1. The primer group for detecting melanoma targeted medication related gene mutation is characterized by consisting of nucleic acids shown in SEQ ID NO.1-28, wherein the nucleic acids shown in SEQ ID NO.1-4 are an amplification primer pair of BRAF genes; the nucleic acid shown in SEQ ID NO.5-22 is an amplification primer pair of the KIT gene; the nucleic acid shown in SEQ ID NO.23-28 is an amplification primer pair of NRAS gene.

2. A kit for detecting melanoma targeted drug administration related gene mutation, which is characterized by comprising the primer group for detecting melanoma targeted drug administration related gene mutation according to claim 1.

3. The kit for detecting melanoma targeted drug related gene mutation according to claim 2, further comprising a positive DNA quality control product, a negative quality control product and a library building reagent.

4. The kit for detecting melanoma targeted drug related gene mutation according to claim 3, wherein the library creating reagent comprises 5 x Ion AmpliSeq^TMHiFi Mix、Pre-ligation Enzyme、Barcording buffer、Barcoding enzyme、Adapter。

5. The kit for detecting melanoma tumor targeting drug related gene mutation according to claim 2, wherein the suitable detection objects are one or more of fresh operation tissue and/or puncture tissue, frozen operation tissue and/or puncture tissue, formalin fixed paraffin embedded tissue samples.

Background

Melanoma usually occurs in skin tissues, but also in external skin tissues such as eyes, nasal cavities, throats, anorectal tracts, central nervous systems, lymph nodes and the like, and has high malignancy degree. Although early patients can be cured by surgery with the development of early diagnosis, surgical operation and adjuvant therapy, the 5-year survival rate is about 95%, the median survival rate of advanced patients is only 2-8 months, and the 5-year survival rate is less than 5%.

With the rapid development of molecular biology, cell biology, tumor immunology and the like, the abnormality of malignant melanoma cell signaling pathway is gradually recognized, and some targeted drugs aiming at melanoma gene mutation are beginning to be applied to clinic, including delafenib, vemurafenib, trametinib, kanafinil and the like.

The current mature gene targets in skin melanoma are BRAF, KIT and NRAS, and are related to prognosis, molecular typing and late treatment. Some clinical features are associated with a higher frequency of incidence of BRAF mutations, e.g., skin, younger age, torso position with intermittent exposure to sunlight. The BRAF V600 mutation is associated with sensitivity to BRAF inhibitors. KIT mutations occur in 10% to 15% of mucous membranes and acromias (i.e., non-load bearing palms and soles, nail beds) melanoma, and also in 2% to 3% of skin exposed to prolonged sunlight, but rarely occur on intermittently sun-irradiated skin. KIT exon 11 and exon 13 mutations are highly sensitive to KIT inhibition. In melanoma, which is exposed to sun, hand and foot surfaces and mucosal surfaces for long periods and intermittently, approximately 15% of patients have NRAS mutations. NRAS mutations may be associated with poor survival in both local and advanced melanoma. MEK inhibitors may be effective in a small number of patients carrying NRAS mutations. The 2020 CSCO malignant melanoma diagnosis and treatment guideline emphasizes the importance of gene detection by I-grade expert recommendation in the diagnosis part, and suggests that melanoma patients carry out gene detection before treatment and then can find treatable targets, wherein the common targets comprise BRAF, CKIT, NRAS and the like.

Therefore, the technical personnel in the field are dedicated to developing primers and kits capable of simultaneously detecting gene mutations related to various melanoma targeting drugs, so that reference is provided for a clinician to select a proper targeting drug for a melanoma patient, the accuracy of melanoma targeting drug selection is further improved, and the primers and kits are helpful for reducing the economic burden of the patient and prolonging the life cycle.

Disclosure of Invention

The invention aims to provide a primer group and a kit for detecting melanoma targeted drug related gene mutation, which provide an auxiliary effect for the selection of melanoma targeted drug.

The first aspect of the invention provides a primer group for detecting melanoma targeted medication related gene mutation, which consists of nucleic acids shown in SEQ ID NO.1-28, wherein the nucleic acids shown in SEQ ID NO.1-4 are BRAF gene amplification primer pairs; the nucleic acid shown in SEQ ID NO.5-22 is an amplification primer pair of the KIT gene; the nucleic acid shown in SEQ ID NO.23-28 is an amplification primer pair of NRAS gene.

The invention provides a kit for detecting melanoma targeted medication related gene mutation, which comprises a primer group for detecting melanoma targeted medication related gene mutation.

