CN112980950B - Kit for detecting 15 gene mutation sites related to rectal cancer chemoradiotherapy sensitivity and application thereof - Google Patents

Abstract

The invention discloses a kit for detecting 15 gene mutation sites related to the sensitivity of radiotherapy and chemotherapy of rectal cancer and application thereof, wherein the kit is a detection kit based on a high-throughput DNA sequencing technology and is used for detecting the DNA mutation sites of 15 genes related to the sensitivity of radiotherapy and chemotherapy of rectal cancer, the kit comprises a specific primer for detecting the gene mutation sites, the nucleotide sequence of an upstream primer of the specific primer is shown as SEQ ID No.1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27 and 29, and the nucleotide sequence of a downstream primer of the specific primer is shown as SEQ ID No.2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 and 30. The tumor tissue specimen detection gene DNA mutation site has good stability, strong clinical operability and simple and convenient detection process. The invention can be used for distinguishing the locally advanced rectal cancer patients sensitive to the neoadjuvant chemoradiotherapy, so that the locally advanced rectal cancer patients can benefit remarkably in the neoadjuvant chemoradiotherapy.

Description

Kit for detecting 15 gene mutation sites related to rectal cancer chemoradiotherapy sensitivity and application thereof

Technical Field

The invention relates to the technical field of biomedical diagnosis, in particular to a kit for detecting 15 gene mutation sites related to sensitivity of radiotherapy and chemotherapy of rectal cancer and application thereof.

Background

In malignant tumors, the incidence rate of colorectal cancer is ranked 3 in the global place and 5 in China; the incidence rate of the cancer shows a rapid rising and young trend, and the cancer is a common malignant tumor affecting the social development of China and seriously harms the human health. Neoadjuvant chemoradiotherapy is a major component of the overall strategy for treatment of rectal cancer, and neoadjuvant chemoradiotherapy sensitivity varies significantly among individuals. The degree of tumor regression after neoadjuvant radiotherapy and chemotherapy is obviously related to the long-term survival rate and the survival quality of patients, about 30 percent of patients can achieve complete pathological relief after the neoadjuvant radiotherapy and chemotherapy during the operation, if the tumor regression can be predicted in advance, a close-looking waiting and observing strategy is adopted after the neoadjuvant radiotherapy and chemotherapy, so that the part of patients can avoid the operation of keeping anus; in addition, even if some patients receive strong neoadjuvant radiotherapy and chemotherapy, the tumor regression is still unsatisfactory, and if the tumor regression can be predicted to be limited in advance, the treatment strategy can be adjusted from the initial treatment and the treatment intensity can be increased, so that the survival rate of the patients can be improved, and the survival time can be prolonged. The rectum is a very special organ, and according to two principles of radically treating tumors to the maximum extent and ensuring the functions of visceral organs to the maximum extent, the rectum can preserve the survival quality of patients to the maximum extent while ensuring the curative effect of the tumors, improves the curative effect of patients with poor prognosis as much as possible, and is a work which is urgently needed to be developed in the field of rectal cancer treatment.

DNA is genetic material and is the most crucial factor for all biological traits. 95% of human diseases are related to genes, and the genetic information of each human is different, so that the genetic information is not only important content of life science research, but also is an important basis for overcoming difficult and complicated diseases of human. High throughput, second-generation genetic sequencing technologies, also known as "next-generation sequencing technologies", allow simultaneous detection of multiple types of genetic variations, including Single Nucleotide Variations (SNVs), small insertions/deletions (indels), and Copy Number Variations (CNVs), or complex genomic rearrangements, based on parallel sequencing of hundreds of thousands to millions of DNA molecule sequences and subsequent data processing and alignment. Currently, the prediction of complete remission of pathology after chemoradiotherapy before colorectal cancer operation at home and abroad lacks the research on DNA level gene sequencing;

in previous researches, clinical factors (initial clinical stage, carcinoembryonic antigen level and the like) have poor prediction effect on the curative effect of the neoadjuvant chemoradiotherapy. At least 40 different molecular indexes which are possibly related to the sensitivity of the new auxiliary radiotherapy and chemotherapy of the rectal cancer, such as TP53, KRAS, TYMS, MTHFR and the like, are discussed in the literature, the consistency of the results is very undesirable, and contradictory results are often existed. The relationship between the mutations of the genes other than TP53 and the sensitivity of the cancer to neoadjuvant radiotherapy and chemotherapy has not been confirmed by many studies. The effectiveness of the current clinical and molecular indexes for predicting the curative effect of the new adjuvant radiotherapy and chemotherapy is very limited, the research objects are selected by theory and experience, and a large number of partial markers which influence the research outcome are difficult to efficiently excavate. Therefore, establishing a new auxiliary therapeutic effect accurate prediction model for the rectal cancer after radiotherapy and chemotherapy by combining clinical pathological characteristics and gene detection is a key factor for determining whether accurate individualized treatment for the rectal cancer is successful.

