plate for PCR reaction, and transfer the reacted 96-well plate to a ddPCR reader to obtain the result value B; the PCR reaction system includes Supermix reagent, amplification primers, reverse transcription samples, 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 primers, 3 μL of reverse transcription samples, and 6 μL of ultrapure water; the amplification primers are miRNA marker amplification primers, and the reverse transcription samples are miRNA marker reverse transcription samples; further, the reaction conditions for 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, for 40 cycles; extension at 95°C for 10 min; storage at 10°C; Step 4: Calculate the miRNA marker concentration according to formula (Ⅰ); (Ⅰ), where A is the volume of the total free RNA sample in Step 1; C is the volume of the plasma sample from which total free RNA is extracted in Step 1; G is the total volume of the reverse transcription reaction system in Step 2; F is the volume of the total free RNA sample in the reverse transcription reaction system of Step 2; B is the reading result of the ddPCR reader in Step 3, in units of copies / μL; D is the total volume of the PCR reaction system in Step 3; E is the volume of the miRNA marker reverse transcription sample in the PCR reaction system of Step 3; A, C, D, E, F, and G are in the same unit.

[0017] Further, in some embodiments of the present invention, the above method further includes the quality control of the reverse transcription reaction and the PCR reaction. Specifically, in Step 1 above, add H μL of a quality control product (concentration: 5×10 4 copies / μL) to the plasma sample with a volume of C μL, and then extract the total free RNA. In Step 2, use the reverse transcription primers of the quality control product to obtain the reverse transcription sample of the quality control product. In Step 3, use the amplification primers and reverse transcription samples of the quality control product to obtain the ddPCR result value. Replace C in formula (Ⅰ) with H to calculate the concentration of the quality control product. If the calculated concentration of the quality control product is within 4×10 4 ~5×10 4If the concentration calculation result of the miRNA marker is valid within the range value of copies / μL, and if it exceeds this range value, re-extraction and detection are required. Through quality control, the reliability of the miRNA marker concentration can be ensured, thus ensuring the accuracy of liver fibrosis staging screening.

[0018] Droplet Digital™ PCR (ddPCR for short), as the third-generation PCR technology, has made significant innovations and breakthroughs in trace detection technology. Based on the droplet digital PCR platform, the present invention provides a set of kits and their usage methods, ensuring the accuracy of the miRNA marker content detection results, and having characteristics such as high sensitivity and strong specificity.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: The miRNA combination and its detection kit for assisting liver fibrosis staging screening provided by the present invention apply the reverse transcription-droplet digital PCR method, which has 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 liver fibrosis staging screening. The reverse transcription primers and amplification primers provided by its detection kit have good specificity. Based on the ddPCR technology and combined with the kit of the present invention, more accurate and reliable miRNA marker concentration values can be obtained. Description of the Drawings

[0020] Figure 1 It is the ROC curve for distinguishing patients in F0-F2 stage and patients in F3-F4 stage in the embodiment of the present invention; Figure 2 It is the ROC curve for distinguishing patients in F3 stage and patients in F4 stage in the embodiment of the present invention. Detailed Embodiments

[0021] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to specific embodiments and drawings. The reagents involved in the embodiments of the present invention are all commercially available products, and can be obtained through commercial channels without special instructions. In the embodiments of the present invention, the quality control products, reverse transcription primers and PCR amplification primers used are all synthesized by a biological company according to the sequences submitted by the applicant of the present invention.

[0022] A miRNA combination for assisting in the screening of liver fibrosis staging, including 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.

[0023] Table 1. Public database information of the 5 miRNA sequences provided by the present invention

[0024] A kit for assisting in the screening of liver fibrosis staging, the kit is used for detecting the content of miRNA markers in a sample by ddPCR, and the miRNA markers include miR-216b-5p, miR-2681-5p, miR-1178-3p, miR-1323, and miR-4418. The kit includes reverse transcription primers for miRNA markers, amplification primers for miRNA markers, reverse transcription primers for the quality control product, and amplification primers. The quality control product is synthetic single-stranded miRNA (cel-miRNA-39) at 5×10 4 copies / μL. The sequences of the quality control product and each reverse transcription primer and amplification primer are shown in Table 2 below.

