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A diagnostic method and diagnostic kit for intrahepatic cholangiocarcinoma

The technology of a kit and detection kit, applied in the field of molecular biology, can solve the undisclosed problem of GPC3 expression in intrahepatic cholangiocarcinoma

Active Publication Date: 2020-10-27
MUDANJIANG MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the application did not disclose the expression of GPC3 in intrahepatic cholangiocarcinoma

Method used

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  • A diagnostic method and diagnostic kit for intrahepatic cholangiocarcinoma

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Experimental program
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Effect test

Embodiment 1

[0030] Example 1: Detection of mutant truncated GPC3 proteoglycans

[0031] With the consent of the patient, the inventor of the present application took a cancer cell tissue sample from a patient diagnosed with intrahepatic cholangiocarcinoma for protein analysis, extracted the total protein by cell disruption technology, and obtained the GPC3 proteoglycan in it by antibody purification technology. Protein structure analysis found that the GPC3 proteoglycan structure was different from the known GPC3 proteoglycan structure in hepatocellular carcinoma. The GPC3 protein was sent out for protein sequencing, and its sequence is shown in SEQ ID No.5. Sequence comparison found that compared with the known high expression of GPC3 protein in hepatocellular carcinoma (as shown in SEQ ID No.4, NCBI database number: XP_018874999.1), the GPC3 proteoglycan in the above intrahepatic cholangiocarcinoma samples There are two mutation sites, Y314S and M316I, and 33 amino acid sequences at th...

Embodiment 2

[0033] Embodiment 2 Specific primers and probe design and PCR amplification method

[0034] In response to the above findings, the inventors confirmed the cDNA sequence of the above-mentioned mutant truncated GPC3 protein through a genetic screening analysis method, and designed primers and probes for the above-mentioned mutant truncated GPC3 proteoglycan mutation site for the sequence, The specific sequences are respectively shown in SEQ ID No.1-3. The two ends of the probe shown in SEQ ID No.3 are respectively labeled with a fluorescent reporter group FAM and a fluorescent quencher group TAMRA. The above primers can amplify the cDNA sequence containing the mutation site, and the probe

[0035] The method for target sequence-specific amplification detection using the above primers and probes is as follows:

[0036] (1) extract the total RNA in the sample tissue (peripheral blood), and reverse transcribe it into cDNA;

[0037] (2) using the cDNA in step (1) as a template, usi...

Embodiment 3

[0043] Embodiment 3 clinical examination test

[0044] With the consent of the patient, the peripheral blood of 783 patients undergoing screening and detection of hepatocellular carcinoma or intrahepatic cholangiocarcinoma was tested using the method of the above-mentioned embodiment 2. Among them, the method described in this application showed positive results in 38 cases and negative results in 745 cases. Example; the above positive and negative results were verified separately, and the final confirmation test results are shown in the following table:

[0045]

[0046] It can be seen that the mutant truncated GPC3 proteoglycan described in the present application has good specificity for intrahepatic cholangiocarcinoma, and only 1 case was detected in 38 positive patients using the primers and probes of the present invention. Hepatocellular carcinoma was finally confirmed, and among the 745 negative patients, 132 were diagnosed as hepatocellular carcinoma, and 15 were ot...

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Abstract

The invention provides a diagnosis method and diagnosis kit for intrahepatic cholangiocarcinoma. The kit comprises primers and probe aiming at mutant type glypican 3 (GPC3) complementary DNA (cDNA). According to the diagnosis method and the diagnosis kit, experiments show that the glypican 3 can mutate in the intrahepatic cholangiocarcinoma; the mutation has specificity in the intrahepatic cholangiocarcinoma, and therefore, the mutant GPC3 cDNA can be used as a specific marker for diagnosing and treating an acute liver injury with drugs. Based on the above, the invention provides a new hepatocyte diagnosis method and kit.

Description

technical field [0001] The invention belongs to the field of molecular biology, in particular to primers and probes for specific detection of markers of human intrahepatic cholangiocarcinoma cells and a detection kit thereof. Background technique [0002] Primary liver cancer is one of the most common malignant tumors in the world and one of the most common types of malignant tumors of the digestive system in clinical practice. Esther, Cidon. Systemic treatment of hepatocellular carcinoma: Past, present and future [J]. World Journal of Hepatology, 2017, 9(18): 797-807.). Intrahepatic cholangiocarcinoma (ICC) refers to adenocarcinoma originating from the epithelium of the secondary bile duct and its branches. ICC is a primary liver malignant tumor whose incidence is second only to hepatocellular carcinoma, and its incidence has been on the rise in recent years. ICC accounts for approximately 10%-15% of primary liver malignancies. Academician Huang Zhiqiang pointed out in t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6886C12Q1/6858
CPCC12Q1/6858C12Q1/6886C12Q2600/158C12Q2600/166C12Q2531/113C12Q2561/101C12Q2521/107C12Q2545/101
Inventor 代海兵王菡连一霏崔江河李雪梅陈丽丽许晓义孙展鹏金红刘侠栾海蓉
Owner MUDANJIANG MEDICAL UNIV
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