WO2018086263A1 - 一种实时荧光定量pcr检测方法及其标准品和检测试剂盒 - Google Patents

一种实时荧光定量pcr检测方法及其标准品和检测试剂盒 Download PDF

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WO2018086263A1
WO2018086263A1 PCT/CN2017/073336 CN2017073336W WO2018086263A1 WO 2018086263 A1 WO2018086263 A1 WO 2018086263A1 CN 2017073336 W CN2017073336 W CN 2017073336W WO 2018086263 A1 WO2018086263 A1 WO 2018086263A1
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gene
real
quantitative pcr
standard
her2
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French (fr)
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唐向荣
陈建鹤
吴思敏
唐志君
姜为为
杨水兵
赵氏璧
娄竞
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三生国健药业（上海）股份有限公司
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    • 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
    • C12Q1/686—Polymerase chain reaction [PCR]

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  • the invention belongs to the technical field of biomedicine, and particularly relates to a real-time fluorescent quantitative PCR detection method, a standard product thereof and a detection kit.
  • Copy Number Variation is an important component of gene structure variation, which is caused by rearrangement of genomes. Generally, the copy number of large genomes with a length of 1 kb or more is increased or decreased, mainly as submicroscopy. Levels are missing and repeated. CNV is closely related to tumorigenesis. Disklin et al found that CNV at 1q21.1 is closely related to neuroblastoma; and CNV of tumor-associated genes has a great influence on tumorigenesis and metastasis, such as CNV on GSTM1 gene. It will lead to an increased risk of bladder cancer; CNV on the tumor suppressor gene WWOX will lead to a decrease in the activity of tumor suppressor genes, making lung cancer more likely to occur.
  • Detection of relevant CNV is critical for targeted therapy in certain cancer patients, and physicians can take personalized treatments for patient-related gene copy number differences, for example by copying the HER2 gene (relative quantification of the HER2 gene)
  • the detection of whether or not to amplify is a key indication for the clinical treatment of metastatic breast cancer for the targeted drug Herceptin.
  • HER2 human epidermal growth factor receptor-2, HER2
  • c-erbB-2 the c-erbB-2 gene
  • HER2 human epidermal growth factor receptor-2
  • HER2 human epidermal growth factor receptor-2
  • c-erbB-2 the c-erbB-2 gene
  • HER2 is located on chromosome 17q12-21.32 and encodes a transmembrane receptor-like sample with a relative molecular mass of 185,000. Protein with tyrosine kinase activity.
  • HER2 is a single-copy gene, but in breast cancer and gastric cancer cells, chromosomal heterogeneity often occurs, such as an increase in the number of chromosome 17, and more commonly the HER2 gene on chromosome 17 and above.
  • Herceptin (trade name of trastuzumab) for HER2 overexpressing breast cancer, HER2 overexpressing metastatic breast cancer, and HER2 overexpressing metastatic gastric cancer.
  • Herceptin is a recombinant humanized monoclonal antibody that specifically acts on the extracellular domain of human epidermal growth factor receptor-2 (HER2) and inhibits HER2 overexpression. Proliferation of tumor cells expressed in degrees.
  • Herceptin has become the first-line drug for clinical HER2-positive breast cancer targeted therapy, with annual global sales of nearly 8 billion US dollars. Clinical trials have shown that patients with HER2 overexpression receive Herceptin treatment more significantly.
  • the Breast Cancer Treatment Guidelines and the Breast Cancer HER2 Detection Guide issued by the American Society of Clinical Oncology and the Chinese Medical Case Association have clearly stated that HER2 detection must be performed before targeted therapy for breast cancer to correctly detect and evaluate HER2 protein expression in breast cancer. And the state of gene amplification is crucial for the clinical treatment and prognosis of breast cancer, not only whether the patient is suitable for targeted therapy of HER2, but also plays a guiding role in the selection of endocrine therapy, chemotherapy regimen and prognosis.
  • IHC immunohistochemistry
  • ISH in situ hybridization
  • FISH fluorescence in situ hybridization
  • CISH CISH
  • SISH in situ hybridization
  • IHC uses the immunochromatographic reaction to detect HER2 overexpression of formalin-fixed paraffin sections at the protein level
  • ISH uses a specially labeled nucleic acid probe and a HER2 gene DNA target sequence immobilized on paraffin sections.
  • the above two methods are cumbersome to operate in clinical testing, which takes a long time and is expensive (the clinical charge is 2500-3000 yuan), which is difficult to standardize in general clinical laboratories.
  • Real-time fluorescent quantitative PCR has many advantages such as large linear detection range, fast detection speed, high throughput, no contamination detection by closed detection, high sensitivity, etc., and has many clinical applications in the quantitative determination of nucleic acid copy number.
  • the first is the ⁇ Ct method represented by the Roche HER2 gene amplification test kit (for research use only, no clinical license), and the detection principle is based on the difference in Ct value between the HER2 gene and the reference gene amplification reaction (via reference sample)
  • the ⁇ Ct correction is the difference in copy number compared with ⁇ Ct).
  • the difference in amplification efficiency between different PCR reactions is difficult to eliminate and the PCR device still has differences between pores. Therefore, this method is difficult to meet the clinical HER2 detection. Stability, precision requirements.
  • the selected reference gene, Gastrin also has amplification in some patients with gastric cancer, the kit has withdrawn from the market after Herceptin has increased the indication for gastric cancer.
  • the second is the relative standard curve method represented by Thermo Fisher, which uses the standard curve to obtain the true amplification efficiency value, which can be corrected to some extent.
  • the specific method is to use a series of different concentration gradients of the target gene and the reference gene standard (one of plasmid, genomic DNA or chemical sequence) to draw a standard curve, and then calculate the target gene and the reference gene in the sample relative to the respective standards. Copy number.
  • the standard product concentration error range is large, which makes the proportional relationship between the target gene and the reference gene difficult to control, which may cause errors in the experimental results.
  • the inventors of the present invention have made it difficult to control the concentration ratio of the target gene and the reference gene due to the high absolute difficulty of the standard product itself and the large error range of the standard product in the relative quantitative detection method of the existing gene copy number.
  • the error of the experimental results and the complicated detection method, high time and cost, and the like, provide a simple and efficient real-time quantitative PCR detection method and its standard and detection kit.
