CN111518901A

CN111518901A - Primer group for detecting EGFR gene mutation and application method thereof

Info

Publication number: CN111518901A
Application number: CN202010365810.2A
Authority: CN
Inventors: 赵方圆; 佟洪梅; 智慧芳; 倪君君
Original assignee: Beijing Harmony Health Medical Diagnostics Co ltd
Current assignee: Beijing Harmony Health Medical Diagnostics Co ltd
Priority date: 2020-04-30
Filing date: 2020-04-30
Publication date: 2020-08-11

Abstract

The invention provides a primer group for detecting EGFR gene mutation and an application method thereof, wherein the primer group comprises at least one of the following 4 primer pairs: the sequence of an upstream primer for detecting the 18 th exon of the EGFR gene is shown as SEQ ID NO.1, and the sequence of a downstream primer is shown as SEQ ID NO. 2; the sequence of the upstream primer for detecting the 19 th exon of the EGFR gene is shown as SEQ ID NO.3, and the sequence of the downstream primer is shown as SEQ ID NO. 4; the sequence of the upstream primer for detecting the No. 20 exon of the EGFR gene is shown as SEQ ID NO.5, and the sequence of the downstream primer is shown as SEQ ID NO. 6; the sequence of the upstream primer for detecting the 21 st exon of the EGFR gene is shown as SEQ ID NO.7, and the sequence of the downstream primer is shown as SEQ ID NO. 8. So that the gene detection flux is improved.

Description

Primer group for detecting EGFR gene mutation and application method thereof

Technical Field

The invention relates to the technical field of gene detection, in particular to a primer group for detecting EGFR gene mutation and an application method thereof.

Background

Epidermal Growth Factor Receptor (EGFR) plays an important role in cell signaling pathways, and once activated, can lead to tyrosine protein kinase activation and receptor autophosphorylation in tumor cells, thereby promoting cell proliferation, differentiation, metastasis, angiogenesis and apoptosis inhibition. Research shows that in malignant tumor tissues such as non-small cell lung cancer NSCLC and the like, EGFR activity is abnormally increased due to EGFR overexpression, gene amplification, activation mutation of EGFR gene and the like, and EGFR gene mutation is mainly concentrated in a tyrosine kinase region, namely 18-2l of exons.

Currently, PCR detection assays can be designed for exons 18, 19, 20, and 21, respectively, of the EGFR gene. However, since each PCR reaction can detect a gene mutation only for one exon of the EGFR gene, the detection throughput of the gene mutation is low.

Disclosure of Invention

The embodiment of the invention provides a primer group for detecting EGFR gene mutation and an application method thereof, which can improve the detection flux of EGFR gene mutation.

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

multiplex PCR is a novel amplification technology developed on the basis of conventional PCR, and 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. Based on this, upstream and downstream primers for specific amplification were designed for exons 18, 19, 20, and 21 of the EGFR gene.

In a first aspect, the present invention provides a primer set for detecting mutations in the EGFR gene, comprising: at least one of the following 4 primer pairs:

the sequence of an upstream primer for detecting the 18 th exon of the EGFR gene is shown as SEQ ID NO.1, and the sequence of a downstream primer is shown as SEQ ID NO. 2;

the sequence of the upstream primer for detecting the 19 th exon of the EGFR gene is shown as SEQ ID NO.3, and the sequence of the downstream primer is shown as SEQ ID NO. 4;

the sequence of the upstream primer for detecting the No. 20 exon of the EGFR gene is shown as SEQ ID NO.5, and the sequence of the downstream primer is shown as SEQ ID NO. 6;

the sequence of the upstream primer for detecting the 21 st exon of the EGFR gene is shown as SEQ ID NO.7, and the sequence of the downstream primer is shown as SEQ ID NO. 8.

So as to relatively maximally improve the detection flux of EGFR gene mutation.

Specifically, when the primer set is used for multiplex PCR amplification reaction, at least one exon among exons 18, 19, 20, and 21 of EGFR gene can be simultaneously amplified in one amplification system. In general, when the 18 th exon of EGFR gene is amplified by using an upstream primer SEQ ID NO.1 and a downstream primer SEQ ID NO.2, the length of a fragment of a corresponding amplification product is 674 bp; when the 19 th exon of the EGFR gene is amplified by using the upstream primer SEQ ID NO.3 and the downstream primer SEQ ID NO.4, the length of the fragment of the corresponding amplification product is 424 bp; when the No. 20 exon of the EGFR 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 539 bp; when the No. 21 exon of EGFR gene is amplified by using the upstream primer SEQ ID NO.7 and the downstream primer SEQ ID NO.8, the length of the fragment of the corresponding amplification product is 264 bp.

