CN112322738A

CN112322738A - BRAFV600EMutation ratio detection kit and detection method

Info

Publication number: CN112322738A
Application number: CN202011299529.XA
Authority: CN
Inventors: 郑仲征; 杜可明; 袁志阳; 徐祥; 邵文涵
Original assignee: Shanghai Dishuobeiken Gene Technology Co ltd; Shanghai Tissuebank Biotechnology Co ltd; Shanghai Tissuebank Medical Laboratory Co ltd; Shenzhen Tissuebank Precision Medicine Co ltd
Current assignee: Shanghai Dishuobeiken Gene Technology Co ltd; Shanghai Tissuebank Biotechnology Co ltd; Shanghai Tissuebank Medical Laboratory Co ltd; Shenzhen Tissuebank Precision Medicine Co ltd
Priority date: 2020-11-18
Filing date: 2020-11-18
Publication date: 2021-02-05

Abstract

The invention provides a BRAF^V600EA mutation ratio detection kit and a detection method, wherein, the BRAF^V600EThe mutation ratio detection kit comprises a specific primer I, a specific primer II, a specific probe I and a specific probe II; the BRAF^V600EThe mutation ratio detection method comprises the following steps: performing digital PCR amplification by using the first primer, the second primer, the first probe and the second probe and ctDNA as a template, and calculating the copy number of nucleic acids of different fluorescent channels to obtain BRAF^V600EMutation ratio of gene locus. BRAF of the invention^V600EThe mutation ratio detection kit and the detection method can overcome tumor heterogeneity and effectively detect BRAF^V600EThe mutation is as low as 0.01%, and the detection accuracy and sensitivity are obviously improved.

Description

BRAFV600EMutation ratio detection kit and detection method

Technical Field

The invention relates to the technical field of molecular biology, in particular to BRAF^V600EMutation ratio detection kit and BRAF^V600EAnd (3) a mutation ratio detection method.

Background

The serine/threonine-specific kinase encoded by the BRAF gene is a key kinase in the Ras-Raf-MEK-ERK pathway and plays a role in signal transduction regulation by activating MEK as a downstream substrate. BRAF proteins usually remain activated only when signaling is delivered, but mutant BRAF proteins are persistently activated. BRAF is thought to be the driver of multiple tumorigenesis, and mutations leading to aberrant activation of these pathways can result in tumor transformation of multiple mutant cells. The most common mutation form of BRAF is the mutation of nucleotide T/A at position l799, which causes the replacement of glutamic acid (V) with valine (E), accounting for about 97 percent of the mutation of BRAF gene, and is named as BRAF^V600E. In recent years, many scholars have repeatedly detected a mutation in the BRAF gene in focal specimens of Langerhans Cell Histocytosis (LCH) patients and considered the mutation as a driver of LCH pathogenesis. LCH is a group of abnormal proliferation of langerhans cells from bone marrow with varying numbers of neutrophils, eosinophils, lymphocytes, plasma cells and multinucleated giant cell infiltrates, causing tissue destruction. The disease has high incidence in the young children, the skin is only affected in the mild cases, and multiple organs are affected in the severe cases to cause important organsFunctional impairment, mortality rate in untreated LCH patients is as high as 92.1%. In addition, many studies have demonstrated that high frequency BRAF is present in populations with malignant melanoma, hairy cell leukemia, papillary thyroid carcinoma, colon cancer, and the like^V600EAnd (4) mutation. Thus, BRAF^V600EMutation detection can not only carry out early screening of tumors and stratification of risks of tumor patients, but also provide guidance for the use of targeted drugs for BRAF mutation tumor patients.

Currently, BRAF^V600EMutation detection is mainly completed by qPCR and first-generation or second-generation sequencing, and the detection accuracy and sensitivity are poor.

Disclosure of Invention

The invention provides a BRAF^V600EMutation ratio detection kit and BRAF^V600EA mutation ratio detection method aiming at improving BRAF^V600EThe accuracy and sensitivity of mutation ratio detection.

