WO2019085250A1

WO2019085250A1 - Primer, probe, and kit for detecting egfr gene mutation

Info

Publication number: WO2019085250A1
Application number: PCT/CN2018/000375
Authority: WO
Inventors: 莫敏俐; 丁凤; 陈钊; 李晖
Original assignee: 嘉兴雅康博医学检验所有限公司
Priority date: 2017-10-31
Filing date: 2018-10-30
Publication date: 2019-05-09

Abstract

Provided are a primer, probe, and kit for detecting an EGFR gene mutation. The primer is as shown in SEQ ID Nos. 1-2 or as shown in SEQ ID Nos. 4-5. The probe is as shown in SEQ ID No. 3 or as shown in SEQ ID No. 6. By using the kit to perform a real-time fluorescence PCR, an EGFR gene mutation with an insertion of ACCCCA bases between positions 2312 and 2313 can be detected, or an EGFR gene mutation with an insertion of CACCCCCAC bases between positions 2319 and 2320 can be detected.

Description

Primers, probes and kits for detecting EGFR 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 mutations in the EGFR gene.

Background technique

The incidence of malignant tumors in China has been increasing year by year in recent years, with more than 1.7 million new patients each year. Lung cancer is one of the most common malignant tumors in the world, and 80%-85% of them are nonsmall cell lung cancer (NSCLC). In China, lung cancer has become the first cause of death in malignant tumors, with a 5-year survival rate of about 15%. With the development of tumor molecular biology, targeted therapy has received much attention. Selecting appropriate patients for targeted therapy is the key to its success. Epidermal growth factor receptor (EGFR) is an important target for cancer therapy. point.

The EGFR gene is located on the short arm of human chromosome 7 (7p12), which is about 118 kb in length and consists of 28 exons. The transcribed mRNA is about 5.6 kb long, encodes a transcellular membrane glycoprotein with a molecular weight of 170 kD, and the intracellular region has tyrosinne kinase (TK) activity, which is responsible for transmitting extracellular signals to the intracellular. Abnormal EGFR activation can promote tumor cell proliferation, migration, differentiation, angiogenesis, and inhibit tumor cell apoptosis.

The EGFR gene mutation mainly occurs in the first 4 exons of the intracellular TK region (18-21 exons). Studies have shown that EGFR gene 18, 19, 21 exon mutation in lung cancer patients taking tyrosine kinase Inhibitors (tyrosine kinase inhibitors, TKIs) have a good effect, and mutations in 20 exons are often associated with TKIs resistance ^[1-4] . In Chinese lung cancer patients, the mutation rate of EGFR is about 30% to 50%, among which the point mutations on the 18 exons account for about 5% of the EGFR mutation types, and the multiple deletion mutations on the 19 exons account for about About 45% of the EGFR mutation types, the L858R and L861Q point mutations on the 21 exons account for about 40% to 45% of the EGFR mutation type, and the T790M point mutations on the 20 exons account for about 5% of the EGFR mutation type. Left and right ^[5-6] .

At present, Sanger sequencing is the main method for detecting EGFR 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 EGFR gene mutation detection technology.

The inventors of the present invention found a new mutation site in the EGFR gene associated with gastrointestinal stromal tumors, namely the 2312-2313 insertion mutation, and 2319- in the routine detection of gastrointestinal stromal tumors (GIST). 2320 insertion mutations. The inventors of the present invention have developed a rapid, highly sensitive, and easy-to-use detection kit and related detection methods for these mutations, which can be used for prognosis of GIST chemotherapy.

Summary of the invention

One of the objects of the present invention is to provide a primer and a probe for detecting an EGFR gene mutation, which is inserted into ACCCCA at 2312-2313 (base mutation: 2312-2313insACCCCA, protein mutation: N771>KPH), or It is inserted into CACCCCCAC at position 2319-2320 (base mutation: 2319-2320insCACCCCCAC, protein mutation: H773_V774insHPH). The primers and probes of the invention comprise the following sequences:

Mutations to insert ACCCCA at positions 2312-2313:

Forward primer (F): GACAAACCCC ACCCCCA (SEQ ID No: 1)

Reverse primer (R): GGACATAGTC CAGGAGGCA (SEQ ID No: 2)

Probe (PB): TGGGCATCTG CCTCACCTCC A (SEQ ID No: 3)

Mutations inserted into CACCCCCAC for positions 2319-2320:

Forward primer (F): TGGACAACCC CCACCAC (SEQ ID No: 4)

Reverse primer (R): GGACATAGTC CAGGAGGCA (SEQ ID No: 5)

Probe (PB): TGGGCATCTG CCTCACCTCC A (SEQ ID No: 6)

Another aspect of the present invention provides a detection kit for detecting a mutation of an EGFR gene, the kit comprising the primer shown in SEQ ID No. 1 - SEQ ID No. 2 and the probe shown in SEQ ID No. 3. Or include the primer shown in SEQ ID No. 4-SEQ ID No. 5 and the probe shown in SEQ ID No. 6.

