Preparation method and application of quality control product or reference product for HER2 gene amplification detection

Abstract

The invention discloses a preparation method and application of a quality control product or a reference product for HER2 gene amplification detection, and relates to the technical field of molecular biology. Compared with a quality control product or a reference product of a clinical sample or a cell line, the synthesized plasmid is adopted for preparing the quality control product and the reference product, biological materials of the synthesized plasmid are easier to obtain, wide in source and easier to prepare on a large scale at low cost, and the prepared quality control product and the reference product have good accuracy and repeatability, small difference between preparation batches and high application value. By designing plasmids, quality control products with different HER2 amplification levels can be prepared according to the repetition times of HER2 gene segments, and the preparation method is simple and convenient, can be used for batch production, is easy to store, can be stored for a long time under the condition of-20 DEG C, and can be used for evaluating the accuracy and repeatability performance of an HER2 gene detection method.

Description

Technical Field

[0001] The present invention relates to the field of molecular biotechnology, and in particular to a preparation method and application of a quality control product or reference product for HER2 gene amplification detection. Background Art

[0002] Breast cancer is the most common cancer in women worldwide, and about 15%-20% of cases are characterized by HER2 overexpression or amplification. The HER2 gene (Human Epidermal Growth Factor Receptor-2) is located on chromosome 17q21, and its encoded product is a 185kD transmembrane receptor-like protein with tyrosine protein kinase activity. In the human body, HER2 gene amplification causes overexpression of HER2 protein receptors, which promotes cell growth and proliferation, leading to tumorigenesis. It also inhibits cell apoptosis, causes chemotherapy resistance, induces angiogenesis, improves cell motility, and enhances tumor infiltration and metastasis. Therefore, HER2 is an important target molecule in tumor immunotherapy research. Studies have shown that 20% to 30% of primary invasive breast cancers have HER2 gene amplification / overexpression, and about 30% of breast cancers have HER2 gene overexpression, which is associated with poor prognosis in patients. Patients with breast cancer with HER2 overexpression have rapid disease progression, short chemotherapy remission period, poor endocrine therapy effect, and low disease-free survival and overall survival rates.

[0003] In recent decades, with the introduction of HER2 targeted drug therapy, mainly trastuzumab, pertuzumab, antibody-drug conjugates and tyrosine kinase inhibitors, the prognosis of patients has been significantly improved. The results of HER2 testing not only involve whether the patient is suitable for HER2 targeted therapy (trastuzumab, etc.), but also guide the selection of endocrine therapy, chemotherapy regimens and prognosis evaluation. Correct detection and evaluation of HER2 protein expression and gene amplification status are crucial for clinical treatment and prognosis judgment.

[0004] Clinically, immunohistochemistry (IHC) is commonly used to detect the expression level of HER2 protein, and in situ hybridization (ISH) is used to detect the level of HER2 gene amplification. With the development of digital PCR technology, HER2 gene amplification detection based on digital PCR technology is simple to operate and the result interpretation is more objective. It can also be applied to the detection of HER2 amplification status in free DNA of plasma samples when tissue samples are not available in the late stage. Therefore, the HER2 detection method based on digital PCR can be a supplement or even a substitute for IHC and FISH, and can assist in guiding the use and efficacy monitoring of tumor targeted drugs for HER2-positive breast cancer.

[0005] With the emergence of HER2 gene amplification detection methods based on digital PCR technology, corresponding quality control products are now needed to evaluate the accuracy and other performance of the detection methods. Currently, quality control products prepared by cell lines are expensive and require cell culture. The quality control product preparation process is complicated, and it is not necessarily possible to obtain cell line raw materials with the expected copy number.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The object of the present invention is to provide a method for preparing a quality control product or a reference product for HER2 gene amplification detection and its application to solve the above technical problems.