  • a real-time quantitative PCR detection method for relatively quantitative gene copy number comprises the following steps:
  • the reference gene is ligated in proportion to the gene fragment to be tested, cloned into the same plasmid vector, and a standard product which can be used for both the reference gene and the amplification of the gene to be tested is constructed, and the obtained standard is obtained. Diluted according to the concentration gradient and used to draw a reference curve and a standard curve of the gene to be tested;
  • the target gene and the reference gene are calculated according to the respective standard curves, and the target gene of the sample to be tested is calculated.
  • the reference gene copy number ratio that is, the copy number of the target gene is relatively quantitatively detected.
  • step (1) is: the reference gene is equated with the gene fragment to be tested, and cloned into the same plasmid vector to construct a standard product which can be simultaneously used for the reference gene and the amplification test of the gene to be tested, and The obtained standard is diluted according to the concentration gradient and used to draw a reference curve and a standard curve of the gene to be tested.
  • the reference gene is a conventional reference gene in the art, preferably a housekeeping gene, preferably an amplified region of RPPH1, wherein the plasmid vector is a conventional plasmid vector in the art, preferably a PCR cloning vector, preferably TA cloning vector, preferably the TA cloning vector is a pMD18-T vector.
  • the nucleic acid sequence of the standard is shown in SEQ ID NO: 6 in the Sequence Listing. The equal ratio is preferably 1:1.
  • the ratio of the target gene to the reference gene in the standard product is 1:1, the problem that the concentration ratio of the target gene and the reference gene in the current relative standard curve method is difficult to control is solved, and the reliability of the HER2 gene amplification detection is improved. .
  • Step (2) is: the sample to be tested and the standard product of step (1) are synchronized for real-time fluorescence quantification After PCR amplification, the target gene and the reference gene are calculated according to their respective standard curves, and the ratio of the target gene to the reference gene copy number of the sample to be tested is calculated, that is, the copy number of the target gene is relatively quantitatively detected.
  • the gene to be tested is a gene to be tested in the field, preferably a tumor or a disease-specific gene, more preferably an amplified region of the HER2 gene, and the fluorescent quantitative PCR amplification is conventional in the art.
  • the fluorescence quantitative PCR amplification method is preferably a multi-channel fluorescent quantitative PCR amplification, more preferably a two-channel fluorescent quantitative PCR amplification.
  • the standard curve is a standard curve obtained by a conventional fluorescent quantitative PCR amplification method in the art, and the drawing method of the standard curve is a conventional drawing method in the art.
  • the term "copy number" as used in the present invention is used in the conventional sense of the art to refer to the number of genes in the genome of a certain organism.
  • the second technical solution adopted by the present invention is: a standard for relative quantitative real-time quantitative PCR detection of gene copy number, the standard is an equal proportion containing reference gene fragments and to be tested A plasmid vector of a gene fragment of interest.
  • the reference gene described therein is a conventional reference gene in the art, preferably a housekeeping gene, more preferably an RPPH1 gene.
  • the gene to be tested is a gene to be tested in the field, preferably a tumor or a disease-specific gene, more preferably a HER2 gene.
  • the plasmid vector described therein is a plasmid vector commonly used in the art, preferably a PCR cloning vector, more preferably a TA cloning vector, and the TA cloning vector is preferably a pMD18-T vector.
  • the nucleic acid sequence of the standard is preferably as shown in SEQ ID NO: 6 in the Sequence Listing.
  • the third technical solution adopted by the present invention is: a real-time fluorescent quantitative PCR detection kit
  • the real-time quantitative PCR detection kit includes: Taq enzyme, PCR reaction buffer, dNTP mixture, target gene And specific upstream and downstream primers and fluorescent probes, standards, negative controls, and positive controls corresponding to each of the reference genes.
  • the target gene is preferably a HER2 gene
  • the corresponding specific upstream primer sequence is preferably as shown in SEQ ID NO: 7
  • the corresponding specific downstream primer is preferably as shown in SEQ ID NO: 8.
  • the corresponding probe is a conventional fluorescent probe in the art, preferably a Taqman fluorescent probe.
  • the probe has a fluorescent luminescent group attached to the 5' end and a fluorescence quenching group attached to the 3' end, and the emitting group is preferably a fluorescent emitting group conventional in the art, preferably
  • the site is HEX
  • the fluorescent quenching group is preferably TARMA
  • the sequence of the fluorescent probe is preferably as shown in SEQ ID NO: 11 of the Sequence Listing.
  • the reference gene is preferably the RPPH1 gene, and the corresponding specific upstream primer sequence is preferably as shown in SEQ ID NO: 9, and the corresponding specific downstream primer is preferably as shown in SEQ ID NO: 10.
  • the corresponding probe is a conventional fluorescent probe in the art, preferably a Taqman fluorescent probe.
  • the probe has a fluorescent luminescent group attached to the 5' end and a fluorescence quenching group attached at the 3' end, preferably a fluorescent emitting group, preferably a FAM, in the art.
  • the fluorescent quenching group is preferably TARMA, and the sequence of the fluorescent probe is preferably as shown in SEQ ID NO: 12 of the Sequence Listing.
  • the standard is a conventional standard in the art, preferably a pMD18-T vector comprising the reference gene RPPH1 gene and the HER2 gene of the target gene, and the sequence thereof is shown in SEQ ID NO: 6 in the Sequence Listing.
  • the Taq enzyme is a conventional Taq enzyme in the art, preferably a hot-start Taq enzyme
  • the PCR reaction buffer is a conventional PCR reaction buffer in the art
  • the formulation of the PCR reaction buffer is a conventional formulation in the field, preferably.
  • the 10X PCR buffer supplied with the hot start Taq enzyme is purchased, wherein the dNTP mixture is a conventional dNTP mixture in the art.
  • the negative control and the positive control are conventional controls in the art, wherein the negative control is preferably a single plasmid having a ratio of HER2 to RPPH1 gene fragment of 1:1, wherein the concentration of the HER2 and RPPH1 gene fragments is preferably 5E6copies/ ⁇ L.
  • the positive control preferably consists of a plasmid containing different proportions of the target gene and the control gene, wherein the ratio of the HER2 to RPPH1 amplified fragment is preferably 4:1, wherein the RPPH1 gene fragment concentration is preferably 5E6copies/ ⁇ L, and the HER2 gene The fragment concentration is preferably 2E7copies/ ⁇ L.
  • the final concentration of each component of the PCR amplification system is: the concentration of the primer corresponding to the target gene and the reference gene is preferably 0.14-0.18 ⁇ M, more preferably 0.16 ⁇ M; and the probe concentration is preferably 0.06-1.0 ⁇ M. More preferably, it is 0.08 ⁇ M; the amount of Taq enzyme is preferably 0.02-0.06 U, more preferably 0.04 U, and the Taq enzyme is more preferably a hot-start Taq enzyme.
