CN115232873A - Detection composition and kit for pan-cancer chemotherapeutics guidance and application thereof - Google Patents
Detection composition and kit for pan-cancer chemotherapeutics guidance and application thereof Download PDFInfo
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Abstract
The application relates to the technical field of gene detection, and particularly provides a composition and a kit for guiding detection of pan-cancer chemotherapy medication. The kit has the advantages of high accuracy, strong specificity, high sensitivity, high flux and the like, also has the advantages of sample saving, short detection period, simplicity in operation, convenience in analysis and the like, and can provide guiding significance for individualized treatment of cancer patients.
Description
Technical Field
The application relates to the technical field of gene detection, in particular to a detection composition and a kit for pan-cancer chemotherapy medication guidance and application thereof.
Background
The main treatment methods of malignant tumors include surgery, radiotherapy, chemotherapy and molecular targeted drug therapy, wherein the surgery refers to a method of surgery to remove cancerous tissues, repair injuries, transplant organs and the like, the radiotherapy refers to a method of indirectly or directly damaging cell DNA by using special radioactive rays, and the chemotherapy refers to a treatment method of inhibiting the growth and reproduction of tumor cells, killing the tumor cells or promoting the differentiation of the tumor cells by using chemical drugs. Chemotherapy currently accounts for about 70% of clinical treatments for tumors, among several approaches, and is the most important and even the only treatment, especially for patients with advanced cancers who have lost the therapeutic interest of surgical treatment. Although there have been great advances in the field of tumor biological research and therapy in recent years, the current clinical use of chemotherapeutic drugs has significant limitations. Most of traditional chemotherapy drugs have great toxicity to normal cells while inhibiting tumor cells, and have serious side effects because the drugs usually have pharmacological effects after entering human bodies through processes of absorption, transportation, metabolism, receptor binding (or binding with target enzymes) and the like, if gene mutation causes differences of functions of related metabolic enzymes, receptors, transporters and the like in the drug metabolism process, and accumulation of the differences finally enables drug effects (therapeutic effect/adverse effect) to be diversified, namely individual differences, so that different patients use the same chemotherapy drug, and the effects and the side effects are different. Therefore, the definition of the individual drug gene types can help clinicians to specifically make treatment strategies, give patients the optimal treatment scheme, reduce a plurality of meaningless treatment time, reduce treatment cost, improve generation quality and prolong life cycle, thereby realizing accurate medication.
The drugs commonly used in chemotherapy comprise two types of anti-tumor drugs and anti-metabolism drugs, mainly comprising cypress, 5-FU/capecitabine, taxoids, anthracyclines, methotrexate, pemetrexed, letrozole/anastrozole, vinblastine, gemcitabine, cyclophosphamide/ifosfamide, irinotecan, etoposide, tamoxifen, azathioprine/mercaptopurine/thioguanine and the like. There are many genes associated with drug sensitivity, each of which is involved in the metabolic processes of one or more chemotherapeutic drugs, for example, studies have shown that in ovarian cancer patients receiving platinum and taxus chemotherapy, the risk of blood toxicity is higher when the GSTP1 (rs 1695) genotype is AA compared to the GG/AG genotype; compared with GG genotype, GSTP1 (rs 1695) genotype is AA, the drug response rate is higher and the drug toxicity risk is reduced in breast cancer patients receiving epirubicin and cyclophosphamide chemotherapy. Compared with GG/AG genotype, MTHFR (rs 1801133) genotype is AA, the medicine response rate is higher in the patients with the advanced non-small cell lung cancer receiving the platinum-based chemotherapy; in patients with Burkitt's lymphoma and T cell precursor cell acute lymphoblastic leukemia who received methotrexate chemotherapy, the risk of drug toxicity was higher when MTHFR (rs 1801133) genotype was AA compared to GG/AG genotype. Therefore, the SNP characteristics of the above-mentioned gene loci of patients are detected before the chemotherapy drugs are used, and then different individualized chemotherapy schemes are adopted according to different drug genotypes: conventional dose chemotherapy may be used for individuals with such sensitive sites; for other combination types, if the curative effect is not obvious, the treatment can be carried out by increasing the dose of the chemotherapeutic drugs or selecting other chemotherapeutic drugs, thereby achieving the purpose of effective treatment.
