WO2018014519A1

WO2018014519A1 - Primer, probe and kit used for detecting c-kit gene mutation

Info

Publication number: WO2018014519A1
Application number: PCT/CN2017/000444
Authority: WO
Inventors: 莫敏俐; 李晖; 陈钊; 丁凤
Original assignee: 北京雅康博生物科技有限公司
Priority date: 2016-07-18
Filing date: 2017-07-14
Publication date: 2018-01-25

Abstract

A primer, probe and kit used for detecting C-KIT gene mutation. The primer is shown in SEQ ID NO: 1-2, 4-5, 7-8 or 10-11, and the probe is shown in SEQ ID NO: 3, 6, 9 and 12. The kit is used for real-time fluorescent polymerase chain reaction (PCR) and may detect mutation of a C-KIT gene.

Description

Primers, probes and kits for detecting C-KIT gene mutations

Technical field

The present invention relates to the field of biotechnology, and in particular to a primer, a probe and a related kit for detecting a mutation of a C-KIT gene.

Background technique

C-KIT is a protooncogene located on chromosome 4q11-12 with a total length of about 80 kb. The C-KIT-encoded C-KIT protein is a member of the type III receptor tyrosine kinase family, a transmembrane protein with a molecular weight of approximately 145 kD, comprising an intracellular tyrosine kinase domain, a transmembrane domain, and ligand binding. Site extracellular region. When the ligand binds to it, it activates its own tyrosine protein kinase activity, and activates the downstream signal transduction pathway through a series of reactions, thereby regulating cell growth and proliferation.

Gastrointestinal stromal tumors (GIST) are the most common mesenchymal tumors of the digestive tract. Studies have shown that the C-KIT gene mutation rate in GIST patients is about 90%.

At present, Sanger sequencing is the main method for detecting C-KIT gene mutation. However, the sensitivity of this method is low, which leads to the occurrence of missed detection and false negatives, and the detection time is long, which cannot meet the actual needs of clinical testing. There is an urgent need to develop a highly sensitive and rapid C-KIT gene mutation detection technology.

The inventors of the present invention found four new mutation sites (mutations 1-4) located in the C-KIT gene associated with gastrointestinal stromal tumors in the routine detection of gastrointestinal stromal tumors (GIST). The inventors of the present invention have developed a rapid, highly sensitive, and easy-to-use detection kit and related detection methods for these mutation sites, which can be used for the prognosis of GIST chemotherapy.

Summary of the invention

It is an object of the present invention to provide a primer and a probe for detecting a mutation of a C-KIT gene, the mutation being at least one of the following four mutations:

Mutation 1:

The 1394 position C was replaced by G (base mutation: 1394C>G, protein mutation: S465C).

Primers and probes directed to mutation 1 include the following sequences:

Forward primer (F): GATGTGCAGACACTAAACTCACG (SEQ ID No: 1)

Reverse primer (R): GCACTAGAATCTATAGAACTCTGAAC (SEQ ID No: 2)

Probe (PB): TGGGCCACCGTTGGAAAAGCTAG (SEQ ID No: 3)

Mutation 2:

A deletion of 39 bases was deleted at 1648-1686 (base mutation: 1648_1686del39, protein mutation: K550_E562del).

Primers and probes for mutation 2 include the following sequences:

Forward primer (F): CCTTTCTCCCCACAGATAAATGG (SEQ ID No: 4)

Reverse primer (R): AAGTCACTGTTATGTGTACCCAA (SEQ ID No: 5)

Probe (PB): CATGGAAAGCCCCTGTTTCATACTGACC (SEQ ID No: 6)

Mutation 3:

A deletion of 36 base mutations at 1654-1689 (base mutation: 1654_1689del36, protein mutation: M552_I563del).

Primers and probes directed to mutation 3 include the following sequences:

Forward primer (F): CCCACAGAAACCCAATGGAA (SEQ ID No: 7)

Reverse primer (R): AAGTCACTGTTATGTGTACCCAA (SEQ ID No: 8)

Probe (PB): CATGGAAAGCCCCTGTTTCATACTGACC (SEQ ID No: 9)

Mutation 4:

The 1675-1681 position was replaced by A (base mutation: 1675_1681>A, protein mutation: V559_E561>A).

