CN104450887B - DNA probe, genetic chip and its application for detecting carcinoma of endometrium - Google Patents

Abstract

The invention belongs to biological technical field, a kind of DNA probe for detecting carcinoma of endometrium is disclosed.The DNA probe of detection carcinoma of endometrium of the present invention has the nucleotide sequence shown in sequence table SEQ ID No.1 to SEQ ID No.26, and it detects carcinoma of endometrium high specificity, and sensitivity is high.The present invention also provides the genetic chip for including the DNA probe.High flux, automation and the quick detection of the sample to carcinoma of endometrium may be suffered from, high specificity can be realized using genetic chip of the present invention, it is ensured that the accuracy of testing result, be with a wide range of applications.

Description

DNA probe, gene chip and application thereof for detecting endometrial cancer

technical field

The invention belongs to the field of biotechnology, and in particular relates to a DNA probe, a gene chip and an application thereof for detecting endometrial cancer.

Background technique

Endometrial cancer, also known as uterine body cancer, refers to a group of epithelial malignant tumors that originate in the endometrium, most of which originate in endometrial glands, and are called endometrial adenocarcinoma or endometrioid adenocarcinoma. It is one of the three most common malignant tumors of the female reproductive tract, accounting for 7% of female systemic malignant tumors and 20% to 30% of female reproductive tract malignant tumors. It is more common in elderly women. In recent years, the incidence rate has been increasing worldwide, second only to cervical cancer, but the 5-year survival rate has been decreasing year by year. The diagnosis and clinical staging of endometrial cancer are mainly based on the results of medical history, clinical examination, pathological examination and various auxiliary examinations. Currently, the main detection methods include B-ultrasound examination, diagnostic dilatation and curettage, hysteroscopy and lymphography. The detection rate is It is not high, and it is easy to cause pain to the patient. Currently, there are no effective early diagnosis and prognostic indicators for endometrial cancer.

Gene chip (Gene Chip) is to immobilize a large number of oligonucleotide molecules on a solid phase carrier to form a DNA microarray, and use the characteristics of nucleotide molecular hybridization pairing to efficiently interpret and analyze the sequence information of DNA samples.

The gene chip originated in the 1980s and has developed rapidly in recent years, but it is mainly used in the fields of medicine and research. It is the first time that it is used in the early detection of endometrial cancer in my country, which will greatly promote the development of endometrial cancer. The development of early diagnosis of cancer.

Contents of the invention

The invention provides a DNA probe for detecting endometrial cancer, a gene chip and applications thereof. The probe and the gene chip are used to detect endometrial cancer with good specificity, high sensitivity and high accuracy.

The DNA probe for detecting endometrial cancer of the present invention includes 27 gene fragments, referring to the sequences shown in SEQ ID No.1 to SEQ ID No.27 in the sequence listing, wherein the 27th gene fragment is a quality control gene. The 27 gene fragments are designed for 27 indicators of endometrial cancer, and the 27 gene fragments are used to detect changes in the gene expression profile of endometrial cancer, and then detect and monitor the prognosis of endometrial cancer. Table 1 shows the names of 27 gene fragments and the corresponding specific primers.

Table 1 27 gene fragments and their corresponding specific primers

The DNA probe of the present invention can be applied to biochip technology (including gene chip technology, protein chip technology, tissue chip technology, cell chip technology, etc.), polymerase chain reaction (PCR), nucleic acid molecular hybridization technology and other pathogen gene diagnosis technology.

Combining the gene chip technology, the present invention can realize the purpose of small sample, high throughput, specificity, rapidity and automatic detection of endometrium cancer.

The present invention provides a gene chip for endometrial cancer detection, which is formed by immobilizing specific oligonucleotide probes on the surface of a solid phase carrier, and the oligonucleotide probes comprise A DNA probe of the sequence shown in SEQ ID No.26.

Preferably, the solid phase carrier is selected from nitrocellulose membrane, nylon membrane, polystyrene, glass sheet, silicon sheet, microparticle or plastic sheet.