At present, the domestic adopted general cancer chemotherapy medication guide gene SNP locus detection technology mainly comprises the following steps: the DNA sequencing method comprises a direct sequencing method and a pyrosequencing method, wherein the former method has low flux and large sample consumption, the average detection of each site needs 100ng, and the latter method has low accuracy on a plurality of single base repeat sequences; a restriction enzyme fragment length polymorphism (RFLP) method, which is used for detecting genes with changed restriction enzyme cutting sites, can directly judge genotypes, but cannot be used for detecting genes without generating new restriction enzyme cutting sites, so that the polymorphism of a part of DNA can be detected, and the method has the advantages of complex experimental operation, long detection period, high cost, lower flux and difficulty in meeting the requirements of clinical detection; the fluorescence quantitative PCR method has high sensitivity, strong specificity and strong automation degree, but can not detect unknown mutation and the price of the probe is high; the single-strand conformation polymorphism (SSCP) method is time-consuming, labor-consuming and difficult to realize automation, and the secondary structure of a DNA chain is easy to cause artificial false positive, so that the result is deviated; the denaturing high performance liquid chromatography (dHPLC) method has expensive equipment and complicated operation; unknown mutations are difficult to detect by both gene chip methods and electrophoretic analysis methods; the in situ hybridization method has too low flux, and is large in workload when a large amount of SNP is typed, and is only suitable for partial SNP typing.
The existing detection method can be really applied to accurate detection of pan-cancer chemotherapeutic drug genomes effectively at low cost, and how to provide a detection means which is convenient and quick in comparison, short in period, strong in pertinence and accurate and reliable in detection result becomes a problem to be solved in the industry.
In view of this, the present application is presented.
Disclosure of Invention
The first purpose of the application is to provide a primer group for the drug-guided detection of pan-cancer chemotherapy.
The second purpose of the application is to provide a detection kit for the drug administration guidance of pan-cancer chemotherapy.
The third purpose of the application is to provide the application of the primer group or the kit in the drug guidance of pan-cancer chemotherapy.
The fourth purpose of the application is to provide a detection method of pan-cancer chemotherapy medication guide.
In order to achieve the above object, the present application specifically provides the following technical solutions:
the application firstly provides a primer group for guiding and detecting the chemotherapy medication of pan-cancer species based on a flight time mass spectrum platform, wherein the primer group is directed at the following genes:
SOD2、MTHFR、DYNC2H1、NQO1、UMPS、ESR1、TYMS、XPC、CYP2C19、DHFR、DPYD、GSTP1、 GGH、CYP19A1、CYP2D6、LRMDA、CBR3、CYP2C8、XRCC1、ABCB1、CDA、TP53、ERCC1、SLCO1B1、 TPMT、TNFSF11、NT5C2、UGT1A1。
further, the primer group is directed at the following sites of the gene:
rs4880, rs1801133, rs716274, rs1800566, rs1801019, rs2234693, rs11280056, rs2228001, rs4244285, rs442767, rs67376798, rs1801265, rs1801159, rs55886062, rs3918290, rs1695, rs11545078, rs4646, rs3892097, rs10509373, rs1056892, rs 92 zxft 3292, rs 62487, rs1045642, rs2072671, rs1042522, rs11615, rs 3592, rs 62376225 zxft 374258, zxft 4258 zxft 4235, rs 42xft 4258 zxft 42xlt 4258.
Further, the primer group comprises 68 amplification primers, and the primer sequences are shown in SEQ ID NO. 1-68.
Further, the primer group also comprises 34 UEP extension primers, and the primer sequences are shown as SEQ ID NO. 69-102.
Furthermore, the primers are divided into two groups, namely group 1, the amplification primer sequence is shown as SEQ ID NO.1-36, and the UEP extension primer sequence is shown as SEQ ID NO. 69-86; group 2: the amplification primer sequence is shown as SEQ ID NO.37-68, and the UEP extension primer sequence is shown as SEQ ID NO. 87-102.
Further, the optimal ratios of the amplification primers are shown in Table 7.
The application also provides a composition for drug-induced detection of pan-cancer chemotherapy, which comprises any one of the primer sets.
The application also provides a product for the drug administration guidance detection of pan-cancer chemotherapy, which comprises any one of the primer sets.
The application also provides a kit for pan-cancer chemotherapy medication guidance detection, which comprises any one of the primer sets.
Further, the kit also comprises conventional reagent components for mass spectrometric detection of MassARRAY nucleic acid.
The application also provides application of any one of the primer sets in preparation of a pan-cancer chemotherapeutic drug guidance detection product.
The application also provides application of any one of the primer sets in the guiding detection of the pan-cancer chemotherapy.
The application also provides a detection method of the pan-cancer chemotherapy medication guide, which comprises the step of utilizing the primer group or the kit.
Further, the optimal amplification conditions for PCR are as follows:
compared with the prior art, the method has the following technical advantages:
1) By exploring and establishing the effective SNP locus combination for pan-cancer chemotherapy medication guidance evaluation, the method can detect 34 loci of 28 genes simultaneously, the coverage of the related genes of the chemotherapy medicaments is comprehensive, and a doctor can be helped to provide the most appropriate treatment scheme for a cancer patient.