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 obtaining a Ct value associated with a mutation in an EGFR gene, comprising the steps of:

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

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

(3) Detecting the FAM fluorescence intensity of the reaction system, and obtaining the number of cycles required for the FAM to reach the set threshold, that is, the Ct value. The Ct value was used as a negative and positive criterion.

The invention adopts specific primer and probe technology to specifically detect EGFR gene mutation. This method has high sensitivity, high specificity and fast detection speed.

DRAWINGS

Figure 1 is a PCR diagram of a negative sample (wild type) detected using the sequence of SEQ ID No. 1-3.

Figure 2 is a PCR diagram for detecting a mutation-positive sample using the sequence of SEQ ID No. 1-3.

Figure 3 is a PCR diagram of a negative sample (wild type) detected using the sequence of SEQ ID No. 4-6.

Figure 4 is a PCR diagram for detecting a mutation-positive sample using the sequence of SEQ ID No. 4-6.

Detailed ways

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 primer and probe of the present invention are combined with the DNA template, Taq DNA polymerase uses deoxynucleotide (dNTP) as a substrate to expand the internal reference gene (IR) and the EGFR gene mutant gene in vitro. increase. Fluorescence PCR was used to detect the release of fluorescence by specific probe hydrolysis, and the PCR reaction was monitored to determine the mutation of EGFR 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 EGFR gene to be detected. 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 invention simultaneously sets an internal control (IC) detection system in the EGFR 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 EGFR gene to be detected. The probe that recognizes the EGFR gene mutation template is modified to a FAM fluorophore, and the probe that recognizes the internal control gene template is modified to a HEX fluorophore. 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 kit test results are judged, the FAM and HEX channel detection in the negative control product (NC) should be non-amplified (not a typical S-shaped curve. The typical S-shaped curve is in turn exponential, straight and platform) Period) or no Ct value, FAM, HEX channel detection in the positive control product (PC) should have amplification (typical S-shaped curve) and Ct value ≤ 35; otherwise, the experiment is considered invalid, and the experiment needs to be repeated.

2. Experimental materials and equipment

1. Design synthetic specific primers and probes for mutation sites

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

The optimized primers and probes are as follows:

Forward primer (F): GACAAACCCC ACCCCCA (SEQ ID No: 1)

Reverse primer (R): GGACATAGTC CAGGAGGCA (SEQ ID No: 2)

Probe (PB): TGGGCATCTG CCTCACCTCC A (SEQ ID No: 3)

Or for:

Forward primer (F): TGGACAACCC CCACCAC (SEQ ID No: 4)

Reverse primer (R): GGACATAGTC CAGGAGGCA (SEQ ID No: 5)

Probe (PB): TGGGCATCTG CCTCACCTCC A (SEQ ID No: 6)

2. Detection of sample processing and DNA extraction

The test sample may be fresh pathological tissue, paraffin embedded tissue, whole blood, plasma or ascites. 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, and not less than 25% of the entire sample; and the DNA sample OD260/OD280=1.8±0.2, OD260/OD230≥1.5; the concentration is 5-10 ng/μl, not exceeding 10 ng/μl.

Paraffin-embedded tissue samples should be stored at room temperature for no more than 3 years, 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 the DNA extraction of the paraffin-embedded tissue samples is completed using an Autostation N16 nucleic acid purification analyzer [Jingyao Medical Machinery (Quasi) 2013 No. 1400574] (produced by Beijing Yakangbo Biotechnology Co., Ltd.). According to the instructions for use of the nucleic acid purification analyzer, each of the 8 tubes in the kit is used to detect 1 sample. The A-site of the detection reagent (corresponding to the tube number 1) contains only the internal reference gene detection system (FAM channel), B to H position. The well contains both the EGFR 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 Autostation N16 Nucleic Acid Purification Analyzer [Kyoto Prescription (No.) 2013 No. 1400574], DNA extraction of paraffin-embedded tissue samples was performed according to the instruction manual.

2. According to the number of DNA samples to be tested (n), take the corresponding number of reagents (n + 2) in the kit and the positive control PC. The test reagent and the positive control product PC were briefly centrifuged and placed on ice.

3. According to the number of DNA samples to be tested (n), take the corresponding number (n + 2) of PCR single tubes in the kit, add Taq DNA polymerase (3 μl / tube) to the bottom of the tube, and place on ice. Add 27 μl of the DNA sample to be tested, the NC control substance (buffer for dissolving DNA) and the positive control PC to the PCR single tube, mix by pipetting, and cover the tube cover and centrifuge briefly. Note: Do not use a vortex oscilloscope when mixing; follow up immediately after mixing.