Disclosure of Invention

In view of the above, in order to overcome the defects in the prior art, the technical problem to be solved by the present invention is to provide a kit for detecting 15 gene mutation sites related to sensitivity of radiotherapy and chemotherapy of rectal cancer, which can stably and effectively predict the sensitivity of neoadjuvant radiotherapy and chemotherapy of local advanced rectal cancer based on gene DNA mutation sites, and screen patients sensitive to radiotherapy and chemotherapy, so that the patients can benefit significantly from neoadjuvant radiotherapy and chemotherapy.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

a kit for detecting 15 gene mutation sites related to the sensitivity of the rectal cancer to chemoradiotherapy is characterized in that: the kit is a detection kit based on a high-throughput DNA sequencing technology and is used for detecting 15 gene DNA mutation sites related to the sensitivity of the rectal cancer chemoradiotherapy, the kit comprises specific primers for detecting the gene mutation sites, the nucleotide sequences of upstream primers of the specific primers are shown as SEQ ID No.1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27 and 29, and the nucleotide sequences of downstream primers of the specific primers are shown as SEQ ID No.2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 and 30.

The specific primers are shown in the following table 1:

further, the mutation sites of the gene DNA are as follows: chr6_33052958_ C _ T, chr9_125316157_ C _ T, chr9_123850770_ G _ A, chr3_122259606_ T _ C, chr1_150679033_ G _ A, chr6_160211646_ GTT _, chr1_160920966_ C _ T, chr8_23294761_ T _ C, chr21_47974582_ a _ G, chr7_122635024_ C _ T, chr6_56420538_ C _ T, chr1_232574921_ T _ C, chr8_21862551_ a _ G, chr11_66254085_ G _ T, chr11_ G _44636833_ G _ a.

Further, the kit also comprises a reagent for detecting the level of the DNA mutation site in the tumor tissue of the gene.

Still further, the reagents for detecting levels in tumor tissue at sites of DNA mutations in the genes are second generation sequencing reagents.

On the other hand, the invention also provides application of the kit in serving as a detection reagent for predicting the sensitivity of the neoadjuvant chemoradiotherapy of the locally advanced rectal cancer.

Specifically, the locally advanced rectal cancer is locally advanced rectal cancer in stage II and stage III recommended by NCCN guidelines and applied with a new auxiliary Arhualu. The gene DNA mutation site has higher gene mutation frequency in tumor tissues of patients sensitive to radiotherapy and chemotherapy than in patients insensitive to radiotherapy and chemotherapy.

In order to realize the application of the kit in serving as a detection reagent for predicting the neoadjuvant chemoradiotherapy sensitivity of the locally advanced rectal cancer, a prediction model for predicting the neoadjuvant chemoradiotherapy sensitivity of the locally advanced rectal cancer, which consists of 15 gene DNA mutation sites, is established according to the following steps:

(1) based on a second-generation sequencing method, screening candidate gene DNA mutation sites of a new auxiliary chemoradiotherapy sensitive patient and a new auxiliary chemoradiotherapy insensitive patient of the local late rectal cancer by sequencing of a whole exon;

(2) target sequencing verifies candidate gene DNA mutation sites;

(3) establishing a gene DNA mutation site combination capable of distinguishing a locally advanced rectal cancer patient sensitive to neoadjuvant chemoradiotherapy in a training group consisting of a locally advanced rectal cancer patient sensitive to neoadjuvant chemoradiotherapy and a patient insensitive to neoadjuvant chemoradiotherapy;

(4) verifying the combination of the gene DNA mutation sites established in the step (3) in two independent verification groups to predict the effect of the combination of the neoadjuvant chemoradiotherapy sensitivity of the locally advanced rectal cancer;

(5) and (4) analyzing the combination of the DNA mutation sites of the genes established in the step (3) to predict the effect of the combination of the neoadjuvant chemoradiotherapy sensitivity of the locally advanced rectal cancer by adopting a logistic regression model.