2) The primer system provided by the application is obtained by design and screening, and comprises the selection and comparison of a primer design region, the self optimization of a primer sequence, the innovative design aiming at a specific site (such as rs3064744 site (TA) n copy number variation amplification detection) and the like, all primers can accurately and specifically detect a sample through sample test and evaluation, and the amplification of multiple DNA samples simultaneously in the same multiple system is realized, and the mutual amplification interference is small.
3) The detection kit has high sensitivity, can detect samples with the concentration of 5 ng/. Mu.L at the lowest, needs 2 holes for each sample, only needs 10ng of DNA for each hole, and only needs 20ng of DNA for one detection.
4) The detection kit is simple to operate, high in detection efficiency, high in accuracy, high in specificity, high in experimental repeatability and high in practicability. The detection result can assist doctors to fully know the expression condition of the chemotherapy medication guide gene of the patients, and gives proper suggestions, so that the most proper treatment scheme is designed for the cancer patients needing chemotherapy, and the method has remarkable social significance.
Drawings
In order to more clearly illustrate the detailed description of the present application or the technical solutions in the prior art, the drawings needed to be used in the detailed description of the present application or the prior art description will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a spectrum of the rs1056892 locus;
FIG. 2 is a cluster map of the rs1056892 site;
FIG. 3.Rs4880 site mass spectrum;
FIG. 4.Rs4880 site clustering plot;
FIG. 5.Rs1695 site mass spectrum;
FIG. 6.Rs1695 site clustering map;
FIG. 7 shows a mass spectrum of primer combination 1 at the rs3064744 site;
FIG. 8 mass spectrum of primer combination 2 at site rs3064744;
FIG. 9.RS3064744 site primer combination 3 mass spectrogram;
FIG. 10.Rs3064744 site primer combination 4 mass spectrum;
FIG. 11.Rs3064744 site primer set 4 cluster map;
FIG. 12.Rs2234693 site mass spectrum;
FIG. 13.Rs2234693 site clustering map;
FIG. 14 is a RS3212986 site mass spectrum;
FIG. 15.rs3212986 site cluster map;
figure 16.Rs1801265 site mass spectrum;
FIG. 17.Rs1801265 site cluster map;
FIG. 18.Rs2228001 site mass spectrum;
FIG. 19.Rs2228001 site clustering map;
FIG. 20.Rs3064744 site (TA) 6 /(TA) 7 Sample first generation sequencing results (top), mass spectrometry results (bottom);
FIG. 21.Rs3064744 site (TA) 6 /(TA) 6 Sample first generation sequencing results (top), mass spectrometry results (bottom);
FIG. 22.Rs1800566 site A/A sample first generation sequencing results (top), mass spectrometry results (bottom);
FIG. 23 shows the sequencing results of the first generation C/C sample at rs1801019 site (top) and the mass spectrometry results (bottom).
Detailed Description
Embodiments of the present application will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present application and should not be construed as limiting the scope of the present application, and that the examples are a part of, but not all of the examples of the present application. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
The main reagents and instrument information used in the examples of the present invention are as follows:
| name of reagent | Specification of | Manufacturer(s) of |
| Nucleic acid extraction or purification kit | 200 times/box | Meiji (American Foundation) |
| Complete iPLEX Pro Genotyping Reagent Set,CPM- | 3840/set | Agena |
| MassARRAY CleaAn Resin,40g-Agena | 3840/set | Agena |
| Name of instrument | Manufacturer of the product |
| High-speed centrifugal machine | Thermo Fisher |
| Medical centrifugal machine | Thermo Fisher |
| Miniature centrifugal machine | TIANGEN BIOTECH (BEIJING) Co.,Ltd. |
| PCR instrument (96-well) | Thermo fisher |
| PCR instrument (384-well) | Thermo fisher |
| Vortex oscillation instrument | ScientificIn dustry |
| Liquid transfer device | Eppendorf |
| Time-of-flight mass spectrometry detection system | Jiangsu Xiansheng Medical Devices Co.,Ltd. |
Experimental example establishment of detection method of the present application
1. DNA sample extraction
The whole blood sample DNA extraction is operated according to the instruction of the nucleic acid extraction or purification kit, and the extracted DNA sample can be stored at the temperature of minus 2 +/-5 ℃.
2. Detection procedure and results analysis (hereinafter, single sample size)
1.PCR amplification reaction
(1) Taking out the required reagent, putting the PCR Enzyme component on an ice box, unfreezing the other components at room temperature, putting the unfrozen components on the ice box for later use, shaking and uniformly mixing for 3-5 s before use, and performing instantaneous centrifugation.
(2) And preparing a PCR reaction system according to a formula table.