Thus, after the primer group is used for carrying out multiplex PCR amplification, DNA fragments with different lengths can be generated, so that the fragments with different lengths can be distinguished by subsequent electrophoresis. Furthermore, the DNA of different fragments can be cut and recovered, and the sequence can be measured.

The invention also provides a kit comprising at least one primer pair shown in SEQ ID No.1 to SEQ ID No. 8.

In a second aspect, based on the above, the present invention provides a method for using the primer set for detecting mutations in EGFR gene according to the first aspect, comprising:

designing the primer set according to the first aspect;

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

preparing a multiple Polymerase Chain Reaction (PCR) reaction system containing the primer group and the amplification template;

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

and determining the gene mutation conditions of the 18 th, 19 th, 20 th and 21 st exons of the EGFR gene of the sample to be detected according to the PCR product.

In one embodiment of the present invention, the determining, according to the PCR product, the gene mutation condition of exons 18, 19, 20, and 21 of the EGFR gene of the sample to be tested includes:

detecting the PCR product through electrophoresis to obtain the amplified fragment size of the PCR product;

and when the amplified fragment of the PCR product is correct in size, carrying out sequence determination on the PCR product to obtain the gene mutation conditions of the 18 th, 19 th, 20 th and 21 st exons of the EGFR gene of the sample to be detected.

Specifically, agarose gel electrophoresis or polyacrylamide electrophoresis can be used to resolve DNA fragments of different lengths.

The application method is a method for non-diagnostic purposes.

In one embodiment of the present invention, the multiplex PCR reaction system further comprises: DNA polymerase, PCR buffer solution corresponding to the DNA polymerase, a mixture of 4 kinds of deoxyribonucleoside triphosphate dNTP and ultrapure water;

specifically, the amount of DNA polymerase used is 0.5-5U, so as to avoid waste due to excessive amounts of DNA polymerase while ensuring that deoxynucleotides can be added to the amplification template.

Specifically, the final concentration of each dNTP is 50-500. mu.M, and the final concentration of each primer in the primer group is 20-300 nM.

For the amount of DNA polymerase, 0.5-5U means any amount in the range of 0.5U to 5U, for example, 0.5U, 1U, 1.5U, 2U, 2.5U, 3U, 3.5U, 4U, 4.5U and 5U.

For the final concentration of each dNTP, 50-500. mu.M refers to any concentration in the range of 50. mu.M to 500. mu.M, such as 50. mu.M, 100. mu.M, 150. mu.M, 200. mu.M, 250. mu.M, 300. mu.M, 350. mu.M, 400. mu.M, 450. mu.M, and 500. mu.M.

For the final concentration of each primer, 20-300nM refers to any concentration in the range of 20nM to 300nM, e.g., 20nM, 50nM, 100nM, 150nM, 200nM, 250nM, and 300 nM.

Specifically, the DNA polymerase comprises: taq polymerase, KOD FX polymerase, Pfu polymerase or Phusion polymerase. The PCR buffer is the concentrated buffer corresponding to the selected DNA polymerase. Wherein the concentration of the PCR buffer solution is selected from 2X, 3X, 4X, 5X, 6X, 7X, 8X, 9X and 10X.

In one embodiment of the present invention, the reaction conditions of the PCR reaction system are as shown in table 1:

TABLE 1

The preservation of the PCR products at 2-8 ℃ is also shown in Table 1.

The temperature of 92-96 ℃ means any temperature in the range of 92 ℃ to 96 ℃ with respect to the temperature of the pre-denaturation under PCR conditions, for example, 92 ℃, 93 ℃, 94 ℃, 95 ℃ and 96 ℃.

For the time of the pre-denaturation under the PCR reaction condition, 1-10 min refers to any time within 1min to 10min, such as 1min, 2min, 3min, 4min, 5min, 6min, 7min, 8min, 9min and 10 min.

The temperature of the denaturation reaction under the PCR reaction conditions is 92-98 ℃ which is any temperature in the range of 92 ℃ to 98 ℃, for example, 92 ℃, 93 ℃, 94 ℃, 95 ℃, 96 ℃, 97 and 98 ℃.

For the time of denaturation under PCR reaction conditions, 5-20 s means any time within the range of 5s to 20s, for example, 5s, 8s, 10s, 14s, 17s, and 20 s.

The temperature of the annealing reaction in the PCR reaction condition is 52-68 ℃ which is any temperature in the range of 52 ℃ to 68 ℃, for example, 52 ℃, 54 ℃, 56 ℃, 58 ℃, 60 ℃, 62 ℃, 64 ℃, 66 ℃ and 68 ℃.

