WO2016106645A1 - Primer for detecting colorectal cancer drug-related gene mutation and detection method - Google Patents

Abstract

Disclosed are a primer for detecting colorectal cancer drug-related gene mutation, a detection method and an application. The primer of the present application comprises 25 specific primer sets and 1 general primer set. The specific primer sets comprise specific primers respectively for detecting 27 mutation sites of 15 colorectal cancer drug-related genes, and the general primer set comprises outer primers commonly used for amplified products of the 25 specific primer sets. The 25 specific primer sets are sequentially shown as Seq ID NO. 1 to Seq ID NO. 50, wherein Seq ID NO. 1 and Seq ID NO. 2 form one set, Seq ID NO. 3 and Seq ID NO. 4 form one set, and so on; and the sequences of the general primer set are shown as Seq ID NO. 51 and Seq ID NO. 52.

Description

Primer and detection method for detecting gene mutation related to colorectal cancer Technical field

The present application relates to the field of gene detection, and in particular to a primer for detecting mutation of a gene related to colorectal cancer, and a detection method thereof.

Background technique

Cancer, also known as a malignant tumor, is a disease caused by a disorder that controls the growth and proliferation of cells. In today's society, cancer has become a major threat to human health. The World Health Organization (WHO) stated in the Global Cancer Report 2014 that the world will face a cancer outbreak. The report predicts that global cancer cases will show rapid growth, from 14 million in 2012 to 19 million in 2025 and 24 million by 2035. The report also showed that in 2007, China added 3.07 million cancer patients and caused about 2.2 million deaths, accounting for 21.9% and 26.8% of the global total, respectively. From the perspective of disease types, the first place in the country for malignant tumors is lung cancer, followed by gastric cancer, colorectal cancer, liver cancer and esophageal cancer. The top 10 malignant tumors accounted for 76.39% of all malignant tumors. The first death of malignant tumors in the country is still lung cancer, followed by liver cancer, stomach cancer, esophageal cancer and colorectal cancer. The top 10 malignant tumors account for 84.27% of all malignant tumors.

Colorectal cancer, also known as colorectal cancer, is the common site of the rectum and the rectum and the sigmoid colon. It is a common malignant tumor of the digestive tract. It is often overlooked because the early symptoms are mild or inconspicuous. According to the American Cancer Society, in 2014, there were 136,830 new cases of colorectal cancer in the United States, with 50,310 deaths, ranking second in cancer deaths. The incidence rate in China has reached 46.8/100,000, second only to lung cancer and stomach cancer. Colorectal cancer is classified according to histopathology and can be divided into adenocarcinoma, mucinous carcinoma and undifferentiated carcinoma. Adenocarcinoma is the most common.

At present, the main treatment methods for colorectal cancer include surgery, radiotherapy, chemotherapy and molecular targeted drug therapy. Commonly used chemotherapy drugs include fluorouracil, capecitabine, platinum and irinotecan, which play an important role in the treatment of colorectal cancer. But clinical results show that not every patient can benefit from a chemotherapy regimen. The efficacy of chemotherapy is related to the patient's drug sensitivity, drug resistance and the toxicity of the drug itself. In recent years, a large number of clinical studies have found that each chemotherapeutic drug has its associated measurable therapeutic related genes, ie, biomarkers, and the efficacy is related to their expression levels. For example, the genetic polymorphisms of XRCC1, ERCC1 and GSTP1 can be used to predict the clinical efficacy of platinum drugs.

In recent years, molecular targeted drugs, such as cetuximab, panitumumab, bevacizumab, etc., have been successively researched and developed, making colorectal cancer have made great progress in drug treatment. A large number of clinical studies have shown that the efficacy of targeted drugs is directly related to the state of the drug target genes. National Cancer Comprehensive Therapy Alliance (NCCN) The Clinical Practice Guidelines for Colorectal Cancer clearly states: (1) All patients with metastatic colorectal cancer should be tested for KRAS gene status; (2) Only KRAS wild-type patients are recommended to receive EGFR inhibitors such as Erbitux and Treatment of panitumumab. It can be seen that the detection of drug-related genes is crucial for the analysis of the effects of drug selection and drug use.