In order to achieve the above object, in a first aspect, the present invention provides a BRAF^V600EThe mutation ratio detection kit comprises:

the nucleotide sequence of the primer I is shown as SEQ ID No. 1;

the nucleotide sequence of the primer II is shown as SEQ ID No. 2;

the probe I comprises a nucleotide sequence I shown as SEQ ID No.3 and a fluorescent group and a quenching group, wherein the fluorescent group and the quenching group are respectively connected with the 5 'end and the 3' end of the nucleotide sequence I; and

and the probe II comprises a nucleotide sequence II shown as SEQ ID No.4 and a fluorescent group and a quenching group, wherein the fluorescent group and the quenching group are respectively connected with the 5 'end and the 3' end of the nucleotide sequence II.

Optionally, the 5 'end of the first nucleotide sequence is connected with a FAM fluorescent group, and the 3' end of the first nucleotide sequence is connected with a MGB quenching group; the 5 'end of the second nucleotide sequence is connected with a HEX fluorescent group, and the 3' end of the second nucleotide sequence is connected with a MGB quenching group.

In a second aspect, the present invention further provides a BRAF^V600EThe mutation ratio detection method comprises the following steps:

(1) obtaining a sample to be detected, and extracting ctDNA of the sample to be detected;

(2) performing digital PCR amplification by using the first primer, the second primer, the first probe and the second probe and taking the ctDNA obtained in the step (1) as a template, and scanning fluorescent signals of FAM and HEX channels;

(3) calculating the copy numbers of nucleic acids of different channels by using the fluorescent signals of the FAM and HEX channels in the step (2) to obtain the mutation ratio of the gene locus;

wherein the nucleotide sequence of the primer I is shown as SEQ ID No. 1; the nucleotide sequence of the primer II is shown as SEQ ID No. 2; the probe I comprises a nucleotide sequence I shown as SEQ ID No.3 and a fluorescent group and a quenching group, wherein the fluorescent group and the quenching group are respectively connected with the 5 'end and the 3' end of the nucleotide sequence I; the probe II comprises a nucleotide sequence II shown as SEQ ID No.4 and a fluorescent group and a quenching group, wherein the fluorescent group and the quenching group are respectively connected with the 5 'end and the 3' end of the nucleotide sequence II.

The BRAF provided by the invention^V600EMutation ratio detection kit and BRAF^V600EThe mutation ratio detection method is developed aiming at ctDNA samples, is based on digital PCR, can overcome tumor heterogeneity and effectively detect BRAF^V600EThe mutation is as low as 0.01 percent, the sensitivity and the accuracy of detection are obviously improved, the kit has the characteristics of strong specificity, high sensitivity and strong practicability, and can realize screening of tumor genes, guide the application of targeted drugs to tumor patients and monitor the minimal residual disease of the tumor patients.

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, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the structures shown in the drawings without creative efforts.

FIG. 1 shows a BRAF in accordance with the first embodiment of the present invention^V600EDetecting the mutation ratio standard substance;

FIG. 2 is a graph showing the correlation between the mutation ratio of the test results of the present invention and the standard in the first example;

FIG. 3 shows a BRAF portion of the third embodiment^V600EExtracting ctDNA from a mutation positive clinical sample;

FIG. 4 is a graph showing the correlation between the second generation sequencing assay results and the present invention in example III.

The implementation, functional features and advantages of the objects of the present invention will be further explained with reference to the accompanying drawings.

Detailed Description

The present invention is further illustrated by the following examples, which are intended to be illustrative, but not limiting, of the present invention. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications also fall into the protection scope of the present invention.

The invention provides a BRAF^V600EThe mutation ratio detection kit comprises a first primer, a second primer, a first probe and a second probe; the nucleotide sequence of the primer I is shown as SEQ ID No.1, and the nucleotide sequence of the primer II is shown as SEQ ID No. 2; the probe I comprises a nucleotide sequence I shown as SEQ ID No.3 and a fluorescent group and a quenching group, wherein the fluorescent group and the quenching group are respectively connected with the 5 'end and the 3' end of the nucleotide sequence I; the probe II comprises a nucleotide sequence II shown as SEQ ID No.4 and a fluorescent group and a quenching group, wherein the fluorescent group and the quenching group are respectively connected with the 5 'end and the 3' end of the nucleotide sequence II.

