WO2023279703A1 - Primer probe for detecting egfr gene l858r mutation, kit, and use thereof - Google Patents

Primer probe for detecting egfr gene l858r mutation, kit, and use thereof Download PDF

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WO2023279703A1
WO2023279703A1 PCT/CN2022/073653 CN2022073653W WO2023279703A1 WO 2023279703 A1 WO2023279703 A1 WO 2023279703A1 CN 2022073653 W CN2022073653 W CN 2022073653W WO 2023279703 A1 WO2023279703 A1 WO 2023279703A1
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mutation
probe
primer
detection
egfr gene
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PCT/CN2022/073653
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Chinese (zh)
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刘国东
黄守程
董涛
邱万伟
张学记
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英飞凌生物科技(江苏)有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the invention relates to the technical field of gene detection, in particular to a primer probe for detection of EGFR gene L858R mutation, a reagent strip and an application thereof.
  • Epidermal growth factor receptor is the expression product of proto-oncogene, located on the cell membrane, and is a transmembrane receptor tyrosine kinase.
  • EGFR gene mutations are mainly concentrated in exons 18-21 of the tyrosine kinase region (TKI), of which exon 21 point mutations account for about 40-45% of all mutations, and exon 21 point mutations are mainly A T-G transition occurs at codon 858, causing the amino acid at this position in the protein to change from leucine to arginine, namely L858R.
  • EGFR gene mutation detection is mainly divided into tissue biopsy technology and liquid biopsy technology.
  • Tissue biopsy techniques are invasive, pose patient safety risks, and are inaccurate due to their inability to capture genetic heterogeneity within tumors and between metastases.
  • the emerging liquid biopsy technology overcomes the detection drawbacks of tissue biopsy technology.
  • Liquid biopsy is a detection method for determining the disease by detecting biomarkers in non-solid biological tissues, mainly blood.
  • clinical practice mainly focuses on detecting biomarkers such as circulating tumor cells (CTC), circulating tumor DNA (ctDNA), and exosomes in samples.
  • CTC circulating tumor cells
  • ctDNA circulating tumor DNA
  • exosomes exosomes in samples.
  • the detection of these biomarkers can also be used to judge disease progression and prognosis, thereby optimizing treatment options.
  • liquid biopsy Compared with traditional methods, liquid biopsy has the following characteristics: (1) non-invasive, with few side effects; (2) no need for imaging support, easy to operate; (3) generally only needs 5-10mL blood, and can be re-sampled; (4) ) real-time detection to effectively deal with tumor heterogeneity; (5) early detection of cancer and prediction in advance; (6) low cost.
  • ctDNA is a tumor marker in liquid biopsy. It is a part of the total amount of circulating free DNA (ccfDNA) in cancer patients. It is a small fragment of nucleic acid released by tumor cells into peripheral blood. Its concentration in the blood mainly depends on the tumor burden of cancer patients. and residual disease. In general, the average amount of ctDNA contained per milliliter in cancer patient plasma ranges from a few copies to 104 copies. The length of ctDNA released by tumor cells is about 145-180bp, while the length of ctDNA produced by tumor cell necrosis can reach 10kbp. Studies have demonstrated that ctDNA appears in the blood earlier than other circulating tumor markers, such as CTCs, in patients with bladder, breast, and colorectal cancers.
  • mutated genes such as murine sarcoma virus oncogene (KRAS), epidermal growth factor receptor (EGFR), tumor suppressor gene (APC), etc.
  • KRAS murine sarcoma virus oncogene
  • EGFR epidermal growth factor receptor
  • APC tumor suppressor gene
  • PCR-based methods or next-generation sequencing technologies are mostly used.
  • Traditional PCR methods such as real-time quantitative PCR, cannot be used for ctDNA detection because they cannot detect small mutations below 20%.
  • PCR-based ctDNA mutation detection techniques have emerged, such as allele-specific PCR (ARMS-PCR), low-temperature denaturing co-amplification PCR (COLD-PCR, digital PCR (dPCR), microdroplet Digital PCR (ddPCR), etc., among which ARMS-PCR has been approved by my country for the detection of ctDNA mutations.
  • next-generation sequencing technology and PCR-based methods are the "gold standard" for detecting ctDNA mutations, the shortcomings of these two types of technologies are also Significantly.
  • the next-generation sequencing technology has a long detection period (2-3 weeks) and high cost, which is difficult to meet the actual clinical needs.
  • the PCR-based method has high operational requirements and is easy to introduce sol contamination, resulting in false negative or false positive results.
  • both of these methods require well-trained professionals and expensive instruments and equipment. Therefore, for the detection of ctDNA mutations, there is an urgent need for a low-cost, easy-to-operate, and expensive instrument that can be directly obtained from plasma or blood Techniques for detecting mutant ctDNA.
  • the present invention provides a primer probe for detecting the L858R mutation of the EGFR gene quickly, easily, accurately and visually.
  • the technical scheme adopted in the present invention is: the invention provides a kind of primer probe of EGFR gene L858R mutation detection, the upstream primer sequence of the L858R mutation is shown in SEQ ID NO: 1; the L858R The mutated downstream primer sequence is shown in SEQ ID NO: 2; the probe of the L858R mutation is a gold-labeled probe, and its sequence is shown in SEQ ID NO: 3.
  • the 3' end of the gold-labeled probe sequence is modified with 18-20nm colloidal gold.
  • the present invention also provides an application of the above-mentioned primer probe for preparing a reagent for detecting the L858R mutation of the EGFR gene.
  • the present invention also provides a kit for detecting the L858R mutation of the EGFR gene, comprising the above-mentioned primers and a lateral flow chromatography test strip, the binding pad on the test strip is sprayed with the above-mentioned probe.
  • the above kit also includes RPA amplification reagents.
  • the kit also includes a lateral flow chromatography buffer solution, the buffer solution includes 5X SSC, 2% BSA and 2% PVP, and the 5X SSC is 4.4% sodium chloride at pH7.0 and 2.2 % sodium citrate solution.
  • the buffer solution includes 5X SSC, 2% BSA and 2% PVP
  • the 5X SSC is 4.4% sodium chloride at pH7.0 and 2.2 % sodium citrate solution.
  • the concentration of the primer is 4 ⁇ M, and the concentration of the gold-labeled probe is 15 nM.
  • streptavidin is drawn on the detection line of the test strip, and a biotin-modified quality control probe is drawn on the quality control line, and the quality control probe sequence is as shown in SEQ ID NO:4.
  • the sample pad on the test strip is soaked with a processing buffer before use, and the processing buffer includes 0.05M Tris-HCl, 0.15mM NaCl, 0.25% Triton X-100 and 2.5% Tween- 20.
  • the processing buffer includes 0.05M Tris-HCl, 0.15mM NaCl, 0.25% Triton X-100 and 2.5% Tween- 20.
  • the present invention also provides a non-diagnostic method for detecting the EGFR gene L858R mutation according to the kit, comprising the following steps:
  • the primer probe and kit for EGFR gene L858R mutation detection provided by the present invention adopt RPA isothermal amplification of tail primers and lateral flow chromatography detection technology, the detection process is easy to operate and the detection time is short, and the entire detection process can be completed within half an hour Complete, greatly reducing the detection time.
  • the kit for detecting the EGFR gene L858R mutation provided by the invention has high specificity, accuracy and sensitivity for detecting the EGFR gene L858R mutation, and the sensitivity reaches 0.78%. Using the kit of the invention to carry out blind test on 45 cases of clinical samples, the test result is consistent with the sequencing result, and the accuracy rate is 100%.
  • both ARMS-PCR and digital PCR currently use fluorescent signal detection and conventional PCR reaction procedures.
  • the detection equipment needs to be equipped with expensive fluorescence detection systems and PCR amplification instruments.
  • the requirements for instruments and equipment High, complicated operation procedures, but the present invention does not require complex precision instruments, and the detection process can be visually detected by using a 37°C constant temperature reaction tank and test strips.
  • the operation is simple, fast, low in cost, and the results are easy to interpret.
  • the invention can directly use the blood of patients with non-small cell lung cancer (NSCLC) as a detection sample, and compared with the tissue biopsy technique commonly used in clinic, the sample acquisition is easier and the detection is more convenient.
  • NSCLC non-small cell lung cancer
  • Fig. 1 is the RPA amplification schematic diagram of tail primer
  • Figure 2 is a PCR electrophoresis diagram of 6 pairs of primers for detecting EGFR gene L858R, wherein MT is a mutant type, and WT is a wild type;
  • Figure 3 is a graph showing the results of flow chromatography test strips optimized for the concentration of primers detecting EGFR gene L858R;
  • Fig. 4 is the result figure of the flow chromatography test strip optimized for the primer time detection of EGFR gene L858R;
  • Figure 5 is a graph showing the results of flow chromatography test strips optimized for primer temperature detection of EGFR gene L858R;
  • Fig. 6 is the detection principle diagram of lateral flow chromatography nucleic acid test strip
  • Figure 7 is a lateral flow chromatography detection sensitivity detection diagram after the isothermal amplification of the method for detecting the EGFR gene L858R mutation;
  • Fig. 8 is the sensitivity detection diagram of the detection of different ratios of the mutant plasmid standard substance of the method for detecting the EGFR gene L858R mutation;
  • Fig. 9 is a graph showing the detection results of 45 clinical samples by the method described in Example 2.