(3) After the preparation is finished, the rest reagent is put back to-20 +/-5 ℃ for storage; and (3) sealing the membrane, shaking and uniformly mixing for 30s, centrifuging at 3000rpm for 3 s, completely throwing the liquid on the tube wall to the tube bottom, and flicking and centrifuging until bubbles are removed if the bubbles exist.
(4) The 384-well plate was placed in a PCR instrument for thermocycling reactions.
(5) And discharging the product, centrifuging at 3000rpm for 30s, completely throwing the liquid on the tube wall to the tube bottom, and placing on an ice box for later use.
2. Enzymolysis reaction
(1) Taking out the required reagents in the following table, putting the SAP Enzyme on an ice box, unfreezing the other components at room temperature, putting the components on the ice box for standby, shaking and uniformly mixing for 3-5 s before use, and performing instantaneous centrifugation.
(2) Preparing an enzymolysis reaction system according to a formula table:
| components | Volume/. Mu.L | x 2/μL |
| Nuclease-Free Water | 1.53 | 3.06 |
| SAP Buffer | 0.17 | 0.34 |
| SAP Enzyme(1.7U/μl) | 0.3 | 0.6 |
| Total | 2 | 4 |
(3) After the preparation is finished, the rest reagent is put back to-20 +/-5 ℃ for storage; adding enzymolysis mixed solution into the amplification product hole of the downward machine according to 2 mul/hole; sealing the membrane, shaking and uniformly mixing for 30s, centrifuging at 3000rpm for 30s, completely throwing the liquid on the tube wall to the tube bottom, and if bubbles exist, flicking and centrifuging until the bubbles are removed.
(4) The 384-well plate was placed in a PCR instrument for thermocycling reactions.
(5) And discharging the product, centrifuging at 3000rpm for 30s, completely throwing the liquid on the tube wall to the tube bottom, and placing on an ice box for later use.
3. Extension reaction
(1) Taking out the required reagents in the following table, putting the component iPLEX Enzyme on an ice box, unfreezing the other components at room temperature, putting the unfrozen components on the ice box for later use, shaking and uniformly mixing for 3-5 s before use, and performing instantaneous centrifugation.
(2) Preparing an extension reaction system according to a formula table:
| components | W1/μL | W2/μL |
| Nuclease-Free Water | 0.62 | 0.62 |
| iPLEX buffer | 0.2 | 0.2 |
| iPLEX Termination mix | 0.2 | 0.2 |
| Extend primer Mix | 0.94 | 0.94 |
| iPLEX Enzyme | 0.04 | 0.04 |
| Total | 2 | 2 |
(3) After the preparation is finished, the rest reagent is put back to-20 +/-5 ℃ for storage; adding extension mixed liquor into the enzymolysis product hole of the lower machine according to the proportion of 2 mul/hole; sealing the membrane, shaking and uniformly mixing for 30s, centrifuging at 3000rpm for 30s, completely throwing the liquid on the tube wall to the tube bottom, and if bubbles exist, flicking and centrifuging until the bubbles are removed.
(4) The 384-well plate was placed in a PCR machine for thermocycling.
(5) And discharging the product, centrifuging at 3000rpm for 30s, completely throwing the liquid on the tube wall to the tube bottom, and standing at room temperature for later use.
4. Water supplement
To the 384 well plate which had been subjected to the extension reaction, nuclean-Free Water was added in an amount of 16. Mu.L/well, and the 384 well plate was sealed with a new sealing plate and centrifuged at 3000rpm for 30 seconds.
5. And (4) detecting by mass spectrometry.
6. And (6) analyzing results.
The detection report from the flight time mass spectrum detection system can directly obtain the genotypes of 34 SNP loci without manual analysis.
Example 1 screening of SNP site of drug-guide gene for pan-cancer chemotherapy
At present, the drugs commonly used for chemotherapy of tumor patients clinically mainly comprise cypress, 5-FU/capecitabine, taxoids, anthracyclines, methotrexate, pemetrexed, letrozole/anastrozole, vinblastine, gemcitabine, cyclophosphamide/ifosfamide, irinotecan, etoposide, tamoxifen, azathioprine/mercaptopurine/thioguanine and the like, a large number of clinical studies show that each chemotherapeutic drug has a corresponding target for evaluating the effect of the chemotherapeutic drug, and the curative effect of the chemotherapeutic drug is mainly related to the expression level of related genes. Such as:
ERCC1 is an important member in an exonucleolytic repair family, participates in cutting and damage recognition of a DNA chain, and platinum drugs inhibit DNA synthesis and replication by causing cross-linking in and among the DNA chains of target cells, so that the production of tumor cells is inhibited. DNA repair is one of the major mechanisms for the development of resistance to platinum-based chemotherapy. Clinical studies have shown that ERCC1 participates in the development of platinum chemotherapy resistance, and the expression level of ERCC1 is negatively correlated with the curative effect and survival time of platinum chemotherapy for various cancers, namely, patients with low expression level are sensitive to platinum drugs, and conversely, patients with high expression level show resistance. Thus, it is well documented in the clinical treatment guidelines for NCCN non-small cell lung cancer: the ERCC1 mRNA expression level detection before the platinum chemotherapy can improve the treatment effective rate and the patient survival rate.