  • the present invention also provides the use of the real-time fluorescent quantitative PCR detection kit as described above for detecting the state of amplification of the HER2 gene of the sample to be tested.
  • the sample to be tested is a conventional sample to be tested in the art, and preferably includes samples such as fresh tissue, paraffin-embedded tissue, and peripheral blood-recovered cells.
  • the reagents and starting materials used in the present invention are commercially available.
  • the positive progress of the present invention is that the standard provided by the present invention comprises the target gene HER2.
  • the plasmid and the nucleic acid sequence of the reference gene RPPH1 gene were ligated in a 1:1 ratio.
  • the standard can solve the problem that the concentration ratio of the target gene and the reference gene in the current relative standard curve method is difficult to control, improve the reliability of the HER2 gene amplification detection, and can correctly detect and evaluate the HER2 gene expansion in breast cancer tissues or cells. Increase the status.
  • the real-time fluorescent quantitative PCR detection method provided by the invention adopts two-channel fluorescent PCR to synchronously amplify the HER2 gene and the reference gene, thereby significantly increasing the detection flux and greatly reducing the detection cost.
  • the detection kit provided by the invention has the advantages of simple operation, good stability, high repeatability and high detection efficiency.
  • the invention has important significance for the diagnosis of HER2-positive breast cancer, research and development, quality control and even clinical application of the drug against the disease.
  • Fig. 1 is a plasmid map of the standard obtained by the construction of Example 3 of the present invention, and the arrow indicates the position of the inserted PCR product sequence in the vector.
  • FIG. 2 is the results of real-time PCR amplification.
  • 2(A) is an amplification curve of single-channel amplification of HER2 gene using the plasmid standard obtained in Example 3 of the present invention as a template
  • FIG. 2(B) is a plasmid standard obtained in Example 3 of the present invention.
  • the template was used to amplify the amplification curve of RPPH1 gene amplification in a single channel
  • FIG. 2(C) is an amplification curve of the two-channel simultaneous amplification of HER2 and RPPH1 of the plasmid standard obtained in Example 3 of the present invention.
  • Figure 3 is a standard curve of HER2 and RPPH1 plotted according to Figure 2(C).
  • Figure 4 is a graph showing the amplification of HER2 and RPPH1 genes in the sample of Example 5.
  • 4(A) is a gene amplification curve of paraffin-embedded tissue numbered 1520438
  • FIG. 4(B) is a gene amplification curve of paraffin-embedded tissue numbered 1601415.
  • Figure 5 is a schematic diagram of the real-time quantitative PCR detection method.
  • the genomic DNA used for the construction of the plasmid standard of the present invention was derived from the K562 cell line cultured in vitro (purchased from ATCC).
  • the samples tested in the present invention are paraffin-embedded human breast cancer tissues (from Changhai Hospital affiliated to the Second Military Medical University).
  • sample DNA extraction step is described using Qiagen's GeneRead DNA FFPE KIT as an example:
  • ribonuclease A 100 mg/ml was added and mixed at room temperature for 2 minutes. Then, 250 ⁇ L of AL buffer was added, and 250 ⁇ L of ethanol was added after high-speed vortexing, and then vortexed again at high speed and centrifuged.
  • the TA cloning vector pMD18-T was purchased from Promega.
  • the gene fragment of the target gene and the reference gene was prepared by a PCR method, and the PCR template was the cell genomic DNA extracted in Example 1, and the reaction system is shown in Table 1 (PCR reaction system 1).
  • the HER2 gene fragment can be prepared by adding the HER2 lateral forward and reverse primer sequences to the PCR amplification system.
  • the RPPH1 gene can be prepared by adding the RPPH1 lateral forward and reverse primer sequences to the amplification system. The sequence is shown in Table 2.
  • the PCR amplification conditions are shown in Table 3.
  • the HER2 gene fragment and the RPPH1 gene fragment were ligated by PCR to prepare an insert, and PCR amplification primers and ligation primer sequences were as shown in Table 2.
  • the PCR sequence of the target sequence was shown in Table 4 (PCR reaction system 2), and the PCR amplification conditions are shown in Table 5.
  • the target sequence is linked to the PCR reaction system (50 ⁇ L, PCR reaction system 2)
  • the target fragment was recovered using an agarose gel recovery kit.
  • the insert was ligated into the pMD18-T vector (purchased from Promega) by TA cloning, see TA cloning method. Carrier system product specification.
  • the newly constructed plasmid was amplified in a large amount in Escherichia coli JM109 strain. After screening for blue and white spots, the monoclonal was picked and sequenced, and the sequence was correctly used as a standard.
  • the nucleotide sequence of the resulting standard is shown in SEQ ID NO: 6 in the Sequence Listing.
  • Figure 1 is a plasmid map of the standard constructed in accordance with the present invention, and the arrow indicates the position of the vector inserted into the PCR product sequence of the vector.
  • the single channel fluorescent quantitative PCR reaction template was the standard prepared in Example 3, and double distilled water was used as a blank control.
  • the single channel fluorescent quantitative PCR reaction system is shown in Table 6 (PCR reaction system 3), and the PCR reaction conditions are shown in Table 8.
  • HER2 gene fluorescence quantitative PCR amplification system needs to add HER2 inner positive and negative primers and HER2 probe; RPPH1 fluorescence quantitative PCR amplification system needs to add RPPH1 inner positive and negative primers and RPPH1 probe, the primers and probes
  • Table 7 The sequence of the needle is shown in Table 7.
  • the fluorescent emitting group at the 5' end of the fluorescent probe labeled HER2 gene is HEX
  • the fluorescent quenching group at the 3' end is TARMA
  • the fluorescent emitting group at the 5' end of the fluorescent probe labeled with RPPH1 gene is FAM
  • 3 The fluorescent quenching group at the 'end' is TARMA.
  • FIG. 2 The results of real-time PCR amplification are shown in Figure 2, in which: in Figure 2 (A), the eight curves are increased by single-channel fluorescence quantification of HER2 gene using different concentrations (4-fold dilution) of plasmid standards as templates.
  • Amplification profile of E4copies/ ⁇ L plasmid standards was 3.2E8copies/ ⁇ L, 8E7copies/ ⁇ L, 2E7copies/ ⁇ L, 5E6, 1.25E6copies/ ⁇ L, 3.12E5copies/ ⁇ L, 7.81E4copies/ ⁇ L, 1.95.