  • the invention provides a primer probe for detecting the L858R mutation of the EGFR gene, the upstream primer sequence of the L858R mutation is 5'-ATGTCAAGATCACAGATTTTGGGTG-3' (SEQ ID NO: 1), and the 5' end of the upstream primer sequence is modified There is biotin, namely 5'-biotin-
  • the downstream primer sequence 5'-CAATACAGCTAGTGGGAAGGCAG-3'(SEQ ID NO: 2) of the L858R mutation the 5' end of the downstream primer sequence is modified with TTTTTTTT-C3, namely 5'-TTTTTTTTTT-C3 -CAATACAGCTAGTGGGAAGGCAG-3'; the gold-labeled probe sequence 5-AAAAAAAAAAAAAA-3' (SEQ ID NO: 3), the probe sequence is modified with a sulfhydryl group, that is, 5'-AAAAAAAAAAAAAAAA-SH-3'.
  • colloidal gold is prepared by trisodium citrate reduction method.
  • the specific operation method is as follows: take 100mL of 0.01% HAuCl4 aqueous solution, heat it to boiling on a magnetic heating stirrer, under vigorous stirring, accurately add a certain amount of freshly prepared 1% trisodium citrate (Na 3 C 6 H 5 O 7 2H 2 O) aqueous solution, continue to heat and boil for 15 minutes.
  • the golden yellow chloroauric acid aqueous solution turns gray quickly after adding sodium citrate, then turns black, and then gradually stabilizes into wine red.
  • the whole process is about After cooling to room temperature for 2 to 3 minutes, restore to the original volume with distilled water, store at 4°C for later use, and use transmission electron microscopy (TEM), zeta potential analyzer, and ultraviolet-visible spectroscopy (UV-Vis) to analyze the colloidal gold morphology, particle size, And the charge and other aspects are characterized, and 18-20nm colloidal gold is obtained.
  • TEM transmission electron microscopy
  • zeta potential analyzer zeta potential analyzer
  • UV-Vis ultraviolet-visible spectroscopy
  • the preparation method of the gold-labeled probe is not particularly limited, and the method known in the art can be adopted.
  • the preparation method of the gold-labeled probe of the present invention includes taking 1 mL of a 10-fold concentrated colloidal gold solution, adding 10 ⁇ L of 1mM dATP, and Shake with a shaker at room temperature for 20 minutes, then add 15 ⁇ L of 1% SDS, shake and incubate for 10 minutes, then slowly add 50 ⁇ L of 2M NaCl solution, then add 1.0OD thiol-modified detection probes, react at 60°C for 3 hours, refrigerate and centrifuge, Centrifuge at 12000rpm for 10min, remove the supernatant, wash 3 times with PBS buffer (pH7.2 ⁇ 7.4), and finally dissolve the precipitate in 1.0mL nanoparticle storage solution, the nanoparticle storage solution is 20mM NaPO4. 12H2O, 5% BSA, 0.25% Twen20, 10% sucrose to obtain the colloidal gold universal DNA detection probe, and store it in a refrigerator at 4°C until use.
  • the present invention also provides an application of the above-mentioned primer probe for preparing a reagent for detecting the L858R mutation of the EGFR gene.
  • the present invention also provides a kit for detecting the L858R mutation of the EGFR gene, comprising the above-mentioned primers and a lateral flow chromatography test strip, the binding pad on the test strip is sprayed with the above-mentioned probe.
  • the kit is low in cost, high in specificity, accuracy and sensitivity, the sensitivity reaches 0.78%, and the accuracy rate is 100%.
  • the operation is simple and the detection time is short, and the entire detection process can be completed within half an hour , which greatly shortens the detection time and makes the results easy to interpret.
  • the above kit also includes RPA amplification reagents.
  • the RPA amplification reagent includes a reaction solution for isothermal amplification, a starting solution for isothermal amplification and enzymes required for the reaction.
  • the isothermal amplification reagent is purchased from AMP Future Biotechnology Co., Ltd., and the product name is DNA constant temperature rapid amplification kit (basic type).
  • the kit contains liquid A, liquid B and dry powder reaction tubes required for the reaction.
  • Solution A is the reaction solution for isothermal amplification
  • solution B is the starting solution for isothermal amplification
  • the dry powder reaction tube is the enzyme required for the reaction, including recombinase, polymerase, and single-chain binding protein.
  • described buffer solution comprises 5X SSC, 2%BSA and 2%PVP
  • described 5X SSC is 4.4% sodium chloride and 2.2% citric acid of pH7.0 sodium solution.
  • the present invention has no special limitation on the preparation method of the buffer solution.
  • the lateral flow chromatography test strip includes a back substrate, on which a sample pad, a binding pad, a nitrocellulose membrane and an absorbent pad are sequentially arranged on the back substrate; the sample pad and the The binding pad is partially overlapped, the binding pad is partially overlapped with the nitrocellulose membrane, one side of the binding pad is located under the sample pad, and the other side of the binding pad is located on the nitrocellulose membrane.
  • a test line and a quality control line are arranged on the top, and the quality control line is close to the absorbent pad.
  • the interval between the detection line and the quality control line on the nitrocellulose membrane is 0.5 cm.
  • the sample pad on the test strip is soaked with a processing buffer before use, and the processing buffer includes 0.05M Tris-HCl, 0.15mM NaCl, 0.25% Triton X-100 and 2.5% Tween-20, the preparation method of the treatment buffer is to weigh Tris-HCl, NaCl, Triton X-100 and Tween-20 of the above-mentioned concentration and quality, dissolve them in double-distilled water, and stir until fully dissolved.
  • the present invention is soaked in the treatment buffer solution, it is dried in a drying box at 37° C. for 4 hours, and stored in a desiccator for future use. In the present invention, soaking in the treatment buffer for 30 minutes can reduce the background signal.
  • the detection line of the test strip is marked with 1 mg/mL streptavidin
  • the quality control line is marked with a biotin-modified quality control probe
  • the quality control probe sequence is as SEQ ID NO:4 shown.
  • concentration of described streptavidin is preferably 1mg/mL
  • described quality control probe sequence is 5'-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-Biotin-3'.
  • the nitrocellulose membrane is Pall vivid 170 (2.5cm ⁇ 50m), purchased from Shanghai Jiening Biotechnology Co., Ltd., the liquid climbing rate (water) of the nitrocellulose membrane: 150 ⁇ 225 seconds /4cm.
  • the fluid migration rate in the nitrocellulose membrane can correspond to the time required to complete the DNA-DNA hybridization reaction during the migration.
  • the present invention also provides a non-diagnostic method for detecting the EGFR gene L858R mutation according to the kit, comprising the following steps:
  • step (2) the concentration of the primer is 4 ⁇ M, the concentration of the gold standard probe is 15 nM; the reaction system of the RPA amplification is 50 ⁇ L: A solution 29.5 ⁇ L, B 2.5 ⁇ L of solution, 2 ⁇ L of template, 2 ⁇ L of each of the above-mentioned upstream primer and downstream primer, and sterilized ultrapure water to 50 ⁇ L; in step (3), the reaction product obtained by the RPA amplification reaction is mixed with the buffer solution , drop on the side flow chromatography test strip sample pad place and detect, the loading volume is preferably 100 ⁇ L, and the volume ratio of described reaction product and buffer solution is preferably 5:95; Adopt side flow chromatography in step (3) Test the amplification product on the test strip.
  • the result is positive, indicating that the blood sample contains the EGFR gene L858R mutation. If there is only a red band in the quality control area of the paper strip and no band in the detection area, the result is negative, indicating that the sample does not contain the EGFR gene L858R mutation.
  • this example is RPA amplification based on tail primers. Due to the modification of the C3 arm between the tail primer-specific extension sequence and the detection sequence, it is ensured that the detection sequence will not be extended by the opposite primer Extend to form a double strand, and only two rounds of amplification are required to form a double-tailed DNA amplification product during RPA amplification (see Figure 1), and then the double-tailed amplification product will be used as the template for the next round of amplification Exponential amplification yields more double-tailed DNA products.
  • No. 2 primers have the best effect on amplification, good band specificity, no non-specific amplification, although other primer pair groups also have certain specificity, the primer pair of the present invention has the best amplification effect for MT, and the band for WT is weaker, and the upstream primer of No. 2 primer pair is 5' -biotin-ATGTCAAAGATCACAGATTTTGGGTG-3', the downstream primer is 5'-TTTTTTTTTT-C3-CAATACAGCTAGTGGGAAGGCAG-3'.
  • the concentration of primers (10 ⁇ M, 8 ⁇ M, 6 ⁇ M, 4 ⁇ M, 2 ⁇ M, 1 ⁇ M, 0.5 ⁇ M), temperature (30 °C, 32 °C, 35 °C, 36 °C, 37 °C) used in the amplification reaction were adjusted respectively.
  • condition of RPA amplification reaction is 37°C water bath for 30min.
  • the detection principle of the visualized lateral flow chromatography test strip is shown in Figure 6.
  • a gold-labeled probe that can bind to one end of the L858R amplification product is sprayed on the binding pad of the lateral flow chromatography test strip, and the nitrocellulose membrane
  • the detection area has streptavidin with a concentration of 1mg/mL, and the streptavidin can combine with the biotin (Biotin) at the other end of the L858R amplification product, and the quality control area has a quality control probe, which can bind to the gold
  • Biotin biotin
  • the isothermal amplification product reaches the detection area of the lateral flow biosensor, it will be specifically captured and developed, and the color depth of the detection area is directly proportional to the amount of colloidal gold captured, that is, with The concentration of the isothermal amplification product is directly proportional.