TP53, the gene most highly correlated with human tumors found to date (P53 gene mutation occurred in more than 50% of human tumors). The mutant p53 protein causing tumor formation or cell transformation is a tumor-promoting factor, while the wild-type p53 gene is a tumor suppressor gene, and its inactivation plays an important role in tumor formation. According to research reports, p53 gene variation is related to platinum drug resistance, but does not influence the sensitivity of taxol drugs. Therefore, p53 gene mutation detection can be used for guiding the individualized administration of chemotherapy for clinical tumor patients.
TYMS, the encoded Thymidylate Synthase (TS) is the rate-limiting enzyme for pyrimidine nucleotide synthesis and an important factor in tumor growth. The fluorine drugs are fluoro derivatives of uracil, and play a role in inhibiting the production of tumor cells by influencing DNA synthesis or interfering protein synthesis in cells. The medicines comprise 5-fluorouracil (5-FU), tegafur, carmofur and the like, and the medicines are converted into the 5-FU in vivo. The clinical research of various tumors shows that the mRNA expression level of the TYMS gene is closely related to the curative effect of 5-FU, and the mRNA expression level comprises colorectal cancer, lung cancer, breast cancer, head and neck squamous cell carcinoma and the like. Research shows that the tumor patients with low TYMS mRNA level have better effect of receiving fluorine chemotherapy and longer median survival time. The efficacy of pemetrexed was also found to be inversely correlated with the level of T YMS mRNA expression.
UGT1A1, encoding uridine diphosphate glucuronosyltransferase. And the Yi Li Tikang enters the body and then is subjected to enzymolysis to generate SN-38, and then is subjected to liver uridine diphosphate glucuronosyltransferase to generate SN-38G, so that healthy cells are protected from the toxic effect of irinotecan. The research shows that the wild type UGT1A1 (6/6) has lower risk of generating toxic and side effects when receiving the illicit treatment, and the mutant homozygote (7/7) has 50 percent of possibility of generating toxic and side effects. Thus, in 2005 the FDA required a warning to be placed on the drug label, suggesting that patients check for UGT1A1 x 28 mutations before using i Li Tikang. Clinical findings from several asian populations demonstrated a significant correlation between the 211g > a mutation and the increased risk of toxic side effects of i Li Tikang. The Yi Li Tikang is mainly used for treating adult patients with advanced/metastatic colorectal cancer, and also has curative effects on small cell and non-small cell lung cancer, cervical cancer and ovarian cancer.
The application refers to clinical practice guidelines about common malignant tumors such as breast cancer, lung cancer, non-Hodgkin lymphoma, stomach cancer, colon cancer, stomach cancer, rectal cancer, cervical cancer, ovarian cancer, kidney cancer, head and neck tumors and the like in NCCN oncology clinical practice guidelines (Chinese edition), and combines databases such as ACMG, exAC,1000genomes, dbSNP, clinVar, HGMD and the like to summarize the corresponding relationship between the common chemotherapeutic drugs and drug sensitivity related genes SNP loci, as shown in Table 1:
TABLE 1 common chemotherapeutic drugs and their drug sensitivity related gene SNP sites
After the analysis and screening, 34 SNP loci of 28 genes are comprehensively determined as the loci detected by the invention, and the specific mutation information is shown in Table 11:
TABLE 11.34 SNP site mutation information
Example 2, primer design and sequence optimization;
for the determined detection sites, the Massarray primer sequences are preliminarily designed, and then sequence optimization is performed, for reasons of space, the primer sequences of 4 sites are optimized as an example in this embodiment.