  • the eight curves that are raised are the amplification curves of single-channel real-time PCR amplification of RPPH1 gene using plasmid standards of different concentrations (4-fold dilution) as templates, from left to right.
  • the standard concentration was 3.2E8copies/ ⁇ L, 8E7copies/ ⁇ L, 2E7copies/ ⁇ L, 5E6, 1.25E6copies/ ⁇ L, 3.12E5copies/ ⁇ L, 7.81E4copies/ ⁇ L, 1.95E4copies/ ⁇ L.
  • Fluorescence quantitative PCR reaction template was the standard prepared in Example 3, using double distilled water as a blank control.
  • the primers and probe sequences required for the two-channel real-time PCR reaction are shown in Table 7.
  • the reaction conditions of the real-time PCR system are shown in Table 8.
  • the two-channel fluorescence quantitative PCR reaction system is shown in Table 9 (PCR reaction system). 4).
  • FIG. 2(C) is a graph showing the amplification curve obtained by simultaneous amplification of HER2 and RPPH1 by two-channel real-time quantitative PCR using the specific standard obtained in Example 3 of the present invention as a template.
  • the concentration of the plasmid standard was 3.2E8copies/ ⁇ L, 8E7copies/ ⁇ L, 2E7copies/ ⁇ L, 5E6, 1.25E6copies/ ⁇ L, 3.12E5copies/ ⁇ L, 7.81E4copies/ ⁇ L, 1.95E4copies/ ⁇ L.
  • the solid line represents the amplification curve of the HER2 gene
  • the dotted line represents the amplification curve of the RPPH1 gene, with a 4-fold gradient of the concentration of the plasmid standard from left to right.
  • the fluorescent quantitative PCR reaction template is the genomic DNA of the breast cancer sample extracted in Example 2,
  • the double-distilled water was used as the blank control, and the negative control was the single plasmid with the ratio of HER2 and RPPH1 gene fragments prepared in Example 3, and the concentration of HER2 and RPPH1 amplified fragments was 5E6copies/ ⁇ L; the positive control was composed of two different plasmids.
  • the ratio of HER2 to RPPH1 amplified fragment was 4:1, wherein the concentration of RPPH1 amplified fragment was 5E6copies/ ⁇ L, and the concentration of HER2 amplified fragment was 2E7copies/ ⁇ L.
  • the primers and fluorescent probe sequences used in the fluorescent quantitative PCR reaction, the amplification reaction conditions and the reaction system are shown in Table 7, Table 8, and Table 9. Simultaneous detection of HER2 and RPPH1 genes using a two-channel real-time PCR method.
  • the HER2-F-2 and HER2-R-2 primers and their fluorescent probes need to be added to the HER2 amplification system.
  • RPPH1-F-2 and RPPH1-R-2 primers and their fluorescent probes need to be added to the RPPH1 amplification system.
  • the amplification curve of HER2 and RPPH1 genes can be further drawn by using a plasmid standard containing an equal proportion of the target gene and the reference gene gene fragment as a template and performing two-channel real-time PCR detection.
  • the standard curve for the corresponding gene is shown in Figure 3 (C), wherein the black square curve represents the standard curve of the HER2 gene, and the white square curve represents the standard curve of the RPPH1 gene.
  • the copy number of HER2 and RPPH1 genes can be separately determined, and the relative value of the copy number of HER2 gene can be calculated by calculating the ratio of the copy number of HER2 and RPPH1 gene.
  • Figure 5 shows the real-time quantitative PCR detection method. schematic diagram.
  • Figure 4 is a graph showing the amplification curves of HER2 and RPPH1 genes in two samples in this example.
  • 4(A) is a gene amplification curve of paraffin-embedded tissue numbered 1520438, and its CT value is 22.57;
  • FIG. 4(B) is a gene amplification curve of paraffin-embedded tissue numbered 1601415, and its Ct value Is 27.42.
  • the CT values of the reference gene RPPH1 were 25.33 and 27.75, respectively.
  • the corresponding copy number was obtained from the corresponding standard curve formula (Fig. 3), and the HER2/RPPH1 was 7.86 (HER2 amplification) and 1.72 (HER2 amplification, respectively). ).
  • the Ct value of the target gene and the reference gene in the unknown sample and the control sample can be obtained by the real-time fluorescent quantitative PCR detection method, and then the amplification factor of HER2 can be calculated according to the formulas 1, 2, and 3.
  • the formulas 1, 2, and 3 are as follows:
  • a single plasmid containing a ratio of HER2 to RPPH1 gene fragment of 1:1 is used as a control sample, and the sample is quantitatively detected by real-time quantitative PCR, and an average ⁇ Ct control sample value is obtained according to the average Ct value of HER2 and RPPH1;
  • Equation 3 Second, using Equation 2 and the existing ⁇ Ct unknown sample value and the ⁇ Ct control sample value, after calculating ⁇ Ct, the fold change relationship can be obtained according to Equation 3.
  • the relative quantitative detection of HER2 gene was performed on 50 paraffin-embedded tissue samples of breast cancer, and compared with the results of fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC).
  • FISH fluorescence in situ hybridization
  • IHC immunohistochemistry
  • the sample processing method of the present invention refers to Qiagen's GeneRead DNA FFPE KIT instructions, primer sequences, amplification systems and amplification conditions are described in Tables 7, 8, and 9, and amplified on the ABI7500 real-time PCR instrument.
  • Two HER2 gene relative quantitative assays were used to evaluate the amplification results.
  • IHC detection antibody was purchased from Abcam
  • FISH detection reagent was purchased from Abbott Vysis, and the experimental operation was carried out according to the instructions.
  • the results of two HER2 gene amplification detection methods and fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) provided by the present invention are shown in Table 10.
  • IHC uses the formalin-fixed paraffin section immunochromatographic reaction to detect HER2 protein overexpression at the protein level, while FISH is a fluorescently labeled nucleic acid probe and DNA of the HER2 gene in the nucleus immobilized on paraffin sections. The target sequence is hybridized to detect its gene amplification on the chromosome. Further confirmation of FISH is needed when the IHC test result is 2+.
  • the whole detection process of the method can be completed within 3-5 hours, and fully automated operation can be realized;
  • the method adopts dual-channel detection, and the operation is simple and convenient, and 40-50 samples can be simultaneously detected at one time, and FISH is completely manual operation, and it is difficult to exceed 10 samples in one test; finally, the method is simple in operation, and thus has good stability and repetition. Sex is far superior to IHC and FISH testing. Therefore, this method can be a new standard for clinical HER2 gene detection and has great economic value.

