CN116694757A - Application of primer group for detecting colorectal cancer genes in sample in preparation of kit for detecting colorectal cancer recurrence risk - Google Patents
Application of primer group for detecting colorectal cancer genes in sample in preparation of kit for detecting colorectal cancer recurrence risk Download PDFInfo
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Abstract
The application belongs to the field of biological medicine, and relates to application of a primer group for detecting colorectal cancer genes in a sample in preparation of a kit for detecting colorectal cancer recurrence risk, which can be used for colorectal cancer 3 gene detection and can be used for obtaining recurrence risk prediction of colorectal cancer by calculating gene expression quantity. The kit is widely applicable, is applicable to scientific research and clinical detection, and can be used for single samples or multiple samples, and the detection result is rapid and accurate.
Description
Technical Field
The application belongs to the field of biological medicine, and relates to application of a primer group for detecting colorectal cancer genes in a sample in preparation of a kit for detecting colorectal cancer recurrence risk.
Background
Colorectal cancer is one of the most common malignant tumors, and the multidisciplinary comprehensive treatment mode taking surgery as a center and chemotherapy and radiotherapy as assistance is developed in a long term in recent 20 years, so that the recurrence and death rate of colorectal cancer are obviously reduced, but an effective method for accurately predicting the recurrence risk of patients and giving corresponding treatment is still lacking.
At present, a kit for multi-gene detection generally adopts a Taqman fluorescent PCR method and a SYBR Green method. The Taqman fluorescent PCR method needs to design a primer and a probe, the simple Taqman method only needs to design a pair of primers and a probe, the multiplex fluorescent PCR needs to design a plurality of pairs of primers and probes, and the primers and the probes of the multiplex PCR are ensured not to interfere with each other, so that the requirements on the primers and the probes are high, the design difficulty is high, and the experiment cost is high. In addition, multiplex PCR has certain requirements for both PCR buffers and QPCR instrumentation. In the SYBR Green method, only a pair of primers is needed to be designed, and SYBRGreen fluorescent dye is needed to be added during the reaction. The method has the greatest advantages of strong universality and suitability for all fluorescent quantitative PCR reactions. And by analyzing the melting curve of the PCR product, the problem of nonspecific amplification can be solved, thereby ensuring the specificity of fluorescent signals. Therefore, we need to find a multi-gene detection kit and method with strong specificity, high sensitivity, simple operation and accurate data.
Disclosure of Invention
The application mainly aims to provide the application of the primer group with strong specificity, high sensitivity, simple operation and accurate data in preparing the kit for detecting colorectal cancer recurrence risk in the detection sample.
In order to achieve the above object, according to one aspect of the present application, there is provided the use of a primer set for detecting colorectal cancer genes in a sample for preparing a kit for detecting a risk of recurrence of colorectal cancer,
wherein the primer sequences for detecting NMU genes are as follows: SEQ ID NO.1 and SEQ ID NO.2, the specific sequences are as follows:
NMU：
F：5‘-CTCAGGCATCCAACGCACT-3’(SEQ ID NO.1)
R：5‘-GACTTGCCCAACTTCTGTGTC-3’(SEQ ID NO.2)
(2) The primer sequences for detecting the DCBLD2 gene are as follows: SEQ ID NO.3 and SEQ ID NO.4, the specific sequences are as follows:
DCBLD2：
F：5‘-ATAATGGAATTGGAGTCAGCAG-3’(SEQ ID NO.3)
R：5‘-TTCCACTCATGAACAGCAATG-3’(SEQ ID NO.4)
(3) The primer sequences for detecting the ZFYVE27 gene are as follows: SEQ ID NO.5 and SEQ ID NO.6, the specific sequences are as follows:
ZFYVE27：
F：5‘-TGCCCTGCCTTTCTCAAGTA-3’(SEQ ID NO.5)
R：5‘-CCCAATTCCTCCTCATCCCA-3’(SEQ ID NO.6)
preferably, the kit comprises 3 primer premix solutions corresponding to 3 colorectal cancer genes respectively.
Preferably, the concentration of the primer set corresponding to detection of the same gene is diluted to 1 to 500. Mu.M.
Preferably, the 3 kinds of primer premix solutions are respectively filled in 3 QPCR reaction tubes.
Preferably, the kit comprises SYBR Green solution; the SYBR Green solution comprises antibody-containing mediated thermal initiation iTaq DNA polymerase, dNTP, mgCl2,Green I dye, enhancer, stabilizer and inert reference dye mixture.