1) rs1056892 locus
Aiming at the locus rs1056892, a plurality of groups of amplification primers are designed, and through tests (the results are shown in figures 1 and 2), the detection result of a heterozygous sample at the locus rs1056892 shows that the performances of the extended G and A products of the primer combination 1 are inconsistent, the high and low peak phenomena occur, and the result is not ideal; the performance of the extension G and A products of the primer combination 2 is basically consistent, and the result is ideal, so the primer combination 2 is selected for system construction, and 2 groups of primer sequences are shown in Table 2:
TABLE 2 RS1056892 site 2 set of primer information
2) rs4880 locus
The detection result of the rs4880 locus heterozygous sample shows (figures 3 and 4), the performances of the primer combination 1 for extending G and A products are inconsistent, the high and low peak phenomena occur, and the result is not ideal; the performance of the extension G and A products of the primer combination 2 is basically consistent, and the result is ideal, so the primer combination 2 is selected for system construction, and 2 groups of primer sequences are shown in Table 3:
TABLE 3 RS4880 site 2 set primer information
3) rs1695 locus
The detection result of the rs1695 site heterozygous sample shows (fig. 5 and 6), the performances of the primer combination 1 extended G and A products are inconsistent, the high-low peak phenomenon occurs, and the result is not ideal; the performance of the extension G and A products of the primer combination 2 is basically consistent, and the result is ideal, so the primer combination 2 is selected for system construction, and 2 groups of primer sequences are shown in Table 4:
TABLE 4 primer information for rs1695 site 2 group
4) rs3064744 locus
The rs3064744 site is a variation in (TA) n copy number. Previous studies showed that the Chinese UGT1A1 gene rs8175347 and (TA) 6 /(TA) 6 Mainly, it accounts for about 73%, (TA) 6 /(TA) 7 And (TA) 7 /(TA) 7 23% and 4% respectively.
Because the site is a TA short tandem repeat sequence and the invention is a multiplex system, the primer of the site is prevented from forming a secondary structure; (2) secondary structures are formed between the primers of the site and other sites; (3) the accuracy and other properties between sites in the same reaction well are not affected each other, so that the design of the amplification primer and the extension primer is very challenging. The method combines ARMS-PCR technology with time-of-flight mass spectrometry technology, and designs 4 groups of primers for performance test (wherein, group 4 relates to degenerate sequence primers, and detection is carried out by equal proportion mixing addition), wherein (TA) 6 /(TA) 6 And (TA) 6 /(TA) 7 For true clinical samples, (TA) 7 /(TA) 7 No clinical samples were collected and replaced with synthetic plasmids.
The results show (FIGS. 7-11): primer combination 1 (TA) 6 /(TA) 6 And (TA) 7 /(TA) 7 The sample mass spectrum results are correct, but (TA) 6 /(TA) 7 The sample mass spectrum results are wrong, so the sample mass spectrum results are unusable; primer combination 2 (TA) 6 /(TA) 6 And (TA) 7 /(TA) 7 The sample mass spectrum results are correct, but (TA) 6 /(TA) 7 Due to different extension efficiencies of the samples, high and low peaks appear in mass spectrum results, the Angles value is in an instrument parameter gray area, and no genotype results exist, so that the samples cannot be used; primer combination 3 (TA) 6 /(TA) 6 Sample Mass Spectrometry results were correct, however (TA) 6 /(TA) 7 And (TA) 7 /(TA) 7 The sample mass spectrum result is wrong, so the sample mass spectrum result is unavailable; primer combination 4 (TA) 6 /(TA) 6 、(TA) 6 /(TA) 7 And (TA) 7 /(TA) 7 The sample mass spectrum result is correct and available; and the primer combination 4 is subjected to multi-clinical sample test, and the mass spectrum result is consistent with the first-generation sequencing result. The sequences of the 4 sets of primers are shown in Table 5.
TABLE 5 primer information for 4 sets of rs3064744 sites
After comprehensive optimization, the primer system finally established in the application is as follows in table 6:
TABLE 6.34 site detection primer information
Example 3 optimization of the detection System
In order to further improve the detection efficiency of the system, the present example also discusses the optimization of the system, such as the optimization of the primer addition ratio (taking 2 sites as an example) and the annealing temperature (taking 2 sites as an example).
1. Primer ratio optimization
Under the condition of primer sequence determination, part of sites have poor detection performance due to insufficient or excessive addition of amplification primers, so that the addition of the amplification primers is properly adjusted to ensure that the detection performance is optimal.
1) rs2234693 locus
Much UEP remains before rs2234693 locus optimization, the product peak is lower than the UEP remaining peak, which indicates that the amplification product is relatively less, so that the amplification primer SEQ ID NO:11 and SEQ ID NO: the primer addition amount is increased to 1.5 times of the original primer addition amount for testing, the result shows that UEP is completely extended into a product, the optimization result is more ideal, and the test result is shown in figures 12-13.
2) rs3212986 locus
Much UEP remains before rs3212986 locus optimization, the product peak is lower than the UEP remaining peak, which indicates that the amplification product is relatively less, so that the amplification primer SEQ ID NO:59 and SEQ ID NO:60 primer addition is increased to 2 times of the original addition for testing, and the result shows that UEP is greatly extended to a product, the optimization result is more ideal, and the test result is shown in figures 14-15:
through multiple experimental tests, the optimal addition ratio of the 34-site detection amplification primers is finally determined, and is shown in table 7 (it needs to be reminded that the ratio is only the optimal scheme, and other related ratios (such as the conventional 1:1 balanced ratio) can also meet the basic detection requirements).