The invention uses Primer Premier 5.0 to BRAF^V600EDesigning the mutation sites of the first primer, the second primer, the first probe and the second probe. The Tm values of the two primers and the two probes are both 70 ℃, so that hairpin structures and dimers can be avoided. Referring to Table 1, the first primer is an upstream primer for amplifying BRAFThe 5' end of SEQ ID No.1 is positioned at 1753bp downstream of the BRAF gene sequence; the second primer is a downstream primer for amplifying BRAF, and the 5' end of SEQ ID No.2 is positioned at 1823bp downstream of the BRAF gene sequence; the first probe is a probe for detecting wild type BRAF, the 5' end of SEQ ID No.3 is positioned at 1794bp downstream of BRAF gene sequence, and the second probe is a probe for detecting mutant BRAF^V600EThe 5' end of SEQ ID No.4 is positioned at 1793bp downstream of the BRAF gene sequence; the I799 site of BRAF gene generates T/A mutation, the site of wild type BRAF is T, and the mutant BRAF^V600EThe position of (A) is thus the corresponding 6 th base of SEQ ID No.3 in the direction from the 5 'end towards the 3' end is A (marked in red in the figure) and the 7 th base of SEQ ID No.4 in the direction from the 5 'end towards the 3' end is T (marked in red in the figure).

TABLE 1 detection primer and Probe sequences

And (4) supplementary notes: primers and probes were synthesized by bioengineering (Shanghai) Inc. and purified by HPLC. Primers and probes for lysis dilution use M ltraPure^TMDNase/RNase-Free Distilled Water (Invitrogen, cat. No. 10977-015), the concentration of the primer and probe mother liquor is 100 pmol/. mu.l, the primer and Taqman probe can be diluted according to specific conditions, and the kit can be stored in a refrigerator at-20 ℃ in a dark place.

Optionally, the 5 'end of the first nucleotide sequence is connected with a FAM fluorescent group, and the 3' end of the first nucleotide sequence is connected with a MGB quenching group; the 5 'end of the second nucleotide sequence is connected with a HEX fluorescent group, and the 3' end of the second nucleotide sequence is connected with a MGB quenching group. Namely, the 5' end of the wild type BRAF detection probe is marked by HEX, and the mutant BRAF^V600EThe 5 'end of the detection probe is marked by FAM, and the 3' ends of the two probes are marked by MGB quenching groups.

The invention also provides a BRAF^V600EThe mutation ratio detection method comprises the following steps:

BRAF of the invention^V600EThe primer I, the primer II, the probe I and the probe II used in the mutation ratio detection method are already introduced in the foregoing, and are not described herein again. The sample to be detected is peripheral blood of a tumor patient, the ctDNA is a tumor genome DNA fragment released by tumor cells into a blood circulation system, and the ctDNA carries gene information of tumor mutation, so that tumor heterogeneity can be overcome.

BRAF^V600EThe mutation ratio detection method comprises the following specific steps:

1) collecting 8ml of peripheral blood sample of a tumor patient by using a Streck free DNA storage tube, centrifuging the sample at 1600g and 4 ℃ for 10min, and transferring plasma in the sample to a new centrifuge tube;

2)16000g, centrifuging the plasma at 4 deg.C for 10min to remove residual cell membranes from the plasma, transferring 3ml of the plasma to a new centrifuge tube;

3) extracting ctDNA in the plasma by using a QIAamp MinElute ccfDNA Mini Kit, and extracting the ctDNA according to Kit operation instructions;

4) concentrating the ctDNA, adding the ctDNA into a droplet type digital PCR reaction solution according to a droplet type digital PCR detection system in the table 2, and detecting the droplet type digital PCR according to a droplet type digital PCR amplification program in the table 3;

5) after the amplification is finished, scanning and detecting the digital PCR chip by a microdroplet detector to obtain fluorescence signals of FAM and HEX channels and obtain the mutation ratio of the gene locus.