[0008] The present invention is achieved in that:

[0009] In a first aspect, the present invention provides a method for preparing a quality control product for HER2 gene amplification detection, comprising the following steps:

[0010] Preparation of negative quality control: digesting the first plasmid including the HER2 gene fragment and the reference gene fragment, and quantifying the copy number of the digestion product to prepare a negative quality control; wherein the copy number ratio of the HER2 gene fragment to the reference gene fragment on the first plasmid is 1:1, and both ends of the HER2 gene fragment and both ends of the reference gene fragment have restriction sites;

[0011] Preparation of positive quality control product: digesting the second plasmid including the HER2 gene fragment and the reference gene fragment, and quantifying the copy number of the digestion product to prepare a positive quality control product; wherein the ratio of the copy number of the HER2 gene fragment on the second plasmid to the copy number of the reference gene fragment is at least 2:1, and on the second plasmid, both ends of each HER2 gene fragment and both ends of the reference gene fragment have restriction sites;

[0012] The reference gene is: the housekeeping gene located on human chromosome 17.

[0013] In a second aspect, the present invention also provides a method for preparing a reference substance for HER2 gene amplification detection, comprising the following steps:

[0014] The negative quality control product and the positive quality control product prepared by the preparation method of the quality control product for HER2 gene amplification detection are mixed according to a preset volume ratio;

[0015] In a preferred embodiment of the present invention, the mixed solution mixed according to a preset volume ratio is diluted.

[0016] In a third aspect, the present invention also provides the use of a quality control product prepared by the method for preparing a quality control product for HER2 gene amplification detection or a reference product prepared by the method for preparing the reference product for HER2 gene amplification detection in preparing a HER2 gene amplification detection kit or evaluating the performance of a HER2 gene detection method.

[0017] In a fourth aspect, the present invention further provides a kit, which includes a quality control product prepared by the above-mentioned method for preparing a quality control product for HER2 gene amplification detection, and / or a reference product prepared by the above-mentioned method for preparing a reference product for HER2 gene amplification detection.

[0018] The present invention has the following beneficial effects:

[0019] The present invention provides a method for preparing quality control products and reference products for HER2 gene amplification detection. Compared with quality control products or reference products of cell lines, the quality control products and reference products are prepared by using synthetic plasmids, and the biological materials thereof are easier to obtain and have a wide source, and are easy to prepare in large quantities at low cost. The prepared quality control products and reference products have good accuracy and consistency, small differences between preparation batches, and high application value.

[0020] By designing the plasmid, quality control products with different HER2 amplification levels are prepared according to the number of repetitions of the HER2 gene nucleic acid fragment. This production method is simple, can be mass-produced, easy to store, and can be stored for a long time at -20°C. It can be used to evaluate the accuracy, repeatability and other performance of the HER2 gene detection method. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0022] Figure 1 It is a simulation diagram of the HER2-negative plasmid and the HER2-positive plasmid of the present invention;

[0023] Figure 2 This is a result diagram of the digital PCR detection of negative quality control products in Example 2;

[0024] Figure 3 This is a graph showing the results of digital PCR testing of positive quality control products in Example 2. DETAILED DESCRIPTION

[0025] References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0026] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. This technique is fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (PCR: The Polymerase Chain Reaction) (Academic Press, Inc., 1987). Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0027] In the present invention, "reference gene" refers to a gene that serves as a benchmark for the amplification state of the target gene, and a gene that is not frequently repeated, deleted, mutated or replaced is selected. It can also be synonymous with "standard gene". In addition, the standard gene can be a DNA sequence that includes not only genes in a specific chromosome region but also introns in the region.

[0028] In the present invention, the reference gene may include a gene or intron sequence on the long arm (q arm) or short arm of chromosome 17 or other chromosomes, but is not limited thereto, and may be any one of EFTUD2, G6PC3 and ALDOC (aldolase C, fructose bisphosphate), and the gene or intron sequence located on the telomeric side and the centromere side based on the position of the HER2 gene may be used as a reference gene.

