CN112708677A - Primer group, kit and method for detecting head and neck tumor gene survival correlation - Google Patents
Primer group, kit and method for detecting head and neck tumor gene survival correlation Download PDFInfo
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- CN112708677A CN112708677A CN202110115509.0A CN202110115509A CN112708677A CN 112708677 A CN112708677 A CN 112708677A CN 202110115509 A CN202110115509 A CN 202110115509A CN 112708677 A CN112708677 A CN 112708677A
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Abstract
The invention discloses a primer group, a kit and a method for detecting head and neck tumor gene survival correlation, wherein the primer group at least comprises nucleotide sequences of two groups of primers, and a plurality of genes are preferably CDKN 2A. According to the invention, when the situation that CDKN2A is highly amplified is detected, the patient can be recommended to receive CDKN2A inhibitor drug treatment, so that the patient suitable for CDKN2A targeted therapy benefits; if the CDKN2A is not highly amplified, other treatment drugs should be considered in time, so that the potential toxic effect and unnecessary economic burden of the drugs are avoided, the optimal treatment opportunity is missed, and the good guiding effect is achieved.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a primer group, a kit and a method for detecting head and neck tumor gene survival correlation.
Background
According to medical statistics, the occurrence of tumors is not random, and a plurality of cancers such as breast cancer, colorectal cancer, prostate cancer, gastric cancer and the like have remarkable familial aggregation. The activation of protooncogenes and the inactivating mutation of cancer suppressor genes play a central biological role in the canceration process. The major risk factors for human cancer are environmental factors, and genetic polymorphisms of the genes associated therewith determine the susceptibility of an individual to these factors. The intensity of tumor susceptibility increases with the increase of patients with tumors in the family and the decrease of the age of onset, and the intensity of the susceptibility increases the risk of cancer for the family members as the family gene polymorphism is inherited.
Genetic factors are classified as Copy Number Aberrations (CNA), non-synonymous mutations. Through counting the occurrence frequency of the mutant genes in the sample, the occurrence frequency of the mutant genes of non-synonymous mutation types in the sample is found to be too low, and the research significance is not great, so that in the research on the influence of genetic factors on the disease-free survival period and the overall survival period of head and neck tumor patients, only the influence of copy number aberration on the overall survival period of the tumor patients is researched.
The personalized targeted therapy of head and neck tumor patients by using CDKN2A inhibitor firstly needs to screen those patients with CDKN2A gene amplification in cancer cells. Therefore, it is necessary to develop a gene diagnosis kit for determining the pathological factors of cancer and making an effective treatment scheme by detecting the amplification of CDKN2A in tumor tissues. If there is a high amplification of CDKN2A, it may be recommended that the patient receive CDKN2A inhibitor drug therapy to benefit patients eligible for CDKN2A targeted therapy; if there is no high amplification of CDKN2a1, other therapeutic drugs should be considered in time to avoid subjecting the patient to the potentially toxic effects of the drug and unnecessary economic burden, and missing the optimal opportunity for treatment.
Disclosure of Invention
In order to solve the technical problems, the invention provides a primer group, a kit and a method for detecting the survival correlation of head and neck tumor genes, which analyze the influence of eIF3A gene amplification on the non-progressive life cycle of head and neck tumors and discover a plurality of gene markers with high information and capable of being used for detecting the life cycle of head and neck tumor patients.
A primer set for simultaneously amplifying a plurality of genes, comprising: the primer group at least comprises nucleotide sequences of two groups of primers, and the nucleotide sequences of the two groups of primers are respectively as follows: the front primer is as follows: 5-tacacgtatatatatatatacacgtatatata-3 and the rear primer: 5-gtatctccccagagatattctatgtata-3, preferably the plurality of genes are CDKN 2A.
The invention also provides a kit for directly amplifying and detecting the head and neck tumor gene survival correlation, which comprises the primer group and a fluorescent probe.
The invention also provides a detection method for detecting head and neck tumor gene survival correlation by direct amplification, which comprises the following steps: the kit as described above was used.
