CN111500688A - Method for synchronously detecting mutation of No.2, 3 and 4 exon genes of NRAS gene - Google Patents

Abstract

The invention provides a primer group and a detection method for synchronously detecting the gene mutation of No.2, 3 and 4 exons of NRAS genes, which are beneficial to improving the detection flux. The invention provides a primer group for synchronously detecting exon gene mutation No.2, 3 and 4 of NRAS gene, which comprises at least two pairs of the following three pairs of primer pairs: the nucleotide sequence of the upstream primer of the first group of primer pairs is shown by SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown by SEQ ID NO. 2; the nucleotide sequence of the upstream primer of the second group of primer pairs is shown by SEQ ID NO.3, and the nucleotide sequence of the downstream primer is shown by SEQ ID NO. 4; the nucleotide sequence of the upstream primer of the third group of primer pairs is shown by SEQ ID NO.5, and the nucleotide sequence of the downstream primer is shown by SEQ ID NO. 6. The mutation of No.2, 3 and 4 exon genes of NRAS gene can be detected by one reaction. Therefore, more than 90 samples can be detected simultaneously, the detection efficiency is improved, and the cost is greatly saved.

Description

Method for synchronously detecting mutation of No.2, 3 and 4 exon genes of NRAS gene

Technical Field

The invention relates to the technical field of gene detection, in particular to a primer group and a detection method for synchronously detecting the gene mutation of No.2, 3 and 4 exons of NRAS genes.

Background

Neuroblastoma rat sarcoma (NRAS) gene is 85kb in length, located on chromosome 1, and encodes a protein of 495 amino acids. Members belonging to the RAS family with gtpase activity are key regulators in cell proliferation and differentiation. The common mutation sites of NRAS are positioned on the No.2, No.3 and No.4 exons of the gene.

A number of metastatic colon cancer studies indicate that NRAS mutations are significantly associated with short overall Survival and their PFS (Progression-Free-Survival, Progression-Free Survival) tends to be shortened. Clinical research shows that the metastatic colorectal patients with NRAS gene mutation have drug resistance to EGFR targeted therapeutic drugs such as cetuximab and panitumumab, and whether the EGFR targeted therapeutic drugs are used as clinical therapeutic measures or not is determined according to detection results. Currently, NRAS gene detection is classified as a routine examination which is necessary before the medical treatment of colorectal cancer patients in European and American areas. NRAS gene mutation detection has been listed as a necessary clinical drug inspection item by the national cancer integration network (NCCN) clinical practice guide for NCCN colon cancer and the NCCN clinical practice guide for rectal cancer.

Data from the FIRE-3 study published at the European cancer society in 2013 show that cetuximab + FO L FIR (5-fluorouracil (5-FU)/calcium folinate (L V) + irinotecan) was used in patients with metastatic colorectal cancer in RAS wild-type (meaning no mutations in NRAS and KRAS exons 2, 3, 4) and that median Overall Survival (OS) was significantly extended by 7.5 months compared to bevacizumab + FO L FIR, whereas in RAS mutant (meaning mutations in at least one of the genes in NRAS and KRAS exons 2, 3, 4) patients the median survival (OS) was similar in both groups.2018 JSMO clinical guidelines molecular testing for colorectal cancer treatment (3 rd edition) several randomized clinical studies showed that anti-EGFR monoclonal antibody treatment did not benefit RAS mutant patients.

PCR (Polymerase Chain Reaction) has been widely used in medicine, genetics, microbiology, and even throughout life sciences. Currently, PCR detection tests can be designed for exons 2, 3, and 4 of NRAS gene respectively to detect the mutation of gene. Since each PCR reaction is directed to only one exon, the detection throughput is low. Multiplex PCR is a novel amplification technique developed on the basis of conventional PCR, i.e., two or more pairs of primers can be added into a reaction system to simultaneously amplify a plurality of nucleic acid fragments. The multiplex PCR has important application in microbe, genetic disease and tumor pharmacogenomics.

Disclosure of Invention

In order to solve the above-mentioned drawbacks, the present invention provides a primer set and a detection method for synchronously detecting mutations in exon genes 2, 3, and 4 of NRAS gene, which are beneficial to increase of detection throughput.

