阅读说明：本技术 K-ras基因突变位点检测试剂盒 (K-ras gene mutation site detection kit ) 是由 *** 于 2019-12-16 设计创作，主要内容包括：本发明涉及分子生物技术和基因检测领域,具体而言,涉及一种K-ras基因突变位点检测试剂盒,该试剂盒包括：a)引物对；b)上游探针、下游探针、发夹探针；以及c)两种纳米金探针。上述引物和探针能够实现在闭管条件下对K-ras基因的突变位点进行高灵敏、高分辨、低成本检测,能够有效的避免扩增产物的交叉污染。其中下游探针既可单独使用,也可互相配合在同一反应体系下进行多重检测,因而本试剂盒能够对最多七种K-ras基因突变位点同时进行检测,检测效率更高。(The invention relates to the field of molecular biotechnology and gene detection, in particular to a K-ras gene mutation site detection kit, which comprises: a) a primer pair; b) an upstream probe, a downstream probe, a hairpin probe; and c) two kinds of nano-gold probes. The primers and the probes can realize high-sensitivity, high-resolution and low-cost detection of the mutation sites of the K-ras gene under a closed tube condition, and can effectively avoid cross contamination of amplification products. The downstream probes can be used independently or matched with each other to carry out multiple detection in the same reaction system, so that the kit can simultaneously detect at most seven K-ras gene mutation sites, and has higher detection efficiency.)

A kit for detecting K-ras gene mutation sites, comprising:

a) SEQ ID NO: 1 and SEQ ID NO: 2, and (b) a primer pair shown in the figure;

b) SEQ ID NO: 3, and the upstream probe shown in SEQ ID NO: 4-10, and SEQ ID NO: 11; and

c) two nanogold probes, each comprising SEQ ID NO: 12 and SEQ ID NO: 13.

2. The kit for detecting K-ras gene mutation site of claim 1, SEQ ID NO: 12 and SEQ ID NO: 13 is independently connected with the gold nanoparticle through a connecting segment, and the connecting segments do not hybridize with each other and a) and b).

3. The kit for detecting K-ras gene mutation site of claim 2, wherein the length of the connecting fragment is 1nt to 15 nt.

4. The kit for detecting K-ras gene mutation site of claim 3, wherein the length of the connecting fragment is 3nt to 5 nt.

5. The K-ras gene mutation site detection kit as claimed in any one of claims 1 to 4, further comprising one or more of DNA polymerase, endonuclease, dNTP, buffer or buffer salt, soluble magnesium salt, Tween-20 and water.

6. The K-ras gene mutation site detection kit of claim 5, wherein the DNA polymerase is selected from any one of Taq, Bst, Vent, Phi29, Pfu, Tru, Tth, Tl1, Tac, Tne, Tma, Tih, Tf1, Pwo, Kod, Sac, Sso, Poc, Pab, Mth, Pho, ES4 DNA polymerase and Klenow fragment.

7. The kit for detecting K-ras gene mutation site according to claim 5, wherein the endonuclease is selected from any one of TaqPol, TthPol, TaqExo, Afufen, PfuFEN, MjaFEN or MthFEN.

8. The kit for detecting K-ras gene mutation site of claim 5, wherein the soluble magnesium salt is MgCl₂。

9. The composition is prepared by combining the K-ras gene mutation site detection kit of any one of claims 1 to 8.

10. The composition of claim 9, wherein the concentration of the primers in a) is independently selected from 0.5 μ Μ to 1.5 μ Μ, the concentration of the probes in b) is independently selected from 0.5 μ Μ to 1.5 μ Μ, the concentration of the nanogold probes in c) is independently selected from 0.03 μ Μ to 0.2 μ Μ, and the composition further comprises the target nucleic acid.

Technical Field

The invention relates to the field of molecular biotechnology and gene detection, in particular to a kit for detecting K-ras gene mutation sites.

Background

K-ras is a small molecule G protein at the downstream of an EGFR signal pathway, and inhibits the activity of GTP enzyme after mutation, so that the K-ras protein is always in an activated state, and the signal pathway is not regulated by an upstream EGFR signal instruction. The mutation rate of K-ras gene in colorectal cancer patients is about 40%, 70% occurs at codon 12, and 30% occurs at codon 13. The K-ras gene mutation state is related to the curative effect of cetuximab, and cetuximab treatment is ineffective when K-ras gene is mutated, so that molecular typing detection is carried out on K-ras gene mutation, and a dosing scheme is very necessary to be established according to the mutation condition. However, in general, in actual clinical samples, mutant genes are often mixed in a large number of wild-type gene sequences, and since the sequences between the two often differ by only one base, it is difficult to detect the mutant genes in a targeted manner. Based on this, most of the current gene locus detection is completed based on a molecular detection method with higher specificity, and more technical platforms are ARMS technology and NGS technology, and the two methods can detect about 1% of gene mutation, but the cost of the kit is higher (or the requirement on an operator is higher), so that a larger economic burden is brought to a patient.