A variety of molecular detection products exist on the market, such as detection kits, but the existing detection kits are expensive, time consuming, and the number of drug-related genes detected at the same time is limited. In practice, tumor patients often have more than one type of gene mutation, and in order to determine which drug is most suitable for patients, multiple detection products are often used, and multiple detections for different gene mutations not only greatly increase the cost, but also cost. A lot of valuable treatment time.

Summary of the invention

The purpose of the present application is to provide a novel primer for detecting a mutation in a drug related to colorectal cancer, and a detection method and application based on the primer.

This application uses the following technical solutions:

In one aspect, the present application discloses a primer for detecting a mutation related to a colorectal cancer drug, the primer comprising 25 pairs of a specific primer set and a pair of universal primer sets, and 25 pairs of specific primer sets for respectively detecting 15 colorectal cancer drugs. The specific primer set of 27 mutation sites of the related gene, the universal primer set is a common primer for the PCR amplification product of 25 pairs of specific primer sets; the primer sequence of 25 pairs of specific primer set is sequentially as Seq ID No. 1 to Seq ID No. 50, wherein Seq ID No. 1 and Seq ID No. 2 are a primer set, Seq ID No. 3 and Seq ID No. 4 are a primer set, and so on; a universal primer set The sequences are shown as Seq ID No. 51 and Seq ID No. 52.

It should be noted that the primers of the present application are designed based on the principle of PCR amplification of the four-primer method. Therefore, among the 25 pairs of specific primer sets, both the upstream primer and the downstream primer have a universal sequence at the 5' end. , such as the sequence shown by Seq ID No. 53 and Seq ID No. 54, the universal sequence of the upstream primer corresponds to the upstream primer of the outer primer, and the universal sequence of the downstream primer corresponds to the downstream primer of the outer primer; it is understood that these universal sequences are In order to facilitate the amplification of the outer primer, it has nothing to do with the target sequence. Therefore, in the case of ensuring that the sequence of about 20 bp of the 3' end of each primer in the pair of specific primer sets is unchanged, the PCR amplification is not affected. In the case of efficiency and accuracy, base deletion or substitution at the 5' end of each primer, or overall replacement of the sequence portions shown in Seq ID No. 53 and Seq ID No. 54 are all in the present application. Within the scope of protection.

It should also be noted that the universal primer, that is, the outer primer, is a PCR amplification target of 25 pairs of specific primer sets, which is realized by 25 universal sequences added to the 5' end of the specific primer set; Therefore, the 3' end of the upstream and downstream primers of the outer primer are respectively upstream and downstream primers with 25 pairs of specific primer sets. The universal sequence added at the 5' end corresponds to the sequence shown in Seq ID No. 53 and Seq ID No. 54; whereas in the outer primer, whether it is the upstream primer or the downstream primer, the 5' end does not affect In the case of PCR amplification, base addition or modification can be performed. In one implementation of the present application, an index sequence of about 10 bp is added to the 5' end of the upstream primer of the external primer, and at the same time, in order to facilitate sequencing, A linker is added to the upstream primer of the outer primer and the 5' end of the downstream primer to facilitate subsequent sequencing detection; it is understood that both the index sequence and the linker are used to facilitate subsequent analysis of the final PCR product of the four-primer method. The sequence can be replaced according to different laboratory conditions, in the case of ensuring that the 3' end of the upstream primer and the downstream primer of the outer primer correspond to the universal sequence added by the 5' end of the upstream primer of the 25 pairs of the specific primer set, In the case of PCR amplification of the outer primer, the base primer is deleted at the 5' end of the upstream primer or the downstream primer of the outer primer, especially for the index sequence or the linker. Deletion or replacement overall, are within the scope of the present disclosure. In addition, it can be understood that if only for the amplification of PCR amplification products of specific primers, regardless of the problem of building a library or using different index sequences for different chips, it is entirely possible to use only Seq ID No. 53 and Seq ID No. The sequence of the forward and reverse primers shown in .54 does not require the design of more complex primers shown in Seq ID No. 51 and Seq ID No. 52.