Further, the logistic regression formula is: the logit [ p ═ PCR ] ═ 2.0298+1.0830 × chr6_33052958_ C _ T-33052958 × chr 33052958_ 33052958_ G _ a + 33052958 × chr 33052958_ T _ C + 33052958 × chr 33052958_ G _ a + 33052958 × chr 33052958_ GTT _+ 33052958 × chr 33052958_ C _ T-33052958 _33052958_ T _ C + 33052958 × chr 33052958_ a _ G + 33052958 × chr 33052958_ C _ T + 33052958 × chr 33052958_ T33052958 _ C _ T _33052958_ C _ T + 33052958_ C _33052958_ T _ C + 33052958_ 33052958_ C _33052958_ T _33052958_ C + 33052958_ C + 33052958_ C _ T _ C _33052958_ C + 33052958_ 33052958_ C _33052958_ x 33052958_ 33052958_ C _33052958_ G + 33052958_ 33052958_ x 33052958_ 33052958_ a _ G + 33052958_ 33052958 x 33052958_ 33052958_ a _33052958 x 33052958_ b _ a 33052958 x 33052958_ p ═ PCR 33052958_ PCR _ p ═ PCR ═ 33052958 x 33052958_ PCR ═ 33052958_ p ═ PCR ═ p ═ PCR ═ p ═ PCR × ═ p × _ PCR ═ p ═ 33052958 x chr _ PCR ═ C _33052958 x chr _33052958_ PCR ═ C _33052958_ C _33052958_ C _33052958_ C _33052958_ C _33052958_ C _33052958_ C _.

Compared with the prior art, the invention has the following beneficial effects:

the tumor tissue specimen detection gene DNA mutation site is stable and effective, strong in clinical operability, convenient to detect, mature in experimental method, simple and convenient in detection process and easy to repeat. The invention can be used for distinguishing the locally advanced rectal cancer patients sensitive to the neoadjuvant chemoradiotherapy, so that the locally advanced rectal cancer patients can benefit remarkably in the neoadjuvant chemoradiotherapy. The method has the following specific advantages:

(1) the invention adopts high-throughput screening, multi-center verification and research in the locally advanced rectal cancer specimens collected prospectively, comprehensively evaluates the effect of gene DNA mutation site combination and a diagnostic kit, ensures the potential application value of the invention in evaluating whether a locally advanced rectal cancer patient is sensitive to new auxiliary radiotherapy and chemotherapy and provides a referable method strategy for the development of other disease biological radiotherapy and chemotherapy sensitivity prediction markers.

(2) The gene DNA mutation site combined diagnostic kit can predict whether a patient with the locally advanced rectal cancer is sensitive to chemoradiotherapy before neoadjuvant chemoradiotherapy in time, so that the patient can benefit significantly from the neoadjuvant chemoradiotherapy. The complicated detection is avoided, the time and labor cost are saved, and the clinician can conveniently adopt an individualized prevention and treatment scheme in time.

Detailed Description

The invention is further illustrated by the following examples. It should be understood that these examples are illustrative and exemplary of the present invention, and are not intended to limit the scope of the present invention in any way.

Example 1

The diagnostic kit for predicting the sensitivity of the neoadjuvant chemoradiotherapy of the locally advanced rectal cancer based on the gene DNA mutation site comprises specific primers for detecting the gene mutation site, the nucleotide sequences of upstream primers of the specific primers are shown as SEQ ID No.1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27 and 29, and the nucleotide sequences of downstream primers of the specific primers are shown as SEQ ID No.2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 and 30.

The gene DNA mutation sites are as follows: chr6_33052958_ C _ T, chr9_125316157_ C _ T, chr9_123850770_ G _ A, chr3_122259606_ T _ C, chr1_150679033_ G _ A, chr6_160211646_ GTT _, chr1_160920966_ C _ T, chr8_23294761_ T _ C, chr21_47974582_ a _ G, chr7_122635024_ C _ T, chr6_56420538_ C _ T, chr1_232574921_ T _ C, chr8_21862551_ a _ G, chr11_66254085_ G _ T, chr11_44636833_ G _ a.