The solid phase carrier used in the gene chip of the present invention is selected from any material that can be used to prepare the gene chip, including but not limited to nitrocellulose membrane, nylon membrane, polypropylene, glass sheet, silicon chip, microparticles and plastic sheet. The solid-phase support has active groups on its surface that can immobilize nucleic acid molecules after various chemical modifications, and these groups include but are not limited to amino (-NH2), aldehyde (-CHO), carboxyl (- COOH) and mercapto (-SH).

The gene chip of the present invention is connected with specific nucleic acid probes for endometrial cancer on the solid phase carrier, and the probes can be single-stranded or double-stranded DNA molecules, which can be combined with endometrial cancer under appropriate conditions. Membrane oncogene-specific hybridization. The probes can be obtained by automatic DNA synthesis or PCR amplification.

Preferably, positive control probes and negative control probes are immobilized on the surface of the solid phase carrier.

Wherein, the positive control probes are human GAPDH and ACTIN gene fragments.

Negative control probes do not contain any gene sequence.

The present invention also provides a kit for detecting endometrial cancer, comprising: a probe specifically binding to endometrial cancer, said probe having the sequence listed in SEQ ID No.1-SEQ ID No.26 The nucleotide sequence shown. The kit of the present invention preferably further comprises primers for amplifying endometrial cancer genes.

The present invention also provides a method for detecting a sample that may suffer from endometrial cancer, comprising the following steps:

Step 1: extract and amplify the nucleic acid sequence of the sample, the amplification will molecular beacon to the amplification product;

Step 2: hybridize the amplified product obtained in step 1 with the probe immobilized on the gene chip;

Step 3: Scanning and analyzing the gene chip with a corresponding detection method according to the beacon molecule.

The probe labeling techniques that can be used in the gene chip include: radionuclide labeling, biotin labeling, digoxin labeling, fluorescein labeling, colloidal iron labeling, colloidal gold labeling, joint labeling and other available labeling techniques.

Preferably, the primer used in the amplification step of step 1 is 5'-AAG CAG TGG TAA CAA CGC AGA GT-3'.

The detection sample of the gene chip can be fresh tissue of endometrial cancer, and can also be exfoliated cells of the uterine cavity of patients with endometrial cancer.

In order to ensure the quality of the gene chip, the following items should be paid attention to:

1. Extraction of sample nucleic acid: the extracted nucleic acid must have high purity (OD ₂₆₀ /OD ₂₈₀ ratio should be greater than 1.50) and a certain amount (should be 10ng-100ng), to ensure the quality and specific amplification of the marker, If the purity is not enough, it may cause non-specific spots in the subsequent hybridization.

2. Labeling of sample nucleic acid: the incorporation of the marker must reach a certain concentration (0.1-1 μg of the incorporation in each reaction) to ensure the sensitivity of the hybridization point. If the concentration is not enough, the hybridization result will often appear false negative.

3. Homologous nucleic acid hybridization: The hybridization temperature should be controlled at about 42°C. If the temperature is too low, non-specific hybridization spots will appear, which will reduce the specificity. If the temperature is too high, false negatives will often appear.

The beneficial effects of the present invention are:

1. Good specificity: The existing diagnosis of endometrial cancer only detects one or several indicators and lacks specificity, so it is easy to cause misdiagnosis, especially the misdiagnosis rate of early endometrial cancer is as high as 70%. The DNA probe and gene chip used for detecting endometrium cancer can simultaneously detect 26 indicators of endometrium cancer pathogens, and the specificity is as high as 95%.

2. High sensitivity and accuracy: the sensitivity reaches 95%, and the clinical coincidence rate (accuracy) reaches 95%.

3. Rapid detection and prognosis monitoring of endometrial cancer.

4. It can be used for the diagnosis and prognosis monitoring of various types of endometrial cancer, especially for the diagnosis of early endometrial cancer.

5. The gene chip is equipped with positive and negative controls, which can avoid wrong diagnosis caused by environmental and human factors, and has a high accuracy rate.

6. Conventional detection takes one week, but the gene chip of the present invention only needs about 8 hours.

7. The sensitivity of the gene chip of the present invention is 0.1 pg, 1000 times higher than that of PCR technology.

8. Large amount of information: PCR only detects one indicator at a time, while the chip can detect 20 to 30 indicators at a time.

