CN114410795A

CN114410795A - Liver cancer early detection based on miRNA (micro ribonucleic acid) feature marker

Info

Publication number: CN114410795A
Application number: CN202210178482.4A
Authority: CN
Inventors: 张建勋; 张茜; 马刘敏; 黄龙妹; 吕宁; 陈一友
Original assignee: Hangzhou New Horizon Health Technology Co Ltd
Current assignee: Hangzhou New Horizon Health Technology Co Ltd
Priority date: 2022-02-25
Filing date: 2022-02-25
Publication date: 2022-04-29

Abstract

A miRNA characteristic marker detection method for early screening of liver cancer and a corresponding kit for detecting liver cancer. The miRNA to be detected comprises one or more miRNAs selected from hsa-miR-33B-3p, hsa-miR-92B-3p, hsa-miR-205-5p, hsa-miR-374C-5p, hsa-miR-4516-3p and hsa-miR-6803-3p, and the samples to be detected comprise a tissue sample and a plasma sample.

Description

Liver cancer early detection based on miRNA (micro ribonucleic acid) feature marker

Technical Field

The invention relates to a liver cancer detection method based on miRNA (micro ribonucleic acid) characteristic markers and a corresponding kit for liver cancer detection, which can be used for screening liver cancer.

Background

Liver cancer is known as "the king of cancer", one of the most common malignant tumors. The new liver cancer patients in China occupy more than half of the world every year, the death rate is the second place in all malignant tumors, and the lives and the health of people are seriously threatened. Liver cancer is very hidden and has no obvious symptoms in early stage. About 80% of liver cancer patients have been diagnosed in the middle and late stages, the 5-year survival rate is almost zero, and the 5-year survival rate of early liver cancer patients can reach more than 60% after treatment. Therefore, early diagnosis and treatment are important means for fighting against liver cancer. Alpha Fetoprotein (AFP) is a serum marker and a common indicator for diagnosing liver cancer. However, the AFP detection sensitivity is low, and only 60% -70% of patients with early liver cancer have positive AFP value. Meanwhile, conventional imaging examinations such as B-mode ultrasonography and CT have difficulty in finding early liver cancer with a tumor diameter of less than 2cm or a density similar to the parenchyma of the liver. Therefore, early liver cancer diagnosis still faces higher false negative rate and missed diagnosis rate.

Therefore, the researchers of various countries have never stopped searching for new and better liver cancer markers. The ideal tumor marker needs to have higher specificity, and can distinguish liver cancer from cirrhosis, hepatitis, liver regeneration nodules and the like; meanwhile, the kit also needs higher sensitivity, can prompt diagnosis in early liver cancer, and has the characteristics of easy detection, repeatability and less invasion.

With the development and progress of science, a small invasive and high-sensitivity tumor diagnosis technology, namely Liquid biopsy (Liquid biopsy), is produced. The detection of circulating miRNA molecular markers is written in 2019 edition of Primary liver cancer diagnosis and treatment Standard, and is incorporated into early detection and diagnosis clinical application of liver cancer.

Micro ribonucleic acid (miRNA) is a single-stranded endogenous non-coding RNA with short length, is confirmed to be widely involved in regulating life activities such as cell growth and development, differentiation, apoptosis and the like, and can positively influence the occurrence and development of tumors. The research shows that human genes above about 1/3 are directly regulated by miRNA, and the biological effect is to suppress or degrade mRNA target transcription and translation into corresponding protein, thereby participating in the process of life activity. Among them, circulating, free miRNAs in peripheral blood are often stably present in blood in the form of nucleic acid, protein complexes, etc., and are resistant to repeated freezing and thawing and extreme pH environments. Thus, these factors determine their biological properties of being less susceptible to degradation. The role of miRNAs in the digestive system is related to development, aging, secretion, metabolism, viral infection, immune response, tumors, etc.

Disclosure of Invention

Specifically, the present invention solves the technical problems in the prior art by the following technical solutions.

1. A kit for the detection of liver cancer comprising detection reagents for detecting one or more mirnas selected from the group consisting of hsa-miR-33B-3p, hsa-miR-92B-3p, hsa-miR-205-5p, hsa-miR-374C-5p, hsa-miR-4516-3p, and hsa-miR-6803-3 p.

2. The kit of item 1, comprising detection reagents for detecting at least two miRNAs selected from the group consisting of hsa-miR-33B-3p, hsa-miR-92B-3p, hsa-miR-205-5p, hsa-miR-374C-5p, hsa-miR-4516-3p, and hsa-miR-6803-3 p.

3. The kit of item 1, comprising detection reagents for detecting at least three mirnas selected from the group consisting of hsa-miR-33B-3p, hsa-miR-92B-3p, hsa-miR-205-5p, hsa-miR-374C-5p, hsa-miR-4516-3p, and hsa-miR-6803-3 p.

4. The kit of item 1, comprising detection reagents for detecting at least four mirnas selected from the group consisting of hsa-miR-33B-3p, hsa-miR-92B-3p, hsa-miR-205-5p, hsa-miR-374C-5p, hsa-miR-4516-3p, and hsa-miR-6803-3 p.

5. The kit of item 1, comprising detection reagents for detecting at least five mirnas selected from the group consisting of hsa-miR-33B-3p, hsa-miR-92B-3p, hsa-miR-205-5p, hsa-miR-374C-5p, hsa-miR-4516-3p, and hsa-miR-6803-3 p.

6. The kit of item 1, comprising a detection reagent for detecting hsa-miR-33B-3p, hsa-miR-92B-3p, hsa-miR-205-5p, hsa-miR-374C-5p, hsa-miR-4516-3p, and hsa-miR-6803-3 p.

7. The kit of any one of claims 1 to 6, wherein the kit comprises at least one reverse transcription primer, at least one amplification primer, and/or at least one probe.

8. The kit of claim 7, wherein the kit comprises a reverse transcription primer selected from the group consisting of at least one of SEQ ID NOs 7,11,15,19,23 and 27.

9. The kit of claim 7 or 8, wherein the kit comprises amplification primers selected from at least one of SEQ ID NOs 8-9,12-13,16-17,20-21,24-25 and 28-29.

10. The kit of any one of claims 7 to 9, wherein the kit comprises a probe selected from the group consisting of at least one of SEQ ID NOs 10,14,18,22,26 and 30.

11. The kit according to any one of items 1 to 10, wherein the detection reagent for detecting hsa-miR-33B-3p comprises a reverse transcription primer shown in SEQ ID NO. 7, amplification primers shown in SEQ ID NO. 8 and SEQ ID NO. 9, and/or a probe shown in SEQ ID NO. 10.

12. The kit according to any one of items 1 to 11, wherein the detection reagent for detecting hsa-miR-92B-3p comprises a reverse transcription primer shown in SEQ ID NO. 11, amplification primers shown in SEQ ID NO. 12 and SEQ ID NO. 13, and/or a probe shown in SEQ ID NO. 14.

13. The kit according to any one of items 1 to 12, wherein the detection reagent for detecting hsa-miR-205-5p comprises a reverse transcription primer shown in SEQ ID NO. 15, amplification primers shown in SEQ ID NO. 16 and SEQ ID NO. 17, and/or a probe shown in SEQ ID NO. 18.

