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Molecular marker for diagnosis and prognosis judgment of canine breast tumor

A technology of molecular markers and mammary gland tumors, which is applied in the field of molecular markers for the diagnosis and prognosis of canine mammary gland tumors, can solve the problems of misdiagnosis or missed diagnosis, insufficient specificity and sensitivity, and difficult popularization of joint diagnosis, etc., and achieve high sensitivity and specificity, the effect of improving the survival rate

Inactive Publication Date: 2018-12-18
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] The tumor markers currently used clinically have many defects: ①The specificity and sensitivity are not high enough, and there are cases of misdiagnosis or missed diagnosis; specific markers

Method used

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  • Molecular marker for diagnosis and prognosis judgment of canine breast tumor
  • Molecular marker for diagnosis and prognosis judgment of canine breast tumor
  • Molecular marker for diagnosis and prognosis judgment of canine breast tumor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 RT-qPCR method to detect the mRNA expression level of ICAM1 in canine mammary gland tumor tissue

[0056] 1. Experimental method

[0057] 1. Sample storage and processing

[0058] The surgically resected fresh tumor tissue was washed with PBS to clean the blood stains and hair on the surface of the tissue, and then the cancer tissue and paracancerous tissue (less than or equal to 2 cm away from the cancer lesion) were separated and decomposed into 1 cm × 1 cm tissue blocks and placed in sterilized EP. Immediately after the tube was frozen with liquid nitrogen, and then stored at -80°C for later use; a part of the tissue from each sample was fixed with 4% paraformaldehyde for subsequent paraffin-embedded sections and sent to Nabok clinical animal clinical testing in Germany Laboratory identification.

[0059] 2. Preparation of pathological sections and HE staining

[0060] The canine breast tumor tissue samples collected clinically were paraffin-embedded, se...

Embodiment 2

[0100] Example 2 RT-qPCR method to detect the mRNA expression level of ICAM1 in CHMm and CHMp cells

[0101] 1. Culture of canine breast tumor cells CHMm and CHMp

[0102] 1. Recovery of canine breast tumor cells CHMm and CHMp

[0103] Take the CHMp and CHMm cells out of the liquid nitrogen and quickly put them into a 37°C water bath and shake them constantly to dissolve them quickly; transfer them to 15mL centrifuge tubes after the cells in the cryopreservation tubes are completely thawed, add 10mL DMEM to each tube and Repeatedly blow and mix; then centrifuge at 2,000rpm for 5min; discard the supernatant, resuspend the cells with 1mL complete cell culture medium (90% DMEM, 10% serum, 1% double antibody) and transfer them to a 25cm2 adherent cell culture flask After adding 4mL of complete medium to each bottle, culture them in an incubator at 37°C containing 5% CO2. After 24 hours, observe under the microscope, if most of the cells adhered to the wall normally, and only a s...

Embodiment 3

[0122] Example 3 A kit for diagnosis and prognosis of canine mammary gland tumors

[0123] 1. A kit for the diagnosis and prognosis of canine mammary gland tumors

[0124] A kit for diagnosing and judging prognosis of canine mammary gland tumors, containing RT-qPCR detection primers for detecting ICAM1 gene expression, the nucleotide sequence of which is shown in SEQ ID NO: 2-3; the internal reference gene is RT-qPCR of β-actin qPCR detection primer, the nucleotide sequence of the RT-qPCR detection primer is shown in SEQ ID NO: 4-5; it also contains ddH 2 O. SYBR GreenⅠMaster.

[0125] 2. How to use the kit

[0126] The method of using the kit is as follows: extract total RNA from the sample, synthesize cDNA, and perform RT-qPCR.

[0127] S1. extract the total RNA of the sample;

[0128] S2. Synthesizing cDNA;

[0129] S3. Perform RT-qPCR.

[0130] Among them, the total volume of the RT-qPCR system is 20 μL, specifically: ddH 2 O 7.2 μL; SYBR GreenⅠMaster 10 μL; forward...

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Abstract

The invention discloses a molecular marker for the diagnosis and prognosis judgment of a canine breast tumor. The molecular marker is an ICAM1 gene with a nucleotide sequence shown in the formula of SEQ ID NO: 1. The invention also discloses RT-qPCR detection primers for detecting ICAM1 gene expression. The primers have nucleotide sequences shown in the formulas of SEQ ID NO: 2-3. The invention also discloses a kit for diagnosis and prognosis determination of the canine breast tumor. The ICAM1 gene only is significantly low expressed in the malignant tumor group. The expression level of the ICAM1 gene in canine mammary gland tumor cells isolated from metastatic lesions is significantly different from that of primary tumor cells so that it is proved that the ICAM1 can identify benign and malignant canine breast tumors and is participated in the infiltration and metastasis of canine mammary tumors. Therefore, ICAM1 can be used as a molecular marker for the diagnosis and prognosis of canine breast tumors. The molecular marker related to canine mammary gland tumor metastasis has high sensitivity and specificity, can identify the nature of the canine breast tumor, can evaluate the prognosis, can determine the treatment plan in time, and can improve the survival rate of the diseased dog.

Description

technical field [0001] The invention relates to the technical field of detection of canine mammary gland tumors, in particular to a molecular marker that can be used for the diagnosis and prognosis of canine mammary gland tumors. Background technique [0002] Canine mammary gland tumors are the most common neoplastic disease in female dogs, mostly in middle-aged and elderly female dogs aged 6 to 11, and about 30% to 50% of them are malignant. Lumps in the breast due to environmental factors or genetic causes are clinically called breast tumors. A large number of studies have shown that hormones can greatly affect the occurrence of canine mammary gland tumors, such as estrogen, progesterone, and growth hormone, all of which can affect the occurrence of malignant tumors. Studies have found that the incidence of mammary gland tumors in dogs increases significantly with the number of estruses, and sterilization after the first estrus can effectively limit the incidence of mamma...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12N15/12C12N15/11
CPCC07K14/47C12Q1/6886C12Q2600/112C12Q2600/118C12Q2600/158
Inventor 贾坤芶思
Owner SOUTH CHINA AGRI UNIV
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