  • the detection results of the present invention are consistent with the sequencing results, with an accuracy rate of 100%. Compared with the sequencing method, the detection method of the present invention is easier to operate and faster in detection speed.

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Abstract

Provided is a primer probe for detecting an EGFR gene L858R mutation, which relates to the technical field of gene detection. An upstream primer sequence of the L858R mutation is shown in SEQ ID NO:1. A downstream primer sequence of the L858R mutation is shown in SEQ ID NO:2. The L858R mutation probe is a gold-labeled probe, and sequence thereof is shown in SEQ ID NO:3. The primer probe used has high specificity, accuracy and sensitivity. For the first time, an RPA amplification technology is used in combination with a lateral flow dipstick to detect the EGFR gene L858R mutation. The detection time is short, the operation is simple, and the cost is low.

Description

EGFR基因L858R突变检测的引物探针、试剂盒及其应用Primer probe, kit and application for detection of EGFR gene L858R mutation
本申请要求于2021年07月07日提交中国专利局、申请号为202110765704.8、发明名称为“EGFR基因L858R突变检测的引物探针、试剂盒及其应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。This application claims the priority of the Chinese patent application submitted to the China Patent Office on July 07, 2021, with the application number 202110765704.8, and the title of the invention is "Primer probe, kit and application for detection of EGFR gene L858R mutation", all of which The contents are incorporated by reference in this application.
技术领域technical field
本发明涉及基因检测技术领域，尤其涉及一种EGFR基因L858R突变检测的引物探针、试剂条及其应用。The invention relates to the technical field of gene detection, in particular to a primer probe for detection of EGFR gene L858R mutation, a reagent strip and an application thereof.
背景技术Background technique
表皮生长因子受体(EGFR)是原癌基因的表达产物，定位于细胞膜上，为一种跨膜受体酪氨酸激酶。EGFR基因突变主要集中在酪氨酸激酶区(TKI)的18～21外显子上，其中外显子21点突变，约占所有突变的40～45％，外显子21的点突变主要是位于第858位密码子出现T～G转换，引起蛋白中该位点的氨基酸由亮氨酸转变为精氨酸，即L858R。Epidermal growth factor receptor (EGFR) is the expression product of proto-oncogene, located on the cell membrane, and is a transmembrane receptor tyrosine kinase. EGFR gene mutations are mainly concentrated in exons 18-21 of the tyrosine kinase region (TKI), of which exon 21 point mutations account for about 40-45% of all mutations, and exon 21 point mutations are mainly A T-G transition occurs at codon 858, causing the amino acid at this position in the protein to change from leucine to arginine, namely L858R.
目前EGFR基因突变检测主要分为组织活检技术和液体活检技术。组织活检技术具有侵入性，存在患者安全风险，且由于无法捕捉瘤内和转移间的遗传异质性，准确性不高。而新兴的液体活检技术克服了组织活检技术的检测弊端，液体活检是对以血液为主的非固体生物组织中的生物标志物进行检测来确定病情的一种检测方法。目前，临床主要以检测样本中循环肿瘤细胞(CTC)、循环肿瘤DNA(ctDNA)、外泌体等生物标志物为主。对于这些生物标志物的检测除了可以用于癌症早期的筛查和患者分级以外，还可以用来判断疾病进展和预后情况，从而优化治疗方案。相较于传统方法，液体活检具有以下特点：(1)无侵入性，副作用小；(2)无需影像学支持，操作简单；(3)一般只需5-10mL血液，可重复取样；(4)实时检测，有效应对肿瘤异质性；(5)可在癌症早期检测，提前预测；(6)成本低。At present, EGFR gene mutation detection is mainly divided into tissue biopsy technology and liquid biopsy technology. Tissue biopsy techniques are invasive, pose patient safety risks, and are inaccurate due to their inability to capture genetic heterogeneity within tumors and between metastases. The emerging liquid biopsy technology overcomes the detection drawbacks of tissue biopsy technology. Liquid biopsy is a detection method for determining the disease by detecting biomarkers in non-solid biological tissues, mainly blood. At present, clinical practice mainly focuses on detecting biomarkers such as circulating tumor cells (CTC), circulating tumor DNA (ctDNA), and exosomes in samples. In addition to being used for early cancer screening and patient grading, the detection of these biomarkers can also be used to judge disease progression and prognosis, thereby optimizing treatment options. Compared with traditional methods, liquid biopsy has the following characteristics: (1) non-invasive, with few side effects; (2) no need for imaging support, easy to operate; (3) generally only needs 5-10mL blood, and can be re-sampled; (4) ) real-time detection to effectively deal with tumor heterogeneity; (5) early detection of cancer and prediction in advance; (6) low cost.
ctDNA是液体活检中的肿瘤标志物，是癌症患者循环游离DNA(ccfDNA)总量的一部分，是肿瘤细胞释放到外周血中的核酸小片段， 它在血液中浓度主要取决于癌症患者的肿瘤负荷和残留病变。一般来说，癌症患者血浆中每毫升平均含有ctDNA的数量从几个拷贝到104个拷贝不等。由肿瘤细胞释放的ctDNA长度约145～180bp，而肿瘤细胞坏死产生的ctDNA长度可达10kbp。研究已证实，在膀胱癌、乳腺癌和结直肠癌患者中ctDNA比其他循环肿瘤标志物(如CTCs)更早在血液中出现。一些存在突变的基因如鼠类肉瘤病毒癌基因(KRAS)、表皮生长因子受体(EGFR)、肿瘤抑制基因(APC)等，都可以通过血液中ctDNA检测到。此外，以ctDNA作为液体活检中的肿瘤标志物能更早的实时、动态反应肿瘤的发生、发展情况，这为癌症患者的治疗带来了极大的便利。ctDNA is a tumor marker in liquid biopsy. It is a part of the total amount of circulating free DNA (ccfDNA) in cancer patients. It is a small fragment of nucleic acid released by tumor cells into peripheral blood. Its concentration in the blood mainly depends on the tumor burden of cancer patients. and residual disease. In general, the average amount of ctDNA contained per milliliter in cancer patient plasma ranges from a few copies to 104 copies. The length of ctDNA released by tumor cells is about 145-180bp, while the length of ctDNA produced by tumor cell necrosis can reach 10kbp. Studies have demonstrated that ctDNA appears in the blood earlier than other circulating tumor markers, such as CTCs, in patients with bladder, breast, and colorectal cancers. Some mutated genes, such as murine sarcoma virus oncogene (KRAS), epidermal growth factor receptor (EGFR), tumor suppressor gene (APC), etc., can all be detected by ctDNA in blood. In addition, using ctDNA as a tumor marker in liquid biopsy can reflect the occurrence and development of tumors earlier in real time and dynamically, which brings great convenience to the treatment of cancer patients.
目前，针对ctDNA的检测，目前大多采用基于PCR的方法或二代测序技术。传统的PCR方法，如实时定量PCR，由于不能检测到20％以下水平的微小突变，因此不能用于ctDNA的检测。近年来，出现了各式各样的基于PCR的ctDNA突变检测技术，如等位基因特异性PCR(ARMS-PCR)、低温变性共扩增PCR(COLD-PCR、数字PCR(dPCR)、微滴式数字PCR(ddPCR)等，其中ARMS-PCR已被我国批准用于ctDNA突变的检测。虽然二代测序技术和基于PCR的方法是检测ctDNA突变的“金标准”，但是这两类技术缺点也很显著。二代测序技术检测周期长(2-3周)且成本高，难以满足临床实际需求。基于PCR的方法操作要求高，易引入溶胶污染，导致出现假阴性或假阳性的结果。此外，这两种方法都需要训练有素的专业人员以及昂贵的仪器设备。因此，对于ctDNA突变的检测，临床迫切需要一种低成本、易操作、无需昂贵仪器，能直接能从血浆或血液中检出突变ctDNA的技术。At present, for the detection of ctDNA, PCR-based methods or next-generation sequencing technologies are mostly used. Traditional PCR methods, such as real-time quantitative PCR, cannot be used for ctDNA detection because they cannot detect small mutations below 20%. In recent years, a variety of PCR-based ctDNA mutation detection techniques have emerged, such as allele-specific PCR (ARMS-PCR), low-temperature denaturing co-amplification PCR (COLD-PCR, digital PCR (dPCR), microdroplet Digital PCR (ddPCR), etc., among which ARMS-PCR has been approved by my country for the detection of ctDNA mutations. Although next-generation sequencing technology and PCR-based methods are the "gold standard" for detecting ctDNA mutations, the shortcomings of these two types of technologies are also Significantly. The next-generation sequencing technology has a long detection period (2-3 weeks) and high cost, which is difficult to meet the actual clinical needs. The PCR-based method has high operational requirements and is easy to introduce sol contamination, resulting in false negative or false positive results. In addition , both of these methods require well-trained professionals and expensive instruments and equipment. Therefore, for the detection of ctDNA mutations, there is an urgent need for a low-cost, easy-to-operate, and expensive instrument that can be directly obtained from plasma or blood Techniques for detecting mutant ctDNA.