The time of the annealing reaction under the PCR reaction conditions is 10 to 60 seconds, for example, 10s, 15s, 20s, 25s, 30s, 32s, 38s, 40s, 45s, 50s, 53s, 56s, and 60 s.

For the temperature of the extension reaction under the PCR reaction conditions, 68-72 ℃ means any temperature in the range of 68 ℃ to 72 ℃, for example, 68 ℃, 69 ℃, 70 ℃, 71 ℃ and 72 ℃.

For the time of the extension reaction under the PCR reaction condition, 0.5-5 min refers to any time within 0.5min to 10min, such as 0.5min, 1min, 1.5min, 2min, 2.5min, 3min, 3.5min, 4min, 4.5min and 5 min.

For the temperature of the final extension reaction under the PCR reaction conditions, 68-72 ℃ refers to any temperature within the range of 68 ℃ to 72 ℃, such as 68 ℃, 69 ℃, 70 ℃, 71 ℃ and 72 ℃.

For the time of the final extension reaction under the PCR reaction condition, 0-30 min refers to any time within the range of 0min to 30min, such as 0min, 5min, 10min, 15min, 20min, 25min and 30 min.

Specifically, the method for extracting DNA from a sample to be tested comprises the following steps: and (3) extracting by hand or extracting by using a kit, and then processing the extracted DNA to obtain the DNA.

Specifically, the sample to be tested is a blood, cell, tissue or buccal swab sample containing human DNA.

Specifically, any one of the primers shown in SEQ ID No.1 to 8 can be applied to preparation of a reagent or a kit for detecting hot spot mutation sites of exons 18, 19, 20 and 21 of the EGFR gene.

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

(1) and (3) improving the detection flux: while the conventional PCR only targets one exon per reaction, the multiplex PCR of the present invention can detect at least 2 exons simultaneously, and the hot spot mutation site of at least one exon in the 18 th, 19 th, 20 th and 21 st exons of EGFR 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.

(2) The cost is reduced: the invention can reduce the PCR reaction system from 4 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

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.

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

FIG. 2 is a partial nucleotide base sequence for a mutation site in the sequencing result of a PCR product according to an embodiment of the present invention;

FIG. 3 is a partial nucleotide base sequence of another mutation site in the sequencing result of the PCR product according to an embodiment of the present invention;

FIG. 4 shows a partial nucleotide base sequence of a further mutation site in the sequencing result of the PCR product according to an embodiment of the present invention.

Detailed Description

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

Design and Synthesis of primer set

Step 1.1: upstream and downstream primers specifically amplifying the region of the gene mutation site were designed based on exons 18, 19, 20 and 21 of the EGFR gene.

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 4 pairs of primers are basically kept consistent.

The primer sets provided in this example cover the hot spot mutation sites of exons 18, 19, 20 and 21 of the EGFR 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 shown in the following table 2 are selected by integrating the fragment length and the site/exon inclusion conditions of products.

TABLE 2

Step 1.2: and (3) synthesizing the primer group designed in the step 1.1.

Example 2

Extraction of DNA from a sample to be tested

Step 2.1: the sample to be tested may be: serum, plasma samples or collected tumor tissue samples isolated with fresh peripheral blood, exfoliated buccal cells collected with buccal swabs or fresh peripheral blood samples.

Step 2.2: the DNA concentration and purity are determined by using Qiagen QIAamp Circulating Nucleic Acid Kit (55114), or using Tiangen blood/cell/tissue genome DNA extraction Kit (DP304), or extracting DNA (ctDNA) from a sample to be detected, or tumor DNA, and NP80-touch (IMPLEN, Germany) to store the DNA.

Example 3

Preparation of PCR reaction System

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 prepare 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 3 below.

It is understood that the proportional scaling up/down of the reaction system is within the scope of the embodiments of the present invention; the amplification can also be achieved by replacing other DNA polymerase systems and adjusting the appropriate proportion.

TABLE 3

Reagent composition	Volume of
		2×PCR buffer for KOD FX	26μl
2mM dNTP	5μl
		Primer Mix (1.25. mu.M each Primer)	5μl
KOD FX(1U/μl)	1μl
		DNA	1μl
Ultrapure water	12μl

Wherein, each primer is mixed in an equimolar way, and the total concentration of the primers is 10 mu M; the amount of the DNA template can be adjusted, and in this example, 5ng of DNA can be used.

Step 3.2: the procedure of the PCR instrument was set according to the multiplex PCR reaction conditions shown in Table 4 below, and a multiplex PCR amplification reaction was performed on the multiplex PCR reaction system prepared in step 3.1 to obtain a PCR product.