[0029] In a first aspect, the present invention provides a method for preparing a quality control product for HER2 gene amplification detection, comprising the following steps:

[0030] Preparation of negative quality control: digesting the first plasmid including the HER2 gene fragment and the reference gene fragment, and quantifying the copy number of the digestion product to prepare a negative quality control; wherein the copy number ratio of the HER2 gene fragment to the reference gene fragment on the first plasmid is 1:1, and both ends of the HER2 gene fragment and both ends of the reference gene fragment have restriction sites;

[0031] Preparation of positive quality control product: digesting the second plasmid including the HER2 gene fragment and the reference gene fragment, and quantifying the copy number of the digestion product to prepare a positive quality control product; wherein the ratio of the copy number of the HER2 gene fragment on the second plasmid to the copy number of the reference gene fragment is at least 2:1, and on the second plasmid, both ends of each HER2 gene fragment and both ends of the reference gene fragment have restriction sites;

[0032] Reference genes are: housekeeping genes located on human chromosomes, such as the IGF1 (insulin-like growth factor 1) gene located on chromosome 12 and the HK1 gene located on chromosome 10.

[0033] The present invention uses plasmid DNA for the preparation of quality control products and reference products, which are consistent with the actual clinical sample gDNA or cfDNA at the nucleic acid level. Among them, the first plasmid (i.e., negative plasmid) contains a nucleic acid sequence of the HER2 gene and a nucleic acid sequence of the reference gene; and the second plasmid (i.e., positive plasmid) contains 2 or n (≥3) repeated HER2 nucleic acid fragments and a nucleic acid sequence of the reference gene, and the HER2 gene fragment of the second plasmid is the same as the HER2 gene fragment in the first plasmid. Both ends of each HER2 gene fragment and both ends of the reference gene fragment have restriction sites; after restriction digestion, the ratio of the HER2 copy number to the reference gene copy number in the negative plasmid is 1, which can simulate the normal state without amplification; the ratio of the HER2 copy number to the reference gene copy number in the positive plasmid is 2 or n (≥3), simulating HER2 amplification by 2 times or n times.

[0034] Therefore, the quality control products and reference products provided by the present invention can be used to evaluate the performance of the HER2 gene detection method, such as the accuracy and repeatability of the detection method.

[0035] The "HER2 gene amplification detection" of the present invention includes but is not limited to HER2 gene amplification detection using digital PCR, fluorescent quantitative PCR, RT-qPCR, capillary electrophoresis, LAMP technology and the like.

[0036] The HER2 copy number in the positive plasmid is, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0037] The reference gene is a housekeeping gene located on human chromosome 17, so that it can be determined that HER-2 amplification is simple HER-2 amplification, rather than overexpression of the HER-2 gene due to chromosome 17 polyploidy.

[0038] In a preferred embodiment of the present invention, the reference gene is selected from the long arm or the short arm of chromosome 17.

[0039] In a preferred embodiment of the present invention, the reference gene is selected from the long arm of chromosome 17;

[0040] In a preferred embodiment of the present invention, the reference gene is selected from the EFTUD2 gene on chromosome 17, the G6PC3 gene located on the telomere side, and the ALDOC gene close to the centromere.

[0041] In a preferred embodiment of the present invention, the reference gene is EFTUD2, and the nucleotide sequence of the reference gene fragment is shown in SEQ ID No.2.

[0042] In a preferred embodiment of the present invention, the length of the reference gene fragment on the first plasmid or the second plasmid is 100 to 1000 bp;

[0043] In a preferred embodiment of the present invention, the length of the HER2 gene fragment on the first plasmid or the second plasmid is 100 to 1000 bp;

[0044] In a preferred embodiment of the present invention, the nucleotide sequence of the HER2 gene fragment is shown as SEQ ID No.1.

[0045] In a preferred embodiment of the present invention, when preparing negative and positive control products, the restriction endonucleases used in the enzyme digestion step are the same or different; for example, the restriction endonucleases set in the negative plasmid can be the same or two. The restriction endonucleases set in the positive plasmid can be the same or two.