TABLE 7 amplification primer Mix formulations
2. Optimizing annealing temperature:
in PCR amplification experiments, the annealing temperature is an important influence factor influencing the PCR amplification efficiency. Particularly, in a multiplex reaction system, the highest amplification efficiency of all PCR primer sets is ensured, and the occurrence of non-specific amplification products is also ensured, so that the selection of an appropriate annealing temperature is particularly important. The initial annealing temperature is 55 ℃, non-specific peaks or high and low peaks appear in the detection results of part of sites, the result is improved after the annealing temperature is increased, and the detection performance of each site is best when the annealing temperature is 60 ℃ after multiple times of debugging.
1) rs1801265 locus
When the annealing temperature of the site rs1801265 =55 ℃, the detection result shows that a small nonspecific peak appears on the left side of UEP; when the annealing temperature is increased to 60 ℃, the non-specific small peak disappears, the amount of the extension product is increased, and the optimization result is ideal (see fig. 16 and 17).
2) rs2228001 locus
When the annealing temperature of the rs2228001 locus =55 ℃, the detection result shows that the extension performance of the G product is inconsistent with that of the T product, and a high peak and a low peak appear; when the annealing temperature is increased to 60 ℃, the two products have consistent peak elongation performance, the UEP elongation is improved, the optimization result is ideal, and the test results are shown in figures 18 and 19.
Based on this, the determined optimal amplification conditions for PCR were as follows:
example 4 verification of System Performance
The system verification of this example includes accuracy, precision, sensitivity and specificity to show the technical advantages of the system of the invention, specifically:
1) Verifying accuracy and specificity: 20 clinical samples with the concentration of 20 ng/mu L are detected at each site, and the mass spectrum detection result is compared with a first generation sequencing result, and the expected target is more than or equal to 95 percent.
And (3) test results: taking sample number 1 as an example, the accuracy and specificity verification results are shown in table 8, the specific detection results of some sites are shown in fig. 20-23, and the results show that the mass spectrometry detection results of 34 SNP sites of 20 clinical samples are 100% consistent with the first-generation sequencing results and meet the expected target.
TABLE 8 verification of accuracy and specificity
2) And (3) sensitivity verification: the concentration of 5 detected samples at each site is 20 ng/muL, 10 ng/muL and 5 ng/muL respectively, and the expected target is more than or equal to 95 percent.
And (3) test results: the sample number 2 is used as an example, and the sensitivity verification results are shown in table 9, and the results show that 5 clinical samples can accurately report the genotypes of 34 SNP sites at 100% under 3 concentrations, and the genotypes meet the expected targets.
TABLE 9 sensitivity verification results
3) Precision validation (both intra and inter batch), expected target > 95%:
internal precision: 5 samples were repeated 3 times in the same batch to compare the in-batch precision;
batch precision: the same operator checks 5 samples of the same sample in 5 batches simultaneously, comparing the precision between batches.
The results show that the intra-batch and inter-batch spectra results of 5 clinical samples are 100% consistent, and the precision accords with the expected target.
By combining the above verification data, the performance of the detection system is summarized in table 10:
TABLE 10 summary of system validation results
| Site of the body | Accuracy of | Specificity of | Sensitivity of the probe | Precision in batch | Inter-batch precision |
| rs1042522 | 100％ | 100％ | 100％ | 100％ | 100％ |
| rs1045642 | 100％ | 100％ | 100％ | 100％ | 100％ |
| rs10509373 | 100％ | 100％ | 100％ | 100％ | 100％ |
| rs1056892 | 100％ | 100％ | 100％ | 100％ | 100％ |
| rs11045879 | 100％ | 100％ | 100％ | 100％ | 100％ |
| rs11280056 | 100％ | 100％ | 100％ | 100％ | 100％ |
| rs1142345 | 100％ | 100％ | 100％ | 100％ | 100％ |
| rs11545078 | 100％ | 100％ | 100％ | 100％ | 100％ |
| rs11572080 | 100％ | 100％ | 100％ | 100％ | 100％ |