14. The kit according to any one of items 1 to 13, wherein the detection reagent for detecting hsa-miR-374C-5p comprises a reverse transcription primer shown in SEQ ID NO:19, amplification primers shown in SEQ ID NO:20 and SEQ ID NO:21, and/or a probe shown in SEQ ID NO: 22.

15. The kit according to any one of items 1 to 14, wherein the detection reagent for detecting hsa-miR-4516-3p comprises a reverse transcription primer shown in SEQ ID NO. 23, amplification primers shown in SEQ ID NO. 24 and SEQ ID NO. 25, and/or a probe shown in SEQ ID NO. 26.

16. The kit of any one of items 1-15, wherein the detection reagent for detecting hsa-miR-6803-3p comprises a reverse transcription primer shown in SEQ ID NO:27, amplification primers shown in SEQ ID NO:28 and SEQ ID NO:29, and/or a probe shown in SEQ ID NO: 30.

17. The kit of any one of claims 1-16, further comprising a detection reagent for detecting an internal reference miRNA, wherein the internal reference miRNA is hsa-miR-16-5 p; preferably, the detection reagent for detecting the internal reference miRNA comprises a reverse transcription primer shown by SEQ ID NO. 31, an amplification primer shown by SEQ ID NO. 32 and SEQ ID NO. 33, and/or a probe shown by SEQ ID NO. 34.

18. The kit of any one of claims 1-17, further comprising a detection reagent for detecting a exo-reference miRNA, wherein the exo-reference miRNA is Cel-miR-39-5 p; preferably, the detection reagent for detecting the external reference miRNA is a reverse transcription primer shown as SEQ ID NO. 35, an amplification primer shown as SEQ ID NO. 36 and SEQ ID NO. 37, and a probe shown as SEQ ID NO. 38.

19. The kit of any one of claims 1 to 18, wherein the kit further comprises miRNA extraction reagents for a plasma or serum sample.

20. The kit according to any one of claims 1 to 19, wherein the liver cancer is early liver cancer.

21. A method for detecting liver cancer, the method comprising detecting the concentration in a sample of one or more mirnas selected from the group consisting of hsa-miR-33B-3p, hsa-miR-92B-3p, hsa-miR-205-5p, hsa-miR-374C-5p, hsa-miR-4516-3p, and hsa-miR-6803-3 p.

22. The method of item 21, comprising detecting the concentration of at least two mirnas selected from the group consisting of hsa-miR-33B-3p, hsa-miR-92B-3p, hsa-miR-205-5p, hsa-miR-374C-5p, hsa-miR-4516-3p, and hsa-miR-6803-3p in the sample.

23. The method of item 21, comprising detecting the concentration of at least three mirnas selected from the group consisting of hsa-miR-33B-3p, hsa-miR-92B-3p, hsa-miR-205-5p, hsa-miR-374C-5p, hsa-miR-4516-3p, and hsa-miR-6803-3p in the sample.

24. The method of item 21, comprising detecting the concentration of at least four mirnas selected from the group consisting of hsa-miR-33B-3p, hsa-miR-92B-3p, hsa-miR-205-5p, hsa-miR-374C-5p, hsa-miR-4516-3p, and hsa-miR-6803-3p in the sample.

25. The method of item 21, comprising detecting the concentration of at least five miRNAs selected from the group consisting of hsa-miR-33B-3p, hsa-miR-92B-3p, hsa-miR-205-5p, hsa-miR-374C-5p, hsa-miR-4516-3p, and hsa-miR-6803-3p in the sample.

26. The method of item 21, comprising detecting the concentration of hsa-miR-33B-3p, hsa-miR-92B-3p, hsa-miR-205-5p, hsa-miR-374C-5p, hsa-miR-4516-3p, and hsa-miR-6803-3p in the sample.

27. The method of any one of claims 21 to 26, wherein at least one reverse transcription primer, at least one amplification primer, and/or at least one probe is used.

28. The method according to claim 27, wherein a reverse transcription primer comprising at least one selected from the group consisting of SEQ ID NO 7,11,15,19,23 and 27 is used.

29. The method of claim 27 or 28, wherein the amplification primer comprises at least one selected from the group consisting of SEQ ID NOs 8-9,12-13,16-17,20-21,24-25 and 28-29.

30. The method of any one of claims 27 to 29, wherein a probe comprising at least one member selected from the group consisting of SEQ ID NOs 10,14,18,22,26 and 30 is used.

31. The method of any one of claims 21-30, wherein the detection reagent for detecting hsa-miR-33B-3p comprises a reverse transcription primer shown in SEQ ID NO. 7, amplification primers shown in SEQ ID NO. 8 and SEQ ID NO. 9, and/or a probe shown in SEQ ID NO. 10.

32. The method of any one of claims 21-31, wherein the detection reagent for detecting hsa-miR-92B-3p comprises a reverse transcription primer set forth in SEQ ID No. 11, amplification primers set forth in SEQ ID NOs 12 and 13, and/or a probe set forth in SEQ ID No. 14.

33. The method of any one of claims 21-32, wherein the detection reagent for detecting hsa-miR-205-5p comprises a reverse transcription primer set forth in SEQ ID No. 15, amplification primers set forth in SEQ ID NOs 16 and 17, and/or a probe set forth in SEQ ID No. 18.

34. The method of any one of claims 21-33, wherein the detection reagent for detecting hsa-miR-374C-5p comprises a reverse transcription primer set forth in SEQ ID No. 19, amplification primers set forth in SEQ ID No. 20 and SEQ ID No. 21, and/or a probe set forth in SEQ ID No. 22.

35. The method of any one of claims 21-34, wherein the detection reagent for detecting hsa-miR-4516-3p comprises a reverse transcription primer shown in SEQ ID NO. 23, amplification primers shown in SEQ ID NO. 24 and SEQ ID NO. 25, and/or a probe shown in SEQ ID NO. 26.

36. The method of any one of claims 21-35, wherein the detection reagent for detecting hsa-miR-6803-3p comprises a reverse transcription primer set forth in SEQ ID No. 27, amplification primers set forth in SEQ ID nos. 28 and 29, and/or a probe set forth in SEQ ID No. 30.

37. The method of any one of claims 21-36, wherein the sample is a plasma or serum sample.

38. The method of any one of claims 21-37, wherein the liver cancer is early stage liver cancer.

39. The method according to any one of items 21 to 38, wherein Ct values obtained by subtracting Ct values of hsa-miR-33B-3p, hsa-miR-92B-3p, hsa-miR-205-5p, hsa-miR-374C-5p, hsa-miR-4516 and hsa-miR-6803-3p and internal reference hsa-miR-16 and external reference Cel-miR-39 according to fluorescence quantitative PCR detection are used as covariates for ROC curve analysis, and the feasibility of liver cancer occurrence is judged by using the following logistic regression equation: p ═ e^K/(1+e^K) Wherein e is a natural constant, and K ═ is (0.15501) + (0.01785) hsa-miR-33B-3p + (-0.02857) hsa-miR-92B-3p + (-0.01955) miR-205-5p + (0.03704) miR-374C-5p + (0.04277) miR-4516-3p + (0.04277) miR-6803-3 p. If the P value is higher than 0.478, the patient is considered as an extremely early liver cancer high-risk patient.