Description of drawings

The accompanying drawings are used to provide a further understanding of the present invention, and constitute a part of the description, and are used together with the embodiments of the present invention to explain the present invention, and do not constitute a limitation to the present invention. In the attached picture:

Fig. 1 is the pattern design drawing of gene chip;

Fig. 2 is the detection result of the gene chip (the normal sample detection result is in the left figure, and the tumor sample detection result is in the right figure);

Fig. 3 is the result of gene chip repeatability detection (each column in the figure detects different genes, and each row is the repetition of the same gene).

detailed description

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores.

Example 1

1. Materials and methods

1.1 Materials

1.1.1 Reagents

Main reagents RNA extraction reagent (Trizol Reagent), saturated phenol, DEPC, DNA marker, Oligod (T) 18 were purchased from Shanghai Sangong. Reverse transcriptase M-MLV (RNase H-), Taq enzyme, Agarose Gel, RNase inhibitor, and agarose (Agarose) were all purchased from Dalian Bao Biological Company. DNA purification kit was purchased from Shanghai Huashun Bioengineering Company; DIG DNA labeling kit and digoxin hybridization detection kit were purchased from Shenzhen Enuojin Biotechnology Co., Ltd.; primers were synthesized by Shanghai Sangon Bioengineering Company; others Reagents were all domestically produced analytically pure.

Preparation of pre-hybridization solution: (6×SSC, 5×Denhardt’s reagent, 0.5% SDS, 50% (v/v) formamide, ddH ₂ O, 100 μg/ml salmon essence is added after DNA denaturation. Note: per square centimeter Nylon membrane requires 0.2ml of pre-hybridization solution.

Preparation of hybridization solution: Add probes to the above-mentioned pre-hybridization solution, and the final concentration of probes is 25 ng/ml.

1.1.2 Clinical data

(1) Fresh tissue samples of endometrial cancer: The tissue samples were taken from the fresh endometrial cancer specimens surgically resected in the Second Clinical Hospital of Lanzhou University between October 2011 and April 2012, without chemotherapy or radiotherapy before. It was confirmed by pathology after operation, and the normal tissue (with no cancer cell invasion confirmed by pathology after operation) was excised from the edge of tumor 5-10cm at the same time as a control. The specimens were put into liquid nitrogen tanks for freezing within 5 minutes after they were separated from the body.

(2) Uterine cavity exfoliated cell samples of patients with endometrial cancer: The test samples were 100 samples of endometrial cancer and normal uterine cavity exfoliated cells from the Second Clinical Hospital of Lanzhou University in Gansu Province between April 2012 and October 2012 Samples for cell detection (including 50 samples of endometrial cancer) were compared and detected by occult blood and microarray methods, and stored at -80°C.

1.1.3 Instruments and equipment

Instruments and equipment: The PCR instrument was purchased from Hangzhou Langji; the spotter and hybridization instrument were purchased from Boao Biological Group Co., Ltd.; the scanner was manufactured by Xinkang Gene Digital Technology Co., Ltd.

2. Primer Design

Determine the sequences of several endometrial cancer tumor markers, and then use DNAStar analysis to design these primers. See Table 1 for specific primers.

1.3 Fabrication of gene chip

1.3.1 Extraction of endometrial cancer nucleic acid (fresh tissue samples of endometrial cancer)

(1) Take out 200 mg of frozen fresh tissue from liquid nitrogen, grind the tissue in a liquid nitrogen pre-cooled mortar, extract the total RNA of the tissue by Trizol method, determine the purity of total RNA and purify mRNA by Oligo dT cellulose chromatography.

(2) Use the reverse transcriptase M-MLV to synthesize the first strand of cDNA by reverse transcription, which is used to prepare the probe template.

1.3.2 Amplification of nucleic acid probes

(1) Using the nucleic acid obtained in 1.3.1 as a template, use the primers designed in Table 1 to amplify fragments respectively as probes; the specific sequences of the amplified fragments are SEQ ID No.1 to SEQ ID in the sequence table The sequence shown in No.27.

(2) PCR reaction system 50.0 μL, including 10×Buffer 5.0 μL, 25mmol dNTPmixture 0.5 μL, TaqDNase 1.0 μL, target primer upstream and downstream 1.0 μL, cDNA template 1.0 μL, triple distilled water 40.5 μL, reaction conditions: 94°C Pre-denaturation for 5 minutes; 30 cycles of denaturation at 94°C for 30s, annealing at 55°C for 30s, and extension at 72°C for 60s; terminate the reaction after 10 minutes at 72°C.

