CN106399527A - Primer group for detecting renal cancer and detecting method thereof - Google Patents

Abstract

The present invention provides a primer set for detecting kidney cancer and a detection method thereof. The primers of the present invention include SEQ ID No.1-SEQ ID No.18, which can be used for MN/CA9, TS, CYP3A4, PD-L1, VEGFR2, cadherin‑6, CK19, CD24, and EPCAM genes were combined for detection. Through PCR or fluorescent quantitative PCR method, the expression and expression level of target genes can be detected sensitively in a short time; using optimized PCR conditions, by increasing the number of cycles of the amplification reaction, the observation results are more significant, and the applicability of the method is further improved. The specificity of the amplification product ensures the high accuracy and sensitivity of PCR and RT-PCR fluorescence quantitative detection. By implementing the primer set used in the detection of kidney cancer in the present invention, the kidney cancer can be diagnosed simply, conveniently and accurately by detecting the expression of target genes in patient samples.

Description

A primer set and detection method for detecting kidney cancer

technical field

The invention relates to the field of tumor genes, in particular to a primer set and a detection method for detecting kidney cancer.

Background technique

Renal cancer is a malignant tumor originating from the urinary tubular epithelial system of the renal parenchyma. With the advancement of science, the incidence of RCC has increased compared to before. The main complaints and clinical manifestations of RCC patients are changeable, and it is easy to be misdiagnosed as other diseases. The location of the kidney is hidden, and the main connection with the outside world is urine. Therefore, hematuria is the most common symptom of renal cancer. However, hematuria can only occur after the tumor invades the renal pelvis, so it is no longer an early symptom. Therefore, early diagnosis of renal cell carcinoma is very necessary.

The current diagnostic methods of RCC include imaging examination, cytopathological and histopathological diagnosis. Imaging examinations include X-rays, CT scans, ultrasound scans, MRI examinations, etc., but their efficiency is low and detection accuracy is not high. Common cell and histopathological tests include cell biopsy, which needs to obtain tumor tissue samples from patients through surgery or puncture. Not only inconvenient, but also will cause bodily harm to the patient, and may even cause tumor metastasis in the puncture tract.

Recently, the detection of circulating tumor cells (Circulating Tumor Cells, CTCs) has brought new hope to the detection of patients. CTCs are tumor cells detached from tumor lesions and translocated into the blood. They are an important cause of postoperative recurrence and distant metastasis in patients with malignant tumors, and are also an important factor leading to the death of tumor patients. Compared with other histological specimens such as bone marrow, peripheral blood specimens are easy to obtain and cause less trauma to patients, making them an ideal source of specimens for routine clinical testing. The detection of CTCs is helpful for early diagnosis of tumors, judging the prognosis of patients, evaluating the efficacy of anti-tumor drugs and formulating individualized treatment plans. Compared with traditional diagnostic methods, CTCs detection can detect changes in diseases more sensitively and has no side effects on patients.

The detection of CTC is usually carried out by drawing a certain volume of blood. Detection is divided into two categories, including direct detection without capture (enrichment) and detection after first capture (enrichment), and the latter is the mainstream detection method. The method of first capture (enrichment) and then detection is also divided into two categories, namely positive selection and negative selection. Positive selection is mainly captured by CTC surface markers or physical properties (size, density) of cells; the former is based on immunomagnetic sphere technology and microfluidic chip technology, and the latter is based on the principles of filtration and centrifugation. Negative selection is the indirect capture of CTCs through leukocyte surface markers. However, the method based on first capture (enrichment) and then detection has obvious defects. It relies heavily on the expression of tumor cell surface markers and thus cannot capture CTC cells that do not express surface tumor markers.

Using RT-PCR to detect the specific genes of CTC cells is another way of CTC cell detection. First, a certain volume of blood is drawn, and CTCs are enriched by density gradient centrifugation, and then the mRNA of a specific gene is detected by RT-PCR to determine the existence of CTCs, and the CTCs are quantified by detecting changes in CT values. This method detects CTCs with low cost, high sensitivity, and easy automation.