[0046] In a preferred embodiment of the present invention, the restriction endonuclease is a blunt-end restriction endonuclease or a sticky-end restriction endonuclease.

[0047] In a preferred embodiment of the present invention, the sticky-end restriction endonuclease is at least one of EcoR V, HpyCH4V, HaeIII, AluI, CvikI-1, BstUI and RsaI.

[0048] For example, if the restriction endonuclease is EcoR V, set the restriction endonuclease cutting site on the plasmid according to the instructions provided by manufacturers such as NEB or Takara, as well as the corresponding enzyme cutting conditions, such as setting the reaction at 37° C. for 2 hours.

[0049] In a preferred embodiment of the present invention, the copy number quantification of the enzyme cleavage product is performed by at least one of the following methods:

[0050] (1) Use an ultraviolet spectrophotometer to measure the mass concentration and then convert the copy number;

[0051] (2) Copy number quantification using digital PCR.

[0052] The two-step detection using UV spectrophotometer and digital PCR is more accurate for quantifying plasmid copy number.

[0053] In a preferred embodiment of the present invention, after the copy number is quantified, the method further comprises: diluting the enzyme cleavage product with a diluent to obtain a quality control product of the target copy number;

[0054] In a preferred embodiment of the present invention, the diluent comprises: Tris EDTA buffer and salmon sperm DNA, the pH of the Tris EDTA buffer is 7.0-8.5, and the final concentration of the salmon sperm DNA in the diluent is 10-100 ng / μL.

[0055] Dissolving and diluting with the above diluent can increase the stability of the solution, improve and stabilize the amplification efficiency of the reaction, and reduce or eliminate batch-to-batch differences.

[0056] For example, the pH of the Tris EDTA buffer in the diluent is 7.0, 7.2, 7.5, 7.8, 8.0 or 8.5, and other specific point values ​​within this numerical range can be selected, which will not be described here one by one.

[0057] The final concentration of salmon sperm DNA is 10-100 ng / μL, for example, it can be 10 ng / μL, 20 ng / μL, 30 ng / μL, 40 ng / μL, 50 ng / μL, 60 ng / μL, 70 ng / μL, 80 ng / μL, 90 ng / μL or 100 ng / μL, etc. Other specific point values ​​within this numerical range can be selected and will not be described here one by one.

[0058] In a preferred embodiment of the present invention, the reaction system of digital PCR includes: a primer pair and a probe for detecting the HER2 gene, a primer pair and a probe for detecting a reference gene, and a PCR premix;

[0059] In a preferred embodiment of the present invention, in the reaction system of digital PCR, the final concentration of the primer pair for detecting the HER2 gene is 600-1000 nM, for example, it can be 600 nM, 650 nM, 700 nM, 750 nM, 800 nM, 850 nM, 900 nM, 950 nM or 1000 nM, etc. The final concentration of the primer pair for detecting the reference gene is 600-1000 nM; for example, it can be 600 nM, 650 nM, 700 nM, 750 nM, 800 nM, 850 nM, 900 nM, 950 nM or 1000 nM, etc. The final concentration of the probe for detecting the HER2 gene is 100-400 nM; for example, it can be 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM or 400 nM, etc. The final concentration of the probe for detecting the reference gene is 100-400 nM; for example, it can be 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM or 400 nM, etc. Other specific point values ​​within the numerical range can be selected, and they will not be described one by one here.

[0060] In a preferred embodiment of the present invention, the sequences of the primer pair for detecting the HER2 gene are shown in SEQ ID Nos. 3 to 4, and the sequence of the fluorescent probe for detecting the HER2 gene is shown in SEQ ID No. 5. The sequences of the primer pair for detecting the reference gene are shown in SEQ ID Nos. 6 to 7, and the sequence of the fluorescent probe for detecting the reference gene is shown in SEQ ID No. 8.