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Abstract

本发明公开了一种实时荧光定量PCR检测方法及其标准品和检测试剂盒。利用特殊标准品制作标准曲线对待测样本HER2基因及参照基因进行实时荧光定量PCR检测，用于指导乳腺癌、胃癌的临床靶向治疗；提供了一种包含等比例参照基因与待测目的基因片段的质粒载体作为检测标准品；还提供了一种检测HER2基因扩增状态的实时荧光定量PCR试剂盒，包括HER2基因扩增状态检测的PCR反应体系、标准品及阴阳性对照品。

Description

一种实时荧光定量PCR检测方法及其标准品和检测试剂盒 技术领域
本发明属于生物医药技术领域，具体涉及一种实时荧光定量PCR检测方法及其标准品和检测试剂盒。
背景技术
拷贝数变异(Copy Number Variation，CNV)是基因结构变异的重要组成部分，由基因组发生重排而导致，一般指长度为1kb以上的基因组大片段的拷贝数增加或者减少，主要表现为亚显微水平的缺失和重复。CNV与肿瘤发生联系密切，Disklin等发现1q21.1位点的CNV与成神经细胞瘤关系密切；而且肿瘤相关基因的CNV会对肿瘤的发生及转移产生很大的影响，例如GSTM1基因上的CNV会导致膀胱癌的发病风险增高；抑癌基因WWOX上的CNV会导致抑癌基因的活性下降，使肺癌更易发生。对相关CNV的检测对于某些肿瘤患者的靶向治疗是至关重要的，医生可以针对患者的相关基因拷贝数差异采取个性化的治疗手段，例如通过对HER2基因拷贝数(HER2基因相对定量)的检测判断其是否扩增是临床治疗转移性乳腺癌是否适用靶向药物赫赛汀的关键指征。
乳腺癌是危害女性健康最主要的恶性肿瘤，在原发性乳腺癌患者中观察到有25％-30％的患者HER2过度表达。HER2(人类表皮生长因子受体2，human epidermal growth factor receptor-2，HER2)基因，即c-erbB-2基因，定位于染色体17q12-21.32上，编码相对分子质量为185000的跨膜受体样蛋白，具有酪氨酸激酶活性。正常人体情况下，HER2是单拷贝基因，但是在乳腺癌及胃癌肿瘤细胞中，常有染色体异质性现象发生，如17号染色体数量增加，更常见的是17号染色体上HER2基因及其上下游300-1000M的DNA序列经过基因重排插入到其他染色体上，由此导致HER2基因的拷贝数增加，进而导致肿瘤细胞表面HER2蛋白过表达。自1997年起，美国食品药品监督局先后批准了赫赛汀(曲妥珠单抗商品名)用于HER2过表达的乳腺癌、HER2过度表达的转移性乳腺癌、HER2过表达的转移性胃癌的治疗。赫赛汀是一种重组人源化单克隆抗体，能够特异性地作用于人表皮生长因子受体-2(HER2)的细胞外部位，可以抑制HER2过 度表达的肿瘤细胞的增殖。目前赫赛汀已成为临床HER2阳性乳腺癌靶向治疗的一线药物，全球年销售额近80亿美元。临床实验证明HER2过度表达患者接受赫赛汀治疗效果更为显著。美国临床肿瘤学会及中华医学病例学会发布的《乳腺癌治疗指南》及《乳腺癌HER2检测指南》均明确提出在乳腺癌靶向治疗前必须进行HER2检测，正确检测和评定乳腺癌的HER2蛋白表达和基因扩增状态对乳腺癌的临床治疗和预后判断至关重要，不仅涉及患者是否适合针对HER2的靶向治疗，并且对内分泌治疗、化疗方案的选择及预后评估起指导作用。
目前，FDA认可的HER2检测方法分为免疫组化(IHC)及原位杂交(ISH包括FISH、CISH、SISH)两类。IHC是利用免疫显色反应在蛋白水平上对福尔马林固定的石蜡切片进行HER2过表达检测，而ISH则是利用特殊标记的核酸探针与石蜡切片上固定的细胞核内HER2基因DNA靶序列杂交，在核酸水平上，检测HER2基因的扩增情况，当IHC检测结果为2+时还需要进一步进行FISH检测确诊。以上两类方法在临床检测中操作繁琐，耗时较长，价格昂贵(临床收费2500-3000元)，在一般的临检实验室中很难进行标准化。
实时荧光定量PCR具有线性检测范围大、检测速度快、通量高、封闭检测无污染、灵敏度高等诸多优点，已经在核酸拷贝数定量方面有了诸多临床应用。目前将荧光定量PCR应用于基因拷贝数变异的检测主要有两种相对定量方法。其一是以罗氏HER2基因扩增检测试剂盒(仅限科研使用，未申请临床许可)为代表的ΔΔCt法，其检测原理是根据HER2基因与参照基因扩增反应的Ct值差异(经参照样本的ΔCt校正后即为ΔΔCt)比较的拷贝数差异，而实际上不同PCR反应的体系之间扩增效率的差异很难消除且PCR设备还存在孔间差异，故而此方法难以满足临床HER2检测对稳定性、精密度的要求。另外由于其选用的参照基因Gastrin在部分胃癌患者中也存在着扩增现象，因此在赫赛汀增加了胃癌适应症后，该试剂盒退出了市场。其二是以赛默飞世尔为代表的相对标准曲线法，利用标准曲线得到真实的扩增效率值，可在一定程度上修正结果。具体方法是利用一系列不同浓度梯度的目的基因及参照基因标准品(质粒、基因组DNA或化学成序列中的一种)绘制标准曲线后，分别计算样本中目的基因、参照基因相对于各自标准品的拷贝数。但由于标准品本身的绝对定量难度高，标准品浓度误差范围较大，使得目的基因与参照基因的浓度比例关系难以控制，会造成实验结果出现误差。
发明内容
本发明的发明人针对现有的基因拷贝数相对定量检测方法中由于标准品本身绝对定量难度高、标准品浓度误差范围较大，导致目的基因与参照基因的浓度比例关系难以控制，从而造成的实验结果误差以及检测方法复杂，耗费时间成本较高等问题，提供了一种简便、高效的实时荧光定量PCR检测方法及其标准品和检测试剂盒。
为了解决上述技术问题，本发明采取的技术方案之一为：一种基因拷贝数相对定量的实时荧光定量PCR检测方法，该实时荧光定量PCR检测方法包括以下步骤：
(1)将参照基因与待测目的基因片段等比例连接，克隆到同一个质粒载体上，构建一种可同时用于参照基因以及待测目的基因扩增检测的标准品，并将所得标准品按照浓度梯度稀释后同时用于绘制参照基因以及待测目的基因的标准曲线；