Disclosure of Invention

The invention realizes the detection of the mutation site by the regular change of the characteristic absorption wavelength caused by the hybridization between the nanogold probe and the template, and is a rapid, simple and economic K-ras gene mutation detection method compared with the prior method because no expensive machine or complex operation is needed.

Specifically, the invention relates to a K-ras gene mutation site detection kit, which comprises:

a) SEQ ID NO: 1 and SEQ ID NO: 2, and (b) a primer pair shown in the figure;

b) SEQ ID NO: 3, and the upstream probe shown in SEQ ID NO: 4-10, and at least one of the downstream probes shown in SEQ ID NO: 11; and

c) two nanogold probes, each comprising SEQ ID NO: 12 and SEQ ID NO: 13.

The primers and the probes can realize high-sensitivity, high-resolution and low-cost detection of the mutation sites of the K-ras gene under a closed tube condition, and can effectively avoid cross contamination of amplification products. The downstream probes can be used independently or matched with each other to carry out multiple detection in the same reaction system, so that the kit can simultaneously detect at most seven K-ras gene mutation sites, and has higher detection efficiency.

According to still another aspect of the present invention, the present invention also relates to a composition prepared by mixing the kit for detecting K-ras gene mutation sites as described above.

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 described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a schematic diagram illustrating the use principle of the kit provided by the present invention;

FIG. 2 shows the results of independent detection of seven mutation sites of K-ras gene in one embodiment of the present invention;

FIG. 3 shows the result of multiplex assay of seven mutation sites of K-ras gene in the same tube according to an embodiment of the present invention;

FIG. 4 shows the amount of the K-ras gene mutation site detection reagent used in the amount of 3X 10 as the template in one embodiment of the present invention⁴Copying a sensitivity detection result;

FIG. 5 shows the amount of the K-ras gene mutation site detection reagent used in the amount of 3X 10 as a template in one embodiment of the present invention³Copying a sensitivity detection result;

FIG. 6 shows the amount of the K-ras gene mutation site detection reagent used in the amount of 3X 10 as a template in one embodiment of the present invention²Copying a sensitivity detection result;

FIG. 7 is a diagram showing the effect of interfering substances in a reagent for detecting a mutation site of K-ras gene according to an embodiment of the present invention;

FIG. 8 is a diagram showing the confirmation of the specificity of a K-ras gene mutation site detection reagent in one embodiment of the present invention;

FIG. 9 shows the results of actual clinical specimens tested by the K-ras gene mutation site assay reagent according to one embodiment of the present invention;

FIG. 10 shows the real-time fluorescence PCR detection results of actual clinical samples of K-ras gene mutation sites in one embodiment of the present invention.

Detailed Description

Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.

It is therefore intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents. Other objects, features and aspects of the present invention are disclosed in or are apparent from the following detailed description. It is to be understood by one of ordinary skill in the art that the present discussion is a description of exemplary embodiments only, and is not intended as limiting the broader aspects of the present invention.

The invention relates to a K-ras gene mutation site detection kit, which comprises:

a) SEQ ID NO: 1 and SEQ ID NO: 2, and (b) a primer pair shown in the figure;

b) SEQ ID NO: 3, and the upstream probe shown in SEQ ID NO: 4-10, and at least one of the downstream probes shown in SEQ ID NO: 11; and

c) two nanogold probes, each comprising SEQ ID NO: 12 and SEQ ID NO: 13.

The K-ras gene mutation site detection kit provided by the invention can realize one-tube detection of hot spot mutations on at most seven K-ras genes, and the whole process can be interpreted without complex operation and complex detection equipment and instruments. The specific principle is as follows. Firstly, a pair of common primers is used for amplifying a hotspot mutation site region to obtain a high-concentration target sequence amplicon. By utilizing the characteristic that hotspot mutation sites of the amplicon are concentrated, a common auxiliary probe and seven detection probes specific to seven mutation targets are designed, when a target sequence exists, the probes are specifically combined with a template, signal molecules are cut under the participation of endonuclease, and different signal differences are formed after the hybridization with the nanogold probe. The signal difference can be interpreted through the visible light difference, and the rapid detection of one or more mutation sites of the K-ras gene in one tube is realized based on the signal difference. Compared with a fluorescent PCR method, the method has the characteristics of sensitivity, simplicity, rapidness and low cost, and is easier to popularize and use.

Obviously, preferably, the kit comprises SEQ ID NO: 4 to 10 in the above range.

In some embodiments, the average particle size of the gold nanoparticles in the gold nanoparticle probe is 1nm to 200 nm.