发明内容Contents of the invention
为了克服现有技术的上述缺点，本发明提供一种快速、简便、准确、可视化地检测EGFR基因L858R突变的引物探针。In order to overcome the above-mentioned shortcomings of the prior art, the present invention provides a primer probe for detecting the L858R mutation of the EGFR gene quickly, easily, accurately and visually.
为了解决上述技术问题，本发明采用的技术方案是：本发明提供了一种EGFR基因L858R突变检测的引物探针，所述L858R突变的上游引物序列如SEQ ID NO:1所示；所述L858R突变的下游引物序列如SEQ ID NO:2所示；所述L858R突变的探针为金标探针，其序列如SEQ ID NO:3所示。In order to solve the above-mentioned technical problems, the technical scheme adopted in the present invention is: the invention provides a kind of primer probe of EGFR gene L858R mutation detection, the upstream primer sequence of the L858R mutation is shown in SEQ ID NO: 1; the L858R The mutated downstream primer sequence is shown in SEQ ID NO: 2; the probe of the L858R mutation is a gold-labeled probe, and its sequence is shown in SEQ ID NO: 3.
优选的，所述金标探针序列3’端修饰18～20nm胶体金。Preferably, the 3' end of the gold-labeled probe sequence is modified with 18-20nm colloidal gold.
本发明还提供了一种上述引物探针用于制备检测EGFR基因L858R突变试剂中的应用。The present invention also provides an application of the above-mentioned primer probe for preparing a reagent for detecting the L858R mutation of the EGFR gene.
本发明还提供了一种检测EGFR基因L858R突变的试剂盒，包括上述引物以及侧流层析试纸条，所述试纸条上的结合垫喷涂有上述探针。The present invention also provides a kit for detecting the L858R mutation of the EGFR gene, comprising the above-mentioned primers and a lateral flow chromatography test strip, the binding pad on the test strip is sprayed with the above-mentioned probe.
优选的，上述试剂盒中还包括RPA扩增试剂。Preferably, the above kit also includes RPA amplification reagents.
优选的，所述试剂盒中还包括侧流层析缓冲溶液，所述缓冲溶液包括5X SSC、2％BSA和2％PVP，所述的5X SSC为pH7.0的4.4％氯化钠和2.2％柠檬酸钠溶液。Preferably, the kit also includes a lateral flow chromatography buffer solution, the buffer solution includes 5X SSC, 2% BSA and 2% PVP, and the 5X SSC is 4.4% sodium chloride at pH7.0 and 2.2 % sodium citrate solution.
优选的，所述引物的浓度为4μM，所述金标探针的浓度为15nM。Preferably, the concentration of the primer is 4 μM, and the concentration of the gold-labeled probe is 15 nM.
优选的，所述试纸条的检测线划有链霉亲和素，质控线上划有生物素修饰的质控探针，所述质控探针序列如SEQ ID NO:4所示。Preferably, streptavidin is drawn on the detection line of the test strip, and a biotin-modified quality control probe is drawn on the quality control line, and the quality control probe sequence is as shown in SEQ ID NO:4.
优选的，所述试纸条上的样品垫在使用前用处理缓冲液浸泡处理，所述的处理缓冲液包括0.05M Tris-HCI、0.15mM NaCl、0.25％Triton X-100和2.5％Tween-20。Preferably, the sample pad on the test strip is soaked with a processing buffer before use, and the processing buffer includes 0.05M Tris-HCl, 0.15mM NaCl, 0.25% Triton X-100 and 2.5% Tween- 20.
本发明还提供了一种非诊断目的的根据所述试剂盒检测EGFR基因L858R突变的方法，包括如下步骤：The present invention also provides a non-diagnostic method for detecting the EGFR gene L858R mutation according to the kit, comprising the following steps:
(1)提取待测血液样本的ctDNA；(1) Extract ctDNA from the blood sample to be tested;
(2)用所述引物以ctDNA为模板进行RPA扩增，RPA扩增反应的条件为：37℃水浴30min；(2) Use the primers to perform RPA amplification using ctDNA as a template, and the conditions for the RPA amplification reaction are: 37°C water bath for 30min;
(3)采用所述测流层析试纸条进行扩增产物检测。(3) Using the flow chromatography test strip to detect the amplification product.
本发明提供的EGFR基因L858R突变检测的引物探针和试剂盒，采用尾引物的RPA等温扩增及侧流层析检测技术，检测过程操作简便、检测时间短，整个检测过程可以在半小时内完成，极大地缩短了检测时间。The primer probe and kit for EGFR gene L858R mutation detection provided by the present invention adopt RPA isothermal amplification of tail primers and lateral flow chromatography detection technology, the detection process is easy to operate and the detection time is short, and the entire detection process can be completed within half an hour Complete, greatly reducing the detection time.
本发明提供的EGFR基因L858R突变检测的试剂盒检测EGFR基因L858R突变特异性、准确度和灵敏度高，灵敏度达到0.78％。采用本发明的试剂盒对45例临床样本进行盲测，检测结果与测序结果一致，准确率为100％。The kit for detecting the EGFR gene L858R mutation provided by the invention has high specificity, accuracy and sensitivity for detecting the EGFR gene L858R mutation, and the sensitivity reaches 0.78%. Using the kit of the invention to carry out blind test on 45 cases of clinical samples, the test result is consistent with the sequencing result, and the accuracy rate is 100%.
另外，在检测设备方面，目前无论是ARMS-PCR还是数字PCR都是采用荧光信号检测和常规的PCR反应程序，检测设备需配备昂贵的荧光 检测系统和PCR扩增仪，对仪器、设备的要求高，操作程序复杂，而本发明无需复杂精密仪器，检测过程利用37℃恒温反应槽和试纸条即可进行可视化检测，操作简便、快捷，成本低廉，结果易判读。In addition, in terms of detection equipment, both ARMS-PCR and digital PCR currently use fluorescent signal detection and conventional PCR reaction procedures. The detection equipment needs to be equipped with expensive fluorescence detection systems and PCR amplification instruments. The requirements for instruments and equipment High, complicated operation procedures, but the present invention does not require complex precision instruments, and the detection process can be visually detected by using a 37°C constant temperature reaction tank and test strips. The operation is simple, fast, low in cost, and the results are easy to interpret.
本发明可以直接以非小细胞肺癌(NSCLC)患者血液为检测样本，较临床常采用的组织活检技术而言，样本的获取更容易，检测更简便。The invention can directly use the blood of patients with non-small cell lung cancer (NSCLC) as a detection sample, and compared with the tissue biopsy technique commonly used in clinic, the sample acquisition is easier and the detection is more convenient.
说明书附图Instructions attached
图1为尾引物的RPA扩增原理图；Fig. 1 is the RPA amplification schematic diagram of tail primer;
图2为检测EGFR基因L858R的6对引物的PCR电泳图，其中MT为突变型，WT为野生型；Figure 2 is a PCR electrophoresis diagram of 6 pairs of primers for detecting EGFR gene L858R, wherein MT is a mutant type, and WT is a wild type;
图3为检测EGFR基因L858R的引物浓度优化的测流层析试纸条结果图；Figure 3 is a graph showing the results of flow chromatography test strips optimized for the concentration of primers detecting EGFR gene L858R;
图4为检测EGFR基因L858R的引物时间优化的测流层析试纸条结果图；Fig. 4 is the result figure of the flow chromatography test strip optimized for the primer time detection of EGFR gene L858R;
图5为检测EGFR基因L858R的引物温度优化的测流层析试纸条结果图；Figure 5 is a graph showing the results of flow chromatography test strips optimized for primer temperature detection of EGFR gene L858R;
图6为侧流层析核酸试纸条检测原理图；Fig. 6 is the detection principle diagram of lateral flow chromatography nucleic acid test strip;
图7为检测EGFR基因L858R突变的方法的等温扩增后，侧流层析检测灵敏度检测图；Figure 7 is a lateral flow chromatography detection sensitivity detection diagram after the isothermal amplification of the method for detecting the EGFR gene L858R mutation;
图8为检测EGFR基因L858R突变的方法的检测不同比例的突变质粒标准品的灵敏度检测图；Fig. 8 is the sensitivity detection diagram of the detection of different ratios of the mutant plasmid standard substance of the method for detecting the EGFR gene L858R mutation;
图9为实施例2所述方法对45例临床样本检测结果图。Fig. 9 is a graph showing the detection results of 45 clinical samples by the method described in Example 2.
具体实施方式detailed description
下面结合实施例和附图对本发明进一步说明。The present invention will be further described below in conjunction with the embodiments and accompanying drawings.