The gene mutation sites are 5 gene DNA mutation sites, and the 15 gene DNA mutation sites form a prediction model for predicting the sensitivity of the local late colorectal cancer in the new auxiliary radiotherapy and chemotherapy:

(1) based on a second-generation sequencing method, screening candidate gene DNA mutation sites of a new auxiliary radiotherapy and chemotherapy sensitive patient and a new auxiliary radiotherapy and chemotherapy insensitive patient of the local late rectal cancer by whole exon sequencing;

(2) target sequencing verifies candidate gene DNA mutation sites;

(3) establishing a gene DNA mutation site combination capable of distinguishing a locally advanced rectal cancer patient sensitive to neoadjuvant chemoradiotherapy in a training group consisting of a locally advanced rectal cancer patient sensitive to neoadjuvant chemoradiotherapy and a patient insensitive to neoadjuvant chemoradiotherapy;

(4) verifying the effect of the combination of the gene DNA mutation sites established in the step (3) on predicting the sensitivity of the neoadjuvant chemoradiotherapy of the locally advanced rectal cancer in two independent verification groups;

(5) analyzing the combination of the DNA mutation sites of the genes established in the step (3) to predict the effect of the combination of the neoadjuvant chemoradiotherapy sensitivity of the locally advanced rectal cancer.

Furthermore, the gene DNA mutation site has higher gene mutation frequency in tumor tissues of patients sensitive to radiotherapy and chemotherapy than in patients insensitive to radiotherapy and chemotherapy.

The diagnostic kit for predicting the sensitivity of the local late colorectal cancer neoadjuvant chemoradiotherapy based on the gene DNA mutation site comprises a reagent for detecting the level of the tumor tissue at the gene DNA mutation site.

The reagent for detecting the level of the gene DNA mutation site in the tumor tissue is a second-generation sequencing reagent.

The logistic regression formula is as follows: a logic [ p ═ PCR ] ═ 2.0298+1.0830 × chr6_33052958_ C _ T-33052958 × chr 33052958_ 33052958_ G _ a + 33052958 × chr 33052958_ T _ C + 33052958 × chr 33052958_ 33052958_ G _ a + 33052958 × chr 33052958_ GTT _. + 33052958 × chr 33052958_ C _ T-33052958 × chr 33052958_ 33052958_ T + 33052958_ C + 33052958 × chr 33052958_ C _33052958_ a _ G + 33052958 × chr 33052958_ C _ T + 33052958_ T33052958 _33052958_ C + 33052958_ C + 33052958_ G _33052958 × 33052958_ 33052958 × 33052958_ 33052958 × 33052958_ 33052958 × 363672 _33052958 × 33052958_ 33052958_ T33052958 _33052958_ T33052958 _33052958_ T33052958 _33052958_ T33052958 _33052958 is less sensitive to a and a of the frequency of the patient is diagnosed as a and the patient is less sensitive to a patient 'a' and a.

The invention discloses application of a diagnostic kit for predicting the new auxiliary chemoradiotherapy sensitivity of locally advanced rectal cancer based on gene DNA mutation sites in a medicine for predicting the new auxiliary chemoradiotherapy sensitivity of locally advanced rectal cancer.

The locally advanced rectal cancer is locally advanced rectal cancer in II stage and III stage recommended by NCCN guidelines and applied by a new auxiliary Arhualu.

Example 2

Collection and preparation of tumor tissue specimen

The inventor collects tumor tissue specimens of local advanced rectal cancer from 09 months to 2017 months in 2005, and the groups meet the grouping standard. The inclusion criteria include four main points: 1. locally advanced rectal patients diagnosed for the first time and not receiving any treatment; 2. patients receiving standard treatment of neoadjuvant radiotherapy and chemotherapy, radical operation and postoperative adjuvant chemotherapy; 3. confirmed by histopathological examination, TGR graded score, PCR group TRG 1 grade, namely: the pathology was completely relieved after neoadjuvant chemoradiotherapy (PCR), and the none-PCR group was TRG grade 2-5, i.e.: the tumor remained in the residual group after the new adjuvant radiotherapy and chemotherapy (None-PCR group); 4. willing to participate and coordinate with the study follower. And setting the samples of the chemoradiotherapy sensitive group and the chemoradiotherapy insensitive group according to the principle of sex and age matching.