In some embodiments, the average particle size of the gold nanoparticles in the gold nanoparticle probe is 5nm to 80 nm.

In some embodiments, the average particle size of the gold nanoparticles in the gold nanoparticle probe is 10nm to 30 nm.

In some embodiments, the nucleic acid sequence of SEQ ID NO: 12 and SEQ ID NO: 13 is independently connected with the gold nanoparticle through a connecting segment, and the connecting segments do not hybridize with each other and a) and b).

In the present invention, the criterion for the evaluation of "hybridization" means that nucleic acids do not hybridize under stringent conditions. Such "stringent conditions" are well known to those skilled in the art and include, for example, hybridization at 60 ℃ for 12 to 16 hours in a hybridization solution containing 400mM NaCl, 40mM PIPES (pH6.4) and 1mM EDTA, followed by washing with a washing solution containing 0.1% SDS and 0.1% SSC at 65 ℃ for 15 to 60 minutes. Alternatively, two nucleic acid fragments are cloned in a molecule such as Sambrook et al: the experimental manuals (1989) (Cold spring Lane laboratory Press, New York, USA) "expression of cloned genes in E.coli" section described under standard hybridization conditions with each other. Such conditions as hybridization at 45 ℃ in 6.0 XSSC, followed by a washing step at 50 ℃ in 2 XSSC. To select stringency, the salt concentration in the washing step can be chosen, for example, between 2.0 XSSC at 50 ℃ for low stringency and 2.0 XSSC at 50 ℃ for high stringency. In addition, the temperature in the washing step may vary between about 22 ℃ for low stringency at room temperature and 65 ℃ for high stringency. In a specific embodiment, the stringent conditions are those in the PCR reaction of the present application.

In some embodiments, the linking fragment is 1nt to 15nt in length, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 nt; preferably 3nt to 5 nt.

In some embodiments, the kit further comprises one or more of a DNA polymerase, an endonuclease, dntps, a buffer or buffer salt, a soluble magnesium salt, Tween-20, and water.

The term "buffer" as used herein refers to an aqueous solution or composition that resists changes in pH when an acid or base is added to the solution or composition. This resistance to pH changes is due to the buffer properties of such solutions. Thus, a solution or composition that exhibits buffering activity is referred to as a buffer or buffer solution. Buffers generally do not have the unlimited ability to maintain the pH of a solution or composition. Rather, they are generally capable of being maintained at a pH within a specified range, for example, pH 7 to pH 9. Generally, the buffer is capable of maintaining a pH at its pKa and within the next logarithm (see, e.g., Mohan, Buffers, agent for the preparation and use of Buffers in biological systems, CALBIOCHEM, 1999). Buffers and buffer solutions are generally prepared from buffered salts or preferably non-ionic buffer components such as TRIS and HEPES. The buffer which can be used in the method of the invention is preferably selected from the group consisting of phosphate buffer, phosphate buffered saline buffer (PBS), 2-amino-2 hydroxymethyl-1, 3-propanediol (TRIS) buffer, TRIS buffered saline solution (TBS) and TRIS/edta (te). The buffer can be obtained after dissolution of the buffer salt in a solvent, usually water.

In some embodiments, the DNA polymerase is selected from any of Taq, Bst, Vent, Phi29, Pfu, Tru, Tth, Tl1, Tac, Tne, Tma, Tih, Tf1, Pwo, Kod, Sac, Sso, Poc, Pab, Mth, Pho, ES4 DNA polymerase, Klenow fragment.

In some embodiments, the endonuclease is selected from any one of tapol, TthPol, TaqExo, AfuFEN, PfuFEN, mqifen, or MthFEN.

In some embodimentsWherein the soluble magnesium salt is MgCl₂。

In some embodiments, the water is generally free of nucleic acids and nucleases, such as double distilled or deionized water. The water is distilled water, deionized water or reverse osmosis water.

The invention also relates to a composition which is prepared by mixing the EGFR gene T790M locus detection kit.

In some embodiments, the composition is a solution, wherein the concentration of the primers in a) is independently selected from 0.5 μ M to 1.5 μ M, the concentration of the probes in b) is independently selected from 0.5 μ M to 1.5 μ M, and the concentration of the nanogold probes in c) is independently selected from 0.03 μ M to 0.2 μ M, and the composition further comprises the target nucleic acid.

In some embodiments, the composition has a pH of 8 to 9, preferably 8.5.

The invention also relates to a method for detecting K-ras gene mutation sites, which comprises the following steps:

a) obtaining a composition as described above;

b) and carrying out PCR reaction, and observing the color change of the reaction system to judge the mutation condition of the K-ras gene mutation site.

The method can be used for the medication guidance of K-ras gene targeted drugs.

Embodiments of the present invention will be described in detail with reference to examples.