At present, the standard of RT-PCR method to detect CTCs in renal cell carcinoma has not been established yet. By detecting the characteristic genes of CTCs in patients with renal cancer, we determined the detection method and standard of renal cancer CTCs. The establishment of these detection methods and standards is helpful for early diagnosis of cancer patients, judgment of patient prognosis, evaluation of anti-tumor drugs, including the efficacy of NK and CAR-T immunotherapy, and formulation of individualized treatment plans.

In view of the above problems, the present invention develops a highly sensitive multi-gene joint detection kit, which is realized by joint detection of 9 genes of MN/CA9, TS, CYP3A4, PD-L1, VEGFR2, cadherin-6, CK19, CD24 and EPCAM Early screening or diagnosis of tumors. The invention designs and uses a target-specific primer set to greatly improve detection sensitivity. The detection rate of the detection method can reach 96%, and at the same time, it has the advantages of high sensitivity, good specificity, rapidity and accuracy.

Contents of the invention

The object of the present invention is to provide a primer set and a detection method for detecting kidney cancer against the deficiencies of the prior art.

The object of the present invention is achieved through the following technical solutions: a primer set for detecting kidney cancer, comprising:

The primer set of the present invention can realize the detection of kidney cancer through the following two methods.

Method 1: PCR method.

(1) 9 pairs of primers described in claim 1 are added respectively in a single PCR reaction tube, and all add nucleic acid extract, reverse transcriptase, RNase inhibitor, DNA polymerase, dNTP; Wherein, PCR reaction The conditions of each cycle are 94 degrees Celsius, 30s; 55 degrees Celsius, 30s; 72 degrees Celsius, 10s;

(2) Carry out reverse transcription and PCR reactions, and detect by agarose gel electrophoresis.

Method 2: Fluorescent quantitative PCR method

(1) 9 pairs of primers described in claim 1 are added respectively in a single PCR reaction tube, and all add nucleic acid extract, reverse transcriptase, RNase inhibitor, DNA polymerase, dNTP; Add 2 * ChamQ respectively SYBR qPCR Master Mix, wherein the conditions of each cycle of the PCR reaction are 95 degrees Celsius, 10s and 60 degrees Celsius, 30s; 40 cycles; three duplicate holes are set up for each tube;

(2) Perform reverse transcription and PCR reactions, and detect fluorescence in real time;

(3) According to the Ct value calculated from the fluorescence detection result, it is judged whether there is marker target gene expressed in the sample.

The beneficial effect of the present invention is that: the present invention develops a combined multi-gene detection method for early detection of kidney cancer by designing specific PCR primers. This method: (1) The established PCR system can jointly detect MN/CA9, TS, CYP3A4, PD-L1, VEGFR2, cadherin-6, CK19, CD24 and EPCAM genes; The detection rate of kidney cancer can reach 96%; (3) high sensitivity, as low as 1-5 copies of gene expression level can be detected; (4) strong specificity, samples from normal people or non-kidney cancer patients are rarely detected Generating specific signals; (5) Fast detection speed, simple operation and low cost.

Description of drawings

Fig. 1 is the negative PCR chart of the peripheral blood samples of healthy people in the embodiment.

Fig. 2 is a negative PCR chart of peripheral blood samples from non-kidney cancer patients in the embodiment.

Fig. 3 is a negative PCR image of the tumor tissue of a non-renal cancer patient in the embodiment.

Fig. 4 is a positive PCR chart of the peripheral blood of the renal cancer patient in the embodiment.

Fig. 5 is a positive PCR image of the tumor tissue of a renal cancer patient in the embodiment.

In the figure, M.100bp marker; 1.B2M, 2.MN/CA9, 3.TS, 4.CYP3A4, 5.PD-L1, 6.VEGFR2, 7.cadherin-6, 8.CK19, 9.CD24, 10. EPCAM.

detailed description

The present invention aims at the main driving mutation genes in tumors at present, and jointly detects MN/CA9, TS, CYP3A4, PD-L1, VEGFR2, cadherin-6, CK19, CD24 and EPCAM9 genes to realize early screening or diagnosis of renal cancer. In view of the low sensitivity of the current detection method, the detection process is complicated and cumbersome, and the detection takes a long time, which cannot meet the actual needs of clinical detection. Aiming at the above problems, a set of specific primer sets for detecting the above nine genes was designed.