Furthermore, the kit for detecting and detecting melanoma targeted medication related gene mutation further comprises a positive DNA quality control product, a negative quality control product and a library establishing reagent.

Further, the library creating reagent comprises 5 × Ion AmpliSeq^TM HiFi Mix、Pre-ligation Enzyme、Barcording buffer、Barcoding enzyme、Adapter。

Furthermore, the kit for detecting melanoma targeted drug related gene mutation is applicable to one or more of fresh operation tissues and/or puncture tissues, frozen operation tissues and/or puncture tissues and formalin-fixed paraffin-embedded tissue samples.

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

the invention can simultaneously detect 82 mutation sites on BRAF, KIT and NRAS genes, the mutations are closely related to melanoma targeted drug selection or related to melanoma prognosis, and the detection result can provide reference for a clinician to select targeted drugs for melanoma patients and improve the treatment effect of melanoma. The invention is based on the second generation high-throughput sequencing technology, and the primer combination, the kit and the detection have the advantages of high sensitivity, good specificity, high repeatability and the like.

Drawings

FIG. 1 is a DNA library fragment distribution map constructed in example 3.

FIG. 2 is a depth map of sequencing of 8 samples from example 4.

FIG. 3 is a depth map of sequencing of the same sample from example 5 by 3 different experimenters.

FIG. 4 is a depth map of sequencing of 3 tests performed on the same sample by the same experimenter of example 6.

Detailed Description

The invention is described in detail below with reference to the figures and the specific embodiments, but the invention should not be construed as being limited thereto. The technical means used in the following examples are conventional means well known to those skilled in the art, and materials, reagents and the like used in the following examples can be commercially available unless otherwise specified.

Example 1

Primer group for detecting melanoma targeted medication related gene mutation

The melanoma targeted drug related gene and site of the embodiment are screened from a COSMIC database, targeted drug related mutation site primer design is carried out according to related gene sequences, and the design range comprises drug selection related mutation in the melanoma targeted drug related gene.

The 82 DNA mutation sites of the 3 genes of this example are shown in table 1, and the corresponding mutation site amplification primers are shown in table 2:

TABLE 1 melanoma targeting drug-related gene DNA mutation sites

TABLE 2 amplification primers for DNA mutation sites of drug-related genes for melanoma targeting

Example 2

Kit for detecting melanoma targeted medication related gene mutation

The kit for detecting melanoma targeted medication related gene mutation in the embodiment comprises:

(1) an amplification primer: the primer sequence is shown as SEQ ID NO.1-28 in Table 2, and is used for amplifying a plurality of target regions on a target gene of a sample to be detected, wherein the amplification range at least covers a hot spot mutation region of the target gene;

(2) positive DNA quality control material, negative quality control material and library establishing reagent, wherein the library establishing reagent comprises 5 Xion AmpliSeq^TMHiFi Mix、Pre-ligation Enzyme、Barcording buffer、Barcoding enzyme、Adapter。

Example 3

Method for detecting melanoma targeted medication related gene mutation

The detection method of the embodiment comprises the following steps:

(1) extracting DNA from a sample to be detected, and detecting the concentration and purity of the DNA, wherein the concentration of the DNA is required to be more than 5 ng/uL; the sample is one or more of fresh operation tissue and/or puncture tissue, frozen operation tissue and/or puncture tissue, and formalin-fixed paraffin-embedded tissue sample;

(2) the DNA extracted in the above steps is used as a template, primers shown in SEQ ID NO.1-28 are used for carrying out first round PCR amplification, and an amplification reaction system is shown in Table 3:

TABLE 3 first round PCR amplification System

(3) After fully shaking and mixing, centrifuging for a short time, and reacting on a PCR instrument according to the following procedures, wherein the reaction procedures are shown in a table 4:

TABLE 4 first round PCR amplification procedure

(4) After the PCR tube was taken out, the tube was centrifuged briefly, and 1. mu.L of LPre-ligation Enzyme was added to 10. mu.L of the PCR product;

(5) fully oscillating and uniformly mixing, carrying out mild centrifugation to collect liquid at the bottom of the tube, putting the PCR tube into a PCR instrument, and operating according to the program shown in Table 5;

TABLE 5 second round PCR amplification procedure

(6) Adding 2 mu L of Barcoding buffer, 1 mu L of Barcoding enzyme and 1 mu L of adapter into the second round of PCR amplification product;

(7) fully oscillating and mixing uniformly, collecting liquid at the bottom of the tube by mild centrifugation, and operating according to the program shown in table 6;