Primers and probes directed to mutation 4 include the following sequences:

Forward primer (F): CCATGTATGAAGTACAGTGGAAGAAG (SEQ ID No: 10)

Reverse primer (R): AAGTCACTGTTATGTGTACCCAA (SEQ ID No: 11)

Probe (PB): CATGGAAAGCCCCTGTTTCATACTGACC (SEQ ID No: 12)

Another aspect of the present invention provides a detection kit for detecting a mutation of a C-KIT gene, the kit comprising the primer shown in SEQ ID No. 1-2 and the probe represented by SEQ ID No. 3, SEQ The primer shown in ID No. 4-5 and the probe shown in SEQ ID No. 6, the primer shown in SEQ ID No. 7-8, and the probe shown in SEQ ID No. 9, and/or SEQ ID The primer shown in No. 10-11 and the probe shown in SEQ ID No. 12.

The kit of the present invention may further comprise an internal reference gene and an internal control gene depending on the PCR method employed.

Another aspect of the invention provides a method of detecting a C-KIT gene mutation comprising the steps of:

(1) extracting genomic DNA in the sample, the detection sample includes fresh pathological tissue, paraffin-embedded tissue, whole blood and plasma;

(2) amplifying DNA using the kit of the present invention;

(3) Judging the detection result according to the Ct value displayed by the fluorescence PCR instrument: detecting the FAM fluorescence intensity of the reaction system, and using the Ct value required for the FAM to reach the set threshold value as a negative and positive determination standard, the Ct value is >38 or No amplification was negative, and Ct value ≤ 38 was positive.

The present invention employs specific primer and probe technologies to specifically detect C-KIT gene mutations. This method has high sensitivity, high specificity and fast detection speed.

DRAWINGS

Figure 1 is a PCR diagram of a negative sample (wild type).

Figure 2 is a PCR diagram of a positive sample for detecting mutation 1.

Figure 3 is a PCR diagram of a positive sample for detecting mutation 2.

Figure 4 is a PCR diagram of a positive sample for detecting mutation 3.

Figure 5 is a PCR diagram of a positive sample for detecting mutation 4.

detailed description

The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention. The experimental methods in the examples which do not specify the specific conditions are in accordance with conventional conditions well known to those skilled in the art or in accordance with the conditions recommended by the manufacturer.

Principle

After the specific primers and probes of the present invention are combined with the DNA template, Taq DNA polymerase uses deoxynucleotides (dNTPs) as a substrate, and internal reference gene (Internal Reference, IR) and C-KIT gene mutant genes are in vitro. Amplification. The detection was carried out by fluorescence PCR, and the fluorescence was released by specific probe hydrolysis, and the progress of the PCR reaction was monitored to determine the mutation of the C-KIT gene.

The kit of the present invention is separately provided with an internal reference gene detection system. The internal reference gene is a housekeeping gene that is different from the C-KIT gene to be examined. By detecting the amplification of the internal reference gene (FAM channel), it can be analyzed whether the DNA to be detected can be normally amplified, thereby eliminating DNA purity, poor concentration, or containing PCR inhibitors and the like to cause PCR detection failure.

The kit of the present invention simultaneously sets an internal control (IC) detection system in the C-KIT gene mutation detection system. Both systems react simultaneously in the same PCR tube. The internal control gene is also a housekeeping gene that is different from the gene C-KIT to be detected. The probe that recognizes the C-KIT gene mutation template is modified to a FAM fluorescent group, and the probe that recognizes the internal control gene template is modified to a HEX fluorescent group. By detecting the internal control gene amplification (HEX channel), it can be analyzed whether the DNA to be detected can be normally amplified, thereby eliminating the possibility of PCR detection failure caused by missing reagents or samples, and samples containing PCR inhibitors.

When the test results of this kit are determined, the FAM and HEX channels in the negative control (NC) should be amplified without a typical S-shaped curve. Note: The typical S-shaped curve is in the exponential phase and straight line. Period and platform period) or no Ct value, FAM, HEX channel detection in positive control products (PC) should have amplification (typical S-shaped curve) and Ct value ≤ 28; otherwise, the experiment is invalid, repeat experiment .