TABLE 2 micro-drop digital PCR detection system

Composition of matter	Volume (μ l)
		2*dPCR Mix	12.5
Primer Mix	2.5
		ctDNA template	2
Sterile water	8

And (4) supplementary notes: the proportion of the Primer Mix is SEQ ID No. 1: SEQ ID No. 2: SEQ ID No. 4: SEQ ID No. 3: sterile water 3: 3: 1: 1: 92.

TABLE 3 micro-droplet digital PCR amplification procedure

Example one, BRAF^V600EDetection of ctDNA standard

In this example, Primer Premier 5.0 software was used for Primer design for BRAF^V600EDesigning the sequences of the first primer, the second primer, the first probe and the second probe by mutation sequences, and comparing the four sequences in an IMGT database to confirm that the first primer, the second primer, the first probe and the second probe can specifically amplify or sequence the required fragment. The sequences of the primer I, the primer II, the probe I and the probe II are shown in Table 1.

Adopting the first primer, the second primer, the first probe and the second probe pair BRAF^V600ETesting ctDNA standards to validate the BRAF^V600EDetection of BRAF by mutation ratio detection method^V600EAccuracy and sensitivity of mutation ratio. In particular, the BRAF^V600EctDNA standards were purchased from AccuraGen (cat No. 002CBF01) and included 4 mutation ratios, respectively: wild type standard, 5% mutation ratio standard, 1% mutation ratio standard, and 0.1% mutation ratio standard. The 0.1% mutant ratio standards were diluted to 0.05% mutant and 0.01% mutant standards using wild-type standards. In this embodiment, the BRAF is utilized^V600EThe mutation ratio detection method is used for detecting the series of standard substances in 3 continuous batches and testing the BRAF^V600ELinear detection range of mutation ratio detection method.

Referring to fig. 1, a, b, c, d, e, and f in the figure are the detection results of 5% mutation ratio standard, 1% mutation ratio standard, 0.1% mutation ratio standard, 0.05% mutation ratio standard, 0.01% mutation ratio standard, and wild-type standard, in that order. The cross dotted lines in the figure respectively indicate the set threshold values of FAM and HEX channels, and the upper left area is the area where only FAM fluorescent signals are detected in the liquid drop, namely only mutant BRAF gene templates exist in the reaction; the lower right region is that only HEX fluorescent signals are detected in the liquid drop, namely only wild type BRAF gene templates are detected in the reaction; the upper right region is that FAM and HEX fluorescent signals are simultaneously detected in the liquid drop, namely a mutant BRAF gene template and a wild BRAF gene template exist in the reaction; the lower left region is background, i.e., there is no mutant BRAF gene template and no wild-type BRAF gene template in the reaction.

Please refer to table 4, the BRAF^V600EActual detection value detected by mutation proportion detection method and BRAF^V600EThe theoretical mutation proportion of the ctDNA standard is relatively close. The correlation curves are plotted according to the data in Table 4, see FIG. 2, where the correlation curves in FIG. 2 show that R²>0.99(R²1); description of the BRAF proposed by the invention^V600EThe mutation ratio detection method is linear in detection within 0.01% -5% of mutation ratio. The above results illustrate the BRAF proposed by the present invention^V600EDetection of BRAF by mutation ratio detection method^V600EThe accuracy and sensitivity of the mutation ratio are high.

TABLE 4 BRAF^V600EDetection results of ctDNA standards

EXAMPLE two clinical sample testing

To further authenticate the BRAF^V600EMutation ratio detection method for 0.01% BRAF^V600EStability of the detection of mutant standards, this example utilizes the BRAF^V600EMutation ratio detection method for 0.01% mutation ratio BRAF^V600EThe mutant standards were tested in 10 consecutive batches, each batch being subjected to 3 technical replicates. Please refer to table 5, the BRAF^V600EMutation ratio detection method for 0.01% mutation ratio BRAF^V600EThe positive detection rate of the mutation standard is 100%, and the average value of the mutation rate detection is 0.014%, which is close to the theoretical mutation rate. The above results confirm that the BRAF^V600EThe mutation ratio detection method can realize the detection of 0.01 percent BRAF^V600ELow frequency mutationThe stable detection of (1).