TABLE 6 third round PCR amplification procedure

(8) Purification of the library

a. The liquid volume was made up to 30ul with water and transferred to 1.5mL EP tubes and 45. mu.l (1.5X sample volume) was added to each libraryXP reagent. Blowing and beating for 5 times by a gun to fully and uniformly mix the DNA and the magnetic bead suspension;

b. the mixture was left at room temperature for 5 minutes;

c. the 1.5mL EP tube was placed on a magnetic stand and allowed to stand for 2 minutes or the solution became clear. Carefully aspirate and discard the supernatant without disturbing the beads;

d. add 150. mu.L of freshly prepared 70% ethanol to the wells, move the EP tube back and forth on the magnetic rack to wash the beads, and then carefully discard the supernatant without disturbing the beads.

e. Repeating the previous step, and carrying out secondary washing;

f. ensure that the ethanol droplets have been completely aspirated from the wells. Place the plate on a magnetic rack and air dry at room temperature for 5 minutes, taking care not to over dry;

g. the EP tube was taken off the magnetic stand, 50. mu.L of Low TE was added to each well to thoroughly soak the beads, thoroughly shaken and mixed, and centrifuged gently to collect the liquid at the bottom of the tube. (more than half of the liquid can be sucked by a gun and blown up and down for at least 5 times to mix evenly);

h. the EP tube was placed on a magnetic stand for 2 minutes. After clarification, the supernatant is taken out to obtain a library;

(9) quantifying the constructed library by using the Qubit, diluting the library to a proper concentration, carrying out template enrichment on an OT instrument according to the operation instruction of life company, and then carrying out Ion one touch^TMRecovering and enriching ISPs with templates on an ES instrument;

(10) performing on-machine sequencing on a PGM instrument according to the instructions of life corporation;

(11) and analyzing and annotating the sequencing result by using biological information analysis software.

Example 4

Sequencing depth performance verification of kit for detecting melanoma targeted medication related gene mutation

8 samples (melanoma paraffin-embedded tissue samples) were tested using the kit of example 2 of the present invention, and the testing procedure was performed according to the procedure of example 3. The detection results show that the kit of the invention has good sequencing depth in 8 different samples, namely 16876X, 21218X, 11823X, 11544X, 14501X, 20437X, 11034X and 18640X, which are all larger than the lowest sequencing depth 5000X set by the kit as shown in FIG. 2.

Example 5

Reproducibility verification of kit for detecting melanoma targeted medication related gene mutation

The kit of example 2 of the present invention was used to detect 3 different subjects in the same sample (melanoma paraffin-embedded tissue sample) according to the method of example 3. The detection results show that the sequencing depths are 34140X, 36583X and 34379X respectively, as shown in FIG. 3. The BRAF V600E locus can be detected, and the detected mutation frequencies are relatively consistent and are respectively 20.1%, 19.5% and 21.3%. Therefore, the kit has better detection reproducibility.

Example 6

Repeatability verification of primer and kit for detecting melanoma targeted medication related gene mutation

The same melanoma sample (melanoma paraffin-embedded tissue sample) was tested 3 times by the same experimenter using the kit of the present invention in example 2, using the protocol according to the method of example 3. The 3-time results showed that the sequencing depths were 37818 ×, 39371 ×, 37281 × (fig. 4), and the frequencies of NRAS mutations were 22.3%, 24.8%, 23.6%, respectively. Therefore, the kit has good detection repeatability for the same sample.

Example 7

Sensitivity verification of primer and kit for detecting melanoma targeted medication related gene mutation

The kit of the embodiment 2 of the invention is used for detecting SNV FFPE standard substance (Jinglian technology) DNA of the same tumor, the standard substance DNA is respectively diluted to 10ng/uL, 5ng/uL and 3ng/uL to be used as initial DNA concentration for library construction, 3uL is respectively taken, namely the input amount of the library DNA is 30ng, 15ng and 9ng, and the library DNA is respectively marked as a standard substance 1, a standard substance 2 and a standard substance 3. The protocol used was according to the procedure of example 3. The detection results show that the results of 4 standards with different concentrations are consistent, and the results of the standard 1 are NRAS (p.Q61K, 6.5%) and BRAF (V600E, 25.2%); results for standard 2 were NRAS (p.q61k, 6.1%), BRAF (V600E, 23.5%); the results for standard 3 were NRAS (p.q61k, 5%), BRAF (V600E, 20.7%). Thus, the present invention can detect gene mutations in a quantity as low as 5% in a DNA sample as low as 9 ng.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

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