Design specific amplification primers based on the reference sequences of the above 9 genes. The optimal PCR product length is between 180-200bp, the annealing temperature is not higher than 60 degrees, and the GC content is between 40-60%, which is in line with general primers design software requirements. By adopting the real-time fluorescent PCR reaction detection system, the combined high-sensitivity detection of multiple genes is realized, and the detection method is as follows.

The present invention will be further described in conjunction with drawings and embodiments. If not otherwise specified, the third edition of "Molecular Colon Experiment Guide" (Cold Spring Harbor Laboratory Press), "Cell Experiment Guide" (Science Press, Beijing, China, 2001) familiar to those skilled in the art can be used. ), "RNA Experimental Technical Manual" (Science Press, Beijing, China, 2004), "Immunoassay Technology" (Science Press, Beijing, China, 1991) and other experimental manuals and the references cited herein method to implement. Among them, the used (labeled) probes and primers can be synthesized by entrusting Shanghai Sangon Bioengineering Technology Service Co., Ltd.

The present invention will be further described below in conjunction with embodiment.

Example 1: Gene selection

Many studies have shown that the sensitivity of single-gene screening is about 20-60%. Most of the single-gene tests used for early tumor screening have low detection sensitivity or specificity, which cannot meet clinical needs. Multi-gene combined detection can effectively solve the problem of low sensitivity and improve the accuracy of early tumor screening and diagnosis. In order to increase the detection rate of early tumors, improve the cure rate of tumor patients, and improve the prognosis of patients, we screened the differentially expressed genes between renal cancer and normal tissues. Through a large number of comparison experiments, we found that the genome composed of MN/CA9, TS, CYP3A4, PD-L1, VEGFR2, cadherin-6, CK19, CD24 and EPCAM has a high detection rate for kidney cancer, reaching 96%, compared to The existing detection technology has produced unexpected technical effects and has made significant progress.

Embodiment 2: primer screening

(1) Design primers a1~a3, b1~b3, c1~c3, d1~d3, e1~e3, f1~f3, g1~g3, h1~h3, i1~i3, specifically: use bioinformatics software to The A-I sequence of the target gene was analyzed, and a specific primer set was designed by using the sequence analysis software, and the specificity of each paired primer in the human genome was detected by the NCBI primer search software, and three pairs of specific amplification primers a1~a3, b1~ b3, c1~c3, d1~d3, e1~e3, f1~f3, g1~g3, h1~h3, i1~i3;

Table 1: PCR Detection Primers

(2) Processing of peripheral blood samples

Peripheral blood was collected from kidney cancer patients admitted to a hospital and confirmed by pathology. At 7:00 in the morning, on an empty stomach, fresh blood was collected through the cubital vein, stored in anticoagulant tubes, shaken well, and stored at room temperature for ≤4 hours.

2.1 Add an equal volume of PBS (pH 7.0) to the whole blood sample in the anticoagulant tube, centrifuge at 3000 rpm for 5 minutes at room temperature, and remove the upper layer of plasma;

2.2 Add an equal volume of ammonium chloride erythrocyte lysate (purchased from Biyuntian), centrifuge at room temperature 200rpm for 8min, mix well, then centrifuge at room temperature for 5min at 3000rpm, and discard the supernatant;

2.3 After mixing the lower blood cells and normal saline at a volume ratio of 1:2 to 2:1, add Ficoll separation solution. The volume ratio of the mixture to Ficoll separation solution is 1:2 to 2:1, and the centrifugal force is 700g. 40min;

2.4 Discard the supernatant, take the cell pellet, and wash to obtain PBMC;

2.5 Take PBMCs for nucleic acid extraction, resuspend excess PBMCs with RNA protection agent, and store at -20 degrees.

(3) Extraction of nucleic acid

3.1 Take no more than 5×10 ⁵ PBMCs, add 250 μL Buffer RLT Plus, and mix by pipetting;

3.2 Transfer the lysate to a gDNA removal spin column and centrifuge at 8000×g (10,000rpm) for 30s. Collect the flow-through and discard the spin column;

3.3 Add 1 volume of 70% ethanol (350 μL) to the flow-through solution, and mix by pipetting;

3.4 Transfer the sample to the RNeasyMinElute spin column, close the lid tightly, centrifuge at 8000×g (10,000rpm) for 15s, and discard the flow-through;