In the 25 pairs of specific primer sets of the present application, a part of the specific primer set contains a plurality of mutation sites, and therefore, 25 pairs of specific primer sets can be used to detect 27 mutation sites.

Based on the primers of the present application, the present application also discloses a gene chip for detecting mutations in a drug related to colorectal cancer, wherein the gene chip contains at least 25 detection sites, and 25 detection sites respectively contain The 25 pairs of specific primer sets of the present application, and a universal primer set is also mixed in each detection site.

It should be noted that the gene chip of the present application actually adds a specific primer set and a universal primer set to each detection site, and each site corresponds to a specific primer set; it can be understood that since this application designs 25 pairs Specific primer sets, therefore, design at least 25 detection sites, but in practice testing, usually each test needs to be repeated 10 times, that is, the actual gene chip contains 250 detection sites; in addition, It is necessary to design some control detection sites, so the number of detection sites of the gene chip can be designed according to the actual test conditions and purposes, and is not specifically limited herein.

Preferably, in the gene chip of the present application, the spotting amount of the specific primer set in the detection site is 5-15×10 ^-6 nmol, and the spot amount of the universal primer set is 10-20×10 ^-6 nmol. It should be noted that the amount of spotting is determined based on the optimized amount of each primer in the PCR amplification of the four primers; the spot amount of the primer set contains an equal amount of the upstream primer and the downstream primer.

Another aspect of the present application discloses a method for detecting a mutation associated with a drug for colorectal cancer, the method comprising: adding a DNA sample extracted from a subject to a detection site of a gene chip of the present application, PCR amplification is performed, and then the PCR amplification product is detected to obtain mutation information.

Preferably, the PCR amplification product is detected, specifically comprising: purifying and recovering the PCR amplification product, and then sequencing the recovered PCR product, and determining the mutation information according to the sequencing result.

It should be noted that there are many ways to detect the PCR product. In order to ensure accuracy, the present invention preferably uses sequencing to detect the PCR product.

Preferably, the purification and recovery of the PCR amplification product is specifically carried out by a magnetic bead method.

Based on the primers and gene chips of the present application, the present application provides a corresponding kit for detecting a mutation related to colorectal cancer drug use; the kit can directly use the primer or gene chip of the present application. It can be understood that the gene chip is only a relatively simple use method of the primer of the present application; in the absence of the gene chip and related instruments, the primer of the present application can also be directly used for testing, and only a conventional PCR instrument can be used. achieve.

The other side of the application also discloses the use of the primer or gene chip of the present application in the preparation of a reagent or device for detecting a mutation in a drug associated with colorectal cancer.

It can be understood that the primer or gene chip of the present application can be made into a kit for convenient use; it can also be integrated into some automatic platform or instrument for detecting mutation of gene related to colorectal cancer, especially for colorectal. Detection of mutations in cancer-related genes; or as a detachable replacement module in an automated platform or instrumentation for the detection of mutations in genes associated with colorectal cancer.

The beneficial effects of the present application are:

In this application, the detection primers of 15 gene 27 mutation sites related to colorectal cancer drugs are designed as a whole, and based on this, gene chips and kits are developed, and 27 mutation sites can be realized at one time. The detection provides an important analytical basis for the effective use of therapeutic drugs. Compared with the existing mutation-by-mutation sites, the primers of the present application can save a lot of time and cost, and lay a foundation for efficiently and quickly determining the best therapeutic drug.