In order to achieve the purpose, the invention is realized by the following technical scheme:

firstly, a primer group for synchronously detecting the mutation of No.2, 3 and 4 exon genes of NRAS genes is provided, which comprises at least two pairs of the following three pairs of primer pairs: the nucleotide sequence of the upstream primer of the first group of primer pairs is shown by SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown by SEQ ID NO. 2; the nucleotide sequence of the upstream primer of the second group of primer pairs is shown by SEQ ID NO.3, and the nucleotide sequence of the downstream primer is shown by SEQ ID NO. 4; the nucleotide sequence of the upstream primer of the third group of primer pairs is shown by SEQ ID NO.5, and the nucleotide sequence of the downstream primer is shown by SEQ ID NO. 6.

The primer group is specially designed for specific amplification of No.2, 3 and 4 exon genes of NRAS genes, wherein the first group of primer pairs are primer pairs for detecting No.2 exons of the NRAS genes; the second group of primer pairs are primer pairs for detecting No.3 exon of NRAS gene; the third group of primer pairs is a primer pair for detecting exon 4 of the NRAS gene. The multiple PCR technology can simultaneously, synchronously, efficiently, specifically and accurately amplify the specific gene segment of the gene, and detect the gene mutation condition through sequencing.

The primer group preferably comprises three groups of primer pairs which are used simultaneously so as to improve the detection flux to the maximum extent, and any two groups of primer pairs can be selected according to the situation and can be used for carrying out targeted amplification reaction on a certain exon gene; when the three primer pairs are used simultaneously to perform multiplex PCR amplification reactions, the No.2, 3 and 4 exon genes of the NRAS gene are amplified simultaneously in one amplification system, i.e., 3 exons containing any mutation are amplified simultaneously.

In general, when the exon No.2 of the NRAS gene is amplified by using an upstream primer SEQ ID NO.1 and a downstream primer SEQ ID NO.2, the fragment length of a corresponding amplification product is 472 bp; when the upstream primer SEQ ID NO.3 and the downstream primer SEQ ID NO.4 are used for amplifying the No.3 exon of the NRAS gene, the segment length of the corresponding amplification product is 566 bp; when the exon 4 of NRAS gene is amplified by using the upstream primer SEQ ID NO.5 and the downstream primer SEQ ID NO.6, the length of the fragment of the corresponding amplification product is 332 bp. The DNA fragment was then recovered by cutting and sequenced.

Multiple tests prove that the primer pair provided by the invention has better specificity and amplification accuracy, can be applied to preparing a kit for synchronously detecting the mutation of No.2, 3 and 4 exon genes of the NRAS gene, can be prepared into a finished kit, and can quickly and accurately obtain the mutation results of the No.2, 3 and 4 exon genes of the NRAS gene after sample collection tests.

The kit mainly comprises the primer group provided by the invention, wherein the final concentration of a single primer is preferably 20-400 nM. Other PCR reagents may be selected according to conventional techniques, for example, a preferred embodiment further comprises a DNA polymerase, a PCR buffer, a mixture of 4 kinds of dNTPs (deoxyribose nucleotide triphosphates), and ultrapure water. Wherein the amount of DNA polymerase is 0.5-5U, and the final concentration of each dNTP is 50-500. mu.M.

The DNA polymerase can be Taq, KOD FX or other DNA polymerase, so the PCR buffer is a concentrated buffer corresponding to the selected DNA polymerase, and the specific concentration degree can be 2 ×, 5 × or 10 ×.

For example, when KOD FX is selected as the DNA polymerase and 2 × is selected as the buffer concentrate, the amount of each component in the PCR reaction system may be 0.5-2. mu.l of the DNA polymerase, 18-30. mu.l of the PCR buffer, 2-12. mu.l of each dNTP mixture, 2-10. mu.l of 3 primer pairs, 5-1000 ng of DNA, and an amount of ultrapure water to replenish water to 50. mu.l, and may be other volume sizes prepared in the same ratio.

When the finished product kit is manufactured, a reagent for extracting the DNA of a sample or a professional DNA extraction kit can be selectively configured according to actual needs; the DNA of the sample to be detected can be obtained more conveniently and quickly, and the convenience and the rapidity of the detection finished product kit are enhanced. The sample to be tested can be any blood, serum, plasma, cell, tissue or buccal swab sample containing human DNA.