2. Experimental materials and equipment

1. Design synthetic specific primers and probes for mutation sites

Specific primers and probes were designed for the C-KIT gene mutation site. Optimized by specific primers and probes for highly sensitive and rapid detection.

The optimized primers and probes are as follows:

For mutation 1:

Forward primer (F): GATGTGCAGACACTAAACTCACG (SEQ ID No: 1)

Reverse primer (R): GCACTAGAATCTATAGAACTCTGAAC (SEQ ID No: 2)

Probe (PB): TGGGCCACCGTTGGAAAAGCTAG (SEQ ID No: 3)

For mutation 2:

Forward primer (F): CCTTTCTCCCCACAGATAAATGG (SEQ ID No: 4)

Reverse primer (R): AAGTCACTGTTATGTGTACCCAA (SEQ ID No: 5)

Probe (PB): CATGGAAAGCCCCTGTTTCATACTGACC (SEQ ID No: 6)

For mutation 3:

Forward primer (F): CCCACAGAAACCCAATGGAA (SEQ ID No: 7)

Reverse primer (R): AAGTCACTGTTATGTGTACCCAA (SEQ ID No: 8)

Probe (PB): CATGGAAAGCCCCTGTTTCATACTGACC (SEQ ID No: 9)

For mutation 4:

Forward primer (F): CCATGTATGAAGTACAGTGGAAGAAG (SEQ ID No: 10)

Reverse primer (R): AAGTCACTGTTATGTGTACCCAA (SEQ ID No: 11)

Probe (PB): CATGGAAAGCCCCTGTTTCATACTGACC (SEQ ID No: 12)

2. Detection of sample processing and DNA extraction

The test sample may be fresh pathological tissue, paraffin embedded tissue, whole blood, plasma, and peritoneal effusion. The following is only an example of a paraffin-embedded tissue sample.

Before the sample collection, the patient was not treated with tyrosine kinase inhibitors (TKIs); since the quality of the DNA sample will affect the test results, it should be determined that the paraffin-embedded tissue sample from the DNA sample contains cancerous tissue. The cells were not less than 25% of the entire sample; and the DNA samples were OD260/OD280=1.8±0.2, OD260/OD230≥1.7; and the concentration was 5-10 ng/μl.

The paraffin-embedded tissue samples should not be stored for more than 12 months at room temperature, and the extracted DNA samples should be stored under -20 °C freezing conditions for a period of not more than 6 months.

3. Applicable instruments

Stratagene Mx3000P.

4. Composition of the kit

The composition of the kit is shown in Table 1. The kit does not contain nucleic acid extraction components, and DNA extraction of paraffin-embedded tissue samples is performed using the QIAamp DNA FFPE Tissue Kit (Qiagen, Cat. No. 56404) kit. The composition of the test reagent and the mutation site of the C-KIT gene to be detected are shown in Table 2. The IR detection reagent contains only the internal reference gene detection system (FAM channel), and the C-KIT detection reagent also contains the C-KIT gene mutation detection system (FAM channel) and the internal control gene detection system (HEX channel).

Table 1: Kit Composition

The specific sequence of the internal reference gene and the internal control gene can be easily determined by a person skilled in the art according to experimental conditions or provided by Beijing Yakambo Biotechnology Co., Ltd.

Three. Detection method

1. Using the QIAamp DNA FFPE Tissue Kit (manufactured by Qiagen) kit, DNA extraction of paraffin-embedded tissue samples was performed according to the instruction manual.

2. Take the test reagent and positive control (PC) in the kit. The test reagent and the positive control product PC were briefly centrifuged and placed on ice.

3. The IR detection reagent and Taq DNA polymerase were mixed at a volume ratio of 17:0.3, and the C-KIT detection reagent and Taq DNA polymerase were mixed at a volume ratio of 17:0.3. The IR detection reagent and C-KIT detection reagent were dispensed into the eight tubes at 17.3/well. Add 2.7 μl of DNA sample to be tested, negative control (NC, buffer for dissolving DNA, user-supplied) to the eight tubes containing IR detection reagent and C-KIT detection reagent, and positive control products. PC, blow and mix, cover the tube cover and centrifuge briefly. Note: Do not use a vortex oscilloscope when mixing; follow up immediately after mixing.