The application relates to a detection method of a kit, which comprises the following steps:
S1、SYBR Green, cDNA and ddH of test sample 2 O is evenly mixed, and then is taken out and respectively added into 3 primer premix solutions for fluorescence quantitative PCR reaction;
s2, calculating through CT values obtained after fluorescent quantitative PCR amplification, modeling data, and predicting recurrence risk of colorectal cancer patients.
Preferably, in step S1, the cDNA of the sample to be tested is prepared by a method comprising the steps of: extracting RNA of colorectal cancer paraffin specimen, and reversely transcribing the extracted RNA into cDNA.
Preferably, in step S1, the reaction conditions of the fluorescent quantitative PCR reaction are: pre-denaturation at 95℃for 3min; denaturation at 95℃for 30 seconds, annealing at 56℃for 30 seconds, and extension at 72℃for 30 seconds, 40-45 cycles.
The kit and the detection method can be used for colorectal cancer 3 gene detection, and recurrence risk prediction of colorectal cancer is obtained by calculating the gene expression quantity. The original PCR data were first regularized with housekeeping genes. And sending the regularized data into a model, and screening out 3 genes for predicting the recurrence risk of colorectal cancer. The bioinformatics analysis method adopts a method for constructing a Cox proportion risk regression model based on Lasso regression screening variables.
The colorectal cancer recurrence risk assessment system is based on an RT-QPCR method, and 3 target gene expressions related to tumor in tumor tissues are detected through accurate calculation, namely colorectal cancer 3 gene detection can be used for effectively predicting recurrence risk of colorectal cancer patients in stage II and stage III. The colorectal cancer 3 gene contains 3 colorectal cancer-related genes (NMU, DCDLB, ZFYVE).
The 3 gene detection needs to extract RNA from paraffin embedded slices, reverse transcribe the RNA into cDNA, and then accurately detect the 3 gene by a gene amplification technology.
The beneficial effects of the application are as follows:
1. the kit adopts a SYBR Green method, and the SYBR Green method only needs to design a pair of primers, and SYBR Green fluorescent dye is added during the reaction. The method has the greatest advantages of strong universality and suitability for all fluorescent quantitative PCR reactions. And by analyzing the melting curve of the PCR product, the problem of nonspecific amplification can be solved, thereby ensuring the specificity of fluorescent signals.
2. Because the RNA of the paraffin embedded sample is partially degraded, 1 pair of primers is designed for each of 3 genes in the kit, and the size of each 1 pair of primers is smaller than 200bp, so that the amplification efficiency of QPCR after reverse transcription of degraded RNA is improved;
3. the 1 group of primers of each gene in the kit are placed in a reaction tube, and only one buffer solution is needed in the QPCR amplification process, so that the experimental operation is simplified; after amplification, each gene can see 1 amplification curve on QPCR to obtain clear and accurate CT value, so that the calculation result is more accurate and reliable;
4. the annealing temperature of the primer related to the kit is about 56 ℃, so that 3 genes can be amplified by using the same procedure; by adding a dissolution curve, the PCR result can be directly observed;
5. the kit is widely applicable, is applicable to scientific research and clinical detection, and can be used for single samples or multiple samples, and the detection result is rapid and accurate.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the application. In the drawings:
fig. 1:3 gene QPCR amplification plots in one embodiment;
fig. 2: statistical graphs of CT values of 3 genes in one example in different colorectal cancer stages; detecting CT values of all genes of the sample A and calculating recurrence risk scores according to the model;
fig. 3: a comparison of the patient survival profile of colorectal cancer stage II recurrence risk in one embodiment with the patient survival profile of the prior art;
fig. 4: the patient survival profile for colorectal cancer stage III relapse risk in one embodiment is compared to the patient survival profile of the prior art.
Detailed Description
The application is described in further detail below with reference to the drawings and examples. It is to be understood that the specific embodiments described herein are merely illustrative of the application and are not limiting thereof. It should be further noted that, for convenience of description, only some, but not all of the test results related to the present application are shown in the drawings.