On the basis of the primer set provided by the invention, the invention further provides a detection method for synchronously detecting the gene mutation of No.2, 3 and 4 exons of the NRAS gene, and the primer pair is adopted for PCR detection.

According to a preferred embodiment, the detection method comprises the following specific steps:

(1) extracting DNA from a sample to be detected as an amplification template;

(2) configuring a multiplex PCR reaction system comprising the primer set and the amplification template;

(3) performing multiple PCR amplification reaction on the multiple PCR reaction system to obtain a PCR product;

(4) and (3) determining the exon gene mutations of No.2, 3 and 4 of the NRAS gene according to the PCR product.

Wherein the most preferable reaction conditions for the PCR amplification reaction are: 94-98 ℃: 1-10 min; 94-98 ℃: 5-60 s, 50-68 ℃: 10-120 s, 68-72 ℃: 20 s-4 min, and 25-45 cycles in total; 68-72 ℃: 0-30 min.

On the basis of the above method and conditions, the detection method of the present invention can simultaneously and synchronously obtain the gene mutations of exons 2, 3 and 4 of NRAS gene of a sample to be detected rapidly, effectively and conveniently, and can be used for non-diagnostic purposes.

According to a preferred embodiment of the present invention, the step (4) includes: electrophoretically detecting the PCR product to verify the amplified fragment size of the PCR product; and after the PCR product is verified to be correct, carrying out sequence determination on the PCR product so as to obtain the mutation conditions of No.2, 3 and 4 exon genes of the NRAS gene of the sample to be detected. In detail, the PCR amplified fragment can be detected by agarose gel electrophoresis or polyacrylamide gel electrophoresis.

Compared with the prior art, the invention at least has the following beneficial effects: (1) and (3) improving the detection flux: each reaction of the ordinary PCR only aims at one exon, while the multiplex PCR can simultaneously detect 3 exons, and the mutation of No.2, 3 and 4 exon genes of the NRAS gene can be detected through one reaction. Therefore, more than 90 samples can be detected simultaneously, the detection efficiency is improved, and the cost is greatly saved. (2) The cost is reduced: the invention can reduce the PCR reaction system from 3 systems/procedures to 1 system/procedure, thereby reducing the use amount of reagents and consumables such as DNA polymerase, dNTP and the like and greatly reducing the detection cost.

Drawings

FIG. 1 shows the result of agarose gel electrophoresis detection according to an embodiment of the present invention;

FIG. 2 shows a portion of the PCR product sequence determination result for a mutation site according to an embodiment of the present invention.

Detailed Description

To further illustrate the present invention, reference is made to the following examples. Specifically, the reagents used in the implementation of the invention are all commercial products, and the databases used in the implementation of the invention are all public online databases. The following examples are illustrative only and are not to be construed as limiting the invention.

Example 1

Designing and synthesizing a primer group, comprising the following steps:

step 1.1: based on the No.2, 3 and 4 exons of the NRAS gene, an upstream primer and a downstream primer for specifically amplifying the No.2, 3 and 4 exon gene mutation regions of the NRAS gene and corresponding sequencing primers are designed.

For designing the primers, Primer Quest and Primer Premier 5.0 are adopted to design the primers and carry out dimer and stem-loop mismatch analysis, the primers are designed at two ends containing mutation sites, and the annealing temperatures of 3 pairs of primers are basically kept consistent.

The primer sets provided in this example cover all gene mutations in exons 2, 3, and 4 of the NRAS gene. Since the small sequence change can cause the primer amplification efficiency to be obviously reduced and the specificity to be poor, multiple PCR primer sets are respectively designed aiming at different sites/exons, and after the screening of a pre-experiment, the primer sets with the best amplification effect are selected according to the length of product fragments and the inclusion conditions of the sites/exons, wherein the primer sets are shown in the following table 1.

TABLE 1

Step 1.2: the primer set designed in 1.1 was synthesized.

Example 2

Extracting genome DNA, tumor genome DNA or tumor free DNA (ctDNA) from a sample to be detected, comprising the following steps:

step 2.1: the sample to be detected is a serum/plasma sample separated from fresh peripheral blood containing DNA, a tumor tissue sample, oral exfoliated cells collected by an oral swab or a fresh peripheral blood sample.

In this example, the sample sources were tumor patients and normal humans.