本发明提供了一种EGFR基因L858R突变检测的引物探针，所述的L858R突变的上游引物序列5’-ATGTCAAGATCACAGATTTTGGGTG-3’(SEQ ID NO:1)，所述上游引物序列的5’端修饰有生物素，即5’-biotin-The invention provides a primer probe for detecting the L858R mutation of the EGFR gene, the upstream primer sequence of the L858R mutation is 5'-ATGTCAAGATCACAGATTTTGGGTG-3' (SEQ ID NO: 1), and the 5' end of the upstream primer sequence is modified There is biotin, namely 5'-biotin-
ATGTCAAGATCACAGATTTTGGGTG-3’；所述L858R突变的下游引物序列5’-CAATACAGCTAGTGGGAAGGCAG-3’(SEQ ID NO:2)，所述下游引物序列的5’端修饰有TTTTTTTTTT-C3，即5’-TTTTTTTTTT-C3-CAATACAGCTAGTGGGAAGGCAG-3’；所述金标 探针序列5-AAAAAAAAAAAAAAA-3’(SEQ ID NO:3)，所述的探针序列修饰有巯基，即5’-AAAAAAAAAAAAAAA-SH-3’。ATGTCAAGATCCAGATTTTGGGTG-3'; the downstream primer sequence 5'-CAATACAGCTAGTGGGAAGGCAG-3'(SEQ ID NO: 2) of the L858R mutation, the 5' end of the downstream primer sequence is modified with TTTTTTTTTT-C3, namely 5'-TTTTTTTTTT-C3 -CAATACAGCTAGTGGGAAGGCAG-3'; the gold-labeled probe sequence 5-AAAAAAAAAAAAAAAA-3' (SEQ ID NO: 3), the probe sequence is modified with a sulfhydryl group, that is, 5'-AAAAAAAAAAAAAAAA-SH-3'.
所述金标探针序列3’端修饰18～20nm胶体金。本发明对胶体金的制备方法没有特殊限定，作为一种可选的实施方式，采用柠檬酸三钠还原法制备胶体金。具体操作方法如下：取100mL 0.01％的HAuCl4水溶液，在磁力加热搅拌器上加热至沸腾，剧烈搅动下，准确加入一定量的新鲜配制的1％柠檬酸三钠(Na 3C 6H 5O 7·2H 2O)水溶液，继续加热煮沸15min，此时可观察到金黄色的氯金酸水溶液在柠檬酸钠加入后很快变灰色，继而转成黑色，随后逐渐稳定成酒红色，全过程约2～3min，冷却至室温后用蒸馏水恢复至原体积，4℃保存备用，采用透射电子显微镜(TEM)、zeta电位分析仪、紫外可见光谱(UV-Vis)对胶体金进行形态、粒径、及所带电荷等方面进行表征，得到18～20nm胶体金。所述金标探针的制备方法没有特殊限定，采用本领域公知的方法即可，本发明金标探针的制备方法包括，取1mL浓缩10倍的胶体金溶液，加入10μL lmM的dATP，在室温下用振荡器振荡20min，接着加入15μL 1％SDS，震荡培养10min后，缓慢加入50μL 2M的NaCl溶液，然后加入1.0OD巯基修饰的检测探针，在60℃下反应3h，冷冻离心机，于转速12000rpm离心10min，去除上清液，用PBS缓冲液(pH7.2～7.4)清洗3次，最后将沉淀溶于1.0mL纳米粒子存储液中，所述的纳米粒子存储液为20mM NaPO4·12H2O，5％BSA，0.25％Twen20，10％蔗糖，即得胶体金通用DNA检测探针，放在4℃冰箱保存待用。 The 3' end of the gold label probe sequence is modified with 18-20nm colloidal gold. The present invention has no special limitation on the preparation method of colloidal gold. As an optional embodiment, colloidal gold is prepared by trisodium citrate reduction method. The specific operation method is as follows: take 100mL of 0.01% HAuCl4 aqueous solution, heat it to boiling on a magnetic heating stirrer, under vigorous stirring, accurately add a certain amount of freshly prepared 1% trisodium citrate (Na 3 C 6 H 5 O 7 2H 2 O) aqueous solution, continue to heat and boil for 15 minutes. At this time, it can be observed that the golden yellow chloroauric acid aqueous solution turns gray quickly after adding sodium citrate, then turns black, and then gradually stabilizes into wine red. The whole process is about After cooling to room temperature for 2 to 3 minutes, restore to the original volume with distilled water, store at 4°C for later use, and use transmission electron microscopy (TEM), zeta potential analyzer, and ultraviolet-visible spectroscopy (UV-Vis) to analyze the colloidal gold morphology, particle size, And the charge and other aspects are characterized, and 18-20nm colloidal gold is obtained. The preparation method of the gold-labeled probe is not particularly limited, and the method known in the art can be adopted. The preparation method of the gold-labeled probe of the present invention includes taking 1 mL of a 10-fold concentrated colloidal gold solution, adding 10 μL of 1mM dATP, and Shake with a shaker at room temperature for 20 minutes, then add 15 μL of 1% SDS, shake and incubate for 10 minutes, then slowly add 50 μL of 2M NaCl solution, then add 1.0OD thiol-modified detection probes, react at 60°C for 3 hours, refrigerate and centrifuge, Centrifuge at 12000rpm for 10min, remove the supernatant, wash 3 times with PBS buffer (pH7.2～7.4), and finally dissolve the precipitate in 1.0mL nanoparticle storage solution, the nanoparticle storage solution is 20mM NaPO4. 12H2O, 5% BSA, 0.25% Twen20, 10% sucrose to obtain the colloidal gold universal DNA detection probe, and store it in a refrigerator at 4°C until use.
本发明还提供了一种上述引物探针用于制备检测EGFR基因L858R突变试剂中的应用。The present invention also provides an application of the above-mentioned primer probe for preparing a reagent for detecting the L858R mutation of the EGFR gene.
本发明还提供了一种检测EGFR基因L858R突变的试剂盒，包括上述引物以及侧流层析试纸条，所述试纸条上的结合垫喷涂有上述探针。The present invention also provides a kit for detecting the L858R mutation of the EGFR gene, comprising the above-mentioned primers and a lateral flow chromatography test strip, the binding pad on the test strip is sprayed with the above-mentioned probe.
在本发明中，所述的试剂盒成本低廉，特异性、准确度和灵敏度高，灵敏度达到0.78％，准确率为100％，同时操作简便、检测时间短，整个检测过程可以在半小时内完成，极大地缩短了检测时间，结果易判读。In the present invention, the kit is low in cost, high in specificity, accuracy and sensitivity, the sensitivity reaches 0.78%, and the accuracy rate is 100%. At the same time, the operation is simple and the detection time is short, and the entire detection process can be completed within half an hour , which greatly shortens the detection time and makes the results easy to interpret.
在本发明中，上述试剂盒中还包括RPA扩增试剂。In the present invention, the above kit also includes RPA amplification reagents.
在本发明中，所述的RPA扩增试剂包括等温扩增时的反应液、等温扩增时的启动液和反应所需的酶。所述的等温扩增试剂购自安普未来生物科技有限公司，产品名称为DNA恒温快速扩增试剂盒(基础型)，该试剂盒中含有反应所需A液、B液以及干粉反应管，其中A液为等温扩增时的反应液，B液为等温扩增时的启动液，干粉反应管是反应所需的酶，包括重组酶、聚合酶、单链结合蛋白。所述试剂盒中还包括侧流层析缓冲溶液，所述缓冲溶液包括5X SSC、2％BSA和2％PVP，所述的5X SSC为pH7.0的4.4％氯化钠和2.2％柠檬酸钠溶液。本发明对所述缓冲溶液的制备方法没有特殊限定。In the present invention, the RPA amplification reagent includes a reaction solution for isothermal amplification, a starting solution for isothermal amplification and enzymes required for the reaction. The isothermal amplification reagent is purchased from AMP Future Biotechnology Co., Ltd., and the product name is DNA constant temperature rapid amplification kit (basic type). The kit contains liquid A, liquid B and dry powder reaction tubes required for the reaction. Solution A is the reaction solution for isothermal amplification, solution B is the starting solution for isothermal amplification, and the dry powder reaction tube is the enzyme required for the reaction, including recombinase, polymerase, and single-chain binding protein. Also include lateral flow chromatography buffer solution in the described test kit, described buffer solution comprises 5X SSC, 2%BSA and 2%PVP, and described 5X SSC is 4.4% sodium chloride and 2.2% citric acid of pH7.0 sodium solution. The present invention has no special limitation on the preparation method of the buffer solution.
在本发明中，所述侧流层析试纸条包括背衬底板，所述的背衬底板上依次设置有样品垫、结合垫、硝酸纤维素膜和吸水垫；所述样品垫与所述结合垫部分重叠，所述结合垫与所述硝酸纤维素膜部分重叠，所述结合垫的一侧边位于所述样品垫的下方，所述的结合垫的另一侧边位于所述硝酸纤维素膜的一侧边的上方；所述硝酸纤维素膜与所述吸水垫部分重叠，所述硝酸纤维素膜的另一侧边位于所述吸样垫的下方；所述的硝酸纤维素膜上设有检测线和质控线，所述的质控线靠近所述吸水垫。所述硝酸纤维素膜上的检测线和质控线之间的间隔为0.5cm。In the present invention, the lateral flow chromatography test strip includes a back substrate, on which a sample pad, a binding pad, a nitrocellulose membrane and an absorbent pad are sequentially arranged on the back substrate; the sample pad and the The binding pad is partially overlapped, the binding pad is partially overlapped with the nitrocellulose membrane, one side of the binding pad is located under the sample pad, and the other side of the binding pad is located on the nitrocellulose membrane. The top of one side of the plain film; the nitrocellulose membrane partially overlaps with the water-absorbing pad, and the other side of the nitrocellulose membrane is located below the sample-absorbing pad; the nitrocellulose membrane A test line and a quality control line are arranged on the top, and the quality control line is close to the absorbent pad. The interval between the detection line and the quality control line on the nitrocellulose membrane is 0.5 cm.