[0025] Table 2. Sequence list of quality control product, reverse transcription primers, and amplification primers

[0026] The specific steps of the example for assisting in the screening of liver fibrosis staging in the present invention are as follows: 1. Collect about 3 ml of venous blood. After centrifuging at 2000 g for 10 min at 4°C, separate the upper plasma part, and then continue to centrifuge at 12000 g for 20 min at 4°C to collect the upper plasma sample. Pipette about 200 μL of the plasma sample, and use the miRNeasy Serum / Plasma Kit from QIAGEN to extract total free RNA (note that 10 μL of the quality control product is added before RNA extraction). After extraction, measure the concentration and adjust the concentration to 20 - 40 ng / μL to obtain the total free RNA sample.

[0027] 2. Configure the reverse transcription reaction system for miRNA markers and the quality control product, and carry out the reaction to obtain the reverse transcription samples of miRNA markers and the quality control product.

[0028] The reverse transcription reaction system for miRNA markers and the quality control product is shown in Table 3 below, and the reverse transcription reaction conditions are shown in Table 4 below (note: each miRNA and the quality control product are detected separately in one tube).

[0029] Table 3. Reverse transcription reaction system

[0030] Table 4. Reverse transcription reaction conditions

[0031] 3. Prepare the digital PCR reaction system, prepare the digital PCR reaction droplets, and then transfer them to a 96-well plate for PCR reaction. Transfer the completed 96-well plate to a QX200™ digital PCR reader to read the results.

[0032] 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 are detected separately in a single tube).

[0033] Table 5. Digital PCR detection reaction system

[0034] Table 6. Digital PCR reaction conditions

[0035] 4. Result calculation: (1) Quality control product calculation: Assume 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). Then the concentration X (copies / μL) of the quality control product is calculated according to the following formula (Ⅱ): (Ⅱ).

[0036] (2) The detection result of the quality control product should meet 4×10 4 ~5×10 4 copies / μL. If it meets the requirement, the miRNA detection result of the sample is valid; otherwise, the experiment is invalid and needs to be retested after re-extraction.

[0037] (3) If the quality control product result is 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): (Ⅲ).

[0038] 5. Risk assessment First, calculate the P1 value according to formula (Ⅳ): (Ⅳ); Among them, S1 = 2.125 + 0.02×m1 + 0.014×m2 - 0.033×m3 + 0.052×m4 + 0.017×m5, where m1 is the content of miR-216b-5p in the sample to be tested; m2 is the content of miR-2681-5p in the sample to be tested; m3 is the content of miR-1178-3p in the sample to be tested; m4 is the content of miR-1323 in the sample to be tested; m5 is the content of miR-4418 in the sample to be tested. The units of m1, m2, m3, m4, and m5 are copies / μL.

[0039] When P1 ≤ 0.39, it indicates that the subject does not have liver fibrosis or only has mild fibrosis (F0 - F2).

[0040] When P1 > 0.39, it indicates that the subject has significant liver fibrosis or cirrhosis (F3 - F4).

[0041] For patients diagnosed with significant liver fibrosis or cirrhosis (F3 - F4), calculate the P2 value according to formula (Ⅴ): (Ⅴ); Among them, S2 = 6.114 + 0.034×m1 + 0.047×m2 - 0.061×m3 + 0.034×m4 + 0.022×m5, where m1 is the content of miR-216b-5p in the sample to be tested; m2 is the content of miR-2681-5p in the sample to be tested; m3 is the content of miR-1178-3p in the sample to be tested; m4 is the content of miR-1323 in the sample to be tested; m5 is the content of miR-4418 in the sample to be tested. The units of m1, m2, m3, m4, and m5 are copies / μL.

[0042] When P2 ≤ 0.44, it indicates that the subject has a large number of septa formation in liver fibrosis but no cirrhosis (F3).

[0043] When P2 > 0.44, it indicates that the subject has cirrhosis (F4).