Drawings

FIG. 1 ROC Curve analysis of 6 targets

Detailed Description

The invention aims to provide a miRNA combination and a kit for diagnosing early liver cancer. In order to overcome the defect of low sensitivity in the existing early liver cancer diagnosis technology, the invention provides a miRNA-based detection product and application of miRNA characteristic markers in preparation of liver cancer screening and diagnosis reagents or kits. In some embodiments, the products thereof comprise primers, probes, detection reagents and kits for detecting miRNA content. In some embodiments, by extracting and detecting the expression level of one or more mirnas from the plasma sample, it can be determined whether the subject to be tested has liver cancer. The method is safe and noninvasive, has high sensitivity and specificity, and can be effectively used for early diagnosis of liver cancer.

In a first aspect of the present invention, there is provided a kit for detecting liver cancer, wherein the kit comprises:

a detection reagent for specifically detecting human hsa-miR-33B-3 p;

a detection reagent for specifically detecting human hsa-miR-92B-3 p;

a detection reagent for specifically detecting human hsa-miR-205-5 p;

a detection reagent for specifically detecting human hsa-miR-374C-5 p;

a detection reagent for specifically detecting human hsa-miR-4516-3 p; and

a detection reagent for specifically detecting human hsa-miR-6803-3 p;

one or more detection reagents. In some embodiments, the selected kit comprises 2, 3, 4, 5, or 6 detection reagents selected from the miRNA detection reagents described above. In some embodiments, the selected kit comprises all 6 miRNA detection reagents selected from above.

In some embodiments, the detection reagents in the kit are RNA reverse transcription primers, DNA amplification primers, and DNA probes.

In some embodiments, the kit comprises a reverse transcription primer shown in SEQ ID NO. 7, an amplification primer shown in SEQ ID NO. 8 and SEQ ID NO. 9, and a probe shown in SEQ ID NO. 10 as a detection reagent for specifically detecting human hsa-miR-33B-3 p;

in some embodiments, the kit comprises a reverse transcription primer shown in SEQ ID NO. 11, an amplification primer shown in SEQ ID NO. 12 and SEQ ID NO. 13, and a probe shown in SEQ ID NO. 14 as a detection reagent for specifically detecting human hsa-miR-92B-3 p;

in some embodiments, the kit comprises a reverse transcription primer shown in SEQ ID NO. 15, an amplification primer shown in SEQ ID NO. 16 and SEQ ID NO. 17, and a probe shown in SEQ ID NO. 18 as a detection reagent for specifically detecting human hsa-miR-205-5 p;

in some embodiments, the kit comprises a reverse transcription primer shown in SEQ ID NO. 19, an amplification primer shown in SEQ ID NO. 20 and SEQ ID NO. 21, and a probe shown in SEQ ID NO. 22 as a detection reagent for specifically detecting human hsa-miR-374C-5 p;

in some embodiments, the kit comprises a detection reagent for specifically detecting human hsa-miR-4516-3p, which is a reverse transcription primer shown in SEQ ID NO. 23, an amplification primer shown in SEQ ID NO. 24 and SEQ ID NO. 25, and a probe shown in SEQ ID NO. 26;

in some embodiments, the kit comprises a detection reagent for specifically detecting human hsa-miR-6803-3p, which is a reverse transcription primer shown in SEQ ID NO:27, an amplification primer shown in SEQ ID NO:28 and SEQ ID NO:29, and a probe shown in SEQ ID NO: 30;

in some embodiments, the kit further comprises:

a detection reagent for specifically detecting an internal reference gene and an external reference gene; preferably, the detection reagent for specifically detecting the internal reference gene is a reverse transcription primer, an amplification primer and a probe aiming at human hsa-miR-16-5 p; more preferably, the detection reagent for specifically detecting the reference gene is a reverse transcription primer shown by SEQ ID NO. 31, an amplification primer shown by SEQ ID NO. 32 and SEQ ID NO. 33, and a probe shown by SEQ ID NO. 34. The detection reagent for specifically detecting the foreign gene is a reverse transcription primer, an amplification primer and a probe aiming at human Cel-miR-39-5 p; more preferably, the detection reagent for specifically detecting the external reference gene is a reverse transcription primer shown by SEQ ID NO. 35, an amplification primer shown by SEQ ID NO. 36 and SEQ ID NO. 37, and a probe shown by SEQ ID NO. 38.

In some embodiments, the probe for each miRNA or internal reference is linked to a specific fluorophore.

In some embodiments, the kit further comprises: negative quality control material and/or positive quality control material.

In some embodiments, the kit further comprises: RNA extraction reagents, reverse transcription reaction reagents, PCR amplification reagents, and/or instructions for use.

In another aspect of the invention, the application of detecting one or more miRNAs selected from hsa-miR-33B-3p, hsa-miR-92B-3p, hsa-miR-205-5p, hsa-miR-374C-5p, hsa-miR-4516-3p and hsa-miR-6803-3p is provided, and the method can be used for detecting liver cancer or preparing a reagent or a kit for detecting liver cancer. In some embodiments, the use comprises detecting 2, 3, 4, 5, or 6 mirnas selected from the mirnas described above. In some embodiments, the use comprises detecting all 6 mirnas described above.

In some embodiments, the detection reagents are RNA reverse transcription primers, DNA amplification primers, and DNA probes.

In some embodiments, the liver cancer is early stage liver cancer, including very early stage liver cancer (earliest stage liver cancer).

In some embodiments, the Liver Cancer staging employs the Barcelona clinical Liver Cancer staging System (BCLC), see Table A below). In some embodiments, the early stage liver cancer comprises stage a of the barcelona stage. In some embodiments, the early stage liver cancer comprises stage 0 and stage a of the barcelona stage.

Table a: clinical staging of Barcelona carcinoma

In some embodiments, the liver cancer staging employs a chinese liver cancer staging protocol (CNLC) comprising: CNLCIa stage, Ib stage, IIa stage, IIb stage, IIIa stage, IIIb stage and IV stage, which are as follows:

CNLC phase Ia: the physical activity status (PS) score is 0-2, the liver function Child-Pugh A/B grade, the diameter of a single tumor is less than or equal to 5cm, and no vascular invasion and extrahepatic metastasis are caused.

CNLC Ib stage: PS is divided into 0-2 parts, liver function Child-Pugh A/B grade, single tumor with the diameter larger than 5cm or 2-3 tumors with the maximum diameter less than or equal to 3cm, and no vascular invasion and extrahepatic metastasis.

CNLC IIa stage: PS is divided into 0-2 parts, liver function Child-Pugh A/B grade, 2-3 tumors, maximum diameter larger than 3cm, no blood vessel invasion and extrahepatic metastasis.

CNLC IIb stage: PS is divided into 0-2, liver function Child-Pugh A/B grade, tumor number is more than or equal to 4, tumor diameter is no matter, vascular invasion and extrahepatic metastasis do not exist.

CNLC stage IIIa: PS is 0-2, liver function Child-Pugh A/B grade, and no matter whether the tumor condition is, vascular invasion and extrahepatic metastasis are avoided.

CNLC stage IIIb: PS is 0-2, liver function Child-Pugh A/B grade, tumor condition, blood vessel invasion and extrahepatic metastasis.

CNLC stage IV: PS 3-4, or liver function Child-Pugh grade C, no matter how the tumor is, no matter how the blood vessel is invaded, no matter how the liver is metastasized.