| rs11598702 | 100％ | 100％ | 100％ | 100％ | 100％ |
| rs11615 | 100％ | 100％ | 100％ | 100％ | 100％ |
| rs1695 | 100％ | 100％ | 100％ | 100％ | 100％ |
| rs1800566 | 100％ | 100％ | 100％ | 100％ | 100％ |
| rs1801019 | 100％ | 100％ | 100％ | 100％ | 100％ |
| rs1801133 | 100％ | 100％ | 100％ | 100％ | 100％ |
| rs1801159 | 100％ | 100％ | 100％ | 100％ | 100％ |
| rs1801265 | 100％ | 100％ | 100％ | 100％ | 100％ |
| rs2072671 | 100％ | 100％ | 100％ | 100％ | 100％ |
| rs2228001 | 100％ | 100％ | 100％ | 100％ | 100％ |
| rs2234693 | 100％ | 100％ | 100％ | 100％ | 100％ |
| rs25487 | 100％ | 100％ | 100％ | 100％ | 100％ |
| rs3064744 | 100％ | 100％ | 100％ | 100％ | 100％ |
| rs3212986 | 100％ | 100％ | 100％ | 100％ | 100％ |
| rs3892097 | 100％ | 100％ | 100％ | 100％ | 100％ |
| rs3918290 | 100％ | 100％ | 100％ | 100％ | 100％ |
| rs4148323 | 100％ | 100％ | 100％ | 100％ | 100％ |
| rs4244285 | 100％ | 100％ | 100％ | 100％ | 100％ |
| rs442767 | 100％ | 100％ | 100％ | 100％ | 100％ |
| rs4646 | 100％ | 100％ | 100％ | 100％ | 100％ |
| rs4880 | 100％ | 100％ | 100％ | 100％ | 100％ |
| rs55886062 | 100％ | 100％ | 100％ | 100％ | 100％ |
| rs67376798 | 100％ | 100％ | 100％ | 100％ | 100％ |
| rs716274 | 100％ | 100％ | 100％ | 100％ | 100％ |
| rs7984870 | 100％ | 100％ | 100％ | 100％ | 100％ |
Finally, it should be noted that: the above embodiments are only used for illustrating the technical solutions of the present application, and not for limiting the same; although the present application has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present application.
Claims (10)
1. A primer group for guiding and detecting the chemotherapy medication of pan-cancer species based on a time-of-flight mass spectrometry platform is characterized in that the primer group is directed at the following sites: rs4880, rs1801133, rs716274, rs1800566, rs1801019, rs2234693, rs11280056, rs2228001, rs4244285, rs442767, rs67376798, rs1801265, rs1801159, rs55886062, rs3918290, rs1695, rs11545078, rs4646, rs3892097, rs10509373, rs 35 zxft 3235, rs 92 zxft 3292, rs25487, rs1045642, rs2072671, rs1042522, rs 3567 zxft 67, rs 3592, rs 373223492, rs 42xft 3723425, rs 42xft 3723452, rs 42zxft 4235 zxft 3487, rs 42xft 3446, rs 42xxxvt 3552, rs 42xft 3552, rs 42zxft.
2. The primer set of claim 1, wherein the primer set comprises 68 amplification primers, and the sequences of the primers are shown as SEQ ID No. 1-68.
3. The primer set of claim 2, wherein the primer set further comprises 34 UEP extension primers, and the primer sequences are shown in SEQ ID nos. 69-102.
4. The primer set of any one of claims 2-3, wherein the primers are divided into two groups, wherein the primer sequence of group 1 is shown as SEQ ID No.1-36, and the primer sequence of UEP extension is shown as SEQ ID No. 69-86; the sequence of the group 2 amplification primer is shown in SEQ ID NO.37-68, and the sequence of the UEP extension primer is shown in SEQ ID NO. 87-102.
5. A composition for drug-directed detection of pan-cancer chemotherapy, comprising the primer set of any one of claims 1-4.
6. A product for use in the drug-directed detection of pan-cancer chemotherapy, comprising the primer set of any one of claims 1-4.
7. A kit for drug-directed detection of pan-cancer chemotherapy, comprising the primer set of any one of claims 1-4.
8. The kit of claim 7, wherein the kit further comprises conventional reagent components for mass spectrometric detection of MassARRAY nucleic acids.
9. Use of the primer set according to any one of claims 1 to 4 for the preparation of a detection product for a pan-cancer chemotherapeutic medication guide.
10. A method for detecting a drug administration guide for pan-cancer chemotherapy, comprising the step of using the primer set according to any one of claims 1 to 4.
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| CN110257523A (en) * | 2019-07-22 | 2019-09-20 | 上海市胸科医院 | A kind of primer sets and detection method detecting chemotherapeutical medicine curative effect and side effect related SNP |
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| CN106554995A (en) * | 2016-07-26 | 2017-04-05 | 北京京诺玛特科技有限公司 | The method and test kit of detection chemotherapy of tumors personalized medicine related gene |
| CN109517900A (en) * | 2018-12-29 | 2019-03-26 | 江苏先声医学诊断有限公司 | It is a kind of to detect primer sets, reagent and/or the kit of lung cancer chemotherapy related gene, system and its application |
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