(2)待测样本与步骤(1)所述标准品经同步进行实时荧光定量PCR扩增后，目的基因与参照基因按照各自的标准曲线计算各自的拷贝数，计算待测样本的目的基因与参照基因拷贝数比值，即得目的基因的拷贝数相对定量检测结果。
其中步骤(1)为：将参照基因与待测目的基因片段等比例连接，克隆到同一个质粒载体上，构建一种可同时用于参照基因以及待测目的基因扩增检测的标准品，并将所得标准品按照浓度梯度稀释后同时用于绘制参照基因以及待测目的基因的标准曲线。
其中所述参照基因为本领域常规参照基因，较佳地管家基因，优选地为RPPH1的扩增区域，其中所述质粒载体为本领域常规质粒载体，较佳地为PCR克隆载体，优选地为TA cloning载体，所述TA cloning载体较佳地为pMD18-T载体。其中所述标准品的核酸序列如序列表中SEQ ID NO:6所示。所述的等比例较佳地为1：1。由于标准品中待测目的基因与参照基因的比例为1:1，解决了目前相对标准曲线法应用中目的基因与参照基因的浓度比例关系难以控制的问题，提高HER2基因扩增检测的可靠性。
步骤(2)为：待测样本与步骤(1)所述标准品经同步进行实时荧光定量 PCR扩增后，目的基因与参照基因按照各自的标准曲线计算各自的拷贝数，计算待测样本的目的基因与参照基因拷贝数比值，即得目的基因的拷贝数相对定量检测结果。
其中所述待测目的基因为本领域常规待测目的基因，较佳地为肿瘤或者疾病的特异性基因，更佳地为HER2基因的扩增区域，所述荧光定量PCR扩增为本领域常规荧光定量PCR扩增方法，所述荧光定量PCR扩增方法较佳地为多通道荧光定量PCR扩增，更佳地为双通道荧光定量PCR扩增。
其中所述标准曲线为本领域常规荧光定量PCR扩增方法所得标准曲线，所述标准曲线的绘制方法为本领域常规绘制方法。本发明所述术语“拷贝数”为本领域常规含义，即指某基因在某一生物的基因组中的个数。
为了解决上述技术问题，本发明采取的技术方案之二为：一种用于基因拷贝数相对定量实时荧光定量PCR检测的标准品，所述标准品是一种等比例包含参照基因片段与待测目的基因片段的质粒载体。
其中所述的参照基因为本领域常规的参照基因，较佳地为管家基因，更佳地为RPPH1基因。其中所述待测目的基因为本领域常规待测目的基因，较佳地为肿瘤或者疾病的特异性基因，更佳地为HER2基因。其中所述的质粒载体为本领域常用质粒载体，较佳地为PCR克隆载体，更佳地为TA cloning载体，所述TA cloning载体优选地为pMD18-T载体。其中所述标准品的核酸序列优选地如序列表中SEQ ID NO:6所示。
为了解决上述技术问题，本发明采取的技术方案之三为：一种实时荧光定量PCR检测试剂盒，该实时荧光定量PCR检测试剂盒包括：Taq酶、PCR反应缓冲液、dNTP混合液、目的基因以及参照基因各自对应的特异性上、下游引物和荧光探针、标准品、阴性对照和阳性对照。
其中所述目的基因较佳地为HER2基因，其对应的特异性上游引物序列较佳地如SEQ ID NO:7所示，其对应的特异性下游引物较佳地如SEQ ID NO:8所示，其对应的探针为本领域常规荧光探针，较佳地为Taqman荧光探针。所述探针较佳地为：在5’端连接有荧光发光基团，在3’端连接有荧光淬灭基团，所述发射基团较佳地为本领域常规荧光发射基团，优选地为HEX，所述荧光淬灭基团较佳地为TARMA，所述荧光探针的序列优选地如序列表中SEQ ID NO:11所示。
其中所述参照基因较佳地为RPPH1基因，其对应的特异性上游引物序列较佳地如SEQ ID NO:9所示，其对应的特异性下游引物较佳地如SEQ ID NO:10所示，其对应的探针为本领域常规荧光探针，较佳地为Taqman荧光探针。所述探针优选在5’端连接有荧光发光基团，在3’端连接有荧光淬灭基团，所述发射基团较佳地为本领域常规荧光发射基团，优选地为FAM，所述荧光淬灭基团较佳地为TARMA，所述荧光探针的序列优选地如序列表中SEQ ID NO:12所示。
其中所述标准品为本领域常规标准品，较佳地为等比例包含参照基因RPPH1基因以及待测目的基因HER2基因的pMD18-T载体，其序列如序列表中SEQ ID NO:6所示。
其中所述Taq酶为本领域常规Taq酶，较佳地为热启动Taq酶；PCR反应缓冲液为本领域常规PCR反应缓冲液，所述PCR反应缓冲液的配方为本领域常规配方，较佳地市售可得，例如购买热启动Taq酶时配套提供的10X PCR缓冲液，其中所述dNTP混合液为本领域常规dNTP混合液。
其中所述阴性对照和阳性对照为本领域常规对照品，其中阴性对照较佳地为HER2与RPPH1基因片段比例为1：1的单一质粒，其中HER2与RPPH1基因片段浓度较佳地为5E6copies/μL。阳性对照较佳地由包含不同比例的目标基因和对照基因的质粒组成，其中HER2与RPPH1扩增片段比例较佳地为4：1，其中RPPH1基因片段浓度较佳地为5E6copies/μL，HER2基因片段浓度较佳地为2E7copies/μL。
所述PCR扩增体系各成分终浓度为：目的基因、参照基因所对应的引物浓度较佳地为0.14-0.18μM，更佳地为0.16μM；探针浓度较佳地为0.06-1.0μM，更佳地为0.08μM；Taq酶用量较佳地为0.02-0.06U，更佳地为0.04U，所述Taq酶更佳地为热启动Taq酶。
本发明还提供了如上所述的实时荧光定量PCR检测试剂盒在检测待测样品HER2基因扩增状态时的用途。待测样品为本领域常规待测样品，较佳地包括：新鲜组织、石蜡包埋组织以及外周血回收细胞等样品。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：本发明提供的标准品是由包含目的基因HER2 基因及参照基因RPPH1基因的核酸序列按照1:1比例连接而成的质粒。该标准品能够解决目前相对标准曲线法应用中目的基因与参照基因的浓度比例关系难以控制的问题，提高HER2基因扩增检测的可靠性，能够正确检测和评定乳腺癌组织或细胞中HER2基因扩增状态。
