Disclosure of Invention
The invention aims to solve the technical problems that firstly, a marker which is suitable for diagnosing canine breast tumor is screened; on the basis, a gene kit for detecting canine breast tumor is developed.
Accordingly, in one aspect, the invention provides a canine breast tumor diagnostic marker, which is CLCP1 and CPRDX1.
In yet another aspect, the invention also provides a canine breast tumor diagnostic kit comprising reagents for detecting CLCP1 and CPRDX1 expression levels.
Preferably, the reagents described in the present invention include primers that specifically amplify CLCP1 and CPRDX1.
Preferably, the primers for specifically amplifying CLCP1 and CPRDX1 according to the present invention are as follows:
CLCP1-F:5’-cttggtacgagctcggatcc-3’;
CLCP1-R:5’-cgtacacccttgctccgatt-3’;
CPRDX1-F:5’-ttgctttcagtgacagggca-3’;
CPRDX1-R:5’-acagagcgaccaacaggaag-3’。
preferably, the PCR reaction solution of the invention further comprises a specific amplification primer of the internal reference gene beta-actin, and the sequences of the primer and the probe are as follows:
β-actin-F:5’-aagtaccccattgagcacgg-3’;
β-actin-R:5’-catacagggacaggacagcc-3’。
in still another aspect, the invention also provides application of the canine mammary gland tumor diagnosis marker in preparing a canine mammary gland tumor diagnosis reagent.
The invention screens the marker for diagnosing the canine breast tumor by the technical means of bioinformatics and existing molecular biology. CLCP1 and CPRDX1 were selected as markers from the large number of screens. On the basis, the invention provides application of detecting the expression levels of CLCP1 and CPRDX1 in preparing products for diagnosing canine breast tumor and a related detection kit.
Compared with the existing detection means of the canine mammary tumor, the kit provided by the invention has the advantages of rapidness and convenience in detection, high detection sensitivity, good specificity, low cost, capability of meeting most detection requirements, wide application range and the like.
Detailed Description
The present invention is further illustrated below with reference to specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art. Reagents and materials used in the following examples are commercially available unless otherwise specified.
Through extensive and intensive research, the inventor detects the expression level of related genes in canine mammary gland tumor through bioinformatics technology and molecular biology technology, discovers genes with obvious expression difference, discusses the relationship between the genes and canine mammary gland tumor occurrence, and thereby searches better ways and methods for canine mammary gland tumor detection and targeted therapy. Through extensive research and screening, 2 proteins similar to human mesolymphocyte cytoplasmic protein 1 (lymphocyte cytosolic protein, LCP 1) (GenBank: DR107736.1, shown in SEQ ID NO. 1) and peroxidase 1 (peroxiredoxin 1, PRDX 1) (GenBank: DR103789.1, shown in SEQ ID NO. 2) were screened. They were designated in this study as canine lymphocyte cytoplasmic protein 1 (Canine lymphocyte cytosolic protein, clcp 1) and canine peroxidase 1 (Canine peroxiredoxin, cprdx 1).
By detection analysis, CLCP1 and CPRDX1 were highly expressed in canine breast tumors. Further experiments prove that the growth of canine breast tumors can be influenced by regulating the expression levels of CLCP1 and CPRDX1, and the CLCP1 and CPRDX1 are suggested to be used as drug targets for diagnosis or treatment of canine breast tumors.
"biomarker" and "marker" can be equivalently replaced, referring to a molecular indicator having a specific biological property, biochemical characteristic, or aspect, that can be used to determine the presence or absence of a particular disease or condition and/or the severity of a particular disease or condition.
Example 1 detection of CLCP1 and CPRDX1 in canine mammary tumors
1.1 sample collection
The method comprises the steps of selecting a canine breast tumor case which is treated in an animal hospital in Guangzhou city and subjected to pathological diagnosis, carrying out aseptic operation, reserving part of cancer tissues and other normal tissues (about 2-5 cm away from the cancer tissues), sub-packaging the cancer tissues and the other normal tissues in a freezing tube, and immediately storing the frozen tissue in liquid nitrogen. All affected animals were subjected to pathological diagnosis by the senior pathologist, did not suffer from other tumors prior to inclusion in the study, and were not treated with radiation or chemotherapy. Of which 5 malignant tumor tissues and paracancerous tissues were selected for the study.
