A gene detection kit and its application

Abstract

The present invention belongs to the field of biodiagnostic technology, and particularly relates to a gene detection kit and its application. The present invention discloses the application of detecting the expression level of FPAX5 in the preparation of products for diagnosing feline lymphoma and a related detection kit. The kit of the present invention can be used as one of the means for diagnosing feline lymphoma, and has the advantages of rapid and convenient detection, high detection sensitivity, good specificity, and low cost, and can meet most detection requirements.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological diagnosis, and particularly relates to a gene detection kit and its application. Background Art

[0002] Lymphoma is a malignant tumor that originates from lymphocytes and their precursor cells. Approximately 50% of feline tumors are hematological tumors. More than 50% of hematological tumors are lymphomas.

[0003] Lymphoma is a common tumor in humans or animals, and the cause of its occurrence has not been explained so far. In some cats, feline leukemia virus (FeLV) may induce this disease. As more cats are immunized or kept indoors, the risk of feline leukemia virus (FeLV) infection has been reduced, and the virus itself has become less common. Environmental factors, such as smoking, can increase the risk of developing LSA, and other environmental factors remain to be studied. There is no breed or gender predisposition for feline lymphoma.

[0004] Clinical symptoms vary depending on the site of infection. There are three common types of lymphoma in cats: generalized multicentric type, mediastinal type, and digestive tract (gastrointestinal) type. Generalized lymph node enlargement may be the only clinical symptom of the multicentric type. The mediastinal type occurs in the anterior chest cavity and may cause difficulty in breathing. The digestive tract type can involve any part of the stomach or intestine, resulting in weight loss, lethargy, loss of appetite, vomiting, and diarrhea. Abdominal masses may be palpable during physical examination. In some cats, an enlarged liver and spleen may be visible.

[0005] If superficial lymph nodes are enlarged, lymphoma is relatively easy to diagnose. If the tumor is present in organs that are not easily observable, the diagnosis will become difficult. The commonly used diagnostic methods are as follows: (1) Complete blood count: If lymphoma invades the bone marrow and causes bone marrow suppression, manifestations such as decreased blood cell count and decreased hemoglobin will occur, presenting anemia. (2) FeLV / FIV test: FeLV positive and FIV positive can support the diagnosis of lymphoma, but positive does not mean a confirmed diagnosis of lymphoma, and negative does not rule out lymphoma either. (3) Biochemical and urine tests: Invading the kidneys can be manifested as azotemia and decreased urine specific gravity. Invading the liver can show elevated liver enzymes. (4) Ultrasonic exploration: In cats with intra-abdominal lymphoma, abnormal manifestations such as an enlarged liver, an enlarged spleen, changes in the echo of the liver or spleen (mixed echo or multiple hypoechoic areas), and thickening of the intestinal wall are often found. (5) Fine needle aspiration: Through this method, evidence can be provided for a confirmed diagnosis from the aspects of cell or tissue pathology. The risk and cost of performing a fine needle aspiration biopsy are smaller than those of a biopsy. The organs and tissues usually sampled include the liver, spleen, mediastinum, kidneys, and lymph nodes. (6) Small intestine biopsy: Sampling is performed through abdominal exploration or endoscopy, but it is necessary to judge whether anesthesia and surgery are appropriate based on the physical condition of the affected cat.

[0006] These above-mentioned diagnostic methods are costly, require highly skilled personnel, and cause relatively great damage to cats. Therefore, there is an urgent need to develop a method for routinely detecting feline lymphoma. Summary of the Invention

[0007] One technical problem to be solved by the present invention is to screen suitable markers for diagnosing feline lymphoma; on this basis, another is to develop a reagent or kit for detecting feline lymphoma.

[0008] Therefore, on the one hand, the present invention provides a diagnostic marker for feline lymphoma, and the marker is FPAX5.

[0009] On the other hand, the present invention further provides a diagnostic kit for feline lymphoma, and the kit includes a reagent for detecting the expression level of FPAX5.

[0010] Preferably, the reagent for detecting the expression level of FPAX5 in the present invention includes a reagent for detecting the expression level of the FPAX5 gene and / or a reagent for detecting the expression level of the FPAX5 protein.

[0011] Preferably, the reagent for detecting the expression level of the FPAX5 gene in the present invention includes primers for specifically amplifying FPAX5 and the internal reference β-actin.

[0012] Preferably, the primers for specifically amplifying FPAX5 and the internal reference β-actin in the present invention are as follows:

[0013] FPAX5-F: 5’-cggacatcttcaccaccaca-3’;

[0014] FPAX5-R: 5’-ctcccggaaaactcactccc-3’;

[0015] β-actin-F: 5’-cactgtgcccatctacgagg-3’;

[0016] β-actin-R: 5’-tcttctccagggaggacgag-3’.

