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MiRNA composition and kit therefore for diagnosis of early hepatocellular carcinoma

A technology of early liver cancer and kits, applied in the field of medical biological detection, can solve the problems of low sensitivity, increased AFP, and low detection throughput

Inactive Publication Date: 2015-12-16
HEPATOBILIARY SURGERY HOSPITAL SECOND MILITARY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this single marker has low sensitivity and often has false positive results, for example, in a large number of patients with liver cirrhosis, AFP is also elevated
In addition, serum AFP can detect only 60% of liver cancer patients
Such sensitivity and specificity are currently unsatisfactory
Although the improved imaging technology makes it possible to find small focal liver lesions, it is still difficult to distinguish benign lesions from tumors, and the detection throughput is low, requiring expensive detection equipment, which is not suitable for clinical screening of large samples in multiple centers. check

Method used

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  • MiRNA composition and kit therefore for diagnosis of early hepatocellular carcinoma
  • MiRNA composition and kit therefore for diagnosis of early hepatocellular carcinoma
  • MiRNA composition and kit therefore for diagnosis of early hepatocellular carcinoma

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1. Cohort screening of specific miRNAs molecular markers that can predict the possibility of liver cancer in high-risk groups of patients with viral hepatitis

[0070] The annual physical examination cohort of nearly 10,000 chronic HBsAg-positive patients was studied, and the serum of patients with liver cancer diagnosed half a year to one and a half years ago was collected to screen for differential expression compared with those without liver cancer miRNAs profile, screening specific miRNAs molecular markers that can predict the possibility of liver cancer in high-risk groups of patients with viral hepatitis.

[0071] The main plasma samples used in the study came from the Eastern Hepatobiliary Surgery Hospital, collected from a cohort of annual physical examinations from April 2007 to March 2014, including 9287 chronic hepatitis B surface antigen (HBsAg) positive (HBsAg-positive) patients . During the physical examination of these participants, HBsAg, alanin...

Embodiment 2

[0086] Example 2. Preparation of detection reagents and detection of training set patient serum

[0087] Based on the five miRNAs identified above, detection reagents were designed to identify early or very early liver cancer by detecting the five miRNAs.

[0088] 1. Primer design

[0089] Design the following reverse transcription primers and upstream and downstream primers and probes:

[0090] For hsa-miR-193a-3p:

[0091] Reverse transcription primer: 5'-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGACTGGGACTTTG-3'(SEQ ID NO: 6);

[0092] Upstream primer: 5'-ACACTCCAGCTGGGAACTGGCCTACAAAGT-3' (SEQ ID NO: 7);

[0093] Downstream primer: 5'-GTACGACTCACTATAGGGACTCAACTGGTGTCGTGGAG-3' (SEQ ID NO: 8);

[0094] Probe: 5'-TTCAGTTGAGACTGGGACTTTG-3' (SEQ ID NO: 9). The probe has FAM at its 5'-end; BHQ at its 3'-end.

[0095] For hsa-miR-369-5p:

[0096] Reverse transcription primer: 5'-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGGCGAATATAACAC-3'(SEQ ID NO: 10);

[0097] Upstream primer: 5'-ACAC...

Embodiment 3

[0149] Embodiment 3, prepare a kind of kit of the present invention

[0150] In this embodiment, with the detection reagent designed in Example 2, a kit comprising the following components or components was prepared: RNA extraction reagents, reverse transcription reagents for each miRNAs to be tested and internal reference, for each miRNAs to be tested and Internal reference PCR amplification upstream primers and downstream primers, RNase-free pure water, negative quality control, positive quality control.

[0151] 1. RNA extraction reagent: Trizol reagent (LifeTechnologies company, U.S.) was used in this embodiment;

[0152] 2. Reverse transcription primers, forward primers, reverse primers, oligonucleotide probes, and positive controls were synthesized by Shanghai Sangon Biotechnology Co., Ltd.;

[0153] 3. Reverse transcriptase M-MLV, reverse transcription reaction system, nucleic acid amplification enzyme and quantitative PCR reaction system were purchased from Life Techn...

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Abstract

The invention relates to a miRNA composition and a kit thereof for diagnosis of early hepatocellular carcinoma. The method discloses a method for the diagnosis of hepatocellular carcinoma, and in particular early hepatocellular carcinoma, through cooperative detection of expression of five miRNAs for the first time; and the invention also provides a reagent for implementing the diagnosis.

Description

technical field [0001] The invention belongs to the technical field of medical biological detection. More specifically, the invention relates to a miRNA combination and kit for diagnosing early liver cancer, and its application in identifying early liver cancer in a population of asymptomatic high-risk individuals. Background technique [0002] Hepatocellular carcinoma (HCC) is one of the most common solid malignancies worldwide and accounts for the second leading cause of cancer-related deaths worldwide. Due to the lack of effective early diagnosis or preclinical screening methods for potentially high-risk groups of liver cancer, only 1 / 3 of patients diagnosed with liver cancer using existing diagnostic methods are suitable for surgical resection. The 5-year survival rate of these patients is also very unsatisfactory, not higher than 14% overall; while the 5-year survival rate of patients with early liver cancer can be nearly doubled, about 27%. Therefore, a powerful and c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6886C12Q2600/166C12Q2600/178
Inventor 王红阳陈磊李亮
Owner HEPATOBILIARY SURGERY HOSPITAL SECOND MILITARY MEDICAL UNIV
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