本发明提供的实时荧光定量PCR检测方法采用双通道荧光PCR进行HER2基因与参照基因的同步扩增，能够显著提高检测通量，大幅降低检测成本。同时，本发明所提供的检测试剂盒具有操作简便、稳定性好、重复性高、检测效率高等优势。本发明对于HER2阳性的乳腺癌的诊断，抗该疾病药物的研究开发、质量控制乃至临床应用都具有重要的意义。
附图说明
图1是本发明实施例3构建所得标准品的质粒图谱，箭头所指即为载体中插入基因PCR产物序列的位置。
图2是实时荧光定量PCR扩增结果。其中，图2(A)是以本发明实施例3所得质粒标准品为模板，单通道进行HER2基因扩增的扩增曲线，图2(B)是以本发明实施例3所得质粒标准品为模板，单通道进行RPPH1基因扩增的扩增曲线，图2(C)是本发明实施例3所得质粒标准品双通道同步扩增HER2与RPPH1的扩增曲线图。
图3是根据图2(C)绘制的HER2及RPPH1的标准曲线。
图4是实施例5所述样本的HER2及RPPH1基因扩增曲线。其中图4(A)是编号为1520438的石蜡包埋组织的基因扩增曲线，图4(B)是编号为1601415的石蜡包埋组织的基因扩增曲线。
图5是所述实时荧光定量PCR检测方法示意图。
具体实施方式
以下的实施例用于为本领域中的普通技术人员提供有关如何实施和使用本发明的完整披露和描述，并且这些例子并非意在对发明人所认为的发明范围进行限制，亦非意指下文的实验是被实施的全部实验而且是仅可实施的实验。实验例中未注明具体条件的实验方法，通常按照常规条件或按照厂商所建议的条件。
实施例1人类细胞系基因组DNA抽提
本发明质粒标准品构建用的基因组DNA来自于体外培养的K562细胞系(购买自ATCC公司)。
以使用Qiagen公司的Allprep DNA mini KIT为例介绍细胞系DNA提取步骤：
(1)对细胞数进行计数，若细胞数＜5×106，加入350μL Buffer RLT Plus；若细胞数≤1×107，加入600μL Buffer RLT Plus(若体积过大，分次抽提)。
(2)混匀后转移到Allprep DNA吸附柱中(置于2mL收集管内)，最高速离心30秒。
(3)换一新2mL收集管，加入500μL Buffer AW1，最高速离心15秒后倒掉收集管中液体。
(4)加入500μL Buffer AW2，最高速离心2分钟。
(5)换一新收集管(1.5mL带盖)，加入100μL Buffer EB，室温放置1分钟。最高速离心1分钟洗脱得到DNA。
实施例2人类石蜡包埋组织基因组DNA抽提
本发明测试的样本的为石蜡包埋的人类乳腺癌组织(来源于第二军医大学附属长海医院)。
以使用Qiagen公司的GeneRead DNA FFPE KIT为例介绍样本DNA提取步骤：
(1)刮取石蜡切片上的组织入1.5mL离心管中。
(2)加入160μL去石蜡溶液，涡旋10s后简单离心，56℃孵育3min。
(3)在室温下恢复平衡后加入新鲜配制的100μL温育缓冲液/蛋白酶K溶液(具体由55μL无RNA酶水，25μL FTB缓冲液，20μL蛋白酶K配制而成)。涡旋离心后，56℃孵育1小时后，直接转入90℃继续孵育1小时。
(4)简单离心后，取清澈底层液体，入新的离心管中，加入115μL无RNA酶水混合。
(5)加入35μL UNG酶涡旋后50℃孵育1小时。
(6)简单离心后加入2μL核糖核酸酶A(100mg/ml)混合，室温下孵育2分钟后，加入250μL AL buffer，高速涡旋震荡后加入250μL乙醇，再次高速涡旋震荡后离心。
(7)往QIAampMinElute柱中转入裂解液(若体积高于700μL，可分次转入)，注意不要触碰到管壁。最高速离心1分钟后，去除所有裂解液。
(8)加入500μL清洗缓冲液1后最高速离心1分钟，去除所有缓冲液。
(9)加入500μL清洗缓冲液2后最高速离心1分钟，去除所有缓冲液。
(10)转移到新的收集管中，最高速离心1分钟以去除所有残留溶液。
(11)转移到新的1.5mL离心管中，开盖，加入20-40μL洗脱液(滴加在膜的中心)。
(12)室温下孵育1-5分钟后，最高速离心1分钟后即得到基因组DNA。
实施例3制备特殊标准品
1、载体的准备
TA克隆载体pMD18-T购自promega公司。
2、插入片段的准备
2.1、目的基因及参照基因扩增区域的准备
使用PCR方法制备目的基因与参照基因的基因片段，PCR模板为经实施例1提取的细胞基因组DNA，反应体系如表1所示(PCR反应体系1)。
表1、PCR扩增制备靶标基因片段的反应体系(50μL/管，PCR反应体系1)
Figure PCTCN2017073336-appb-000001
PCR扩增体系中加入HER2外侧正、反向引物序列，可制备得到HER2基因片段；扩增体系中加入RPPH1外侧正、反向引物序列，可制备得到RPPH1基因片段，所述序列如表2所示，PCR扩增条件如表3所示。
表2、PCR反应体系的外侧引物和连接引物序列
Figure PCTCN2017073336-appb-000002
表3、制备基因片段的PCR扩增条件
Figure PCTCN2017073336-appb-000003
2.2连接目的基因及参照基因的基因片段
使用琼脂糖凝胶DNA回收试剂盒(购自天根公司)回收目标片段后，利用PCR方法连接HER2基因片段与RPPH1基因片段，制备成插入片段，PCR扩增引物及连接引物序列如表2所示，靶标序列连接PCR反应体系如表4所示(PCR反应体系2)，PCR扩增条件如表5所示。
表4、靶标序列连接PCR反应体系(50μL，PCR反应体系2)
Figure PCTCN2017073336-appb-000004
表5、靶标序列连接PCR体系反应条件
Figure PCTCN2017073336-appb-000005
使用琼脂糖凝胶回收试剂盒回收目标片段。
3、载体的构建与鉴定
通过TA克隆将插入片段连接入pMD18-T载体(购自promega公司)，所述TA克隆方法请参见
Figure PCTCN2017073336-appb-000006
载体系统产品说明书。新构建的质粒在大肠杆菌JM109菌株中进行大量扩增，进行蓝白斑筛选后挑取单克隆送测序，测序正确后作为标准品。所得标准品的核苷酸序列如序列表中SEQ ID NO:6所示。
