阅读说明：本技术 一种用于检测braf基因k601e突变的引物、探针及试剂盒 (Primer, probe and kit for detecting mutation of BRAF gene K601E ) 是由 张宇清 裴婷婷 赵艳伟 于 2019-12-31 设计创作，主要内容包括：本发明涉及分子生物技术领域,公开了一种用于检测BRAF基因K601E突变的引物、探针及试剂盒。本发明独特之处在于通过在BRAF基因K601E突变检测引物的3’末端连续设计了2个错配碱基,大大提高了该突变位点检测的特异性,减少了周围临近突变位点对检测的干扰,获得了很好的检测效果。本发明还公开了一种利用上述引物和探针检测BRAF基因K601E突变的检测试剂盒,能够快速、灵敏、准确地检测BRAF基因K601E突变。本发明具有特异性好、灵敏度高、操作简便、成本低廉、结果准确、判读方便等优点,具有良好的临床应用前景。(The invention relates to the technical field of molecular biology, and discloses a primer, a probe and a kit for detecting mutation of BRAF gene K601E. The invention is characterized in that 2 mismatched bases are continuously designed at the 3' end of the BRAF gene K601E mutation detection primer, thereby greatly improving the specificity of the detection of the mutation site, reducing the interference of the adjacent mutation sites around on the detection and obtaining good detection effect. The invention also discloses a detection kit for detecting the mutation of the BRAF gene K601E by using the primer and the probe, and the mutation of the BRAF gene K601E can be quickly, sensitively and accurately detected. The invention has the advantages of good specificity, high sensitivity, simple and convenient operation, low cost, accurate result, convenient interpretation and the like, and has good clinical application prospect.)

1. A primer and a probe for detecting mutation of BRAF gene K601E are characterized in that the primer comprises nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2, the probe comprises a nucleotide sequence shown in SEQ ID NO.3, the 5 'end of the probe is modified with FAM fluorescent group, and the 3' end of the probe is modified with NFQ-MGB non-fluorescent quenching group.

2. The primers and probes as claimed in claim 1, further comprising quality control primers, wherein the nucleotide sequences of the quality control primers are shown in SEQ ID No.4 and SEQ ID No. 5.

3. A kit for detecting mutation of BRAF gene K601E, comprising the primer and probe of claim 1 or 2.

4. The kit according to claim 3, characterized by comprising a positive control and a negative control, wherein the positive control is a mixed solution of the BRAF gene K601E mutation-containing genomic DNA and the BRAF wild-type genomic DNA, and the negative control is a BRAF gene wild-type genomic DNA-containing genomic DNA solution.

5. The kit according to claim 3, further comprising a PCR reaction solution and 50 XROX.

6. The kit according to claim 3, characterized in that the method of use comprises the following steps:

(1) extracting sample DNA;

(2) preparing a PCR reaction system;

(3) setting PCR amplification conditions;

(4) and (3) carrying out real-time fluorescent PCR amplification reaction, and judging whether the BRAF gene K601E mutation occurs in the sample according to the amplification result of the fluorescent PCR instrument and the quality control standard.

7. The kit according to claim 3, wherein the kit can be used for detecting the mutation of BRAF gene K601E.

Technical Field

The invention relates to the technical field of molecular biology, in particular to a primer, a probe and a kit for detecting mutation of BRAF gene K601E.

Background

BRAF gene mutations are common in some malignant tumors, such as melanoma, non-small cell lung cancer, non-hodgkin lymphoma, colorectal cancer, and the like. The mutation frequency of the BRAF gene is also high in thyroid cancer, particularly the mutation frequency of the BRAF gene V600E is the highest, and because the mutation is often related to poor clinical prognosis, the BRAF gene V600E mutation is an important target point for clinical detection.

However, besides the V600E mutation, the K601E mutation occurs in BRAF gene, the V600E mutation is mainly related to thyroid papillary carcinoma (PTC), but the K601E mutation is a specific mutation in thyroid follicular adenoma or follicular thyroid papillary carcinoma (FVPTC). Clinical data prove that, different from the mutation of the BRAF gene V600E, the mutation of the BRAF gene K601E is one of indexes with better patient prognosis. Therefore, as a molecular diagnostic marker for clinical benign prognosis, the K601E mutation site needs to be distinguished from other mutation sites on the BRAF gene, especially from the V600E mutation site, but the detection of BRAF gene mutation in clinic at present mainly focuses on the BRAF gene V600E mutation, and the detection method and detection product for the BRAF gene K601E mutation are rare.