DRAWINGS

Figure 1 is a graph showing the results of agarose gel electrophoresis of genomic DNA in the examples of the present application;

Figure 2 is an agarose gel electrophoresis pattern of genomic DNA in another embodiment of the present application.

detailed description

In order to realize the combined detection of multiple drug-related genes supported by clinical experiments, the present application develops a method for detecting multiple drug-related genes based on second-generation sequencing technology, and the method is directed to WaferGen's SmartChip TE platform, based on the BGISEQ-100 sequencer of Huada Gene, designed specific primers, which can simultaneously detect 27 mutation sites of 15 genes, which can be completed within 48 hours. Automated, only a small amount of manual operation, easy to operate, wide detection spectrum, easy to arrange and so on. Under such a research framework, this application proposes a sequence of Seq ID No.1 to Seq ID No.50, which detects 27 mutation sites of 15 colorectal cancer drug-related genes, respectively, and 25 pairs of specificities. Primer set; and a pair of universal primer sets corresponding to the sequences indicated by Seq ID No. 51 and Seq ID No. 52; for the purpose of achieving combined detection of the above plurality of drug-related genes. Based on the above 25 pairs of specific primer sets and one pair of universal primer sets, the present application further makes them into gene chips to facilitate detection and use, and at the same time, is more advantageous for automation.

It should be noted that, in the 25 pairs of specific primer sets of the present application, a universal sequence added at the 5' end thereof, that is, a sequence such as Seq ID No. 53 and Seq ID No. 54 is used according to the present application. The sequencing platform is added. It can be understood that if other sequencing platforms, such as Roche/454FLX, Illumina/Solexa Genome Analyzer, Applied Biosystems SOLID system, Life Technologies' new generation Ion Proton sequencer, and Hiseq series sequencer, are used, another segment and their respective platforms are required. The corresponding universal sequence, that is, the entire sequence of the sequence shown in Seq ID No. 53 and Seq ID No. 54 is required; this is only a routine replacement of the sequencing linker, and 3 of each primer in the specific primer set is guaranteed. In the case where the sequence of about 20 bp is unchanged, or in the case of PCR amplification of the 3' end of each primer in the 25-pair specific primer set, it is within the scope of protection of the present application. It should be added that if the sequence parts shown in Seq ID No. 53 and Seq ID No. 54 are replaced as a whole, the corresponding part of the universal primer set also needs to be changed in applicability; similarly, in the universal primer set The index sequence and the linker added in the 5' segment of the universal primer set also need to be changed according to the sequencing platform. For details, refer to the instructions for use of each sequencing platform, which will not be described here.

The present application will be further described in detail below by way of specific embodiments and the accompanying drawings. The following examples are only intended to further illustrate the present application and are not to be construed as limiting the invention.

Example

1. Design specific primers for the mutation site

In this case, we selected the 17 genes of the 17 genes associated with colorectal cancer. These sites are related to the drug, and all of them have achieved remarkable results in clinical trials of more than 100 people, and have been obtained by large medical institutions and experts. accepted. In this example, Primer3 and Primer premier5 were used to design primers for these sites. In order to be more suitable for BGISEQ100 sequencing, the amplification products were limited to 200 bp when designing primers, and then aligned using Prime-Blast to obtain 25 pairs of specific primers. group. In combination with the four-primer method and subsequent sequencing, 1 external primer was designed, and 5' of the upstream and downstream primers of the 25 pairs of specific primer sets. The universal sequence corresponding to the external primer was added to the end, and the sequence of the final 25 pairs of specific primer set and 1 external primer is shown in Table 1.

Table 1 Detection of drug-related gene mutation detection primers

2. Custom chip

In order to simplify the steps and improve the efficiency, this example uses a four-primer scheme, in which a universal sequence is added to the outside of the conventional primer to form an internal primer, and then an external primer, that is, a universal sequence primer, is used for PCR reaction, and an index is added to the external primer. The linker sequence can directly index and sequence the product during the PCR process, eliminating the need to interrupt the database. In order to achieve this, this example uses the following scheme: 24 sets of primers and external primers are arranged on the chip, each group is repeated 10 times, and each set is sprayed with a set of inner primers and outer primers for detecting one site. Each outer primer was spotted at 8×10 ^-6 nmol, the inner primer was spotted at 5×10 ^-6 nmol, all the inner primers shared the same external primer, and the “GTATCGTCGT” in the forward external primer sequence was a 10-bit index sequence, each chip Different indexes are needed to be merged on the machine during sequencing. For the index sequence, see the instructions for use of BGI-SE100. Commissioned by Wafergen to make Smartchip TE chips according to the above scheme.