(3) Purify the PCR products of 27 gene fragments obtained by using the small fragment DNA purification kit.

The aforementioned nucleic acid probes can also be obtained by means of automatic DNA synthesis.

1.3.3 Point film

The obtained probes were spotted on the pretreated nylon membrane by a microspotter.

According to the chip pattern design diagram shown in Figure 1, use a manual sampler to spot the chip. Operating procedure: (1) Denature the spotting solutions of 27 genes such as GAPDH, ACTIN, CYCLIND1, etc. at 100°C for 5 minutes, put them on ice quickly, and keep them cold for more than 10 minutes. (2) Nylon membrane (purchased from Amersco, positively charged) was soaked in sterilized double-distilled water for 10 min, and hung to dry. (3) Spot the above-mentioned probe fragments on a nylon membrane (each nylon membrane has an area of about 20 cm ² ) with 1 μl per spot. (4) The wet film was exposed to ultraviolet light for 5 minutes, and then the film was taken out and allowed to dry naturally.

1.4 Processing of samples

1.4.1 Extraction of sample nucleic acid (exfoliated cell samples from uterine cavity of patients with endometrial cancer)

(1) Fresh tissue samples of endometrial cancer: Take out 200 mg of frozen fresh tissue from liquid nitrogen, grind the tissue in a liquid nitrogen precooled mortar, extract the total RNA of the tissue by the Trizol method, determine the purity of the total RNA and analyze it with Oligo dT The mRNA was purified by cellulose chromatography. Using primers 5'-AAG CAG TGG TAA CAA CGC AGA GTA CGC GGG-3' and 5'-AAGCAG TGG TAA CAA CGC AGA GTA CT ₍₃₀₎ n _-1 n-3, the first strand of cDNA was synthesized by reverse transcription.

(2) Trizol method was used to extract total RNA from uterine cavity exfoliated cells of patients with endometrial cancer, using primers 5'-AAGCAG TGG TAA CAA CGC AGA GTA CGC GGG-3' and 5'-AAG CAG TGG TAA CAA CGC AGA GTACT _{( 30)} n _-1 n-3' reverse transcription to synthesize the first strand of cDNA.

1.4.2 Amplification of sample nucleic acid

(1) Using the nucleic acid obtained in 1.4.1 as a template, use the designed universal primer 5'-AAG CAG TGG TAA CAACGC AGA GT-3 to amplify a gene fragment as a detection sample.

(2) PCR reaction system 25.0 μL, including 10×Buffer 2.5 μL, 10mmol dNTPmixture 0.5ul, digoxin-11-dUTP 0.5ul, TaqDNase 0.5μL, universal primer upstream and downstream 0.5μL, cDNA template 5.0μL, three 15 μL of distilled water, reaction conditions: pre-denaturation at 94°C for 5 min; 30 cycles of denaturation at 94°C for 30 s, annealing at 65°C for 30 s, and extension at 68°C for 500 s; the reaction was terminated after 10 min at 72°C, and the product did not need to be purified.

1.5 Utilize the probe of the present invention to carry out the detection of sample

Hybridization conditions and methods described in the present invention are known in the art. Specifically: Add 500 μl of prehybridization solution (6×SSC, 5×Denhardt’s reagent, 0.5% SDS, 50% (v/v) formamide, ddH ₂ O, 100 μg/ml salmon sperm DNA denatured) on the gene chip , 42°C for 40 minutes. Take 20 μl of the product of 1.4.2 in a 1.5ml centrifuge tube, denature at 100°C for 10 minutes, cool at -20°C for 5 minutes, add 450 μl of hybridization solution (probes are added to the above pre-hybridization solution, the final concentration of the probe is 25ng/ml), Mix well, hybridize on the gene chip at 42°C for 1h. Wash three times with 2×SSC, 0.1×SDS and 0.1×SSC, 0.1×SDS respectively, add 400 μl 3% BSA, block at 42°C for 30 minutes, add 1.6 μl AV-AP to 450 μl buffer, enzyme-link at 42°C for 20 minutes, wash with The membrane was washed three times with liquid solution, and then the color developing solution (450 μl substrate solution + 1.6 μl BCIP + 1.6 μl NBT) was added, and the color was developed at room temperature or 42°C for 3-5 minutes. Rinse with distilled or tap water to terminate color development.