[0017] Preferably, the reagent for detecting the expression level of the FPAX5 protein in the present invention includes a monoclonal antibody that specifically binds to the FPAX5 protein.

[0018] Preferably, the monoclonal antibody that specifically binds to the FPAX5 protein in the present invention is monoclonal antibody 2B4, and the sequences of the heavy chain variable region and the light chain variable region encoding monoclonal antibody 2B4 are shown in SEQ ID NO.3 and SEQ ID NO.4 respectively.

[0019] In one aspect, the present invention also provides an application of the feline lymphoma diagnostic marker in the preparation of a diagnostic reagent for feline lymphoma.

[0020] Through bioinformatics and existing molecular biology techniques, the present invention screens markers for feline lymphoma diagnosis. Through a large number of screenings, FPAX5 is selected as a diagnostic marker. On this basis, the present invention provides an application of detecting the expression level of FPAX5 in the preparation of products for diagnosing feline lymphoma and related detection kits.

[0021] The kit of the present invention can be used as one of the means for diagnosing feline lymphoma. Compared with the existing detection means for feline lymphoma, the kit of the present invention has the advantages of rapid and convenient detection, high detection sensitivity, good specificity, and low cost, and can meet most detection needs. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Immunohistochemical detection results of monoclonal antibody 2B4 on feline lymphoma. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0023] The following specific embodiments are used to further illustrate the present invention, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field. Unless otherwise specified, the reagents and materials used in the following embodiments are all commercially available.

[0024] Example 1: Detection of FPAX5 in feline lymphoma

[0025] 1. Sample collection: Select cases of feline lymphoma that are treated and diagnosed in a certain animal hospital in Guangzhou. Under aseptic conditions, collect some cancer tissues and normal tissues adjacent to the cancer (about 2 - 5 cm away from the cancer tissue) and aliquot them into cryopreservation tubes, and immediately store them in liquid nitrogen. All diseased animals have been pathologically diagnosed by senior pathologists, have not suffered from other tumors before being included in this study, and have not received radiotherapy or chemotherapy. Five cases of malignant tumor tissues and adjacent tissues are selected for this study.

[0026] 1.1 Extraction of total RNA from tissues: Use a commercial RNA extraction kit to extract total RNA from tissues according to the instructions, or extract it by the conventional Trizol method. The general operation steps of the Trizol method are as follows:

[0027] (1) Lyse 50 - 100 mg of tissue with 1 ml of Trizol reagent (Takara) for each tissue;

[0028] (2) Transfer the Trizol lysate of the above tissue into an EP tube and place it at room temperature for 5 minutes;

[0029] (3) In each of the above EP tubes, add 0.2 ml of chloroform, cover the lids of the EP tubes, shake vigorously by hand for 30 seconds, place at room temperature for 2 - 3 minutes, and then centrifuge at 12,000 g at 4°C for 15 minutes;

[0030] (4) Transfer the upper aqueous phase to a new EP tube, add 0.5 ml of isopropanol to each tube, place at room temperature for 10 minutes, and then centrifuge at 12,000 g at 4°C for 10 minutes;

[0031] (5) Discard the supernatant, add 1 ml of 75% ethanol to each tube for washing, vortex mix, centrifuge at 12,000 g at 4°C for 5 minutes, and discard the supernatant;

[0032] (6) Allow the precipitated RNA to air-dry at room temperature;

[0033] (7) Dissolve the precipitated RNA with DEPC water;

[0034] (8) Measure the concentration of the extracted RNA samples and detect the ratio of OD260 / OD280 to control the sample quality. Select samples with an OD260 / OD280 ratio between 1.8 and 2.0 for subsequent experiments.

[0035] 1.2 Primer design Based on the FPAX5 (SEQ ID NO.1) sequence, the Primer Primer 5.0 software was used for primer design. Multiple pairs of specific primers were obtained. After comparison and screening, a set of optimal primers was finally determined respectively, with β-actin (as shown in SEQ ID NO.2) as the internal reference. Specifically as follows:

[0036] FPAX5-F: 5’-cggacatcttcaccaccaca-3’;

[0037] FPAX5-R: 5’-ctcccggaaaactcactccc-3’;

[0038] β-actin-F: 5’-cactgtgcccatctacgagg-3’;

[0039] β-actin-R: 5’-tcttctccagggaggacgag-3’.

[0040] 1.3 Reverse transcription reaction A commercial kit was used for the RNA reverse transcription reaction. For example, for a 20 μl reverse transcription reaction system, refer to the instructions of EasyScript First-Strand cDNA Synthesis SuperMix (Catalog No. AE301-02, TransGen Biotech Co., Ltd., Beijing).