3. Extract DNA from sample tissue

In this example, DNA was extracted using the Qiagen DNA Kit (DNeasy Blood & Tisue Kit). After extraction, it was detected by agarose gel electrophoresis, with a bright band at 23,000 bp and no diffuse band at around 250 bp. The concentration was then checked using a Qubit fluorometer to ensure a concentration of 10-100 ng/μL.

4. Prepare the PCR system for target region amplification

Using the sample DNA that passed the quality control, take 350 ng of DNA as a template, and use KAPA's kit (KAPA2G Robust Hotstar Readymix) to configure the mixture. The system is as follows: 2xPCR Master Mix 175 μL, DNA 350 ng, and add DEPC water to 350 μL to obtain a PCR reaction solution.

The mixed PCR reaction solution was dispensed into the customized SmartChip TE chip using the SmartChip TE Nanodispenser. After the liquid separation was completed, the surface of the chip was sealed with a matching membrane and centrifuged to prevent the liquid from overflowing. The PCR reaction was carried out on a chip using a SmartChip TE PCR cycler, and the reaction conditions are shown in Table 2.

Table 2 Chip PCR reaction conditions

5. Collect PCR products and purify:

After completion of the reaction, the product was collected using an Eppendorf plate centrifuge and purified using Agencourt Ampure XP magnetic beads to remove small fragments of non-specific amplified PCR products and primer dimers.

6. Use BGISEQ-100 for sequencing on the machine:

The purified DNA was sequenced on a BGISEQ-100 sequencer. See the BGISEQ-100 product description for specific procedures.

7. Analyze the data to report

After the sequencing is completed, the BGI-PCT-V2.0 automated analysis process is used to analyze the data of the lower machine, and the pre-set mutation sites are detected and interpreted to generate an interpretation report.

Test 1

Using this example, cell lines containing specific mutations were tested and DNA extracts from Horizon's four cell lines were purchased to assess the sensitivity and specificity of this assay. In these 4 cell lines, the mutation frequency of 4 sites was determined by Digital Droplet PCR and Sanger sequencing, and these 4 sites were all in the site to be tested of the present application. Each cell line has 2 positive sites and 2 negative sites. Using the specific primers and detection methods of this example, the drug-related genes in the cells were detected.

(1) DNA extraction

DNA extraction was carried out in accordance with step 3 above. The results of agarose gel electrophoresis of the extracted DNA samples are shown in Fig. 1. From left to right in the figure, lane 1 is λ-HindIII digest DNAMarker of Takara, lane 6 is DL2000 DNA Marker of Takara, and lanes 2 to 5 are Horizon. The DNA of the company's four cell lines showed that all samples had a bright band at 23,000 bp and no diffuse band around 250 bp, which was in line with expectations. The concentration was then measured using a Qubit fluorometer at an average concentration of 50 ng/μL, which was prepared at 10 ng/μL for subsequent use.

(2) Test the drug-related genes according to steps 4-7 above.

After the sequencing is completed, the BGI-PCT-V2.0 automated analysis process is used to analyze the data of the lower machine, and the pre-set mutation sites are detected and interpreted to generate an interpretation report. For the use of the process, refer to the instructions for use. The results showed that among the 16 loci of the four cell lines, 8 were positive and 8 were negative, which was consistent with the results of Digital Droplet PCR and Sanger sequencing, and the agreement was 100%.

Test 2

The primers and methods of this example were used to detect the drug-related genes in colorectal cancer patients from the Huada Gene Sample Center. At the same time, the same DNA samples were tested using sanger sequencing and the most widely used second-generation sequencing platform, the Illumina Hiseq platform, as a control.