3.5 Add 700μL Buffer RW1 to the RNeasyMinElute spin column, close the cap tightly, centrifuge at 8000×g (10,000rpm) for 15s, and discard the flow-through;

3.6 Add 500μL Buffer RPE to the RNeasyMinElute spin column, close the cap tightly, centrifuge at 8000×g (10,000rpm) for 15s, and discard the flow-through;

3.7 Place the RNeasyMinElute spin column in a new 2mL collection tube, open the lid, and centrifuge at full speed for 5 minutes to evaporate the ethanol;

3.8 Put the RNeasyMinElute spin column in a new 1.5mL collection tube, add 20μL RNase-freeH ₂ O, close the cap tightly, and centrifuge at full speed for 1min to elute RNA.

(4) Acquisition of template cDNA

4.1 Accurately measure the RNA concentration of the sample;

4.2 Prepare the reverse transcription system on ice in strict accordance with the instructions of the cDNA reverse transcription kit;

Element volume 5×Mix 8μL RNA 500ng RNase-free H ₂ O to 40μL

4.3 Gently mix the above reaction system, and use a centrifuge to briefly centrifuge, 55 degrees for 20 minutes, 85 degrees for 2 minutes, to obtain the template cDNA;

(5) Ordinary PCR amplification

Configure the reaction system with Taq DNA polymerase, use primers a-i and human internal reference gene (B2M) to perform PCR amplification of each sample, and detect the product with 1% gel;

Amplification system

Element volume 2×Mix 10 μL cDNA 1μL Primer-F 0.8μL Primer-R 0.8μL RNase-free H ₂ O 7.4μL

The PCR reaction conditions were pre-denaturation at 95°C for 3 minutes, 1 cycle; denaturation at 95°C for 20 seconds, annealing at 55°C for 20 seconds, extension at 72°C for 10 seconds, 35 cycles; extension at 72°C for 7 minutes.

(6) Fluorescence real-time quantitative PCR amplification

According to the designed specific primer set a-i, amplify by fluorescent quantitative PCR, the system is as follows:

Pre-denaturation at 95°C for 20s, 1 cycle; 95°C for 10s, 60°C for 30s, 40 cycles; set up three replicate wells for each tube;

According to the pcr results of steps 5 and 6, the following primer sets were selected as the primer sets for detecting kidney cancer in the present invention.

Example 3: Effect Verification

According to the screening results in Example 2, 100 tumor samples were tested using the primer sets described in the table below.

Among them, the forward primer of CD10 is TGATGATAAGAATTCTGTGA, the reverse primer is GCAAGCTGGTTTTCATCGAT, the forward primer of N-cadherin is CCTTAACTGAGGAGTCAGTG, the reverse primer is CAGACCTGATCCTGACAAGC, the forward primer of Vimentin is CGTGACGTACGTCAGCAATA, and the reverse primer is AAGGGCATCCACTTCACAGG.

The detection rates of primer sets 1 to 8 are shown in the table above. It can be seen from the table that, in the primer set, as the number of primers increases, the detection rate can be improved to a certain extent. Further, by comparing primer sets No. 5 to No. 8, it can be found that the combination in the primer set has an important influence on the detection rate. At the same time, under the same number of detected genes, the detection rate of primer set No. 5 was higher than that of primer sets No. 6-8. Therefore, we prefer the gene combination of the No. 5 primer set to be used in the detection of renal cancer. Further research found that in the primer group No. 5, the expression of CK19 can indicate tumors derived from epithelial cells, which is considered to have diagnostic value for tumor metastasis; the expression of EPCAM can indicate malignant tumor cells of epithelial origin, and MN/CA9 is a carbonic anhydrase family TS is a key enzyme in DNA biosynthesis, which is closely related to the malignant biological behavior of tumors; CYP3A4 is the main member of the CYP3A subfamily and is also a member of adult The most important component of CYP450 in liver microsomes, CYP3A4 has been found to participate in the metabolism of more than 150 drugs (about 50% of all drugs) in about 38 categories, and has a guiding role in the treatment of tumors; activated T cells and tumors The interaction between related proteins PD-L1 leads to programmed cell death, and clinical studies have shown that PD-L1 expression is associated with poor prognosis and/or disease progression; VEGFR-1 interacts with vascular endothelial growth factor (VEGF) to affect tumor blood vessels and lymphatic vessels; cadherin-6 plays an important role in the growth and development of tissues, and is related to the invasion and metastasis of tumor cells; CD24 plays an important role in promoting tumor cell proliferation, adhesion and metastasis. associated with poor prognosis. It can be seen from the above that the present invention does not simply stack and combine 9 pairs of primers. The 9 pairs of primers complement each other and support each other in function, so that the detection rate is greatly improved and unexpected technical effects have been achieved. To sum up, the genes selected in this patent are related to tumor metabolism, proliferation, invasion, etc., and can comprehensively detect the cell biological characteristics of renal cancer.