[0061] The 5' end of the fluorescent probe is labeled with a fluorescence generating group, and the 3' end is labeled with a fluorescence quenching group. The fluorescence generating group includes but is not limited to any one selected from HEX, FAM, TET, CF532, JOE, TAMRA, ROX, CY3, CY5, Texas Red, NED, AlexaFlour or VIC. The fluorescence quenching group includes but is not limited to any one of MGB, BHQ1, BHQ2 or BHQ3 or QSY. In other embodiments, the fluorescence generating group and the quenching group of the probe label can also be adaptively adjusted as needed, and are not limited to the several types defined above.

[0062] PCR master mix includes buffer, DNA polymerase and dNTPs.

[0063] In a preferred embodiment of the present invention, the reaction procedure of digital PCR includes: pre-denaturation: 94-98°C, 8-12 min; cyclic amplification: 94-98°C, 20-40 s; 50-60°C, 60-80 s; and 40-50 cycles.

[0064] Pre-denaturation:

[0065] 94~98℃, 8~12min, the temperature can be, for example, 94℃, 94.5℃, 95℃, 95.5℃, 96℃, 96.5℃, 97℃, 97.5℃ or 98℃, and the time can be, for example, 8min, 8.5min, 9min, 9.5min, 10min, 10.5min, 11min, 11.5min or 12min, and other specific point values ​​within the numerical range can be selected, which will not be described here one by one.

[0066] Cyclic Amplification:

[0067] 94~98℃, 20~40s, the temperature can be, for example, 94℃, 94.5℃, 95℃, 95.5℃, 96℃, 96.5℃, 97℃, 97.5℃ or 98℃, and the time can be, for example, 20s, 22s, 24s, 26s, 28s, 30s, 32s, 34s, 36s, 38s or 40s, and other specific point values ​​within the numerical range can be selected, which will not be described here one by one.

[0068] 50-60°C, 50-70s, the temperature can be, for example, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C or 60°C, and the time can be, for example, 50s, 52s, 54s, 56s, 58s, 60s, 62s, 64s, 66s, 68s or 70s, and other specific point values ​​within the numerical range can be selected, which will not be described here one by one.

[0069] The method may be cycled 35 to 45 times, for example, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 times.

[0070] The present invention uses a synthetic plasmid to prepare a quality control product, which can be used as a substitute for a quality control product prepared by a cell line, and has the advantages of large sample volume, good consistency between preparation batches, high accuracy, low cost, and simple preparation process, and quality control products with different HER2 amplification levels are prepared by designing the plasmid, that is, the number of repetitions of the HER2 nucleic acid fragment. The preparation method provided by the present invention is simple, can be mass-produced, is easy to store, can be stored for a long time at -20°C, and can be used for indoor quality control and inter-laboratory quality evaluation.

[0071] In a second aspect, the present invention also provides a method for preparing a reference substance for HER2 gene amplification detection, comprising the following steps:

[0072] The negative quality control product and the positive quality control product prepared by the preparation method of the quality control product for HER2 gene amplification detection are mixed according to a preset volume ratio;

[0073] In a preferred embodiment of the present invention, the mixed solution mixed according to a preset volume ratio is diluted.

[0074] In an optional embodiment, the negative control product and the positive control product are mixed in a volume ratio of 1:0, 1:1, and 0:1 to obtain a reference product.

[0075] In a third aspect, the present invention also provides the use of a quality control product prepared by the method for preparing a quality control product for HER2 gene amplification detection or a reference product prepared by the method for preparing the reference product for HER2 gene amplification detection in preparing a HER2 gene amplification detection kit or evaluating the performance of a HER2 gene detection method.

[0076] In a fourth aspect, the present invention further provides a kit, which includes a quality control product prepared by the above-mentioned method for preparing a quality control product for HER2 gene amplification detection, and / or a reference product prepared by the above-mentioned method for preparing a reference product for HER2 gene amplification detection.

[0077] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0078] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0079] Example 1

[0080] This example provides a method for preparing quality control products and reference products for HER2 gene amplification detection.