在本发明中，所述试纸条上的样品垫在使用前用处理缓冲液浸泡处理，所述的处理缓冲液包括0.05M Tris-HCI、0.15mM NaCl、0.25％Triton X-100和2.5％Tween-20，所述处理缓冲液的制备方法为称取上述浓度质量的Tris-HCI、NaCl、Triton X-100和Tween-20溶于双蒸水中，搅拌至充分溶解，即得。本发明用处理缓冲液浸泡处理后，在干燥箱中37℃下干燥4h，置于干燥器中保存备用。在本发明中，所述处理缓冲液浸泡30min，能够降低背景信号。In the present invention, the sample pad on the test strip is soaked with a processing buffer before use, and the processing buffer includes 0.05M Tris-HCl, 0.15mM NaCl, 0.25% Triton X-100 and 2.5% Tween-20, the preparation method of the treatment buffer is to weigh Tris-HCl, NaCl, Triton X-100 and Tween-20 of the above-mentioned concentration and quality, dissolve them in double-distilled water, and stir until fully dissolved. After the present invention is soaked in the treatment buffer solution, it is dried in a drying box at 37° C. for 4 hours, and stored in a desiccator for future use. In the present invention, soaking in the treatment buffer for 30 minutes can reduce the background signal.
在本发明中，所述试纸条的检测线划有1mg/mL的链霉亲和素，质控线上划有生物素修饰的质控探针，所述质控探针序列如SEQ ID NO:4所示。所述的链霉亲和素的浓度优选为1mg/mL，所述的质控探针序列为5’-TTTTTTTTTTTTTTT-3’(SEQ ID NO:4)，所述的质控探针序列3’端修饰有生物素，即5’-TTTTTTTTTTTTTTT-Biotin-3’。In the present invention, the detection line of the test strip is marked with 1 mg/mL streptavidin, and the quality control line is marked with a biotin-modified quality control probe, and the quality control probe sequence is as SEQ ID NO:4 shown. The concentration of described streptavidin is preferably 1mg/mL, and described quality control probe sequence is 5'-TTTTTTTTTTTTTTTT-3' (SEQ ID NO:4), and described quality control probe sequence 3' The end is modified with biotin, that is, 5'-TTTTTTTTTTTTTTTT-Biotin-3'.
在本发明中，所述硝酸纤维素膜为Pall vivid 170(2.5cm×50m)，购自上海捷宁生物科技有限公司，所述硝酸纤维素膜的液体爬升速率(水)：150～225秒/4cm。在本发明中，所述硝酸纤维素膜中的流体迁移速率能够与迁移期间完成DNA-DNA杂交反应所需的时间对应起来。In the present invention, the nitrocellulose membrane is Pall vivid 170 (2.5cm × 50m), purchased from Shanghai Jiening Biotechnology Co., Ltd., the liquid climbing rate (water) of the nitrocellulose membrane: 150～225 seconds /4cm. In the present invention, the fluid migration rate in the nitrocellulose membrane can correspond to the time required to complete the DNA-DNA hybridization reaction during the migration.
本发明还提供了一种非诊断目的的根据所述试剂盒检测EGFR基因L858R突变的方法，包括如下步骤：The present invention also provides a non-diagnostic method for detecting the EGFR gene L858R mutation according to the kit, comprising the following steps:
(1)提取待测血液样本的ctDNA；(1) Extract ctDNA from the blood sample to be tested;
(2)用所述引物以ctDNA为模板进行RPA扩增，RPA扩增反应的条件为：37℃水浴30min；(2) Use the primers to perform RPA amplification using ctDNA as a template, and the conditions for the RPA amplification reaction are: 37°C water bath for 30min;
(3)采用所述测流层析试纸条进行扩增产物检测。(3) Using the flow chromatography test strip to detect the amplification product.
在本发明的上述方法中，步骤(2)中，所述引物的浓度为4μM，所述金标探针的浓度为15nM；所述RPA扩增的反应体系为50μL：A液29.5μL，B液2.5μL，模板2μL，上述的上游引物和下游引物各2μL，灭菌超纯水加至50μL；步骤(3)中，所述RPA扩增反应得到的反应产物与所述的缓冲溶液混匀，滴加于侧流层析试纸条样品垫处进行检测，上样体积优选为100μL，所述反应产物与缓冲溶液的体积比优选为5:95；步骤(3)中采用侧流层析试纸条进行扩增产物检测，当试纸条出现两条红色条带，一条位于质控区内，一条位于检测区内，则结果为阳性，表明血液样本中含有EGFR基因L858R突变，当试纸条只有质控区出现一条红色条带，检测区没有条带，则结果为阴性，表明样本中不含有EGFR基因L858R突变。In the above method of the present invention, in step (2), the concentration of the primer is 4 μM, the concentration of the gold standard probe is 15 nM; the reaction system of the RPA amplification is 50 μL: A solution 29.5 μL, B 2.5 μL of solution, 2 μL of template, 2 μL of each of the above-mentioned upstream primer and downstream primer, and sterilized ultrapure water to 50 μL; in step (3), the reaction product obtained by the RPA amplification reaction is mixed with the buffer solution , drop on the side flow chromatography test strip sample pad place and detect, the loading volume is preferably 100 μ L, and the volume ratio of described reaction product and buffer solution is preferably 5:95; Adopt side flow chromatography in step (3) Test the amplification product on the test strip. When two red bands appear on the test strip, one in the quality control area and one in the detection area, the result is positive, indicating that the blood sample contains the EGFR gene L858R mutation. If there is only a red band in the quality control area of the paper strip and no band in the detection area, the result is negative, indicating that the sample does not contain the EGFR gene L858R mutation.
下面结合实施例对本发明提供的EGFR基因L858R突变检测的引物探针、试剂条及其应用的描述，但不能将它们理解为对本发明保护范围的限定。The following describes the primer probes, reagent strips and their applications for the detection of the EGFR gene L858R mutation provided by the present invention in conjunction with the examples, but they should not be interpreted as limiting the protection scope of the present invention.
实施例1Example 1
1、检测EGFR基因L858R引物与探针1. Detection of EGFR gene L858R primers and probes
与常规RPA扩增引物不同，本实施例是基于尾引物的RPA扩增，由于尾引物特异性延伸序列与检测序列之间C3间臂的修饰，保证了检测序列不会被对面延伸过来的引物延伸形成双链，在进行RPA扩增时只需要经过两轮扩增就能形成双尾结构DNA扩增产物(参见图1)，随后双尾的扩增 产物将作为下一轮扩增的模板以指数形式扩增得到更多的双尾结构DNA产物。Different from conventional RPA amplification primers, this example is RPA amplification based on tail primers. Due to the modification of the C3 arm between the tail primer-specific extension sequence and the detection sequence, it is ensured that the detection sequence will not be extended by the opposite primer Extend to form a double strand, and only two rounds of amplification are required to form a double-tailed DNA amplification product during RPA amplification (see Figure 1), and then the double-tailed amplification product will be used as the template for the next round of amplification Exponential amplification yields more double-tailed DNA products.
本实施例针对EGFR基因L858R的保守区设计了3对引物，其中按照本发明的等温扩增过程，对所有引物进行等温扩增后，取10μL产物进行丙烯酰胺凝胶电泳，最后拍胶分析，结果如图2所示。In this example, 3 pairs of primers were designed for the conserved region of the EGFR gene L858R. According to the isothermal amplification process of the present invention, after isothermal amplification of all the primers, 10 μL of the product was taken for acrylamide gel electrophoresis, and finally the gel was taken for analysis. The result is shown in Figure 2.
由图2的结果表明，本发明设计的3对引物经扩增后，通过琼脂糖凝胶电泳检测，2号引物对扩增的效果最好，条带特异性好，无非特异性扩增，而其他引物对组虽然也具有一定的特异性，但本发明所述的引物对对于MT的扩增效果最好，且对于WT的条带较弱，其中2号引物对的上游引物为5’-biotin-ATGTCAAGATCACAGATTTTGGGTG-3’，下游引物为5’-TTTTTTTTTT-C3-CAATACAGCTAGTGGGAAGGCAG-3’。Shown by the result of Fig. 2, after 3 pairs of primers designed by the present invention are amplified, detect by agarose gel electrophoresis, No. 2 primers have the best effect on amplification, good band specificity, no non-specific amplification, Although other primer pair groups also have certain specificity, the primer pair of the present invention has the best amplification effect for MT, and the band for WT is weaker, and the upstream primer of No. 2 primer pair is 5' -biotin-ATGTCAAAGATCACAGATTTTGGGTG-3', the downstream primer is 5'-TTTTTTTTTT-C3-CAATACAGCTAGTGGGAAGGCAG-3'.