In some embodiments, the early stage liver cancer comprises stage Ia of chinese liver cancer staging (CNLC). In some embodiments, the early stage liver cancer comprises stage Ib of chinese liver cancer staging (CNLC). In some embodiments, the early stage liver cancer comprises stage Ia and stage Ib of liver cancer staging in china (CNLC).

In some embodiments, the kit comprises free nucleic acid extraction reagents. In some embodiments, the free nucleic acid extraction reagent extracts and purifies plasma free nucleic acids by a magnetic bead method.

In some embodiments, the plasma-free nucleic acid is plasma-free DNA. In some embodiments, the plasma-free nucleic acids are plasma-free RNA.

Advantages of the invention include, but are not limited to: the method firstly proposes that the accuracy of diagnosing hepatocellular carcinoma, particularly identifying early liver cancer in a group of asymptomatic high-risk individuals is more than 85 percent by detecting the copy number of specific miRNAs molecular markers in plasma (particularly in a circulatory system) of a patient and judging the risk of the viral hepatitis patient to generate liver cancer by using a diagnosis model.

The inventor of the present invention has made intensive studies to disclose for the first time a method for diagnosing liver cancer, particularly early liver cancer, by synergistically detecting the expression of six miRNAs, and also provides a reagent or a kit for carrying out the diagnosis.

As used herein, a "test sample", "analyte" or "test nucleic acid (e.g., DNA, RNA) sample" refers to a sample to be tested which contains a nucleic acid or nucleic acids in which it is desired to know whether a target nucleic acid is present. In the present invention, the sample to be tested may be: plasma, serum, etc.

As used herein, "target nucleic acid" refers to a nucleic acid fragment of interest. In some embodiments, the target nucleic acid is one or more of hsa-miR-33B-3p, hsa-miR-92B-3p, hsa-miR-205-5p, hsa-miR-374C-5p, hsa-miR-4516-3p, and hsa-miR-6803-3p of human origin.

As used herein, a "probe" refers to a single-stranded nucleic acid having a known nucleotide sequence, which has a nucleotide sequence structure substantially complementary to a target nucleic acid and can form a double strand with the "target nucleic acid". The "probe" may carry a label. For example, the label can be attached to the 5 'end or the 3' end of the probe.

As used herein, the term "miRNA combination" refers to a set of miRNAs comprising one or more (e.g., all six) of hsa-miR-33B-3p, hsa-miR-92B-3p, hsa-miR-205-5p, hsa-miR-374C-5p, hsa-miR-4516-3p and hsa-miR-6803-3p, which can be used to detect liver cancer, particularly early stage liver cancer.

The inventor collects plasma of patients with liver cancer before the patients are diagnosed half a year to half a year, and obtains a specific miRNAs molecular marker capable of predicting the possibility of liver cancer occurrence of high-risk groups of patients with viral hepatitis by screening miRNAs spectrums which are differentially expressed compared with those without liver cancer.

Based on the new findings of the inventor, the application of the detection reagent for specifically detecting the hsa-miR-33B-3p, hsa-miR-92B-3p, hsa-miR-205-5p, hsa-miR-374C-5p, hsa-miR-4516-3p and hsa-miR-6803-3p is provided, and the reagent is used for preparing a kit for detecting liver cancer, particularly early liver cancer.

The presence or expression of hsa-miR-33B-3p, hsa-miR-92B-3p, hsa-miR-205-5p, hsa-miR-374C-5p, hsa-miR-4516-3p and hsa-miR-6803-3p can be detected using a variety of techniques known in the art, and are encompassed by the present invention. For example, the conventional techniques such as Polymerase Chain Reaction (PCR), Southern blotting, in situ hybridization, DNA sequencing, etc. can be used, and these techniques can be used in combination.

The invention also provides reagents for detecting the presence of expression of hsa-miR-33B-3p, hsa-miR-92B-3p, hsa-miR-205-5p, hsa-miR-374C-5p, hsa-miR-4516-3p and hsa-miR-6803-3p in an analyte. As a preferred mode, the amount of the miRNA can be determined by the RCR method using its specific amplification primer or specific probe. The design of specific primers and probes for mirnas is a technique well known to those skilled in the art.

As a preferred mode of the invention, hsa-miR-33B-3p, hsa-miR-92B-3p, hsa-miR-205-5p, hsa-miR-374C-5p, hsa-miR-4516-3p and hsa-miR-6803-3p are detected in plasma or serum.

In some embodiments, the unit concentration/content of hsa-miR-205-5p in the liver cancer sample is lower than that in a normal sample. In some embodiments, the unit concentration/content of hsa-miR-205-5p in the liver cancer sample is at least 5%, at least 10%, at least 20%, at least 30%, at least 50%, at least 70%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000% lower than that in a normal sample.

In some embodiments, the unit concentration/content of hsa-miR-374C-5p in the liver cancer sample is higher than that in a normal sample. In some embodiments, the unit concentration/content of hsa-miR-374C-5p in the liver cancer sample is at least 5%, at least 10%, at least 20%, at least 30%, at least 50%, at least 70%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000% higher than in a normal sample.

In some embodiments, the unit concentration/content of hsa-miR-33B-3p in the liver cancer sample is lower than that in a normal sample. In some embodiments, the unit concentration/content of hsa-miR-33B-3p in the liver cancer sample is at least 5%, at least 10%, at least 20%, at least 30%, at least 50%, at least 70%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000% lower than that in a normal sample.

In some embodiments, the unit concentration/content of hsa-miR-92B-3p in the liver cancer sample is higher than that in a normal sample. In some embodiments, the unit concentration/content of hsa-miR-92B-3p in the liver cancer sample is at least 5%, at least 10%, at least 20%, at least 30%, at least 50%, at least 70%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000% higher than in a normal sample.

In some embodiments, the unit concentration/content of hsa-miR-4516-3p in the liver cancer sample is lower than that in a normal sample. In some embodiments, the unit concentration/content of hsa-miR-4516-3p in the liver cancer sample is at least 5%, at least 10%, at least 20%, at least 30%, at least 50%, at least 70%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000% lower than that in a normal sample.

In some embodiments, the unit concentration/content of hsa-miR-6803-3p in the liver cancer sample is lower than that in a normal sample. In some embodiments, the unit concentration/content of hsa-miR-6803-3p in the liver cancer sample is at least 5%, at least 10%, at least 20%, at least 30%, at least 50%, at least 70%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000% lower than in a normal sample.

The present invention also provides a kit for detecting liver cancer, particularly early liver cancer, comprising: a detection reagent for specifically detecting human hsa-miR-33B-3 p; a detection reagent for specifically detecting human hsa-miR-92B-3 p; a detection reagent for specifically detecting human hsa-miR-205-5 p; a detection reagent for specifically detecting human hsa-miR-374C-5 p; a detection reagent for specifically detecting human hsa-miR-4516-3p and a detection reagent for specifically detecting human hsa-miR-6803-3 p. In a preferred embodiment, the detection reagent is an RNA reverse transcription primer, a DNA amplification primer and a DNA probe.

The kit can also comprise an RNA extraction reagent, a reverse transcription reagent, a nucleic acid amplification reagent, pure water without RNA enzyme, a negative quality control material, a positive quality control material and the like.