Preferably, the method uses PRC amplification and the detection method comprises the steps of:
s1: taking out the kit, balancing to room temperature, fully dissolving all components, and quickly centrifuging for 10 s;
s2: the number of reactions (n) required for the current experiment was counted, and each reaction solution was dispensed into n reaction tubes at a dispensing rate of 23. mu.L/well:
n ═ sample number + blank control + weak positive control
S3: extracting DNA for sample adding treatment, and adding the genome DNA, the weak positive control and the blank control of a sample to be detected into a reaction tube filled with 2 PCR reaction mixed liquids respectively, namely detecting each sample to be detected by using the 2 PCR reaction mixed liquids respectively, wherein the adding amount is 2 mu L/hole; the DNA concentration of the sample to be detected is 10-25 ng/mu L;
s4: covering the PCR reaction tube cover, recording the sample adding condition, sending the sample to a machine for detection, and if the sample cannot be immediately loaded on the machine, placing the PCR reaction tube with the template at 4 ℃ and loading the PCR reaction tube as soon as possible within 24 hours;
s5: the pre-amplification reaction procedure of PCR amplification is as follows: the first step is as follows: at 37 ℃ for 10 min; the second step is as follows: 95 ℃ for 5 min;
s6: the PCR amplification and dissolution reaction program is as follows:
circulating for 40 times at 95 ℃ for 15 s;
circulating for 40 times at 62 deg.C for 1 min;
hold, cycle 40 times at 4 ℃.
Compared with the prior art, the invention has the following beneficial effects:
(1) by the detection of the CDKN2A gene, if the detection result is the high amplification condition of CDKN2A, the patient can be recommended to receive CDKN2A inhibitor drug treatment, so that the patient suitable for CDKN2A targeted treatment benefits; if the CDKN2A is not highly amplified, other treatment drugs should be considered in time, so that the potential toxic effect and unnecessary economic burden of the drugs are avoided, the optimal treatment opportunity is missed, and the good guiding effect is achieved.
Drawings
FIG. 1 is a schematic diagram showing quality control of the CDKN2A gene of the present invention without amplification.
FIG. 2 is a schematic diagram showing quality control in amplification of CDKN2A gene of the present invention.
FIG. 3 is a schematic diagram of covariate coding of SPSS gene CDKN2A according to the present invention.
FIG. 4 is a diagram illustrating the final result of Cox regression according to the present invention.
FIG. 5 is a summary of mutant genes with a frequency of more than 10% that has a significant effect on overall survival in the prior art.
Detailed Description
The invention is further described with reference to the following drawings and detailed description.
The invention comprises the following steps:
s1: taking out the kit, balancing to room temperature, fully dissolving all components, and quickly centrifuging for 10 s;
s2: the number of reactions (n) required for the current experiment was counted, and each reaction solution was dispensed into n reaction tubes at a dispensing rate of 23. mu.L/well:
n ═ sample number + blank control + weak positive control
S3: extracting DNA for sample adding treatment, and adding the genome DNA, the weak positive control and the blank control of a sample to be detected into a reaction tube filled with 2 PCR reaction mixed liquids respectively, namely detecting each sample to be detected by using the 2 PCR reaction mixed liquids respectively, wherein the adding amount is 2 mu L/hole; the DNA concentration of the sample to be detected is 10-25 ng/mu L;
s4: covering the PCR reaction tube cover, recording the sample adding condition, sending the sample to a machine for detection, and if the sample cannot be immediately loaded on the machine, placing the PCR reaction tube with the template at 4 ℃ and loading the PCR reaction tube as soon as possible within 24 hours;
s5: the pre-amplification reaction procedure of PCR amplification is as follows: the first step is as follows: at 37 ℃ for 10 min; the second step is as follows: 95 ℃ for 5 min;
s6: the PCR amplification and dissolution reaction program is as follows:
circulating for 40 times at 95 ℃ for 15 s;
circulating for 40 times at 62 deg.C for 1 min;
hold, cycle 40 times at 4 ℃.
A primer set for simultaneously amplifying a plurality of genes, the primer set at least comprises nucleotide sequences of two groups of primers, and the nucleotide sequences of the two groups of primers are respectively as follows: the front primer is as follows: 5-tacacgtatatatatatatacacgtatatata-3 and the rear primer: 5-gtatctccccagagatattctatgtata-3, preferably CDKN2A
As shown in fig. 4, Cox regression was used to investigate the effect of 7 mutant genes on disease-free survival, and it was found that two genes CDKN2A — DeepDel were the genes actually playing a major role in influencing overall survival among the 7 mutant genes. As shown in FIG. 3, the data was re-encoded by COX regression, FIG. 4 shows the result of Cox regression, and from FIG. 4, the regression coefficient for MIR3664_ AMP is-0.564 and the regression coefficient for CDKN2A _ DeepDel is-0.335.