Screening group: tumor tissue samples from patients in the chemoradiotherapy sensitive group (8 cases) and patients in the chemoradiotherapy insensitive group (7 cases).

Training group: tumor tissue samples from patients in the chemotherapy-sensitive group (41) and patients in the chemotherapy-insensitive group (161).

Internal verification group: tumor tissue samples from patients in the chemoradiotherapy sensitive group (18 cases) and patients in the chemoradiotherapy insensitive group (59 cases).

External authentication group: tumor tissue samples from patients in the chemoradiotherapy sensitive group (17 cases) and patients in the chemoradiotherapy insensitive group (56 cases). And simultaneously detecting the CEA, CA199 and other biochemical indexes and MR and other imaging examinations.

When a patient is in initial diagnosis, fresh tumor tissues and tissues beside the cancer of the patient, which are reserved in an enteroscope biopsy, are frozen, the sizes of the tissues are 0.2 x 0.2cm, and after the tissues are washed by physiological saline, the tissues are placed into a special tissue freezing tube and transported by liquid nitrogen and stored in a refrigerator at minus 80 ℃.

Example 3

Whole exon sequencing and data analysis thereof

The inventor selects tumor tissue specimens of 8 neoadjuvant chemoradiotherapy sensitive patients and 7 neoadjuvant chemoradiotherapy insensitive patients for whole exon sequencing screening.

The invention adopts a whole exon sequencing method of Thermor Fisher company to screen differential gene mutation sites between a new auxiliary radiotherapy and chemotherapy sensitive patient and new auxiliary radiotherapy and chemotherapy insensitivity, and exon sequencing can detect the mutation frequency of DNA mutation sites of all protein coding gene exon regions. See the ther Fisher website for specific steps. After the obtained original data are calibrated, the inventor selects DNA mutation sites of the difference genes by adopting an Ion Torrent information platform Ion Reporter analysis method, and finally obtains 1385 candidate gene DNA mutation sites for subsequent verification by screening.

Example 4

Targeted sequencing detection of DNA mutation site level of training set specimen gene

Step 1, extracting tumor tissue DNA, and constructing library

1. Extracting the genome DNA of the tumor tissue,

2. utilizes the multiplex PCR primer designed by the inventor to capture the target gene segment,

3.20 μ L reaction: 5 XPPhusion HF buffer 4. mu.L, 10mM dNTPs 0.4. mu.L, Phusion DNA

Polymerase 2U, NIPT-Library F and NIPT-Library R (10. mu.M) were each 1. mu.L, and DNA template was 40 ng.

4. The reaction procedure is as follows: 2min at 98 ℃, 15s at 62 ℃ and 1min at 70 ℃ for 10 cycles, and the extension time at 70 ℃ is 5 min.

5. And removing enzymes, primers and the like added in the reaction process by using a magnetic bead purification method, and purifying the target DNA.

Agilent 2100Bioanalyzer analysis of the library for fragment distribution with a main peak of 140 bp.

7. The concentration of the DNA library was analyzed with a Qubit 2.0.

Qubit concentration of qPCR quantitative standards

Step 2 sequencing on machine

1. And (2) adopting the library constructed in the step (1), performing template preparation, template enrichment and on-machine sequencing, wherein the operation is performed according to the Thermor Fisher website specification, and the used instruments mainly comprise DA8600 and matched instruments. And (3) diluting the obtained library DNA to 100pM respectively according to a qPCR quantitative method and a Qubit quantitative method, uniformly mixing, and then carrying out on-machine sequencing to obtain the numbers of Reads.

2. The loading rate (namely the coverage rate of the DNA positive magnetic beads in the micropores of the chip) of the sequencing result is 92%, the DNA Reads number is 87.12M, and the total Reads number reaches 80M, which indicates that the concentration on-machine sequencing is suitable, and the higher the total Reads is after the sequencing of the standard, the higher the Reads number of each sample is, the higher the Reads number of the standard is, the more the requirement can be met.

Step 3 data analysis

1. The mutation frequency of the genes in the samples was analyzed using the sequencing data mutation analysis software variantCaller supplied by the Theror Fisher.