1.2 extraction of Total RNA from tissues
(1) 1ml Trizol reagent (Takara Co.) was used to lyse the tissue per 50-100 mg of tissue;
(2) Transferring Trizol lysate of the above tissue into an EP tube, and standing at room temperature for 5 min;
(3) In the above EP tube, chloroform was added in an amount of 0.2ml of chloroform per 1ml of Trizol, the EP tube was capped, the tube was shaken vigorously in the hand for 15 seconds, and after leaving at room temperature for 2 to 3 minutes, 12000g (2 to 8 ℃) was centrifuged for 15 minutes;
(4) The upper aqueous phase was removed and placed in a fresh EP tube, isopropanol was added in an amount of 0.5ml of isopropanol per 1ml of Trizol, and after 10 minutes at room temperature, 12000g (2-8 ℃) was centrifuged for 10 minutes;
(5) Discarding the supernatant, adding 1ml of 75% ethanol into each 1ml of Trizol for washing, mixing by vortex, centrifuging for 5 minutes at 12000g (2-8 ℃), and discarding the supernatant;
(6) Allowing the precipitated RNA to naturally dry at room temperature;
(7) Dissolving the RNA precipitate by using RNase-free water;
(8) The extracted RNA samples were assayed for concentration and OD260/OD280 ratio to control sample quality, and samples with OD260/OD280 ratios between 1.8 and 2.0 were selected for subsequent testing.
1.3 primer design
Primer design was performed using Primer 5.0 software based on the CLCP1 (SEQ ID NO. 1) and CPRDX1 (SEQ ID NO. 2) sequences. And (3) obtaining a plurality of pairs of specific primers, and finally respectively determining a group of optimal primers through comparison and screening. The method comprises the following steps:
CLCP1-F:5’-cttggtacgagctcggatcc-3’;
CLCP1-R:5’-cgtacacccttgctccgatt-3’;
CPRDX1-F:5’-ttgctttcagtgacagggca-3’;
CPRDX1-R:5’-acagagcgaccaacaggaag-3’;
β-actin-F:5’-aagtaccccattgagcacgg-3’;
β-actin-R:5’-catacagggacaggacagcc-3’。
1.4 reverse transcription reaction
The reverse transcription assay system was carried out at 20. Mu.l, with reference to EasScript First-Strand cDNA Synthesis SuperMix (catalog number AE301-02, beijing full gold Biotechnology Co., ltd.).
1.5 fluorescent quantitative PCR reactions
The reverse transcription reaction is taken for real-time fluorescence quantitative PCR operation, and the reaction system is 20 μl: mu.l SYBR Premix, 2. Mu.l cDNA template, 0.6. Mu.l upstream and downstream primer each and 6.8. Mu.l DEPC water. Experiments were performed using 7500 fluorescent quantitative PCR from ABI, usa, with reaction settings: pre-denaturation at 95℃for 30s, annealing at 60℃for 20s, elongation at 72℃for 30s,40 cycles.
Wherein 2 is -△△Ct The expression of the target genes of the experimental group and the control group is expressed by the following formula: Δct=ct (target gene) -Ct (reference), ΔΔct= Δct Experimental group -△Ct Control group . Ct is the number of diffusion cycles that pass when the real-time fluorescence intensity of the reaction reaches a set threshold, at which time diffusion isIs growing in logarithmic phase. The expression levels of CLCP1 and CPRDX1 were calculated. Wherein the internal reference is beta-actin (shown as SEQ ID NO. 3).
1.6 detection results
The specific results are shown in FIG. 1. From the figure, it can be seen that CLCP1 and CPRDX1 were significantly up-regulated in canine breast tumor expression levels.
Example 2 detection of clinical samples by the kit
The expression levels of CLCP1 and CPRDX1 in the serum of 30 dogs and 30 healthy dogs were detected using the detection kit of example 2. Regression analysis was performed on the detection results using origin software, wherein single detection index results, two index combinations, and three index combinations of CLCP1 and CPRDX1 are shown in table 1.
The results show that the P value of each index and the P value of the combined index are less than 0.05, which indicates that each detection index is obviously related to the canine breast tumor prediction. The combined prediction accuracy of CLCP1 and CPRDX1 reaches 96% (area under ROC curve) and is higher than that of a single detection index. Thus, the combined detection of CLCP1 and CPRDX1 has significant advantages in canine breast tumors.
TABLE 1 analysis of test results
Detecting a variable
|
Sensitivity of
|
Specificity (specificity)
|
Area under ROC curve
|
CLCP1
|
0.72
|
0.79
|
0.83
|
CPRDX1
|
0.78
|
0.75
|
0.84
|
CLCP1+CPRDX1
|
0.95
|
0.96
|
0.96 |
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.