4. Carefully open the 8-tube cap with the detection reagent, and mix the DNA sample to be tested, the NC control product, and the positive control PC, which are mixed with Taq DNA polymerase in step 2, at 3 μl/well. , respectively, added to the different reaction wells of the detection reagent (each 8 tubes are used to detect 1 sample). Close the fluorescent PCR tube cover, mix gently and mix briefly. Note: When replacing or capping the cap, replace it with new PE or rubber gloves; do not use a vortex oscilloscope when mixing; after centrifugation, keep the reagent at the bottom of the tube and immediately check it on the machine.

5. Fluorescence PCR instrument (Stratagene Mx3000P) detection channel setting must select FAM, HEX channel (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 the FAM and HEX channel detection in NC (not a typical S-shaped curve. The typical S-shaped curve is in the exponential phase, straight phase and plateau phase) or no Ct value. In the PC, the FAM and HEX channel detections are amplified (typical S-shaped curve) and the Ct value is ≤35, then the analysis can be continued; 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 22 ≤ Ct value ≤ 30, 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 ≤ 35, 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 ≤ 39, 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 >39, 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 the mutation site detection (FAM channel) in the sample is amplified and the Ct value is ≤36, the mutation result is determined to be positive; if there is no amplification or Ct value> 39, it is determined that the sample mutation result is negative; if 36 < Ct value ≤ 39, the experiment can be repeated, if the Ct value is still within this range, it is determined that the sample mutation result is suspected positive (may be due to the low mutation content causing Ct value fluctuation).

In the experiment, multiple mutations may exist simultaneously in the same DNA sample.

Figures 1 and 3 are PCR plots of samples with negative test results, and Figures 2 and 4 are PCR plots of samples with positive test results.

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 the mutation of the EGFR gene 2312-2313 into the ACCCCA base mutation, or detect the insertion of the 2319-2320 insertion into the CACCCCCAC mutation, and the detection is convenient, rapid and accurate, and can meet the requirement of rapid detection of EGFR gene mutation. .

【references】

1. Paez J.G., Janne P.A., Lee J.C., et al. EGFR mutations in lung cancer: association with clinical response to gefitinib treatment [J]. Science, 2004, 304 (5676): 1497-1500.

2. Lynch TJ, Bell DW, Sordella R., etc., activating mutations in epidermal growth factor receptor in response to gefitinib in non-small cell lung cancer [J].N Engl J Med,2004,350(21) :2129-2139.

3. Mok T.S., Wu Y.L., Thongprasert S. et al., Gefitinib or carboplatin-paclitaxel in lung adenocarcinoma [J]. N Engl J Med, 2009, 361(10): 947-957.

4. Kobayashi S., Boggon T. J., Dayaram T. et al. Resistance of EGFR to gefitinib in non-small cell lung cancer [J]. N Engl J Med, 2005, 352(8): 786-792.

5. Li Qi, Zhao Yali, Hao Haojie, Li Xianghong, Mutation of EGFR gene in Chinese lung cancer patients [J], Chinese Journal of Oncology, 2007, 29: 270-273.

6. Song Guohong, Yan Lijun, Ren Jun, Zhang Lijian, Yu Jing, Study on epidermal growth factor receptor gene mutation in Chinese patients with non-small cell lung cancer [J], Modern Oncology Medicine, 2008, 16: 553-556.

Claims

A primer for detecting a mutation in an EGFR gene, wherein the primer is represented by SEQ ID No. 1 - SEQ ID No. 2, or as shown in SEQ ID No. 4-SEQ ID No. 5.
A probe for detecting a mutation in an EGFR gene, wherein the probe is as shown in SEQ ID No. 3 or as shown in SEQ ID No. 6.
A kit for detecting a mutation of an EGFR gene, which comprises the primer shown in SEQ ID No. 1 - SEQ ID No. 2 and the probe shown in SEQ ID No. 3, or comprises The primer shown in SEQ ID No. 4-SEQ ID No. 5 and the probe shown in SEQ ID No. 6.
The kit according to claim 3, further comprising an internal reference gene and an internal control gene.
A method of obtaining a Ct value associated with a mutation in an EGFR gene, comprising the steps of:

(1) extracting genomic DNA from the sample;

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

(3) The FAM fluorescence intensity of the reaction system is detected to obtain the number of cycles required when the FAM reaches the set threshold.
The method of claim 5 wherein said sample is fresh pathological tissue, paraffin embedded tissue, whole blood or plasma sample.
The method of claim 5 wherein said sample is a paraffin embedded tissue sample.