From the coding rule of fig. 3, the regression result of Cox can be interpreted that, without MIR3664_ AMP and CDKN2A _ DeepDel genes, the overall survival of the patient population is prolonged, i.e. both mutant genes play a role in shortening the overall survival of the patient population, so that a detection method and a kit of CDKN2A are provided, and if the detection result shows that the CDKN2A is highly amplified, the patient can be recommended to receive the CDKN2A inhibitor drug treatment, so that the patient suitable for the CDKN2A targeted therapy benefits; if the CDKN2A is not highly amplified, other treatment drugs should be considered in time, so that the potential toxic effect and unnecessary economic burden of the drugs are avoided, the optimal treatment opportunity is missed, and the good guiding effect is achieved.
The above-mentioned embodiments are only preferred embodiments of the present invention, and the scope of the present invention is not limited thereto, and therefore, modifications, equivalent changes, improvements, etc. made in the claims of the present invention are still included in the scope of the present invention.
Claims (4)
1. A primer set for simultaneously amplifying a plurality of genes, comprising: the primer group at least comprises nucleotide sequences of two groups of primers, and the nucleotide sequences of the two groups of primers are respectively as follows: the front primer is as follows: 5-tacacgtatatatatatatacacgtatatata-3 and the rear primer: 5-gtatctccccagagatattctatgtata-3, preferably the plurality of genes are CDKN 2A.
2. A kit for detecting head and neck tumor gene survival correlation is characterized in that: the primer set of claim 1, further comprising a fluorescently labeled probe.
3. A detection method for detecting head and neck tumor gene survival correlation is characterized in that: a kit according to claim 2 is used.
4. The method for detecting the survival correlation of the head and neck tumor by direct amplification according to claim 3, wherein the method comprises the following steps: the method uses PRC amplification, and the detection method comprises the following steps:
s1: taking out the kit, balancing to room temperature, fully dissolving all components, and quickly centrifuging for 10 s;
s2: the number of reactions (n) required for the current experiment was counted, and each reaction solution was dispensed into n reaction tubes at a dispensing rate of 23. mu.L/well:
n ═ sample number + blank control + weak positive control
S3: extracting DNA for sample adding treatment, and adding the genome DNA, the weak positive control and the blank control of a sample to be detected into a reaction tube filled with 2 PCR reaction mixed liquids respectively, namely detecting each sample to be detected by using the 2 PCR reaction mixed liquids respectively, wherein the adding amount is 2 mu L/hole; the DNA concentration of the sample to be detected is 10-25 ng/mu L;
s4: covering the PCR reaction tube cover, recording the sample adding condition, sending the sample to a machine for detection, and if the sample cannot be immediately loaded on the machine, placing the PCR reaction tube with the template at 4 ℃ and loading the PCR reaction tube as soon as possible within 24 hours;
s5: the pre-amplification reaction procedure of PCR amplification is as follows: the first step is as follows: at 37 ℃ for 10 min; the second step is as follows: 95 ℃ for 5 min;
s6: the PCR amplification and dissolution reaction program is as follows:
circulating for 40 times at 95 ℃ for 15 s;
circulating for 40 times at 62 deg.C for 1 min;
hold, cycle 40 times at 4 ℃.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103031369A (en) * | 2011-09-30 | 2013-04-10 | 广州益善生物技术有限公司 | CDKN2A gene mutation detection specific primer and liquid chip |
| CN105671187A (en) * | 2016-04-08 | 2016-06-15 | 南方医科大学 | Set of genes for head and neck squamous cell carcinoma (HNSCC) molecular typing and application thereof |
| CN105779465A (en) * | 2016-04-15 | 2016-07-20 | 广东医学院 | CDKN2A gene fragment and application of primers of CDKN2A gene fragment in diagnosing tumors |
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- 2021-01-28 CN CN202110115509.0A patent/CN112708677A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103031369A (en) * | 2011-09-30 | 2013-04-10 | 广州益善生物技术有限公司 | CDKN2A gene mutation detection specific primer and liquid chip |
| CN105671187A (en) * | 2016-04-08 | 2016-06-15 | 南方医科大学 | Set of genes for head and neck squamous cell carcinoma (HNSCC) molecular typing and application thereof |
| CN105779465A (en) * | 2016-04-15 | 2016-07-20 | 广东医学院 | CDKN2A gene fragment and application of primers of CDKN2A gene fragment in diagnosing tumors |
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