The test method comprises the following steps:
extracting RNA of paraffin samples;
extracting RNA of the colorectal cancer sample to obtain a sample A to be detected;
1. placing colorectal cancer samples into a 1.5ml centrifuge tube, adding 300-500 mu l of PKD and 10-30 mu l of proteinase K, mixing the samples uniformly in a reverse manner, then placing the samples into a vibrating metal bath at 50-60 ℃ and 1000-2000rpm, and incubating for 2 hours;
2. taking down 1.5ml centrifuge tube in oscillating metal bath, adjusting the temperature of the oscillating metal bath to 70-90 ℃, putting 1.5ml centrifuge tube into the oscillating metal bath when the temperature is heated, and incubating for 15-20min;
3. placing 1.5ml centrifuge tube in a refrigerator at-20deg.C for 3-5min;
4. taking out, placing into a 12700rpm centrifuge, and centrifuging for 15-20min;
5. preparing a 2ml centrifuge tube, and transferring supernatant in the centrifuge tube of which the volume is 1.5ml after centrifugation into the 2ml centrifuge tube;
6. then 20-50 mu l of DNase Booster Buffer equivalent solution and 5-20 mu l of DNase I stock solution are respectively added, and the mixture is left at room temperature for 15min;
7. adding 300-400 mu l of RBC and 800-1200 mu l of absolute ethyl alcohol, and mixing the materials reversely;
8. the above mixture was transferred into RNeasy MinElute spin columns 700-800. Mu.l each time, and centrifuged at 10000rpm for 30s, and the centrifuged liquid was discarded. Until the mixture is completely transferred to RNeasy MinElute spin column;
9. adding 400-600 μl of RPE solution into RNeasy MinElute spin column, centrifuging at 10000rpm for 30s, discarding the centrifuged liquid, and repeating for 1 time;
10. placing RNeasy MinElute spin column into clean collecting tube, and centrifuging at 12000rpm for 5min;
11. putting RNeasy MinElute spin column into new 1.5ml centrifuge tube, adding proper amount of RNase-free water, and centrifuging at 12000rpm for 1min to obtain eluted RNA solution;
12. detecting the concentration of the RNA solution, 260/280 and 260/230;
the extracted RNA was reverse transcribed into cDNA.
Dnase master mix preparation: according to DNase: DNase solution = 1:3 to prepare a DNase master mix, 2. Mu.l of DNase master mix was mixed with 14. Mu.l of RNA template (total RNA 1pg-1 ug)
2. Mixing by shaking, and centrifuging briefly, and reacting according to the following procedures:
| step (a) | Temperature (. Degree. C.) | Time |
| DNA digestion | 25 | 5min |
| DNase inactivation | 75 | 5min |
| Storage device | 4 | To room temperature |
Preparing cDNA synthesis mixed solution:
4. reverse transcription reaction procedure:
| step (a) | Temperature (temperature) | Time |
| Start-up | 25 | 5 |
| Reverse transcription | 46 | 40 |
| Reverse transcription inactivation | 95 | 1 |
| Storage of | 4 | - |
a. Preparing primer premix:
designing primer sequences according to mRNA sequences of 3 genes, wherein target fragments are smaller than 100bp, and the specific sequences are as follows:
NMU：
F：5‘-CTCAGGCATCCAACGCACT-3’(SEQ ID NO.1)
R：5‘-GACTTGCCCAACTTCTGTGTC-3’(SEQ ID NO.2)
DCBLD2：
F：5‘-ATAATGGAATTGGAGTCAGCAG-3’(SEQ ID NO.3)
R：5‘-TTCCACTCATGAACAGCAATG-3’(SEQ ID NO.4)
ZFYVE27：
F：5‘-TGCCCTGCCTTTCTCAAGTA-3’(SEQ ID NO.5)
R：5‘-CCCAATTCCTCCTCATCCCA-3’(SEQ ID NO.6)
b. diluting the concentration of 3 pairs of primers to 1-500 mu M;
c. 1 pair of primers F and R for each of the 3 genes were subjected to 1:1, mixing, taking out 1u1, adding to the bottoms of the 3 QPCR reaction tubes, covering a cover, centrifuging, placing into an aluminum foil plastic package bag, taking out when in use, and preserving the other materials at-20 ℃.
Preparing a reaction solution:
reaction system (10 μl)
| Reagent name | Dosage (Single, μl) |
| SYBR Green | 5 |
| Primers-F(1-500μM) | 0.5 |
| Primers-R(1-500μM) | 0.5 |
| cDNA template | 2 |
| Water and its preparation method | 2 |
4.1, directly taking SYBR Green and primer premix out of the kit, and centrifuging;
4.2, taking out the cDNA of the sample to be tested after reverse transcription;
4.3 calculating the number of reactions to be detected, and taking out the SYBR Green and ddH in the corresponding amounts according to the reaction system 2 O and cDNA of the sample to be tested, then fully and uniformly mixing, centrifuging, and subpackaging 9 mu l of each reaction tube;
4.4, covering the reaction tube with a cover, which is necessarily closed (preventing evaporation in the PCR process), and centrifuging for several seconds;
4.5 opening the fluorescent quantitative PCR instrument and placing the added reaction tube on the fluorescent quantitative PCR instrument.