2.2, extracting tumor free DNA (ctDNA) from a sample to be detected by adopting a Qiagen QIAamp Circulating Nucleic Acid Kit (55114), or extracting a root of heaven blood/cell/tissue genome DNA extraction Kit (DP304), or extracting tumor genome DNA from a sample to be detected by adopting a root of heaven oral swab genome DNA extraction Kit (DP322), or extracting genome DNA from a sample to be detected by adopting a blood/cell/tissue genome DNA extraction Kit (DP304), determining the concentration and purity of the DNA by adopting NP80-touch (IMP L EN) and storing the DNA.

Example 3

The multiplex PCR detection method for synchronously detecting the exon gene mutation No.2, 3 and 4 of NRAS gene comprises the following steps:

step 3.1: and (3) taking the DNA obtained in the step 2.2 as an amplification template, and adopting the primer group synthesized in the step 1.2 to configure a multiple PCR reaction system.

In this example, a multiplex PCR amplification system was prepared by using DNA polymerase and buffer as basic raw materials in KOD FX enzyme system (cat. KFX-101) manufactured by Toyobo, Inc., and adjusting the primer concentration, dNTP concentration, buffer concentration and enzyme amount based on the amplification system in the enzyme system specification, and the specific composition of this reaction system is shown in Table 2 below. Of course, the equal scale enlargement/reduction of the reaction system is within the protection scope of the embodiment of the invention; the amplification can also be achieved by replacing other DNA polymerase systems and adjusting the appropriate proportion.

TABLE 2

Reagent composition	Volume of
		2×PCR buffer for FX	25μl
2mM dNTP	12.5μl
		Primer Mix	4μl
KOD FX(1U/μl)	1.5μl
		DNA	2μl
Water (W)	5μl

The primers were mixed equimolar, the primer concentration being 10 micromolar; the amount of the DNA template can be adjusted, and 40ng of genomic DNA can be used in this example.

Step 3.2: the PCR apparatus was programmed according to the multiplex PCR reaction conditions shown in Table 3 below.

TABLE 3

Step 3.3: and (3) carrying out multiple PCR amplification reaction on the multiple PCR reaction system configured in the step (3.1) by using a programmed PCR instrument to obtain a PCR product.

Example 4

Electrophoresis detection, comprising the following steps:

step 4.1: the PCR product obtained in step 3.3 was examined by agarose gel electrophoresis to verify the size of the PCR product fragment.

The detection results are shown in fig. 1, wherein 883, 890, DJ L, and JYX shown in fig. 1 are mainly used for distinguishing different samples to be detected, the rightmost column of fig. 1 shows a ruler bar, the left column of the ruler bar shows the electrophoresis results of PCR products of the blank control group, and the middle columns show the electrophoresis results of PCR products of different samples.

According to the comparison between the position of each product bright band and the left scale bar, the amplification product of which exon corresponds to each product bright band can be identified. For example, 3 bright bands from top to bottom are usually PCR amplification products corresponding to exons NRAS-3, NRAS-2, and NRAS-4, respectively.

Referring to fig. 1, it can be seen from the electrophoresis results of the blank set that the environmental factors have no adverse effect on the electrophoresis detection results of the sample to be detected. According to the electrophoresis result of each sample to be detected, 3 bright bands respectively correspond to the PCR amplification products of the NRAS-3, NRAS-2 and NRAS-4 exons, and the number of the bright bands is consistent with the theory; 3 bright bands are clear and have obvious intervals, different bright bands have no overlapping, no smear and the like, and the bright band effect is good. Thus, it can be shown that when the PCR amplification primer set designed in step 1.1 is used for PCR amplification, only the expected target product is generated, but no other irrelevant product is generated, and the design of the primer set is reasonable.

Step 4.2: after the size of the PCR product fragment is verified to be correct, the sequence of the PCR product can be determined.

Example 5

Sequence determination comprising the steps of:

step 5.1: and (4) after the sizes of the PCR product fragments are verified to be correct in the step 4.2, sending the PCR product obtained in the step 3.3 to a sequencing company for sequence determination to obtain a sequencing result in the format of ab 1.

Step 5.2: analyzing the sequencing result obtained in the step 5.1 by using Chromas sequence analysis software to obtain the gene mutation conditions of the No.2, 3 and 4 exons of the NRAS gene.

The partial sequencing results are shown in FIG. 2.