2、RPA扩增反应的条件优化2. Condition optimization of RPA amplification reaction
按表2所示的反应体系，分别对扩增反应所用引物浓度(10μM、8μM、6μM、4μM、2μM、1μM、0.5μM)、温度(30℃、32℃、35℃、36℃、37℃、38℃、39℃、40℃、42℃)、时间(10min、20min、30min、40min、50min)进行优化，其中所述的C液为上游引物和下游引物的混合液，等温扩增反应结束后，将反应管中液体震荡混匀，取5μL反应产物与95μL缓冲溶液混匀，滴加于侧流层析试纸条样品垫上，5分钟后，观察侧流层析试纸条显色情况。According to the reaction system shown in Table 2, the concentration of primers (10 μM, 8 μM, 6 μM, 4 μM, 2 μM, 1 μM, 0.5 μM), temperature (30 °C, 32 °C, 35 °C, 36 °C, 37 °C) used in the amplification reaction were adjusted respectively. , 38°C, 39°C, 40°C, 42°C) and time (10min, 20min, 30min, 40min, 50min) were optimized, wherein the liquid C was a mixture of upstream primers and downstream primers, and the isothermal amplification reaction ended Finally, shake and mix the liquid in the reaction tube, take 5 μL of the reaction product and 95 μL of buffer solution, mix it evenly, and add it dropwise on the sample pad of the lateral flow chromatography test strip. After 5 minutes, observe the color development of the lateral flow chromatography test strip .
表2 RPA扩增试剂反应体系Table 2 RPA amplification reagent reaction system
Figure PCTCN2022073653-appb-000001
Figure PCTCN2022073653-appb-000001
由图3中的A和B图可知，通过不同浓度的引物对野生型(阴性)与突变型(阳性)进行扩增，发现当引物浓度为4μM时，阳性扩增后检测条带，阴性无条带，当引物浓度提高后，阴性出现了背景信号。As can be seen from A and B diagrams in Figure 3, the wild-type (negative) and mutant (positive) were amplified by different concentrations of primers, and it was found that when the primer concentration was 4 μM, the band was detected after positive amplification, and the negative had no Bands, when the concentration of primers increased, background signals appeared in the negatives.
由图4可知，当反应时间到30min时，信号达到了平台期，因此反应时间为30min效果最佳。It can be seen from Figure 4 that when the reaction time reaches 30 minutes, the signal reaches a plateau, so the reaction time is 30 minutes and the effect is the best.
由图5可知，在反应温度为37℃时，反应的条带颜色最深，因此反应温度为37℃时效果最佳。It can be seen from Figure 5 that when the reaction temperature is 37°C, the color of the reaction band is the darkest, so the effect is the best when the reaction temperature is 37°C.
因此，RPA扩增反应的条件为37℃水浴30min。Therefore, the condition of RPA amplification reaction is 37°C water bath for 30min.
实施例2：Example 2:
一种非诊断目的的根据所述试剂盒检测EGFR基因L858R突变的方法，具体步骤如下：A non-diagnostic method for detecting the EGFR gene L858R mutation according to the kit, the specific steps are as follows:
(1)提取待测血液样本的ctDNA；(1) Extract ctDNA from the blood sample to be tested;
(2)根据所述的引物，以ctDNA为模板，进行RPA扩增；(2) according to the primers, use ctDNA as a template to perform RPA amplification;
(3)提前30min将试剂盒所需组分取出，室温融化，震荡混匀；(3) Take out the required components of the kit 30 minutes in advance, melt at room temperature, shake and mix;
(4)每个干粉管中加入10μL A液，即为反应管；(4) Add 10 μL of solution A to each dry powder tube, which is the reaction tube;
(5)每个反应管分别加入6μL C液；(5) Add 6 μL of solution C to each reaction tube;
(6)向反应管中加入2μL模板；(6) Add 2 μL template to the reaction tube;
(7)向反应管中加入2μL B液并充分混匀；(7) Add 2 μL of solution B to the reaction tube and mix well;
(8)混匀后，将反应液甩(或快速离心)至管子底部，然后立即将反应管放入恒温反应槽中37℃孵育30min；(8) After mixing, shake (or quickly centrifuge) the reaction solution to the bottom of the tube, then immediately put the reaction tube into a constant temperature reaction tank and incubate at 37°C for 30 minutes;
(9)反应结束后，将反应管中液体震荡混匀，取5μL反应产物与95μL缓冲溶液混匀，滴加于侧流层析试纸条样品垫处；(9) After the reaction, oscillate and mix the liquid in the reaction tube, take 5 μL of the reaction product and mix with 95 μL buffer solution, and add dropwise to the sample pad of the lateral flow chromatography test strip;
(10)5分钟后，观察侧流层析试纸条显色情况，当试纸条出现两条红色条带，一条位于质控区内，一条位于检测区内，则结果为阳性，表明血液样本中含有EGFR基因L858R突变，当试纸条只有质控区出现一条红色条带，检测区没有条带，则结果为阴性，表明样本中不含有EGFR基因L858R突变。(10) After 5 minutes, observe the color development of the lateral flow chromatography test strip. When two red bands appear on the test strip, one in the quality control area and one in the detection area, the result is positive, indicating that blood The sample contains the EGFR gene L858R mutation. If there is only a red band in the quality control area of the test strip and no band in the detection area, the result is negative, indicating that the sample does not contain the EGFR gene L858R mutation.
可视化侧流层析试纸条检测原理如图6所示，先在侧流层析试纸条的结合垫上喷有能够与L858R扩增产物一端尾部结合的金标探针，在硝酸纤维素膜检测区划有浓度为1mg/mL的链霉亲和素，链霉亲和素能够与L858R扩增产物的另一端的生物素(Biotin)结合，质控区划有质控探针，它能够与金标检测探针结合，这样当等温扩增产物到达侧流式生物传感器的检测区时，就会被特异性的捕获显色，检测区颜色的深浅与捕获的胶体金的量呈正比，即与等温扩增产物的浓度呈正比。The detection principle of the visualized lateral flow chromatography test strip is shown in Figure 6. First, a gold-labeled probe that can bind to one end of the L858R amplification product is sprayed on the binding pad of the lateral flow chromatography test strip, and the nitrocellulose membrane The detection area has streptavidin with a concentration of 1mg/mL, and the streptavidin can combine with the biotin (Biotin) at the other end of the L858R amplification product, and the quality control area has a quality control probe, which can bind to the gold In this way, when the isothermal amplification product reaches the detection area of the lateral flow biosensor, it will be specifically captured and developed, and the color depth of the detection area is directly proportional to the amount of colloidal gold captured, that is, with The concentration of the isothermal amplification product is directly proportional.
实施例3Example 3
对上述检测EGFR基因L858R突变的方法的灵敏度进行了检测，采用突变型标准品(购自生工生物工程(上海)股份有限公司)进行灵敏度检测。The sensitivity of the above-mentioned method for detecting the L858R mutation of the EGFR gene was tested, and a mutant standard product (purchased from Sangon Bioengineering (Shanghai) Co., Ltd.) was used for sensitivity testing.
由图7可知，经过RPA等温扩增后使用试纸条进行检测，发现灵敏度可达600copies。为了进一步验证该检测方法灵敏度，在野生型标准品(购自生工生物工程(上海)股份有限公司)中加入不同比例的突变型标准品并进行扩增检测，发现该方法检测灵敏度可达0.78％，其中存在细微背景信号加标阴性造成(参见图8)。It can be seen from Figure 7 that after RPA isothermal amplification, the test strip is used for detection, and the sensitivity can reach 600copies. In order to further verify the sensitivity of the detection method, different proportions of the mutant standard were added to the wild-type standard (purchased from Sangon Bioengineering (Shanghai) Co., Ltd.) and amplified for detection. It was found that the detection sensitivity of this method can reach 0.78%. , where there is a slight background signal resulting from a negative spike (see Figure 8).
实施例4Example 4
根据实施例2的检测EGFR基因L858R突变的方法，对45例临床样本进行盲测，同时对45例临床样本进行了测序，所述的测序为使用PCR技术对45例临床样本进行扩增，将扩增产物送至生工生物工程(上海)股份有限公司进行测序，测序结果见表3。According to the method for detecting the EGFR gene L858R mutation in Example 2, 45 clinical samples were tested blindly, and 45 clinical samples were sequenced simultaneously. The amplified products were sent to Sangon Bioengineering (Shanghai) Co., Ltd. for sequencing, and the sequencing results are shown in Table 3.
表3 45例临床样本不同检测方法的结果对比Table 3 Comparison of the results of different detection methods for 45 clinical samples
Figure PCTCN2022073653-appb-000002
Figure PCTCN2022073653-appb-000002
Figure PCTCN2022073653-appb-000003
Figure PCTCN2022073653-appb-000003
Figure PCTCN2022073653-appb-000004
Figure PCTCN2022073653-appb-000004
Figure PCTCN2022073653-appb-000005
Figure PCTCN2022073653-appb-000005
Figure PCTCN2022073653-appb-000006
Figure PCTCN2022073653-appb-000006
Figure PCTCN2022073653-appb-000007
Figure PCTCN2022073653-appb-000007
Figure PCTCN2022073653-appb-000008
Figure PCTCN2022073653-appb-000008
由图9和表3可知，本发明的检测结果和测序结果一致，准确率达100％，与测序方法相比本发明的检测方法操作更加简便，检测速度更快。As can be seen from Figure 9 and Table 3, the detection results of the present invention are consistent with the sequencing results, with an accuracy rate of 100%. Compared with the sequencing method, the detection method of the present invention is easier to operate and faster in detection speed.