The invention designs and optimizes a detection system mainly based on the principle of amplification-retardation mutation system PCR (ARMS-PCR). Through searching NCBI database, the sequence of 25bp before and after the mutation site of BRAF gene K601E is found to contain 5 continuous identical base sequences which are respectively AAAAA, TTT, AAA, GGG and CCC, and the sequence at the mutation site is AAAT. According to the basic rules of PrimerPremier 5.0 and other design software, when designing a primer probe, it is desirable to avoid the occurrence of an excessive number of identical bases, particularly 4 or more identical bases in succession, and to avoid A at the 3' end of the primer, 3 or more identical bases in succession. Meanwhile, since there are many other types of mutations, such as V600E, V600K, V600R, etc., near the site of BRAF gene K601E, the mutations near these sites also interfere with the detection of BRAF gene K601E mutation. Due to the limitation of the design conditions of the primer probes, and the multi-round primer probe test results designed aiming at the region in the experiment show that the primer probes designed according to the conventional principle have poor combination specificity and obvious non-specific amplification, and can not effectively distinguish the wild type of the BRAF gene from the mutant type of the K601E. The design of the primer probe of the site needs to find a new technical scheme to solve the existing technical problems.

Therefore, the 3 ' end of the primer is set as a complementary base of the K601E mutation site, 2 mismatched bases are continuously introduced, the primer extension is started from the 3 ' end in the PCR process, the base pair primer extension of the 3 ' end is important, after 2 mismatched bases are added, only the mutant template can realize stable amplification depending on the base pair complementary of the 3 ' end, and the wild template cannot carry out effective amplification due to the fact that 3 continuous bases of the 3 ' end are not paired, so that the wild type and K601E mutant of the BRAF gene are effectively distinguished, and meanwhile, the method can also reduce the incidence rate of false positives in the detection result. However, the optimal detection effect can be achieved only by specifically introducing which two mismatched bases, and the optimal combination scheme and PCR reaction conditions of the invention are finally obtained by continuously optimizing and adjusting a detection reaction system and reaction conditions, so that the mutation of the BRAF gene K601E can be stably detected, the lower limit of the detected mutation frequency is stabilized at 0.5%, the method is superior to the common ARMS-PCR detection method, and the requirement of clinical detection is met.

In the aspect of probe selection, the Taqman MGB probe with good specificity and high sensitivity is adopted, the 3' end of the probe is modified by the NFQ-MGB non-fluorescence quenching group, the group does not generate fluorescence, the intensity of a background signal can be greatly reduced, meanwhile, the Tm value of the probe can be improved by about 10 ℃ through the modification, and the specificity of the probe is improved. Meanwhile, the probe is a universal probe for mutation system detection and quality control system detection, and the detection cost is reduced on the basis of ensuring the detection specificity and sensitivity.

Finally, the method of the invention does not need to calculate the value of Delta CT, and the final detection result can be interpreted only by detecting the existence or nonexistence of the specific amplification product, namely the existence or nonexistence of the mutation detection amplification curve, which is very simple and intuitive.

In summary, the invention discloses a primer, a probe and a kit for detecting mutation of BRAF gene K601E, which are simple and convenient to operate, low in cost, accurate in result and convenient to interpret. The kit takes DNA as a detection object, detects whether the BRAF gene K601E mutation exists in a sample, can obtain an accurate detection result within 60 minutes, can reach the detection lower limit of mutation frequency of 0.5 percent, has the advantages of good specificity, high sensitivity, simple and convenient operation, low cost, accurate result, convenient interpretation and the like, and has good clinical application prospect.

Disclosure of Invention

The invention aims to provide a primer, a probe and a kit for detecting the mutation of the BRAF gene K601E, which are simple and convenient to operate, low in cost, accurate in result and convenient to interpret, and meet the clinical detection requirement on the mutation of the BRAF gene K601E.

The invention provides a primer and a probe for detecting BRAF gene K601E mutation, which are characterized in that the primer comprises nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2, wherein the 3 ' end of the primer sequence SEQ ID NO.1 is a mutation site complementary base C, then continuous 2 mismatched bases AC are introduced, 2 mismatched bases are introduced for improving the specificity of the mutation detection primer, the probe comprises the nucleotide sequence shown in SEQ ID NO.3, the 5 ' end of the probe is modified with FAM fluorescent group, the 3 ' end is modified with NFQ-MGB non-fluorescent quenching group, the group does not generate fluorescence, on one hand, the intensity of a background signal can be greatly reduced, on the other hand, when the probe is complete, the fluorescence emitted by a reporter group is absorbed by the quenching group, the signal can not be detected by an instrument, along with the PCR, Taq enzyme meets the probe combined with a template in the process of DNA chain extension, the 5 '→ 3' exonuclease activity cleaves the probe and the reporter moves away from the fluorescence quencher, producing a fluorescent signal. Meanwhile, the MGB modifying group on the specific probe can improve the Tm value of the probe by about 10 ℃ and increase the specificity of the probe.

Further, the kit also comprises a quality control primer, and the nucleotide sequence of the quality control primer is shown as SEQ ID NO.4 and SEQ ID NO. 5.

The primers with the nucleotide sequences of SEQ ID NO.1 and SEQ ID NO.2 and the probe with the nucleotide sequence of SEQ ID NO.3 form a mutation detection reaction system, and the primers with the nucleotide sequences of SEQ ID NO.4 and SEQ ID NO.5 and the probe with the nucleotide sequence of SEQ ID NO.3 form a quality control detection reaction system.

The primer and the probe can be applied to the preparation of a kit for detecting mutation of BRAF gene K601E.