2. The samples in the training group are arranged from large to small according to the mutation frequency of the sites, the samples are sequentially taken (only once if repeated values exist), the samples are judged to be positive or negative groups according to the values, the sensitivity and the specificity of each value are obtained by combining the established type analysis of the samples, and an ROC Curve (Receiver Operating characterizing Curve) is further drawn. And (3) searching a point which maximizes the value of (sensitivity + specificity)/2, wherein the corresponding expression value is the discretization threshold value of the locus. And further assigning the samples with the values higher or lower than the threshold value as 1 or 0 respectively, so as to realize discretization for further model construction. The DNA discretization threshold value adopted by the invention is used for discretization of corresponding DNA data in the training set and the verification set, so that continuous variables are converted into binary variables. Site mutation frequencies were discretized to 0 or 1 using ROC. The specific method is to select each point, and the point with the optimal classification effect AUC is used as a discretization threshold point to discretize each locus.

3. And (3) screening indexes, namely utilizing chi-square test to reserve sites (P is less than or equal to 0.05) which can be obviously distinguished in radiotherapy and chemotherapy sensitive and non-sensitive patients in chi-square test.

4. And (3) substituting a stepwise method into logistic regression to further screen the sites, and constructing a classifier model by using the logistic regression.

Example 5

Determination of optimal DNA mutation site combinations in training set

The modeling result shows that the combination of 15 DNA mutation sites constructed by the logistic regression model is the optimal combination, and the formula for evaluating the sensitivity of the local advanced rectal cancer patients to radiotherapy and chemotherapy is as follows: logic [ p ═ PCR ] — -2.0298+1.0830 × chr6_33052958_ C _ T-0.4217 × chr9_125316157_ C _ T-1.9672 × chr9_123850770_ G _ a + 123850770 × chr 123850770_ 123850770_ T _ C + 123850770 × chr 123850770_ 123850770_ G _ a + 123850770 × chr 123850770_ GTT _. + 123850770 × chr 123850770_ C _ T-123850770 × chr 123850770_ 123850770_ T _ C + 123850770 × chr 123850770_ a _ G + 123850770 × chr 123850770_ C _ T + 123850770_ C _ T123850770 _ C + 123850770_ C + 123850770 × 123850770_ a _ G _123850770_ a _123850770_ G _123850770_ C _123850770 × 123850770_ 123850770_ G123850770 × 36363672 _123850770 × 36363672 _ G. Wherein, the bit point information in the formula is a numerical value obtained after the DNA mutation frequency of the biopsy tissue of the corresponding patient is discretized.

The combination can distinguish local advanced colorectal cancer radiotherapy and chemotherapy sensitive population in a training group, and has good liver cancer diagnosis effect, which is shown in that AUC of DNA mutation site combination is larger than AUC of CA199 or CEA (P is less than or equal to 0.05) (Table 2).

In the verification group, the combination of the DNA mutation sites is verified to predict patients sensitive to radiotherapy and chemotherapy. The combination of DNA mutation sites established in the training set predicts patients susceptible to radiotherapy and chemotherapy. The combination was still chemoradiotherapeutic sensitive in both the validation internal and external validation groups and had a very good predictive effect in that the AUC of the DNA mutation site combination was greater than that of CA199 or CEA (Table 2). The classification effect of the classifier and clinical indicators is shown in table 2:

TABLE 2

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the foregoing description only for the purpose of illustrating the principles of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the invention as defined by the appended claims, specification, and equivalents thereof.

Sequence listing

<110> Guangzhou Ruiz Biotechnology GmbH

<120> kit for detecting 15 gene mutation sites related to sensitivity of radiotherapy and chemotherapy of rectal cancer and application thereof

<160> 30

<170> SIPOSequenceListing 1.0

<210> 1

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

cactccagca tcactcactt tg 22

<210> 2

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

gggagctgaa ggactactac ct 22

<210> 3

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ctacaccagt tttatcatct tttgctcatc 30

<210> 4

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

caaaaatctg tttctaccac ttaccttgag 30

<210> 5

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ttcagaaaac caagaagctc tacattga 28

<210> 6

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gaaccatttc ttcataagac tcattgcttt 30