TABLE 50.01% mutation Rate of BRAF^V600ETest results of standards

Test batches	Test results (%)	Test results (%)	Test results (%)
				1	0.020	0.015	0.007
2	0.021	0.030	0.010
				3	0.010	0.015	0.030
4	0.015	0.010	0.010
				5	0.009	0.005	0.006
6	0.010	0.020	0.009
				7	0.020	0.012	0.021
8	0.005	0.008	0.019
				9	0.008	0.010	0.023
10	0.012	0.014	0.017

EXAMPLE III clinical sample testing

The invention then uses a plurality of clinical samples as templates, and respectively uses a second generation sequencing detection method and the BRAF^V600EA mutation ratio detection method for detecting a plurality of clinical samples to verify the BRAF^V600EMutation ratio detection method for BRAF^V600EAnd (3) detection accuracy of mutation ratio.

In this example, 30 BRAFs were extracted according to the QIAamp MinElute ccfDNA Mini Kit instruction^V600EAnd (3) mutating ctDNA (plasma input amount is 3ml) of the positive clinical sample, selecting ctDNA samples with the serial numbers of D20GP001, D20GP002, D20GP003, D20GP004, D20GP005 and D20GP006, and detecting the quality of the ctDNA extracted from the samples by using an Agilent 2200 electrophoresis working platform. The detection results are shown in fig. 3, wherein a, b, c, D, e and f respectively represent electrophoresis results of ctDNA extraction by D20GP001, D20GP002, D20GP003, D20GP004, D20GP005 and D20GP006, and the results show that each sample has a distinct peak (indicated by an arrow in the figure) at 170bp, and thus, the ctDNA extraction of each sample is confirmed.

Please refer to table 6, the BRAF^V600EThe detection result of the mutation ratio detection method is consistent with that of the second generation sequencing detection method, namely, the BRAF^V600EMutation ratio detection method and second-generation sequencing detection method for 20 BRAFs^V600EThe detection results of the mutated ctDNA samples were all negative. Referring to fig. 4, the correlation curve analysis of the data in table 6 shows that the BRAF is a new candidate for the analysis of the correlation curve^V600EThe correlation between the detection result of the mutation ratio detection method and the detection result of the next generation sequencing is high, and the correlation coefficient R²0.962. The above results illustrate the BRAF^V600EThe mutation ratio detection method has accurate and reliable detection result and higher accuracy.

TABLE 6 BRAF^V600EClinical sample test results

Claims

1. BRAF^V600EThe mutation ratio detection kit is characterized by comprising:

the nucleotide sequence of the primer I is shown as SEQ ID No. 1;

the nucleotide sequence of the primer II is shown as SEQ ID No. 2;

2. The BRAF of claim 1^V600EThe mutation ratio detection kit is characterized in that the 5 'end of the first nucleotide sequence is connected with a FAM fluorescent group, and the 3' end of the first nucleotide sequence is connected with a MGB quenching group; the 5 'end of the second nucleotide sequence is connected with a HEX fluorescent group, and the 3' end of the second nucleotide sequence is connected with a MGB quenching group.

3. BRAF^V600EThe mutation ratio detection method is characterized by comprising the following steps of:

4. The BRAF of claim 3^V600EThe mutation ratio detection method is characterized in that the 5 'end of the first nucleotide sequence is connected with an FAM fluorescent group, and the 3' end of the first nucleotide sequence is connected with an MGB quenching group; the 5 'end of the second nucleotide sequence is connected with a HEX fluorescent group, and the 3' end of the second nucleotide sequence is connected with a MGB quenching group.