[0081] 1. Preparation of Plasmid

[0082] The plasmid was commissioned to Suzhou Jinweizhi Biotechnology Co., Ltd., a gene synthesis company, to prepare HER2 negative plasmid and HER2 positive plasmid, respectively. The HER2 negative plasmid is shown in SEQ ID No.9, and the nucleotide sequence of the HER2 positive plasmid is shown in SEQ ID No.10. Figure 1 As shown, in this embodiment, the HER2-negative plasmid includes the EcoR V restriction site base sequence, one copy of the HER2 gene nucleic acid fragment, the EcoR V restriction site base sequence, one copy of the reference gene nucleic acid fragment (EFTUD2 gene) and the EcoR V restriction site base sequence; the HER2-positive plasmid includes the EcoR V restriction site base sequence, one copy of the HER2 gene nucleic acid fragment, the EcoR V restriction site base sequence, one copy of the HER2 gene nucleic acid fragment, the EcoR V restriction site base sequence, one copy of the reference gene nucleic acid fragment and the EcoR V restriction site base sequence.

[0083] 2. Plasmid Dissolution and Concentration Determination

[0084] The HER2-negative plasmid and the HER2-positive plasmid were dissolved in protective diluents respectively; wherein the protective diluents included Tris EDTA buffer with a pH of 8.0 and salmon sperm DNA with a final concentration of 50 ng / μL; then, the concentration of the plasmid was measured using an ultraviolet spectrophotometer and diluted to 20 ng / μL for later use.

[0085] 3. Plasmid digestion

[0086] Restriction endonuclease EcoR V was purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd., with the catalog number Code No. 1024S. Take a certain amount of the above-mentioned dissolved HER2 positive plasmid and HER2 negative plasmid, dilute them to 1 ng / μL with dilution protection solution, prepare the enzyme digestion reaction system according to the following table, cover the tube tightly, mix gently and centrifuge instantly, place in PCR instrument (Bio-Rad T100 PCR amplifier), set at 37°C for 2 hours, perform enzyme digestion treatment, and take out and immediately place at 4°C or on ice after 2 hours to complete the enzyme digestion reaction.

[0087] Components Volume (μL) UltraPure Distilled Water 52 EcoR V 4 10x H Buffer 8 Plasmids 16 Total volume 80

[0088] 4. Digital PCR Quantification

[0089] The concentrations of the HER2-negative plasmid and HER2-positive plasmid after enzyme digestion were both 0.2 ng / μL (200 pg / μL). 5 μL of each was diluted 10 times with diluent to 0.02 ng / μL, and the copy number was detected by digital PCR:

[0090] The digital PCR reaction system includes a primer pair with a final concentration of 800 nM, a fluorescent probe at 250 nM, and a premix;

[0091] The sequence of the primer pair for amplifying the HER2 gene nucleic acid fragment is shown in SEQ ID No.3-4, the sequence of the fluorescent probe for detecting the HER2 gene nucleic acid fragment is shown in SEQ ID No.5, the fluorescence generating group of the fluorescent probe is FAM, and the fluorescence quenching group is BHQ1; the sequence of the primer pair for amplifying the reference gene nucleic acid fragment is shown in SEQ ID No.6-7, the sequence of the fluorescent probe for detecting the reference gene nucleic acid fragment is shown in SEQ ID No.8, the fluorescence generating group of the fluorescent probe is VIC, and the fluorescence quenching group is MGB.

[0092] The premix includes buffer, DNA polymerase and dNTPs. The PCR reaction system is as follows:

[0093] Components Dosage (μL) Master mix 15 HER2 forward primer (20 μM) 1.2 HER2 reverse primer (20 μM) 1.2 HER2 probe (10 μM) 0.75 Reference gene forward primer (20 μM) 1.2 Reference gene reverse primer (20 μM) 1.2 Reference gene probe (10 μM) 0.75 Template (plasmid) 2 water 6.7 Total volume 30

[0094] The reaction procedure of digital PCR is as follows:

[0095]