<210> 7

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ctgttggaga cactcagcac a 21

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

gcctggttcc gagtccaatc 20

<210> 9

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

cagggaacac ttctacctgg g 21

<210> 10

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

ggctgtccta gcagttgtgg 20

<210> 11

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

gcaggaagga ctgtcctcac t 21

<210> 12

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gagcacatgg aagccttacc t 21

<210> 13

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

catgatctgc gcgttgatgt 20

<210> 14

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

ggctgtgctt tctcgtcttc a 21

<210> 15

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

tctgcatcag ctggactgtt g 21

<210> 16

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

acctagtgaa cagtcagttc ctatatcc 28

<210> 17

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

gattgttcta catggcatat tcacatcc 28

<210> 18

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

ccagtcagct ttagcccaga atg 23

<210> 19

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

cgagtctacc tggtcgtgta gt 22

<210> 20

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

gcggctatga gaggttttcc tc 22

<210> 21

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

tcaccaccac attctgcatg tac 23

<210> 22

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

tgctgtgaca gaaagtgagt gag 23

<210> 23

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

ggccaagttt ctcagctgtt ga 22

<210> 24

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

gaacccatca ggaaagttcc agtt 24

<210> 25

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

ggaaccattc tggtggtcat ga 22

<210> 26

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

agagagacaa caaggaggtg tga 23

<210> 27

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

tgagcaagtt ttaatgacgt tgttacattc 30

<210> 28

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

gctctcattc ctaactgaac atcagaaaat 30

<210> 29

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

aagaagccga agaatcccag tac 23

<210> 30

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

ccaggatgat cctgtctaac acca 24

Claims (5)

1. A kit for detecting 15 gene mutation sites related to the sensitivity of the radiotherapy and chemotherapy of rectal cancer is characterized in that: the kit is a detection kit based on a high-throughput DNA sequencing technology and is used for detecting 15 gene DNA mutation sites related to the sensitivity of the rectal cancer chemoradiotherapy, the kit comprises a specific primer for detecting the gene mutation sites, the nucleotide sequence of an upstream primer of the specific primer is shown as SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19, SEQ ID NO.21, SEQ ID NO.23, SEQ ID NO.25, SEQ ID NO.27 and SEQ ID NO.29, the nucleotide sequence of a downstream primer of the specific primer is shown as SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.26, SEQ ID NO.28, and SEQ ID NO. 30.

2. The kit of claim 1, wherein: the kit also comprises a reagent for detecting the level of the gene DNA mutation site in tumor tissues.

3. The kit of claim 2, wherein: the reagent for detecting the level of the gene DNA mutation site in the tumor tissue is a second-generation sequencing reagent.

4. Use of the kit of claim 1 for the preparation of a detection reagent for predicting neoadjuvant chemoradiotherapy sensitivity of locally advanced rectal cancer;

the locally advanced rectal cancer is locally advanced rectal cancer in II stage and III stage recommended by NCCN guidelines and applied by new auxiliary radiotherapy and chemotherapy;

the gene DNA mutation site has higher gene mutation frequency in tumor tissues of patients sensitive to radiotherapy and chemotherapy than in patients insensitive to radiotherapy and chemotherapy.

5. Use according to claim 4, characterized in that: in the practical prediction process of the application, a prediction model for predicting the sensitivity of the local advanced colorectal cancer neoadjuvant chemoradiotherapy, which consists of 15 gene DNA mutation sites, is established according to the following steps:

(1) based on a second-generation sequencing method, screening candidate gene DNA mutation sites of a new auxiliary radiotherapy and chemotherapy sensitive patient and a new auxiliary radiotherapy and chemotherapy insensitive patient of the local late rectal cancer by whole exon sequencing;

(2) target sequencing verifies candidate gene DNA mutation sites;

(3) establishing a gene DNA mutation site combination capable of distinguishing patients with the locally advanced rectal cancer sensitive to the neoadjuvant chemoradiotherapy in a training group consisting of patients with the neoadjuvant chemoradiotherapy sensitivity and patients with the neoadjuvant chemoradiotherapy insensitivity;

(4) verifying the combination of the gene DNA mutation sites established in the step (3) in two independent verification groups to predict the effect of the combination of the neoadjuvant chemoradiotherapy sensitivity of the locally advanced rectal cancer;

(5) and (4) analyzing the combination of the DNA mutation sites of the genes established in the step (3) to predict the effect of the combination of the neoadjuvant chemoradiotherapy sensitivity of the locally advanced rectal cancer by adopting a logistic regression model.