Optionally, the reagent for extracting RNA is a Trizol reagent, a reagent for extracting RNA by a magnetic bead method, or a reagent for extracting RNA by a column method.

The reverse transcription reagent in the kit comprises: and detecting a reverse transcription primer, a reverse transcriptase and a reverse transcription reaction system of each miRNA in the miRNA combination.

The reverse transcriptase may be M-MLV reverse transcriptase or AMV reverse transcriptase.

The reverse transcription reaction system may include: reverse transcription buffers, dNTPs, RNase-free purified water, RNase inhibitor, and control or extracted RNA.

The nucleic acid amplification reagent according to the present invention may include: forward primer, reverse primer, oligonucleotide probe, nucleic acid amplification enzyme and quantitative nucleic acid amplification reaction system.

The nucleic acid amplification enzyme may be a DNA amplification enzyme;

the quantitative nucleic acid amplification reaction system can comprise quantitative buffer solution, dNTPs, pure water and cDNA obtained through reverse transcription.

The negative quality control product can be normal human plasma, plasma of chronic hepatitis B patients without liver cancer, bovine serum albumin solution or normal saline solution; the positive quality control product can be plasma of early liver cancer patients which is determined to be positive, or hsa-miR-33B-3p, hsa-miR-92B-3p, hsa-miR-205-5p, hsa-miR-374C-5p, hsa-miR-4516-3p and hsa-miR-6803-3p oligonucleotides with specific copy numbers.

In order to avoid errors caused by amplification, an internal reference can also be arranged. In the embodiment of the invention, hsa-miR-16-5p is used as an internal reference. It will be appreciated that the selection of other similar genes as internal controls is also feasible.

In order to avoid errors caused by amplification, an external reference can be arranged. In the embodiment of the invention, Cel-miR-39-5p is used as the external reference. It will be appreciated that the selection of other similar genes as a foreign reference is also possible.

The invention further provides a detection method of plasma small molecule nucleic acid related to the very early diagnosis of liver tumor, which comprises the following steps:

(1) extraction of RNA from plasma samples:

extracting RNA in a plasma sample by using the mimic of plasma, negative control plasma, positive control plasma and synthesized external reference gene Cel-39-5p of a patient to be detected according to the standard operation method of the selected RNA extraction kit or the instruction of the used nucleic acid extraction kit;

(2) the kit is used for detection:

and simultaneously carrying out quantitative determination on the internal reference, the external reference and the miRNA combination. Adding the extracted RNA into a reaction tube which is premixed and contains a reverse transcription primer, reverse transcriptase and a reverse transcription reaction system, and carrying out reverse transcription; then adding cDNA into a quantitative nucleic acid amplification reaction tube containing a forward primer, a reverse primer, an oligonucleotide probe, a nucleic acid amplification enzyme and a quantitative nucleic acid calculation amplification reaction system, amplifying, and detecting by using a fluorescent quantitative PCR detector.

(3) Joint analysis of multiple circulating systems by logistic regression (logistic regression) modelsThe specific small molecule nucleic acid is used for judging the feasibility of liver cancer generation: p ═ e^K/(1+e^K)。

In the above formula, P is a composite index, and the range is 0< P < 1; e is a natural constant; k ═ 0.15501) + (0.01785) hsa-miR-33B-3p + (-0.02857) hsa-miR-92B-3p + (-0.01955) miR-205-5p + (0.03704) miR-374C-5p + (0.04277) miR-4516-3+ (0.04277) miR-6803-3 p. Wherein, the expression levels of hsa-miR-33B-3p, hsa-miR-92B-3p, hsa-miR-205-5p, hsa-miR-374C-5p, hsa-miR-4516-3p and hsa-miR-6803-3p are detected by taking miR-16-5p as an internal reference and Cel-miR-39-5p as an external reference. And determining a judgment threshold value according to the distribution of the detection data (P value) of the clinical samples, wherein the detection result is judged to be positive when P is more than or equal to the threshold value, and the detection result is judged to be negative when P < the threshold value.

The invention firstly proposes that the accuracy of diagnosing hepatocellular carcinoma, particularly identifying early liver cancer in a group of asymptomatic high-risk individuals is more than 85 percent by detecting the copy number of specific miRNAs molecular markers in plasma (particularly in a circulatory system) of a patient and judging the risk of the viral hepatitis patient to generate liver cancer by using a diagnosis model.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The invention is described in detail below with reference to the figures and the embodiments. The specific embodiments do not represent limitations to the scope of the invention, and insubstantial modifications and adaptations of the invention by others with respect to the inventive concepts are intended to be within the scope of the invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers.

Example 1: screening of specific miRNAs molecular markers capable of predicting liver cancer occurrence possibility of high-risk population of viral hepatitis patients

Plasma of patients with liver cancer in the patients is collected before half a year to half a year, miRNAs with different expressions compared with those without liver cancer are screened, and specific miRNAs molecular markers capable of predicting the possibility of liver cancer occurrence of high-risk groups of patients with viral hepatitis are screened.

In the plasma samples for the study, 70 chronic hepatitis B surface antigen (HBsAg) positive (HBsAg-positive) patients, 40 hepatitis C patients and 80 liver cancer patients;

second generation sequencing was performed on the above samples to screen the spectrum of differentially expressed miRNAs between the two, resulting in 10 differentially expressed miRNAs (see table 1).

TABLE 1

By further enlarging the sample size, 30 chronic hepatitis B virus infected patients and 40 very early liver cancer patients are obtained as training sets, and 10 candidate miRNAs are further verified by a quantitative RT-PCR method. The results found that the following 6 miRNAs could significantly distinguish the interfering group from the liver cancer group: the up-regulation or down-regulation of the expression of hsa-miR-33B-3p, hsa-miR-92B-3p, hsa-miR-205-5p, hsa-miR-374C-5p, hsa-miR-4516 and hsa-miR-6803-3p in early liver cancer patients is consistent with that in Table 1, and the results show that the 6 circulating miRNAs are novel markers for screening liver cancer.

The sequences of the mirnas were as follows:

hsa-miR-4516-3p：GGGAGAAGGGUCGGGGC(SEQ ID NO:1)

hsa-miR-374c-5p：AUAAUACAACCUGCUAAGUGCU(SEQ ID NO:2)

hsa-miR-92b-3p：UAUUGCACUCGUCCCGGCCUCC(SEQ ID NO:3)

hsa-miR-205-5p：UCCUUCAUUCCACCGGAGUCUG(SEQ ID NO:4)

hsa-miR-6803-3p：UCCCUCGCCUUCUCACCCUCAG(SEQ ID NO:5)

hsa-miR-33b-3p：CAGUGCCUCGGCAGUGCAGCCC(SEQ ID NO:6)

example 2 detection reagent preparation and detection of patient plasma

1. Primer design

The following reverse transcription primers, upper and lower primers and probes were designed:

for hsa-miR-33b-3p:

reverse transcription primer: 5'-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGGGGCTGCA-3' (SEQ ID NO:7)

An upstream primer: 5'-ATTCATCAGTGCCTCGGCAG-3' (SEQ ID NO:8)

A downstream primer: 5'-CTCAACTGGTGTCGTGGAGT-3' (SEQ ID NO:9)

And (3) probe: 5'-TTCAGTTGAGGGGCTGCA-3' (SEQ ID NO: 10). The 5' end of the probe is provided with FAM; the 3' end carries MGB.