TABLE 4

It should be noted that the obtained PCR product can be stored at 4 ℃ for use.

Example 4

Electrophoretic detection

Step 4.1: and (3) detecting the PCR product obtained in the step 3.2 by agarose gel electrophoresis to obtain the size of the PCR product fragment.

The detection results are shown in fig. 1, wherein the labels of YL-1911, YL-1912, YL-1913, N-1806, N-THM and "empty" shown in fig. 1 are mainly used for distinguishing the PCR products of different samples, the left-most column of fig. 1 shows a ruler strip for characterizing the length, the right-most column of fig. 1 shows the electrophoresis results of the PCR products of the blank control group, and the middle columns show the electrophoresis results of the PCR products of different samples to be detected.

According to the comparison between the position of the bright band of each PCR product and the left scale bar in FIG. 1, it can be identified which exon amplification product corresponds to each bright band of the PCR product. For example, the bottom-up 4 bright bands are usually PCR amplification products corresponding to exons 21, 19, 20, and 18, respectively, of the EGFR gene.

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 the PCR product of each sample, 4 bright bands exist and respectively correspond to the PCR amplification products of the 21, 19, 20 and 18 exons of the EGFR gene, and the number of the bright bands is consistent with the theory; the 4 bright bands are clear and have obvious intervals, and different bright bands have no overlapping, no smear and the like, so that 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.

Example 5

Step 5.1: after determining that the size of the PCR product fragment obtained in step 4.1 is correct, the PCR product obtained in step 3.2 is sent to a sequencing company for sequence determination, and a sequencing result in the format of.ab 1 is obtained.

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 hot spot mutation sites of 21, 19, 20 and 18 exons of the EGFR gene.

The partial sequencing results are shown in FIGS. 2 to 4.

Referring to FIG. 2, FIG. 2 shows the c.2156G > ap.Gly719Ala site in exon 18 of the EGFR gene and the nucleotide base sequences upstream and downstream of the site. Referring to the box line part in FIG. 2, it can be seen that the sample DNA is mutated from G to A at the base at position 2156.

Referring to FIG. 3, FIG. 3 shows the nucleotide base sequence at and upstream of the c.del2235-2249 p.delGlu746-Ala750 site in exon 19 of the EGFR gene. Referring to the box line part in FIG. 3, it can be seen that the sample DNA has not undergone base deletion at the 2235-2249 th base.

Referring to FIG. 4, FIG. 4 shows the nucleotide base sequence of c.2582T > ap.Leu861Gln site of exon 21 of EGFR gene and upstream and downstream thereof. Referring to the frame line part in FIG. 2, it can be seen that the sample DNA is mutated from T to A at base 2582.

It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, 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 a" does not exclude the presence of other similar elements in a process, method, article, or apparatus that comprises the element.

Finally, it is to be noted that: the above description is only a preferred embodiment of the present invention, and is only used to illustrate the technical solutions of the present invention, and not to limit the protection scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention shall fall within the protection scope of the present invention.

SEQUENCE LISTING

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Claims

1. Primer set for detecting mutations in the EGFR gene, comprising at least one of the following 4 primer pairs:

2. The method for using the primer set for detecting mutations in EGFR gene according to claim 1, comprising:

designing the primer set of claim 1;

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

3. The method of claim 2,

determining the gene mutation conditions of the 18 th, 19 th, 20 th and 21 st exons of the EGFR gene of the sample to be detected according to the PCR product, wherein the gene mutation conditions comprise the following steps:

4. The method of claim 2,

the multiplex PCR reaction system further comprises: DNA polymerase, PCR buffer solution corresponding to the DNA polymerase, a mixture of 4 kinds of deoxyribonucleoside triphosphate dNTP and ultrapure water;

wherein the dosage of the DNA polymerase is 0.5-5U, the final concentration of each dNTP is 50-500 mu M, and the final concentration of each primer in the primer group is 20-300 nM.

5. The method of claim 4,

the DNA polymerase comprises: taq polymerase, KOD FX polymerase, Pfu polymerase or Phusion polymerase.

6. The method of claim 4,

the reaction conditions of the PCR reaction system comprise: pre-denaturation at 92-96 ℃ for 1-10 min; denaturation is carried out for 5-20 s at the temperature of 92-98 ℃; annealing at 52-68 ℃ for 10-60 s; extending for 0.5-5 min at 68-72 ℃; and (3) performing denaturation, annealing and extension for 25-40 times at 68-72 ℃ for final extension for 0-30 min.