1.6 Analysis of hybridization results

Scan and analyze the hybridized and dried DNA array with a chip scanner. According to the analysis of a large number of samples, the gray value of the negative control is less than 0.05, and the gray value of the positive control GAPDH is greater than 0.3. The value change judges the sample result.

Samples were simultaneously subjected to PCR or RT-PCR and other detection methods for comparison. See Table 2 for the results.

Table 2 applies gene chip of the present invention and fluorescent quantitative PCR to detect sample result simultaneously

Among them, the positive control is human GAPDH and ACTIN gene fragments; the negative control does not contain any gene sequence, and is added to the reaction system to monitor whether the labeling, hybridization, and color development systems are effective.

2 results

2.1 The results of the accuracy test

We specifically detected 27 genes such as GAPDH and ACTIN, and the hybridization results: 27 gene-specific hybridization points such as GAPDH and ACTIN appeared in 30 chips, and the detection results were shown in Figure 2, and there were no non-specific hybridization points. .

2.2 Repeatability test results

At the same time, 1 sample was taken for 3 repeatability tests. The result is shown in Figure 3.

2.3 Detection results of detection rate

Using the endometrial cancer gene diagnosis chip developed by us to detect the exfoliated cells of the uterine cavity of 50 patients, we found that 48 cases were detected as tumors, 1 case was suspicious, and 1 case was normal; the specificity reached 95%, and the sensitivity reached 95%. 95%, and the clinical compliance rate (accuracy rate) reaches 95%, basically meeting the design requirements.

Finally, it should be noted that: the above is only a preferred embodiment of the present invention, and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, for those skilled in the art, it still The technical solutions recorded in the foregoing embodiments may be modified, or some technical features thereof may be equivalently replaced. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

sequence listing

<110> Lanzhou Baiyuan Gene Technology Co., Ltd.

<120> DNA probes, gene chips and their applications for detecting endometrial cancer

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ggcgaagaga agagacacat tgttggaaga agcagcccat gacagctccc cttcctggga 60

ctcgccctca tcctcttcct gctccccttc ctggggtgca gcctaaaagg acctatgtcc 120

tcacaccatt gaaaccacta gttctgtccc cccaggagac ctggttgtgtgtgtgtgtgagt 180

ggttgacctt cct 193

<210> 26

<211> 188

<212>DNA

<213> HLA-E

<400> 26

cccacagaga gcctccacta gagtgatgct aagtggaaat gtgaggtgca gctgccacag 60

agggccccca ccagggaaat gtctagtgtc tagtggatcc aggccacagg agagagtgcc 120

ttgtggagcg ctgggagcag gacctgacca ccaccaggac cccagaactg tggagtcagt 180

ggcagcat 188

<210> 27

<211> 138

<212>DNA

<213> GAPDH

<400> 27

gcaccgtcaa ggctgagaac gggaagcttg tcatcaatgg aaatcccatc accatcttcc 60

aggagcgaga tccctccaaa atcaagtggg gcgatgctgg cgctgagtac gtcgtggagt 120

ccactggcgt cttcacca 138

Claims (7)

1. A DNA probe set for detecting endometrial cancer, characterized in that: said DNA probe set consists of sequences shown in SEQ ID No.1 to SEQ ID No.26. 2. A gene chip for endometrial cancer detection, formed by immobilizing oligonucleotide probes on the surface of a solid phase carrier, characterized in that, said oligonucleotide probes comprise the DNA probe sets. 3. The gene chip according to claim 2, wherein the oligonucleotide probes further comprise positive control probes and negative control probes. 4. The gene chip according to claim 3, wherein the positive control probe is human GAPDH, ACTIN gene fragment. 5. The gene chip according to claim 3, wherein the negative control does not contain any gene sequence. 6. A kit for detecting endometrial cancer, comprising the DNA probe set of claim 1. 7. The application of the DNA probe set according to claim 1, the gene chip according to any one of claims 2-5, and the kit according to claim 6 in the preparation of reagents for detecting endometrial cancer.