For hsa-miR-92b-3p:

reverse transcription primer: 5'-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGGGAGGCCG-3' (SEQ ID NO:11)

An upstream primer: 5'-ACAGCGTATTGCACTCGTCC-3' (SEQ ID NO:12)

A downstream primer: 5'-CTCAACTGGTGTCGTGGAGT-3' (SEQ ID NO:13)

And (3) probe: 5'-TTCAGTTGAGGGAGGCCG-3' (SEQ ID NO: 14). The 5' end of the probe is provided with FAM; the 3' end carries MGB.

For hsa-miR-205-5p:

reverse transcription primer: 5'-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCAGACTCC-3' (SEQ ID NO:15)

An upstream primer: 5'-GCCGAGTCCTTCATTCCACC-3' (SEQ ID NO:16)

A downstream primer: 5'-CTCAACTGGTGTCGTGGAGT-3' (SEQ ID NO:17)

And (3) probe: 5'-TTCAGTTGAGCAGACTCC-3' (SEQ ID NO: 18). The 5' end of the probe is provided with FAM; the 3' end carries MGB.

For hsa-miR-374c-5p:

reverse transcription primer: 5'-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGAGCACTTA-3' (SEQ ID NO:19)

An upstream primer: 5'-CGTCCGAGATAATACAACCTGC-3' (SEQ ID NO:20)

A downstream primer: 5'-CTCAACTGGTGTCGTGGAGT-3' (SEQ ID NO:21)

And (3) probe: 5'-TTCAGTTGAGAGCACTTA-3' (SEQ ID NO: 22). The 5' end of the probe is provided with FAM; the 3' end carries MGB.

For hsa-miR-4516-3p:

reverse transcription primer: 5'-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGGCCCCGAC-3' (SEQ ID NO:23)

An upstream primer: 5'-AACTATCGTACCGGGAGAAGG-3' (SEQ ID NO:24)

A downstream primer: 5'-CTCAACTGGTGTCGTGGAGT-3' (SEQ ID NO:25)

And (3) probe: 5'-TTCAGTTGAGGCCCCGAC-3' (SEQ ID NO: 26). The 5' end of the probe is provided with FAM; the 3' end carries MGB.

For hsa-miR-6803-3p:

reverse transcription primer: 5'-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCTGAGGGT-3' (SEQ ID NO:27)

An upstream primer: 5'-ATAGAGTCCCTCGCCTTCTC-3' (SEQ ID NO:28)

A downstream primer: 5'-CTCAACTGGTGTCGTGGAGT-3' (SEQ ID NO:29)

And (3) probe: 5'-TTCAGTTGAGCTGAGGGT-3' (SEQ ID NO: 30). The 5' end of the probe is provided with FAM; the 3' end carries MGB.

has-miR-16-5p is used as an internal reference, and the related primers are as follows:

reverse transcription primer: 5'-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCGCCAATA-3' (SEQ ID NO:31)

An upstream primer: 5'-GCCGAGTAGCAGCACGTAAA-3' (SEQ ID NO:32)

A downstream primer: 5'-CTCAACTGGTGTCGTGGAGT-3' (SEQ ID NO:33)

And (3) probe: 5'-TTCAGTTGAGCGCCAATA-3' (SEQ ID NO: 34). The 5' end of the probe is provided with FAM; the 3' end carries MGB.

Cel-miR-39-5p is used as an external reference, and relevant primers are as follows:

reverse transcription primer: 5'-CTCAACTGGTGTC GTGGAGTCGGCAATTCAGTTGAGCAAGCTGA-3' (SEQ ID NO:35)

An upstream primer: 5'-GCCGAGTCACCGGGTGTAAA-3' (SEQ ID NO:36)

A downstream primer: 5'-CTCAACTGGTGTCGTGGAGT-3' (SEQ ID NO:37)

And (3) probe: 5'-TTCAGTTGAGCAAGCTGA-3' (SEQ ID NO: 38). The 5' end of the probe is provided with FAM; the 3' end carries MGB.

2. Total RNA extraction

The method comprises the following steps of (1) extracting total RNA from patient blood plasma by using a column extraction method:

a) using a 2.0mL centrifuge tube, taking 0.2mL plasma sample, adding 0.3mL binding solution and 0.3mL lysis solution (please note to see whether to add beta-mercaptoethanol), and mixing uniformly for 15s by vortex;

b) incubating in an electrothermal constant temperature air-blast drying oven at 60 deg.C for 10 min;

c) taking out after the incubation is finished, adding 0.75mL of absolute ethyl alcohol, and uniformly mixing for 15s in a vortex manner;

d) centrifuging at 1250rpm for 1min, and carefully removing supernatant to ensure that the silicon carbide is not discarded;

e) adding 0.3mL of lysis solution (please note to see whether to add beta-mercaptoethanol), and mixing by vortex for 15 s;

f) repeating the step 3.2;

g) taking out after the incubation is finished, adding 0.3mL of absolute ethyl alcohol, and uniformly mixing for 15s in a vortex manner;

h) adding 650 μ L of the above mixture into RNA purification column, centrifuging at 14000rpm for 1min, and discarding the waste solution;

i) repeating for 3.8 times until the mixed solution is completely transferred to an RNA purification column;

j) adding 400 μ L of cleaning solution, centrifuging at 14000rpm for 1min, and discarding the waste liquid;

k) repeat 3.10 times;

l) centrifuging at 14000rpm for 3min, and discarding the collection tube;

m) the RNA purification column was placed in a 1.5mL EP tube, 30. mu.L of the eluate was added, centrifuged at 2000rpm for 2min and subsequently 14000rpm for 3min, and the RNA purification column was removed to give an RNA eluate.

3. Sample RNA reverse transcription reaction

And respectively taking 5 mu L of extracted sample RNA, positive control RNA and negative control, adding 5 mu L of reverse transcription buffer solution into the samples, and shaking and uniformly mixing the samples for later use. Then 10. mu.L of the reverse transcriptase mix was added. The reverse transcription reaction system is shown in Table 2.

TABLE 2

Material(s)	Recommendation system
		RNase-free dd H₂O	4.11μL
100mM dNTPs (with dTTP)	0.2μL
		Reverse transcriptase, 50U/. mu.L	1.0μL
10 Xreverse transcription buffer	1.5μL
		RNase inhibitor, 20U/. mu.L	0.19μL
Stem loop primer	3.0μL

Wherein, the conditions in the reverse transcription process are as follows: 16-30 min, 42-30 min and 85-5 min.

4. Fluorescent quantitative PCR detection of sample cDNA

And carrying out reverse transcription on the obtained cDNA fragments, carrying out fluorescent quantitative PCR detection on hsa-miR-33B-3p, hsa-miR-92B-3p, hsa-miR-205-5p, hsa-miR-374C-5p, hsa-miR-4516-3p, hsa-miR-6803-3p, has-miR-16 and Cel-miR-39 in independent reaction holes respectively, and analyzing the results by using Applied Biosystems 7500Real-Time PCR system software.

The fluorescence quantitative PCR detection system in each reaction well is shown in Table 3.