(1) DNA extraction

Follow the above procedure 3 to extract DNA from tumor tissue using the Qiagen DNA Kit (DNeasy Blood & Tisue Kit). After extraction, it was detected by agarose gel electrophoresis. There was a bright band at 23,000 bp, and there was no diffused band around 250 bp, as shown in Figure 2, from left to right in the figure, and Lane 1 was from Takara. λ-Hind III digest DNA Marker, Lane 3 is the DL2000 DNA Marker of Takara, and Lane 2 is the DNA extracted from the tumor tissue of the patient. Then, it was subjected to concentration detection using a Qubit fluorescence meter at a concentration of 93 ng/μL, which was formulated into 10 ng/μL.

(2) Test the drug-related genes according to steps 4-7 above.

After the sequencing is completed, the BGI-PCT-V2.0 automated analysis process is used to analyze the data of the lower machine, and the pre-set mutation sites are detected and interpreted to generate an interpretation report. The results showed that a total of 5 of the 27 sites in the sample were mutated, consistent with the results of the sanger sequencing method.

Based on the above research, this example also made the primer and the gene chip into kits for convenient use. Using the method of this example, 27 mutation sites can be detected at one time, which greatly saves the detection time and cost, and provides an important basis for the treatment of colorectal cancer, and lays a foundation for the subsequent research of targeted drugs.

The above content is a further detailed description of the present application in conjunction with the specific embodiments, and the specific implementation of the present application is not limited to the description. It will be apparent to those skilled in the art that the present invention can be made in the form of the present invention without departing from the scope of the present invention.

Claims (10)

1. A primer for detecting a mutation related to a colorectal cancer drug-related gene, characterized in that the primer comprises 25 pairs of a specific primer set and a pair of universal primer sets, and the 25 pairs of specific primer sets respectively detect 15 colorectal cancers a specific primer set of 27 mutation sites of a drug-related gene, wherein the universal primer set is a universal primer common to PCR amplification products of 25 pairs of specific primer sets; and the primer sequences of the 25 pairs of specific primer sets are sequentially ordered as Seq ID No. 1 to Seq ID No. 50, wherein Seq ID No. 1 and Seq ID No. 2 are a primer set, Seq ID No. 3 and Seq ID No. 4 are a primer set, and so on. The sequence of the universal primer set is shown as Seq ID No. 51 and Seq ID No. 52.
2. The use of the primer according to claim 1 for the preparation of a reagent or device for detecting a mutation associated with a drug for colorectal cancer.
3. A gene chip for detecting a mutation related to a drug for colorectal cancer, characterized in that the gene chip contains at least 25 detection sites, and each of the 25 detection sites respectively contains the 25 pairs of specificity according to claim 1. A primer set, and the universal primer set of claim 1 is also mixed in each detection site.
4. The gene chip according to claim 3, wherein in the detection site, the spotting amount of the specific primer set is 5-15×10 ^-6 nmol, and the spot amount of the universal primer set is 10-20×. 10 ^-6 nmol.
5. The use of the gene chip according to claim 3 or 4 for the preparation of a reagent or device for detecting a mutation associated with a drug for colorectal cancer.
6. A method for detecting a mutation related to a drug for colorectal cancer, characterized in that the method comprises: adding a DNA sample extracted from a subject to a detection site of the gene chip according to claim 3 or 4, and performing PCR Amplification, and then PCR amplification products were detected to obtain mutation information.
7. The method according to claim 6, wherein the detecting the PCR amplification product comprises: purifying and recovering the PCR amplification product, and then sequencing the recovered PCR product, and determining the mutation information according to the sequencing result. .
8. The method according to claim 7, wherein the PCR amplification product is purified and recovered by using magnetic beads.
9. A kit for detecting a mutation related to a drug for colorectal cancer, characterized in that the kit contains the primer of claim 1.
10. A kit for detecting a mutation related to a drug for colorectal cancer, characterized in that the kit contains the gene chip of claim 3 or 4.

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