The kit for detecting mutation of BRAF gene K601E is characterized by comprising the following reagents:

1) PCR reaction and 50 × ROX: the PCR reaction solution contains DNA polymerase and Mg ²⁺PCR reaction buffer, dATP, dTTP, dCTP and dGTP, wherein 50 XROX is a reference dye containing ROX fluorescence;

2) the nucleotide sequence of the mutation detection primer probe premix is shown as SEQ ID No.: 1. 2 and 3, wherein the concentration of each primer is 10 μ M and the concentration of the probe is 10 μ M. The mother liquors of the two mutation detection primers and probes were run at 2: 2: 1 in a volume ratio;

3) the quality control detection primer probe premix has a nucleotide sequence shown as SEQ ID No.: 3. 4, and 5, wherein the concentration of each primer is 10 μ M, and the concentration of the probe is 10 μ M. The mother liquor of two quality control detection primers and probes is mixed in a proportion of 2: 2: 1 in a volume ratio;

4) positive control: the method comprises the following steps of mixing a mixed solution of the BRAF gene K601E mutant genomic DNA and the BRAF wild genomic DNA, wherein the BRAF gene K601E mutation frequency is 5%, and the sample concentration is 10 ng/muL;

5) negative control: the BRAF gene K601E mutation frequency is 0%, and the sample concentration is 10 ng/. mu.L.

The application of the kit for detecting the mutation of the BRAF gene K601E in the detection of the mutation gene of the BRAF gene K601E comprises the following steps:

1) extracting sample DNA:

according to the sample properties, the sample DNA extraction was performed using a corresponding DNA extraction kit, and the extracted DNA was diluted to 10 ng/. mu.L using nuclease-free water.

2) Preparing a PCR reaction system:

PCR reaction solution 10. mu.L, 50 XROX 0.4. mu.L, primer probe premix 1. mu.L, sample DNA template 2. mu.L, nuclease-free water 6.6. mu.L, total 20. mu.L.

3) Setting PCR amplification conditions:

2min at 37 ℃; 5min at 95 ℃; 10s at 95 ℃, 30s at 65 ℃ and 45 cycles; FAM channel fluorescence was collected at 65 ℃ over a 30s period.

4) And (3) carrying out real-time fluorescent PCR amplification reaction, and judging whether the BRAF gene K601E mutation occurs in the sample according to the amplification result of the fluorescent PCR instrument and the quality control standard.

The quality control standards comprise:

a) positive control: the quality control detection system and the mutation detection system both have normal smooth amplification curves, as shown in FIG. 1;

b) negative control: the quality control detection system has a normal smooth amplification curve, and the mutation detection system has no amplification curve, as shown in FIG. 2;

c) sample quality control: the quality control detection system has a normal smooth amplification curve.

If the conditions are met, the quality control is qualified.

Interpretation of the test results of the samples:

a) on the premise of qualified quality control, if a sample mutation detection system detects a normal smooth amplification curve, the sample is positive for BRAF gene K601E mutation;

b) and on the premise of qualified quality control, if the sample mutation detection system does not detect an amplification curve, the sample is negative to the BRAF gene K601E mutation.

The invention has the beneficial effects that: (1) 2 mismatched bases are continuously introduced into the 3' end, and special primer design is carried out, so that the detection specificity is effectively improved, the interference of other possible mutation conditions around the mutation site of the BRAF gene K601E on the detection result is reduced, and the occurrence of false positive is avoided; (2) the lower limit of the detection of the mutation frequency can reach 0.5 percent, is 1 percent higher than that of the common ARMS-PCR method, and has higher sensitivity; (3) the mutation detection system and the quality control detection system share one Taqman probe, so that the accuracy of quality control is improved, and the detection cost is reduced; (4) the kit disclosed by the invention is based on a real-time fluorescent PCR system, realizes the rapid detection of the mutation of the BRAF gene K601E, is high in sensitivity, strong in specificity and short in detection time, and can complete the detection in 60 minutes; (5) the detection result is convenient to interpret, the result can be judged according to the existence or nonexistence of the amplification signal, the delta CT value does not need to be calculated, and the difficulty of result interpretation is greatly reduced; (6) the sample has wide application range, and can be whole blood, fresh pathological tissue, paraffin embedded tissue or frozen pathological section.

Drawings

FIG. 1 is a graph showing the effect of the kit on the detection of a positive control for BRAF gene K601E.

FIG. 2 is a diagram showing the effect of detecting wild type BRAF gene with the kit.

Fig. 3 is a graph showing the effect of the primer and probe combination of the present invention in detecting BRAF gene K601E mutation positive control in combination with other primers and probes.

FIG. 4 is a graph showing the effect of the kit on detection of the standard BRAF gene K601E with different mutation frequencies.

Fig. 5 is a graph showing the effect of 1 example of the kit for detecting the BRAF gene K601E mutation positive clinical specimen.

Fig. 6 is a graph of the primary sequencing results of 1 case of BRAF gene K601E mutation-positive clinical samples.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. Materials, reagents and the like used in the examples are, unless otherwise specified, conventional products commercially available.