TABLE 3

Components	1T
		2 xTaq Pro HS Universal Probe Master mix	10μL
Primer F (10. mu.M)	0.4μL
		primer-R (10. mu.M)	0.4μL
TaqMan probe (10. mu.M)	0.2μL
		cDNA	3μL
ddH₂O	7μL

The cycle number of the fluorescent quantitative PCR was 45, specifically, pre-denaturation at 95 ℃ for 4 minutes, denaturation at 95 ℃ for 15 seconds, and renaturation at 60 ℃ for 30 seconds.

The result of the fluorescence quantitative PCR shows that the amplification curve of the positive control is a typical S-shaped curve, and the Ct values are all less than 40, which is considered as a positive amplification curve; the amplification curve of the negative control is not S-shaped, and the Ct value is more than 40, so that the negative control is considered as a negative amplification curve.

And subtracting the Ct value of the early liver cancer specific molecular marker from the Ct values of the internal reference and the external reference to obtain a difference value (delta Ct) serving as a covariate of subsequent ROC curve analysis.

5. Feasibility of sample joint analysis for judging liver cancer occurrence through logical model

Subtracting Ct values of has-miR-16 and Cel-miR-39 from hsa-miR-33B-3p, hsa-miR-92B-3p, hsa-miR-205-5p, hsa-miR-374C-5p, hsa-miR-4516-3p and hsa-miR-6803-3p in the independent reaction holes, and taking the obtained difference as a covariate for ROC curve analysis.

The logistic regression calculation formula is as follows: p ═ e^K/(1+e^K)。

In the above formula, P is a composite index, and the range is 0< P < 1; e is a natural constant; k ═ K (0.15501) + (0.01785) hsa-miR-33B-3p + (-0.02857) hsa-miR-92B-3p + (-0.01955) miR-205-5p + (0.03704) miR-374C-5p + (0.04277) miR-4516-3p + (0.04277) miR-6803-3 p. The cutoff value of the P value in the model is between 0 and 1, preferably between 0.3 and 0.5. And after the sample to be detected is subjected to model analysis, if the P value is higher than the cutoff value, the sample is regarded as an extremely early liver cancer high-risk patient.

Example 3 preparation of a kit of the invention

In this example, a kit comprising the following components or modules was prepared with the detection reagent designed in example 2: RNA extraction reagent, reverse transcription reagent aiming at each miRNAs to be detected and internal reference, PCR amplification upstream primer and downstream primer aiming at each miRNAs to be detected and internal and external reference, pure water without RNA enzyme, negative quality control product and positive quality control product.

1) RNA extraction reagent: used in this example are column extraction reagents (Norgen, canada);

2) a reverse transcription primer, a forward primer, a reverse primer, an oligonucleotide probe, and a positive control, which were entrusted to the synthesis by EnxWeijie trading Limited;

3) the reverse transcriptase M-MLV, the reverse transcription reaction system, the nucleic acid amplification enzyme and the quantitative PCR reaction system were purchased from Saimei Fei.

4) The negative quality control product is sterilized physiological saline, and the positive quality control products are hsa-miR-33B-3p, hsa-miR-92B-3p, hsa-miR-205-5p, hsa-miR-374C-5p, hsa-miR-4516-3p, hsa-miR-6803-3p, has-miR-16 and Cel-miR-39 oligonucleotides with copy numbers of 1 multiplied by 106 respectively.

Each reagent is placed in an independent reagent bottle or tube, and the reagent bottles or tubes are effectively separated and are packaged in a packaging box in a centralized way.

Example 4 specificity and sensitivity of application of further sample validation kit to detection of early liver cancer

In order to further verify the specificity and sensitivity of the kit prepared in example 3 applied to the identification of the very early liver cancer of a population of asymptomatic high-risk individuals, plasma samples of 100 subjects (verification set, 60 chronic hepatitis B patients and 40 very early liver cancer patients) are obtained, and the specificity and sensitivity of the miRNA combination (hsa-miR-33B-3p, hsa-miR-92B-3p, hsa-miR-205-5p, hsa-miR-374C-5p, hsa-miR-4516-3p and hsa-miR-6803-3p) applied to the detection of the early liver cancer are verified. Results of parallel AFP detection were used as a comparison. The predicted probabilities are used to build a subject working profile (see figure 1). The results of the comparison of the miRNA multi-target combined diagnostic model performance and the AFP single-marker detection performance are shown in Table 4.

TABLE 4

	Sensitivity of the probe	Specificity of	Joden index
				MiRNA joint detection	82.5％	88.3％	0.705
AFP Single Mark	62.5％	85.0％	0.475

EXAMPLE 5 clinical application of the test kit

For chronic HBsAg-positive patients followed by hospital, serum of the patients was collected, RNA reverse transcription was extracted as in example 2, and quantitative PCR detection was performed using the above-mentioned miRNA combinations (hsa-miR-33B-3p, hsa-miR-92B-3p, hsa-miR-205-5p, hsa-miR-374C-5p, hsa-miR-4516-3p and hsa-miR-6803-3p) as markers, has-miR-16 and Cel-miR-39 as internal and external references, and primers and probes designed in example 2.

The cutoff value is set to 0.438. Model analysis was performed on the samples as in example 2, and if the cutoff value measured was higher than 0.438, it was considered as a very early liver cancer high risk patient, and follow-up positive follow-up examination and treatment were recommended.

After reading the above teachings of the present invention, one of ordinary skill in the art may make various changes and modifications to the invention, and such equivalents are intended to fall within the scope of the invention as defined by the appended claims.

Although claims have been formulated in this application to particular combinations of features, it should be understood that the scope of the disclosure also includes any novel feature or any novel combination of features disclosed herein either explicitly or implicitly or any generalisation thereof, whether or not it relates to the same invention as presently claimed in any claim and whether or not it mitigates any or all of the same technical problems as does the present invention. The applicants hereby give notice that new claims may be formulated to such features and/or combinations of features during the prosecution of the present application or of any further application derived therefrom.

Although a few embodiments have been shown and described, it would be appreciated by those skilled in the art that changes may be made in these embodiments without departing from the principles of the invention, the scope of which is defined in the claims.

It will be appreciated by those skilled in the art that modifications (additions and/or deletions) may be made to the components, methods, steps, structures, motions, and arrangements described herein without departing from the full scope and spirit of the invention, which encompasses such modifications and any and all equivalents thereof.

Claims

2. The kit of claim 1, wherein the kit comprises detection reagents for detecting at least two mirnas selected from the group consisting of hsa-miR-33B-3p, hsa-miR-92B-3p, hsa-miR-205-5p, hsa-miR-374C-5p, hsa-miR-4516-3p, and hsa-miR-6803-3 p.

3. The kit of claim 1, wherein the kit comprises detection reagents for detecting at least three mirnas selected from the group consisting of hsa-miR-33B-3p, hsa-miR-92B-3p, hsa-miR-205-5p, hsa-miR-374C-5p, hsa-miR-4516-3p, and hsa-miR-6803-3 p.

4. The kit of claim 1, wherein the kit comprises detection reagents for detecting at least four mirnas selected from the group consisting of hsa-miR-33B-3p, hsa-miR-92B-3p, hsa-miR-205-5p, hsa-miR-374C-5p, hsa-miR-4516-3p, and hsa-miR-6803-3 p.