Example 4: Specificity Analysis

According to the screening result of embodiment 2, adopt the primer set described in the following table to normal people's peripheral blood sample (Fig. 1), the peripheral blood (Fig. 2) of non-renal cancer patient and tissue sample (Fig. Peripheral blood (Figure 4) and tissue samples (Figure 5) were tested. The results are shown in Figures 1-5, and it can be seen from the figure that the described primer set is not detected in normal human peripheral blood (Fig. 1), non-kidney cancer patient's peripheral blood (Fig. 2) and tissue samples (Fig. 3) to stronger gene expression. In the peripheral blood (Fig. 4) and tissue samples (Fig. 5) of kidney cancer patients, the strong expression of multiple genes can be detected, which are 6/9 and 9/9 respectively, indicating that the primer set described in the present invention has a fairly high specificity.

Claims (3)

1. a kind of primer sets for detecting kidney are it is characterised in that described primer sets can comprise a～i 9 to primer, described The forward primer of primer a as shown in SEQ ID No.1, the reverse primer of primer a as shown in SEQ ID No.2, the forward direction of primer b Primer as shown in SEQ ID No.3, the reverse primer of primer b as shown in SEQ ID No.4, the forward primer such as SEQ of primer c Shown in ID No.5, the reverse primer of primer c as shown in SEQ ID No.6, the forward primer such as SEQ ID No.7 institute of primer d Show, the reverse primer of primer d as shown in SEQ ID No.8, the forward primer of primer e as shown in SEQ ID No.9, primer e's Reverse primer as shown in SEQ ID No.10, the forward primer of primer f as shown in SEQ ID No.11, the reverse primer of primer f As shown in SEQ ID No.12, the forward primer of primer g as shown in SEQ ID No.13, the reverse primer such as SEQ ID of primer g Shown in No.14, the forward primer of primer h as shown in SEQ ID No.15, the reverse primer such as SEQ ID No.16 institute of primer h Show, as shown in SEQ ID No.17, the reverse primer of primer i is as shown in SEQ ID No.18 for the forward primer of primer i.

2. a kind of PCR detection method for detecting kidney is it is characterised in that comprise the steps：

(1) 9 pairs of primers described in claim 1 are added separately in single PCR reaction tube, and all add nucleic acid extractive, Reverse transcriptase, RNase inhibitor, archaeal dna polymerase, dNTP；Wherein, PCR reaction each circulation condition be 94 degrees Celsius, 30s；55 degrees Celsius, 30s；72 degrees Celsius, 10s；

(2) carry out reverse transcription and PCR reaction, detected with agarose gel electrophoresis method.

3. a kind of fluorescent quantitative PCR detection method for detecting kidney is it is characterised in that comprise the steps：

(1) 9 pairs of primers described in claim 1 are added separately in single PCR reaction tube, and all add nucleic acid extractive, Reverse transcriptase, RNase inhibitor, archaeal dna polymerase, dNTP；It is separately added into 2 × ChamQ SYBR qPCR Master Mix, its In, the condition of each circulation of PCR reaction is 95 degrees Celsius, 10s and 60 degree Celsius, 30s；40 circulations；Often pipe sets up three Multiple holes；

(2) reverse transcription and PCR reaction are carried out, and real-time detection fluorescence；

(3) the Ct value being calculated according to fluoroscopic examination result, determines whether mark expression of target gene in sample.