[0096] After PCR amplification is completed, the test results are analyzed using a digital PCR analyzer to obtain the copy number (copy / μL) of the HER2-negative plasmid and HER2-positive plasmid after enzyme digestion and dilution. Based on this test result and the dilution multiple, the copy number concentration of the plasmid after enzyme digestion is calculated, as shown in the following table:

[0097] Plasmid name HER2 copy number Reference gene copy number HER2 / reference gene copy number ratio HER2 negative plasmid <![CDATA[1.48×10 8 ]]> <![CDATA[1.50×10 8 ]]> 0.99 HER2 positive plasmid <![CDATA[2.02×10 8 ]]> <![CDATA[1.02×10 8 ]]> 1.98

[0098] 5. Preparation of Quality Control and Reference Materials

[0099] Use the protection diluent in step 2 to dilute the HER2-negative plasmid and HER2-positive plasmid after enzyme digestion to a final concentration of 5000 copies / μL of the EFTUD2 gene to obtain negative control products and positive control products, respectively.

[0100] The negative control and the positive control were mixed in a volume ratio of 1:0, 1:1, and 0:1, and diluted to 1500 copies / μL with dilution protection solution to obtain reference 1 with an amplification multiple (ratio of the copy number of HER2 gene to the reference gene) of 1, reference 2 with 1.5, and reference 3 with 2, respectively.

[0101] Example 2

[0102] The accuracy of the quality control and reference materials for HER2 gene amplification detection prepared in Example 1 was tested.

[0103] The mature Covis HER2 gene amplification detection kit (digital PCR method) was used for verification, and the quality control products and reference products prepared above were tested according to the instructions of the kit.

[0104] The test results are as follows:

[0105]

[0106] Reference to the results of digital PCR testing of negative quality control products Figure 2 As shown, the result of digital PCR detection of positive quality control products is shown in Figure 3 The test results show that the detection results of the quality control product and the reference product prepared by the present invention using the mature HER2 amplification detection kit are consistent with the expected theoretical results.

[0107] Example 3

[0108] A method for detecting HER2 gene amplification.

[0109] In combination with the digital PCR detection system constructed in Examples 1 and 2, as well as the prepared quality control products and reference products, a HER2 gene detection method was tested. This detection method was used to detect three clinical samples (S1, S2 and S3) with known HER2 amplification results and three reference products (including reference product 1 (R1), reference product 2 (R2) and reference product 3 (R3)) to confirm the accuracy; the reference products were tested 10 times to confirm the repeatability; at the same time, negative quality control products (NC) and positive quality control products (PC) were tested for positive and negative comparison. The test results are shown in the following table:

[0110]

[0111]

[0112] From the above test results, it can be seen that, in combination with the reference and quality control products prepared by the method of the present invention, it can be verified that this HER2 gene amplification detection method has good accuracy and repeatability.

[0113] In summary, the preparation method of the HER2 gene amplification detection quality control product and reference product prepared by the present invention is simple and has high repeatability. The prepared quality control product and reference product have high accuracy and can be used to evaluate the accuracy, repeatability and other performance of the HER2 gene detection method.

[0114] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a quality control product for HER2 gene amplification detection, characterized in that: It includes the following steps: Preparation of negative quality control product: digesting the first plasmid including the HER2 gene fragment and the reference gene fragment, and quantifying the copy number of the digestion product to obtain a negative quality control product; wherein the copy number ratio of the HER2 gene fragment to the reference gene fragment on the first plasmid is 1:1, and both ends of the HER2 gene fragment and both ends of the reference gene fragment have restriction sites; Preparing a positive quality control product: digesting a second plasmid including the HER2 gene fragment and the reference gene fragment, and quantifying the copy number of the digestion product to obtain a positive quality control product; wherein the ratio of the copy number of the HER2 gene fragment on the second plasmid to the copy number of the reference gene fragment is at least 2:1, and on the second plasmid, both ends of each HER2 gene fragment and both ends of the reference gene fragment have restriction sites; The reference gene is a housekeeping gene located on a human chromosome.