[0044] Forty-six liver fibrosis patients with relatively good expected prognosis in F0 - F2 stage, forty-two liver fibrosis patients with medium expected prognosis in F3 stage, and forty-five cirrhotic patients with poor expected prognosis in F4 stage were included. The results were calculated and risk assessment was carried out according to the above implementation steps for auxiliary liver fibrosis staging screening (collect plasma, extract total miRNA in plasma, perform digital PCR reaction, and after the reaction, calculate the P1 or P2 value according to the above steps), and then data analysis was carried out. The data analysis was performed using GraphPad Prism software, and the ROC curve for the test results control was obtained, as shown in Figure 1 and Figure 2 shown. Figure 1The ROC curve for differentiating patients in F0 - F2 stage and those in F3 - F4 stage has an AUC = 0.91, sensitivity = 89.5%, and specificity = 78.2%; Figure 2 The ROC curve for differentiating patients in F3 stage and those in F4 stage has an AUC = 0.85, sensitivity = 84.4%, and specificity = 73.8%.

[0045] In summary, the miRNA combination, kit and usage method provided by the present invention can assist in the screening of liver fibrosis staging, with high sensitivity, strong specificity, simple, rapid, non - invasive operation, and few external influencing factors, and has good prospects in clinical applications.

[0046] The above are only the preferred embodiments of the present invention. It should be noted that the above - mentioned preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. For those of ordinary skill in the art in this technical field, without departing from the spirit and scope of the present invention, several improvements and modifications can also be made, and these improvements and modifications should also be regarded as within the protection scope of the present invention.

Claims

1. A miRNA combination for assisting staging screening of liver fibrosis, characterized in that: Including miR-216b-5p, miR-2681-5p, miR-1178-3p, miR-1323 and miR-4418.

2. A kit for assisting 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 include miR-216b-5p, miR-2681-5p, miR-1178-3p, miR-1323 and miR-4418.

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

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

5.

5. The kit according to claim 3, characterized in that: The miRNA marker amplification primers include: Primer pair used to amplify miR-216b-5p: SEQ ID No: 6 and SEQ ID No: 7; Primer pair used to amplify miR-2681-5p: SEQ ID No: 8 and SEQ ID No: 9; Primer pair used to amplify miR-1178-3p: SEQ ID No: 10 and SEQ ID No: 11; Primer pair used to amplify miR-1323: SEQ ID No: 12 and SEQ ID No: 13; Primer pair used to amplify miR-4418: SEQ ID No: 14 and SEQ ID No:

15.

6. The kit according to claim 3, characterized in that: The kit also includes a quality control product and a reverse transcription primer and an amplification primer for the quality control product. The RNA sequence of the quality control product is shown in SEQ ID No:

16.

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

17.

8. The kit according to claim 6, 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.

9. A method for obtaining the concentration of miRNA markers using the kit according to any one of claims 2 to 8, characterized in that: The following steps are involved: Step 1, obtain a blood sample and centrifuge to obtain a plasma sample, aspirate a C μL volume of the plasma sample to extract total free RNA, and after extraction, adjust the total free RNA concentration to 20-40 ng / μL to obtain a total free RNA sample with a volume of A μL; Step 2, configuring a reverse transcription reaction system to perform a reverse transcription reaction to obtain a reverse transcription sample; the reverse transcription reaction system comprises dNTPs, reverse transcriptase, reverse transcription buffer, RNase inhibitor, reverse transcription primer, total free RNA sample and 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; Step 3, configuring a PCR reaction system to prepare PCR reaction droplets, transferring the PCR reaction droplets to a 96-well plate for PCR reaction, and transferring 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; the amplification primer is a miRNA marker amplification primer, and the reverse transcription sample is a miRNA marker reverse transcription sample; Step 4, calculate the concentration of miRNA markers according to formula (I); （Ⅰ）， Where A is the volume of the total free RNA sample in step 1; C is the volume of the plasma sample from which total free RNA was extracted in step 1; G is the total volume of the reverse transcription reaction system in step 2; F is the volume of the total free RNA sample in the reverse transcription reaction system in step 2; B is the reading result of the ddPCR reader in step 3, in copies / μL; D is the total volume of the PCR reaction system in step 3; E is the volume of the miRNA marker reverse transcription sample in the PCR reaction system in step 3; The units used in A, C, D, E, F, and G are consistent.

10. The method according to claim 9, characterized in that: The method also includes quality control of reverse transcription reaction and PCR reaction.

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