4. The fluorescence PCR instrument detection channel setting needs to select FAM and HEX channels at the same time (the reference dye is set to “None”). The reaction procedure is set as follows (Table 2):

Table 2:

4. Explanation of test results

1. Ct value determination: First set the baseline of the Stratagene MX3000P fluorescence PCR instrument: select the fluorescence signal when the "Adaptive baseline" setting is selected, and the threshold setting principle is just above the threshold line just above the normal negative control. The highest point of the NC amplification curve (random noise line) is that the NC control curve of the negative control shows "No Ct". The Ct value of each sample detected at each point is read from the software.

2. Qualitative analysis:

(1) Evaluation of experimental quality: If there is no amplification in FAM and HEX channel detection in NC (not a typical S-shaped curve. Note: typical S-shaped curve appears as exponential phase, straight phase and plateau) or no Ct Value, FAM, HEX channel detection in PC is amplified (typical S-shaped curve) and Ct value ≤ 28, then the analysis can continue; otherwise, the experiment is considered invalid, and the experiment needs to be repeated.

(2) Evaluation of the internal reference gene (IR) in the DNA sample to be tested: If the internal reference gene detection (FAM channel) has amplification and 19 ≤ Ct value ≤ 25, the analysis can be continued; if the Ct value is small, it is considered The concentration of the DNA sample is too high; if the Ct value is large or no amplification, the DNA sample concentration is considered to be too low, degradation occurs, or a PCR inhibitor may be contained therein.

(3) Evaluation of internal control gene detection in DNA samples to be tested: If the internal control gene detection (HEX channel) is amplified and the Ct value is ≤25, the analysis can be continued; if the internal control gene detection (HEX channel) has a large Ct value or no Amplification, but mutation site detection (FAM channel) has amplification and Ct value ≤ 38, then continue to analyze (may inhibit the amplification of internal control genes due to mutation site amplification); if internal control gene detection (HEX channel) The Ct value is large or no amplification, and the mutation site detection (FAM channel) has no amplification or amplification but the Ct value is >38, then the analysis cannot be continued, and the experiment needs to be repeated (may be due to degradation of the DNA sample, which contains PCR Inhibitor or no sample added).

(4) Judging the gene mutation in the DNA sample to be tested: if a mutation site detection (FAM channel) in the sample is amplified and the Ct value is ≤38, the mutation result is determined to be positive; if the Ct value is >38 or none Amplification determines that the sample mutation result is negative.

Note: Multiple mutations may be present in the same DNA sample.

Figure 1 is a PCR diagram of a sample with a negative test result (wild-type sample), and Figure 2-5 is a PCR chart of a sample with a positive test result (a mutant sample containing mutations 1-4).

By comparison test, it was confirmed that the results of the fluorescent PCR method of the present invention and the conventional sequencing method are consistent, and the sensitivity and selectivity of the fluorescent PCR method of the present invention are higher than those of the conventional sequencing method.

Therefore, the fluorescent PCR reaction system of the invention can detect C-KIT gene mutations 1-4, and the detection is convenient, rapid and accurate, and can meet the requirement of rapid detection of C-KIT gene mutation.

Claims

A primer for detecting a mutation in a C-KIT gene, characterized in that the primer is as shown in SEQ ID No. 1-2, 4-5, 7-8 or 10-11.
A probe for detecting a mutation in a C-KIT gene, characterized in that the probe is represented by SEQ ID No. 3, 6, 9, or 12.
A kit for detecting a mutation of a C-KIT gene, characterized in that the kit comprises the primer shown in SEQ ID No. 1-2 and the probe shown in SEQ ID No. 3, SEQ ID No. The primer shown in 4-5 and the probe shown in SEQ ID No. 6, the primer shown in SEQ ID No. 7-8, and the probe shown in SEQ ID No. 9, and/or SEQ ID No. 10. The primer shown in -11 and the probe shown in SEQ ID No. 12.
The kit according to claim 3, further comprising an internal reference gene and an internal control gene.
A method for detecting a mutation in a C-KIT gene, comprising the steps of:

(1) extracting genomic DNA from the sample;

(2) amplifying the DNA using the kit of claim 3;

(3) The detection result is judged based on the Ct value displayed by the fluorescence PCR machine.