5. The kit of claim 1, wherein the kit comprises detection reagents for detecting at least five mirnas selected from the group consisting of hsa-miR-33B-3p, hsa-miR-92B-3p, hsa-miR-205-5p, hsa-miR-374C-5p, hsa-miR-4516-3p, and hsa-miR-6803-3 p.

6. The kit of claim 1, wherein the kit comprises detection reagents for detecting hsa-miR-33B-3p, hsa-miR-92B-3p, hsa-miR-205-5p, hsa-miR-374C-5p, hsa-miR-4516-3p, and hsa-miR-6803-3 p.

8. The kit of claim 7, wherein the kit comprises a reverse transcription primer selected from the group consisting of at least one of SEQ ID NOs 7,11,15,19,23, and 27.

10. The kit of any one of claims 7 to 9, wherein the kit comprises a probe selected from at least one of SEQ ID NOs 10,14,18,22,26 and 30.

11. The kit of any one of claims 1-10, wherein the detection reagent for detecting hsa-miR-33B-3p comprises a reverse transcription primer shown in SEQ ID No. 7, amplification primers shown in SEQ ID No. 8 and SEQ ID No. 9, and/or a probe shown in SEQ ID No. 10.

12. The kit of any one of claims 1-11, wherein the detection reagent for detecting hsa-miR-92B-3p comprises a reverse transcription primer shown in SEQ ID No. 11, amplification primers shown in SEQ ID No. 12 and SEQ ID No. 13, and/or a probe shown in SEQ ID No. 14.

13. The kit of any one of claims 1-12, wherein the detection reagent for detecting hsa-miR-205-5p comprises a reverse transcription primer shown in SEQ ID No. 15, amplification primers shown in SEQ ID No. 16 and SEQ ID No. 17, and/or a probe shown in SEQ ID No. 18.

14. The kit of any one of claims 1-13, wherein the detection reagent for detecting hsa-miR-374C-5p comprises a reverse transcription primer shown in SEQ ID No. 19, amplification primers shown in SEQ ID No. 20 and SEQ ID No. 21, and/or a probe shown in SEQ ID No. 22.

15. The kit of any one of claims 1-14, wherein the detection reagent for detecting hsa-miR-4516-3p comprises a reverse transcription primer shown in SEQ ID No. 23, amplification primers shown in SEQ ID No. 24 and SEQ ID No. 25, and/or a probe shown in SEQ ID No. 26.

16. The kit of any one of claims 1-15, wherein the detection reagent for detecting hsa-miR-6803-3p comprises a reverse transcription primer shown in SEQ ID No. 27, amplification primers shown in SEQ ID No. 28 and SEQ ID No. 29, and/or a probe shown in SEQ ID No. 30.

18. The kit of any one of claims 1-17, further comprising a detection reagent that detects a exo-reference miRNA, wherein the exo-reference miRNA is Cel-miR-39-5 p; preferably, the detection reagent for detecting the external reference miRNA is a reverse transcription primer shown as SEQ ID NO. 35, an amplification primer shown as SEQ ID NO. 36 and SEQ ID NO. 37, and a probe shown as SEQ ID NO. 38.

19. The kit of any one of claims 1-18, wherein the kit further comprises miRNA extraction reagents for a plasma or serum sample.

20. The kit of any one of claims 1 to 19, wherein the liver cancer is early stage liver cancer.

22. The method of claim 21, comprising detecting the concentration of at least two mirnas selected from the group consisting of hsa-miR-33B-3p, hsa-miR-92B-3p, hsa-miR-205-5p, hsa-miR-374C-5p, hsa-miR-4516-3p, and hsa-miR-6803-3p in the sample.

23. The method of claim 21, comprising detecting the concentration of at least three mirnas selected from the group consisting of hsa-miR-33B-3p, hsa-miR-92B-3p, hsa-miR-205-5p, hsa-miR-374C-5p, hsa-miR-4516-3p, and hsa-miR-6803-3p in the sample.

24. The method of claim 21, comprising detecting the concentration of at least four mirnas selected from the group consisting of hsa-miR-33B-3p, hsa-miR-92B-3p, hsa-miR-205-5p, hsa-miR-374C-5p, hsa-miR-4516-3p, and hsa-miR-6803-3p in the sample.

25. The method of claim 21, comprising detecting the concentration of at least five mirnas selected from hsa-miR-33B-3p, hsa-miR-92B-3p, hsa-miR-205-5p, hsa-miR-374C-5p, hsa-miR-4516-3p, and hsa-miR-6803-3p in the sample.

26. The method of claim 21, comprising detecting the concentration of hsa-miR-33B-3p, hsa-miR-92B-3p, hsa-miR-205-5p, hsa-miR-374C-5p, hsa-miR-4516-3p, and hsa-miR-6803-3p in the sample.

27. The method of any one of claims 21-26, wherein at least one reverse transcription primer, at least one amplification primer, and/or at least one probe is used.

28. The method of claim 27, wherein a reverse transcription primer comprising at least one member selected from the group consisting of SEQ ID NO 7,11,15,19,23 and 27 is used.

29. The method of claim 27 or 28, wherein amplification primers comprising at least one selected from the group consisting of SEQ ID NOs 8-9,12-13,16-17,20-21,24-25 and 28-29 are used.

30. The method of any one of claims 27-29, wherein a probe comprising at least one member selected from the group consisting of SEQ ID NOs 10,14,18,22,26 and 30 is used.

31. The method of any one of claims 21-30, wherein the detection reagent for detecting hsa-miR-33B-3p comprises a reverse transcription primer set forth in SEQ ID No. 7, amplification primers set forth in SEQ ID nos. 8 and 9, and/or a probe set forth in SEQ ID No. 10.

35. The method of any of claims 21-34, wherein the detection reagent for detecting hsa-miR-4516-3p comprises a reverse transcription primer set forth in SEQ ID No. 23, amplification primers set forth in SEQ ID nos. 24 and 25, and/or a probe set forth in SEQ ID No. 26.

36. The method of any of claims 21-35, wherein the detection reagent for detecting hsa-miR-6803-3p comprises a reverse transcription primer set forth in SEQ ID No. 27, amplification primers set forth in SEQ ID nos. 28 and 29, and/or a probe set forth in SEQ ID No. 30.

39. The method of any one of claims 21-38, wherein hsa-miR-33B-3p, hsa-miR-92B-3p, hsa-miR-205-5p, hsa-miR-374C-5p, hsa-miR-4516, and hsa-miR-6803-3p are reacted with hsa-miR-6803-3pSubtracting Ct values obtained by the internal reference hsa-miR-16 and the external reference Cel-miR-39 according to fluorescent quantitative PCR detection, taking the obtained difference value as a covariate for ROC curve analysis, and judging the feasibility of liver cancer occurrence by using the following logistic regression equation: p ═ e^K/(1+e^K) Wherein e is a natural constant, and K ═ is (0.15501) + (0.01785) hsa-miR-33B-3p + (-0.02857) hsa-miR-92B-3p + (-0.01955) miR-205-5p + (0.03704) miR-374C-5p + (0.04277) miR-4516-3p + (0.04277) miR-6803-3 p. If the P value is higher than 0.478, the patient is considered as an extremely early liver cancer high-risk patient.