Referring to FIG. 2, FIG. 2 shows nucleotide base sequences at and upstream and downstream of the mutation sites c.182A > G of the gene. Referring to the box line part in FIG. 2, it can be seen that the site at position 182 is G, but not A, i.e., a gene mutation occurs at the site.

Example 6: configuration of the kit

According to the above experimental results, the present example provides a preferred kit for synchronously detecting mutations of exon 2, exon 3 and exon 4 of NRAS gene, wherein the kit comprises the following reagents:

1. multiplex PCR primer set: the first set of primer pairs: f upstream primer (10. mu.M), R downstream primer (10. mu.M); a second set of primer pairs: f upstream primer (10. mu.M), R downstream primer (10. mu.M); third set of primer pairs: f upstream primer (10. mu.M), R downstream primer (10. mu.M);

2、KOD FX(1U/μl)；

3、2×PCR buffer for FX；

4、dNTP(2mM)；

5. a sample DNA extraction reagent;

6. sample DNA collection kits (e.g., containing buccal swabs and swab kits);

7. ultrapure water.

The reagent is reasonably placed in the kit, the instructions of the kit are put in, the instructions comprise the step of collecting the DNA of the sample to be detected, the collected sample is put in a storage box, the step of extracting the DNA is carried out, and finally the multiple PCR amplification process is carried out according to the detection method.

It should be noted that, in this document, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other similar elements in a process, method, article, or apparatus that comprises the element.

The above-mentioned embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements made to the technical solution of the present invention by those skilled in the art without departing from the spirit of the present invention shall fall within the protection scope defined by the claims of the present invention.

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Claims (10)

1. A primer set for synchronously detecting exon 2, exon 3 and exon 4 mutations of NRAS gene, wherein the primer set comprises at least two of the following three primer pairs: the nucleotide sequence of the upstream primer of the first group of primer pairs is shown by SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown by SEQ ID NO. 2; the nucleotide sequence of the upstream primer of the second group of primer pairs is shown by SEQ ID NO.3, and the nucleotide sequence of the downstream primer is shown by SEQ ID NO. 4; the nucleotide sequence of the upstream primer of the third group of primer pairs is shown by SEQ ID NO.5, and the nucleotide sequence of the downstream primer is shown by SEQ ID NO. 6.

2. The primer set of claim 1, wherein the first set of primer pairs is a primer pair for detecting exon 2 of NRAS gene; the second group of primer pairs are primer pairs for detecting No.3 exon of NRAS gene; the third group of primer pairs is a primer pair for detecting exon 4 of the NRAS gene.

3. Use of the primer set according to claim 1 or 2 for the preparation of a kit for simultaneous detection of exon 2, exon 3, and exon 4 mutations in NRAS gene.

4. A kit for simultaneous detection of exon 2, exon 3, and exon 4 mutations in NRAS gene, comprising the primer set of claim 1 or 2.

5. The kit according to claim 4, further comprising a DNA polymerase, a PCR buffer, a mixture of 4 dNTPs, and ultrapure water.

6. The kit according to claim 5, wherein the DNA polymerase is used in an amount of 0.5 to 5U, the final concentration of each dNTP is 50 to 500. mu.M, and the final concentration of each primer is 20 to 400 nM.

7. The kit according to any one of claims 4 to 6, further comprising a test sample DNA extraction reagent or DNA extraction kit.

8. A method for synchronously detecting the gene mutation of exons 2, 3 and 4 of NRAS gene, which is characterized in that the primer set of claim 1 or 2 is used for carrying out multiplex PCR detection on a sample to be detected.

9. The detection method according to claim 8, characterized by comprising the steps of:

(1) extracting human DNA from a sample to be detected as an amplification template;

(2) preparing a multiplex PCR reaction system comprising the primer group and the amplification template;

(3) performing multiple PCR amplification reaction on the multiple PCR reaction system to obtain a PCR product;

(4) and (3) determining the exon gene mutations of No.2, 3 and 4 of the NRAS gene according to the PCR product.

10. The detection method according to claim 9, wherein the reaction conditions of the PCR amplification reaction are: 94-98 ℃: 1-10 min; 94-98 ℃: 5-60 s, 50-68 ℃: 10-120 s, 68-72 ℃: 20 s-4 min, and 25-45 cycles in total; 68-72 ℃: 0-30 min.

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