[0041] 1.4 Fluorescent quantitative PCR reaction The reverse transcription reaction product cDNA was taken for real-time fluorescent quantitative PCR reaction, and the conventional dye method was used.

[0042] The reaction system was 20 μl: 10 μl SYBR Premix, 2 μl cDNA template, 0.5 μl each of upstream and downstream primers, and 7.0 μl DEPC water. Experiments were carried out using the 7500 type fluorescent quantitative PCR instrument of ABI Company in the United States or other fluorescent quantitative PCR instruments (such as Roche, etc.). The reaction set conditions were: pre-denaturation at 95 °C for 30 s, denaturation at 95 °C for 30 s, annealing at 60 °C for 20 s, extension at 72 °C for 30 s, for 40 cycles.

[0043] The difference between the CT value of the detected gene FPAX5 and the CT value of the internal reference gene β-actin was used to represent the result of gene differential expression. The smaller the result, the higher the expression level. The results showed (Table 1) that the expression level of FPAX5 was significantly up-regulated in feline lymphoma.

[0044] Table 1 Detection results of FPAX5 in feline lymphoma

[0045] Item FPAX5 CT value β-actin CT value CT value difference Cancer tissue 14.65 10.32 4.33 Tissue adjacent to cancer 28.15 10.45 17.7

[0046] Example 2: Detection of clinical samples by the kit

[0047] The detection kit of Example 2 was used to detect the expression levels of FPAX5 in the sera of 20 cases of feline lymphoma and 20 cases of healthy cats. The detection results were analyzed by regression using origin software.

[0048] The results showed (Table 2) that the P value of this index detection was < 0.05, indicating that the detection index was significantly correlated with the prediction of feline lymphoma.

[0049] Table 2 Analysis results of detection results

[0050] Sensitivity Specificity Area under the ROC curve 0.81 0.86 0.85

[0051] Example 3: Preparation and testing of monoclonal antibody against FPAX5

[0052] According to the results of Example 2, the accuracy of predicting feline lymphoma by detecting the expression level of FPAX5 was not very high (both the sensitivity and specificity did not reach 0.9 or above). To further improve the detection accuracy, this study suggested adding protein-level detection, that is, detecting both the gene level and the protein level of FPAX5. To detect the FPAX5 protein, this study developed a monoclonal antibody against the FPAX5 protein.

[0053] In this study, a conventional mouse hybridoma cell technology was used (for the specific experimental procedures, refer to Example 1 of CN 111925436B), and a good monoclonal antibody that can specifically bind to the FPAX5 protein was screened and named monoclonal antibody 2B4.

[0054] The specificity and affinity of monoclonal antibody 2B4 were detected (immunohistochemistry of feline lymphoma cancer tissues and adjacent tissues), and the results showed ( Figure 1 ), that this monoclonal antibody has good specificity and affinity.

[0055] Meanwhile, the sequence of this monoclonal antibody was analyzed (for the specific experimental procedures, refer to Example 1 of CN 111925436B), and the results showed that the sequences of the heavy chain variable region and the light chain variable region encoding this monoclonal antibody are shown in SEQ ID NO.3 and SEQ ID NO.4 respectively.

[0056] The serum samples were detected using the kit of Example 2, and for the suspicious cats, living tissues were collected for immunohistochemical detection (detected using the monoclonal antibody 2B4 of this study) to further determine whether it is feline lymphoma, which can further improve the sensitivity and specificity of this kit and reduce false negatives. After data analysis, after adding immunohistochemistry, the accuracy can be increased by at least 5% - 10%.

[0057] The above embodiments are the preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A cat lymphoma diagnostic kit, characterized in that, The kit includes reagents for detecting the expression level of FPAX5; the reagents for detecting the expression level of FPAX5 include reagents for detecting the expression level of the FPAX5 gene and reagents for detecting the expression level of the FPAX5 protein; The reagents for detecting the expression level of the FPAX5 gene include primers for specifically amplifying FPAX5 and the internal reference β-actin; the primers for specifically amplifying FPAX5 and the internal reference β-actin are shown as follows: FPAX5-F: 5’-cggacatcttcaccaccaca-3’; FPAX5-R: 5’-ctcccggaaaactcactccc-3’; β-actin-F: 5’-cactgtgcccatctacgagg-3’; β-actin-R: 5’-tcttctccagggaggacgag-3’; The reagents for detecting the expression level of the FPAX5 protein include monoclonal antibodies that specifically bind to the FPAX5 protein; the monoclonal antibody that specifically binds to the FPAX5 protein is monoclonal antibody 2B4, and the sequences of the heavy chain variable region and the light chain variable region encoding monoclonal antibody 2B4 are shown as SEQ ID NO.3 and SEQ ID NO.4 respectively.

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