以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。对这些实施例的多种修改对本领域的专业技术人员来说是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。The descriptions of the above embodiments are only used to help understand the method and core idea of the present invention. It should be pointed out that for those skilled in the art, without departing from the principle of the present invention, some improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention will not be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (10)

  1. EGFR基因L858R突变检测的引物探针，其特征在于，所述L858R突变的上游引物序列如SEQ ID NO:1所示；所述L858R突变的下游引物序列如SEQ ID NO:2所示；所述L858R突变的探针为金标探针，其序列如SEQ ID NO:3所示。The primer probe of EGFR gene L858R mutation detection is characterized in that, the upstream primer sequence of the L858R mutation is as shown in SEQ ID NO:1; the downstream primer sequence of the L858R mutation is as shown in SEQ ID NO:2; the The probe for the L858R mutation is a gold-labeled probe, and its sequence is shown in SEQ ID NO:3.
  2. 根据权利要求1所述的引物探针，其特征在于，所述金标探针序列3’端修饰18～20nm胶体金。The primer probe according to claim 1, wherein the 3' end of the gold-labeled probe sequence is modified with 18-20nm colloidal gold.
  3. 权利要求1或2所述引物探针用于制备检测EGFR基因L858R突变试剂中的应用。The application of the primer probe described in claim 1 or 2 in preparing a reagent for detecting the EGFR gene L858R mutation.
  4. 一种检测EGFR基因L858R突变的试剂盒，其特征在于，包括权利要求1或2所述的引物以及侧流层析试纸条，所述试纸条上的结合垫喷涂有权利要求1所述探针。A kit for detecting the EGFR gene L858R mutation, characterized in that it comprises the primers described in claim 1 or 2 and a lateral flow chromatography test strip, and the binding pad on the test strip is sprayed with the one described in claim 1 probe.
  5. 根据权利要求4所述的试剂盒，其特征在于，所述试剂盒中还包括RPA扩增试剂。The kit according to claim 4, characterized in that, the kit also includes an RPA amplification reagent.
  6. 根据权利要求4所述的试剂盒，其特征在于，所述试剂盒中还包括侧流层析缓冲溶液，所述缓冲溶液包括5X SSC、2％BSA和2％PVP，所述的5X SSC为pH7.0的4.4％氯化钠和2.2％柠檬酸钠溶液。The test kit according to claim 4, wherein said kit also includes a lateral flow chromatography buffer solution, said buffer solution comprising 5X SSC, 2%BSA and 2%PVP, and said 5X SSC is 4.4% sodium chloride and 2.2% sodium citrate solution at pH 7.0.
  7. 根据权利要求4所述的试剂盒，其特征在于，所述引物的浓度为4μM，所述金标探针的浓度为15nM。The kit according to claim 4, wherein the concentration of the primer is 4 μM, and the concentration of the gold-labeled probe is 15 nM.
  8. 根据权利要求4所述的试剂盒，其特征在于，所述试纸条的检测线划有链霉亲和素，质控线上划有生物素修饰的质控探针，所述质控探针序列如SEQ ID NO:4所示。The test kit according to claim 4, wherein streptavidin is drawn on the detection line of the test strip, and biotin-modified quality control probes are drawn on the quality control line, and the quality control probes The needle sequence is shown in SEQ ID NO:4.
  9. 根据权利要求4所述的试剂盒，其特征在于，所述试纸条上的样品垫用处理缓冲液浸泡处理，所述的处理缓冲液包括0.05M Tris-HCI、0.15mM NaCl、0.25％Triton X-100和2.5％Tween-20。The test kit according to claim 4, wherein the sample pad on the test strip is soaked with a processing buffer, and the processing buffer includes 0.05M Tris-HCl, 0.15mM NaCl, 0.25% Triton X-100 and 2.5% Tween-20.
  10. 一种非诊断目的的根据权利要求1或2所述的引物探针或权利要求4～9任意一项所述试剂盒检测EGFR基因L858R突变的方法，其特征在于，包括如下步骤：A non-diagnostic method for detecting the EGFR gene L858R mutation according to the primer probe according to claim 1 or 2 or the kit according to any one of claims 4 to 9, characterized in that it comprises the following steps:
    (1)提取待测血液样本的ctDNA；(1) Extract ctDNA from the blood sample to be tested;
    (2)用所述引物以ctDNA为模板进行RPA扩增，RPA扩增反应的 条件为：37℃水浴30min；(2) Carry out RPA amplification with described primer with ctDNA as template, the condition of RPA amplification reaction is: 37 ℃ of water baths 30min;
    (3)采用所述测流层析试纸条进行扩增产物检测。(3) Using the flow chromatography test strip to detect the amplification product.
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Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103282515A (en) * 2010-12-22 2013-09-04 霍夫曼-拉罗奇有限公司 Methods and compositions for detecting mutation in the human epidermal growth factor receptor gene
CN104818320A (en) * 2014-12-03 2015-08-05 厦门艾德生物医药科技有限公司 Primers, probes, detection system and kit for one time detection of lung cancer multiple genes
CN105349654A (en) * 2015-11-23 2016-02-24 厦门艾德生物医药科技股份有限公司 Probes, primers, detection system and kit for detecting mutations of EGFR gene
CN108841953A (en) * 2018-06-05 2018-11-20 北京雅康博生物科技有限公司 The kit of 22 kinds of EGFR gene mutation is detected using digital pcr technology
CN111500780A (en) * 2020-05-07 2020-08-07 安徽科技学院 Kit for detecting novel coronavirus and preparation method thereof
CN111518952A (en) * 2020-05-07 2020-08-11 安徽科技学院 Kit for nucleic acid-antibody dual detection of novel coronavirus and preparation method thereof
CN111518669A (en) * 2020-05-07 2020-08-11 安徽科技学院 Nucleic acid detection micro-fluidic chip and application thereof
CN113355423A (en) * 2021-07-07 2021-09-07 安徽科技学院 Primer probe and kit for detecting mutation of EGFR gene L858R and application of primer probe and kit

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010279264A (en) * 2009-06-03 2010-12-16 Tottori Univ Egfr gene mutation-detecting primer set, egfr gene mutation-detecting kit containing the same, and nucleic acid amplification device for performing egfr gene mutation detection therewith
CN105624309B (en) * 2016-02-23 2020-04-21 深圳华大生命科学研究院 Primer, probe and kit for detecting EGFR and/or K-ras gene mutation
CN111534595A (en) * 2020-05-20 2020-08-14 青岛科技大学 Method for amplifying photoelectrochemical circulating tumor DNA liquid biopsy by recombinase polymerase

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103282515A (en) * 2010-12-22 2013-09-04 霍夫曼-拉罗奇有限公司 Methods and compositions for detecting mutation in the human epidermal growth factor receptor gene
CN104818320A (en) * 2014-12-03 2015-08-05 厦门艾德生物医药科技有限公司 Primers, probes, detection system and kit for one time detection of lung cancer multiple genes
CN105349654A (en) * 2015-11-23 2016-02-24 厦门艾德生物医药科技股份有限公司 Probes, primers, detection system and kit for detecting mutations of EGFR gene
CN108841953A (en) * 2018-06-05 2018-11-20 北京雅康博生物科技有限公司 The kit of 22 kinds of EGFR gene mutation is detected using digital pcr technology
CN111500780A (en) * 2020-05-07 2020-08-07 安徽科技学院 Kit for detecting novel coronavirus and preparation method thereof
CN111518952A (en) * 2020-05-07 2020-08-11 安徽科技学院 Kit for nucleic acid-antibody dual detection of novel coronavirus and preparation method thereof
CN111518669A (en) * 2020-05-07 2020-08-11 安徽科技学院 Nucleic acid detection micro-fluidic chip and application thereof
CN113355423A (en) * 2021-07-07 2021-09-07 安徽科技学院 Primer probe and kit for detecting mutation of EGFR gene L858R and application of primer probe and kit

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
LI XIAN-YING, ZHANG CHAO, ZHANG QIN-LU, ZHU JUAN-LI, LIU QIAN, CHEN MING-WEI, YANG XUE-MIN, HUI WEN-LI, CUI YA-LI: "Sensitive genotyping of mutations in the EGFR gene from NSCLC patients using PCR-GoldMag lateral flow device", SCIENTIFIC REPORTS, vol. 7, no. 1, XP093020431, DOI: 10.1038/s41598-017-08210-8 *
LIU, JING ET AL.: "An Improved Allele-Specific PCR Primer Design Method for SNP Marker Analysis and Its Application", PLANT METHODS, vol. 8, 24 August 2012 (2012-08-24), XP021122370, DOI: 10.1186/1746-4811-8-34 *
QIU MANTANG, WANG JIE, XU YOUTAO, DING XIANGXIANG, LI MING, JIANG FENG, XU LIN, YIN RONG: "Circulating Tumor DNA Is Effective for the Detection of EGFR Mutation in Non–Small Cell Lung Cancer: A Meta-analysis", CANCER EPIDEMIOLOGY, BIOMARKERS & PREVENTION, AMERICAN ASSOCIATION FOR CANCER RESEARCH, vol. 24, no. 1, 1 January 2015 (2015-01-01), pages 206 - 212, XP093020430, ISSN: 1055-9965, DOI: 10.1158/1055-9965.EPI-14-0895 *
WANG, ZHIYING ET AL.: "A Strip of Lateral Flow Gene Assay Using Gold Nanoparticles for Point-of-Care Diagnosis of African Swine Fever Virus in Limited Environment", ANALYTICAL AND BIOANALYTICAL CHEMISTRY, vol. 413, 21 May 2021 (2021-05-21), pages 4665 - 4672, XP037502761, DOI: 10.1007/s00216-021-03408-2 *

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