Amplification procedure
qPCR detection results
The risk assessment value is calculated by the amplification curve obtained after fluorescent quantitative PCR amplification and the obtained CT value.
The amplification curve of the results is shown in FIG. 1.
The CT values (cycle numbers) in the different samples are shown in fig. 2, "II/no" for the non-recurrent sample of colorectal cancer of stage II, "II/there" for the recurrent sample of colorectal cancer of stage II, "III/no" for the non-recurrent sample of colorectal cancer of stage III, "III/there" for the recurrent sample of colorectal cancer of stage III. The 3 genes are genes with strong correlation with colorectal cancer progress time, and have better colorectal cancer risk prediction effects on different periods.
The results of colorectal cancer stage II and stage III recurrence prediction using the primers of the present application were compared with those of the OncotypeDx (Genomic Health Inc company) product of the prior art, and a survival graph (also called Kaplan-Meier curve) commonly used in clinical studies was made. The results show that the primers of the application are significantly better than the prior art primers (e.g., FIGS. 3, 4) when used for low, medium and high risk prediction, both in phase II and in phase III results.
It will be understood by those skilled in the art that the present application is not limited to the embodiments described herein, but is capable of various obvious changes, rearrangements and substitutions as will now become apparent to those skilled in the art without departing from the scope of the application. Therefore, while the application has been described in connection with the above embodiments, the application is not limited to the embodiments, but may be embodied in many other equivalent forms without departing from the spirit or scope of the application, which is set forth in the following claims.
Sequence listing
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<120> application of primer set for detecting colorectal cancer gene in sample in preparation of kit for detecting colorectal cancer recurrence risk
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Claims (8)
1. The application of a primer group for detecting colorectal cancer genes in a sample in preparing a kit for detecting colorectal cancer recurrence risk,
wherein the method comprises the steps of
a. The primer sequences for detecting NMU genes are as follows: SEQ ID NO.1 and SEQ ID NO.2, the specific sequences are as follows:
NMU：
F：5‘-CTCAGGCATCCAACGCACT-3’(SEQ ID NO.1)
R：5‘-GACTTGCCCAACTTCTGTGTC-3’(SEQ ID NO.2)
b. the primer sequences for detecting the DCBLD2 gene are as follows: SEQ ID NO.3 and SEQ ID NO.4, the specific sequences are as follows:
DCBLD2：
F：5‘-ATAATGGAATTGGAGTCAGCAG-3’(SEQ ID NO.3)
R：5‘-TTCCACTCATGAACAGCAATG-3’(SEQ ID NO.4)
c. the primer sequences for detecting the ZFYVE27 gene are as follows: SEQ ID NO.5 and SEQ ID NO.6, the specific sequences are as follows:
ZFYVE27：
F：5‘-TGCCCTGCCTTTCTCAAGTA-3’(SEQ ID NO.5)
R：5‘-CCCAATTCCTCCTCATCCCA-3’(SEQ ID NO.6)
2. the use according to claim 1, wherein the kit comprises 3 primer premixes corresponding to 3 genes of colorectal cancer respectively.
3. The use according to claim 2, wherein the concentration of the primer set corresponding to the detection of the same gene is diluted to 1-500 μm.
4. The use according to claim 2, wherein the 3 primer premixes are contained in 3 QPCR reaction tubes, respectively.
5. The use according to claim 1, wherein the kit comprises a SYBR Green solution; the SYBR Green solution comprises antibody-containing mediated thermal initiation iTaq DNA polymerase, dNTP, mgCl2,Green I dye, enhancer, stabilizer and inert reference dye mixture.
6. The method for detecting a kit according to any one of claims 1 to 5, wherein the method for detecting comprises:
s1, SYBR Green, cDNA and ddH of a sample to be tested 2 O is evenly mixed, and then is taken out and respectively added into 3 primer premix solutions for fluorescence quantitative PCR reaction;
s2, calculating through CT values obtained after fluorescent quantitative PCR amplification, modeling data, and predicting recurrence risk of colorectal cancer patients.
7. The method for detecting a kit according to claim 6, wherein the method for detecting comprises:
in step S1, the cDNA of the sample to be tested is prepared by a method comprising the steps of: extracting RNA of colorectal cancer paraffin specimen, and reversely transcribing the extracted RNA into cDNA.
8. The method of claim 7, wherein the method of detecting comprises:
in step S1, the reaction conditions of the fluorescent quantitative PCR reaction are: pre-denaturation at 95℃for 3min; denaturation at 95℃for 30 seconds, annealing at 56℃for 30 seconds, and extension at 72℃for 30 seconds, 40-45 cycles.
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