2. The method for preparing a quality control product for HER2 gene amplification detection according to claim 1, characterized in that: The reference gene is: a housekeeping gene located on human chromosome 17; Preferably, the reference gene is selected from the long arm or short arm of chromosome 17; Preferably, the reference gene is selected from the long arm of chromosome 17; Preferably, the reference gene is selected from the group consisting of the EFTUD2 gene on chromosome 17, the G6PC3 gene located on the telomeric side, and the ALDOC gene near the centromere; Preferably, on the first plasmid or the second plasmid, the length of the reference gene fragment is 100 to 1000 bp; Preferably, the nucleotide sequence of the reference gene fragment is as shown in SEQ ID No.

2.

3. The method for preparing a quality control product for HER2 gene amplification detection according to claim 1, characterized in that: On the first plasmid or the second plasmid, the length of the HER2 gene fragment is 100 to 1000 bp; Preferably, the nucleotide sequence of the HER2 gene fragment is as shown in SEQ ID No.

1.

4. The method for preparing a quality control product for HER2 gene amplification detection according to claim 1, characterized in that: When preparing the negative quality control product and the positive quality control product, the restriction endonucleases used in the enzyme digestion step are the same or different; Preferably, the restriction endonuclease is a blunt-end restriction endonuclease or a sticky-end restriction endonuclease; Preferably, the blunt-end restriction endonuclease is at least one of EcoR V, HpyCH4V, HaeIII, AluI, CvikI-1, BstUI and RsaI.

5. The method for preparing a quality control product for HER2 gene amplification detection according to claim 1, characterized in that: The copy number quantification of the enzyme digestion product is performed by at least one of the following methods: (1) Use an ultraviolet spectrophotometer to measure the concentration and then convert the copy number; (2) Copy number quantification using digital PCR.

6. The method for preparing a quality control product for HER2 gene amplification detection according to claim 5, characterized in that: After the copy number is quantified, the method further includes: diluting the enzyme cleavage product with a diluent to obtain a quality control product of the target copy number; Preferably, the diluent comprises: Tris EDTA buffer and salmon sperm DNA, the pH of the Tris EDTA buffer is 7.0-8.5, and the final concentration of the salmon sperm DNA in the diluent is 10-100 ng / μL.

7. The method for preparing a quality control product for HER2 gene amplification detection according to claim 5, characterized in that: The digital PCR reaction system includes: a primer pair and a probe for detecting the HER2 gene, a primer pair and a probe for detecting the reference gene, and a PCR premix; Preferably, in the reaction system of the digital PCR, the final concentration of the primer pair for detecting the HER2 gene is 600-1000 nM, the final concentration of the primer pair for detecting the reference gene is 600-1000 nM; the final concentration of the probe for detecting the HER2 gene is 100-400 nM; the final concentration of the probe for detecting the reference gene is 100-400 nM; Preferably, the reaction procedure of the digital PCR includes: pre-denaturation: 94-98°C, 8-12 min; cyclic amplification: 94-98°C, 20-40 s; 50-60°C, 60-80 s; and 40-50 cycles.

8. A method for preparing a reference substance for HER2 gene amplification detection, characterized in that: It includes the following steps: Mixing the negative quality control product and the positive quality control product prepared by the method for preparing a quality control product for HER2 gene amplification detection according to any one of claims 1 to 7 according to a preset volume ratio; Preferably, the mixed solution mixed according to a preset volume ratio is diluted.

9. Use of a quality control product prepared by the method for preparing a quality control product for HER2 gene amplification detection according to any one of claims 1 to 7 or a reference product prepared by the method for preparing a reference product for HER2 gene amplification detection according to claim 8 in preparing a HER2 gene amplification detection kit or evaluating the performance of a HER2 gene detection method.

10. A kit, characterized in that: It includes the quality control product prepared by the method for preparing the quality control product for HER2 gene amplification detection according to any one of claims 1 to 7, and / or the reference product prepared by the method for preparing the reference product for HER2 gene amplification detection according to claim 8.

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