图1是本发明构建所得标准品的质粒图谱，箭头所指即为载体插入基因PCR产物序列的位置。
实施例4单、双通道荧光定量PCR扩增结果的比较
1、目的基因与参照基因的单通道荧光定量PCR扩增结果
1.1、单通道荧光定量PCR反应模板为实施例3制备的标准品，以双蒸水为空白对照。
1.2、单通道荧光定量PCR反应体系如表6所示(PCR反应体系3)，PCR反应条件如表8所示。
表6、单通道荧光定量PCR反应体系(25μL/管，PCR反应体系3)
Figure PCTCN2017073336-appb-000007
HER2基因荧光定量PCR扩增体系中需要加入HER2内侧正、反向引物及HER2探针；RPPH1荧光定量PCR扩增体系中需要加入RPPH1内侧正、反向引物及RPPH1探针，所述引物和探针的序列如表7所示。所述探针中，标记HER2基因荧光探针5’端的荧光发射基团为HEX，3’端的荧光淬灭基团为TARMA，标记RPPH1基因荧光探针5’端的荧光发射基团为FAM，3’端的荧光淬灭基团为TARMA。
表7、内侧引物和荧光探针的核苷酸序列
Figure PCTCN2017073336-appb-000008
表8、荧光定量PCR体系反应条件
Figure PCTCN2017073336-appb-000009
荧光定量PCR扩增结果如图2所示，其中：图2(A)中，上升的8条曲线是以不同浓度(4倍梯度稀释)的质粒标准品为模板，进行HER2基因单通道荧光定量PCR扩增所得的扩增曲线，自左到右质粒标准品浓度依次为3.2E8copies/μL、8E7copies/μL、2E7copies/μL、5E6、1.25E6copies/μL、3.12E5copies/μL、7.81E4copies/μL、1.95E4copies/μL的质粒标准品的扩增曲线。图2(B)中，上升的8条曲线是以不同浓度(4倍梯度稀释)的质粒标准品为模板，进行RPPH1基因单通道荧光定量PCR扩增所得的扩增曲线，自左到右质粒标准品浓度依次为3.2E8copies/μL、8E7copies/μL、2E7copies/μL、5E6、1.25E6copies/μL、3.12E5copies/μL、7.81E4copies/μL、1.95E4copies/μL。
2、目的基因与参照基因的双通道荧光定量PCR扩增结果
2.1荧光定量PCR反应模板为实施例3制备的标准品，以双蒸水为空白对照。
2.2、双通道荧光定量PCR反应所需引物和探针序列如表7所示，荧光定量PCR体系的反应条件如表8所示，双通道荧光定量PCR反应体系如表9所示(PCR反应体系4)。
表9、双通道荧光定量PCR反应体系(25μL/管，PCR反应体系4)
Figure PCTCN2017073336-appb-000010
根据系列梯度稀释的标准品所得CT值结果绘制标准曲线。图2(C)是以本发明实施例3所得特殊标准品为模板，双通道荧光定量PCR同步扩增HER2与RPPH1所得的扩增曲线图。质粒标准品浓度依次为3.2E8copies/μL、8E7copies/μL、2E7copies/μL、5E6、1.25E6copies/μL、3.12E5copies/μL、7.81E4copies/μL、1.95E4copies/μL。在图中，实线代表HER2基因的扩增曲线，虚线代表RPPH1基因的扩增曲线，自左到右质粒标准品浓度4倍梯度降低。
3、单、双通道荧光定量PCR扩增结果的比较
从图2所示结果可以看出，双通道荧光定量PCR检测方法所得结果的准确性与单通道检测系统的高度一致，且具有提高检测通量，节约检测成本的优势。
实施例5双通道荧光定量PCR检测人类石蜡包埋组织样本中HER2基因扩增状态
1、荧光定量PCR反应模板为实施例2提取的乳腺癌样本基因组DNA，以 双蒸水为空白对照，阴性对照为实施例3制备所得HER2与RPPH1基因片段比例为1：1的单一质粒，HER2与RPPH1扩增片段浓度均为5E6copies/μL；阳性对照品由两种不同质粒组成，调整HER2与RPPH1扩增片段比例为4：1，其中RPPH1扩增片段浓度为5E6copies/μL，HER2扩增片段浓度为2E7copies/μL。
2、荧光定量PCR反应所用引物和荧光探针序列，扩增反应条件以及反应体系参见表7、表8、表9内容。使用双通道荧光定量PCR方法同时进行HER2与RPPH1基因检测。HER2扩增体系中需要加入HER2-F-2与HER2-R-2引物及其荧光探针。RPPH1扩增体系中需要加入RPPH1-F-2与RPPH1-R-2引物及其荧光探针。
3、绘制标准曲线
如图2(C)所示，以含有等比例目的基因与参照基因基因片段的质粒标准品为模板，进行双通道荧光定量PCR检测同步得到HER2及RPPH1基因的扩增曲线后，可进一步绘制得到相应基因的标准曲线。图3为根据图2(C)绘制的HER2及RPPH1的标准曲线，其中黑色方块曲线代表的是HER2基因的标准曲线，白色方块曲线代表的是RPPH1基因的标准曲线。
4、利用基因拷贝数相对定量检测方法评估样本中HER2基因的扩增状态
根据绘制的标准曲线，可分别求出HER2与RPPH1基因拷贝数，进而通过计算HER2与RPPH1基因拷贝数的比值关系实现对HER2基因拷贝数的相对定量，图5为所述实时荧光定量PCR检测方法示意图。
图4是本实施例中两例样本HER2及RPPH1基因扩增曲线。其中图4(A)是编号为1520438的石蜡包埋组织的基因扩增曲线，其CT值为22.57；图4(B)是编号为1601415的石蜡包埋组织的基因扩增曲线，其Ct值为27.42。参照基因RPPH1的CT值分别为25.33与27.75，分别由对应的标准曲线公式(图3)求得各自的拷贝数后，得到HER2/RPPH1分别为7.86(HER2扩增)与1.72(HER2不扩增)。
5、利用ΔΔCt方法评估样本中HER2基因的扩增状态
如上文所示，通过实时荧光定量PCR检测方法，能够获得未知样本及对照样本中目的基因及参照基因的Ct值，之后可依据公式1、2、3计算出HER2的扩增倍数。所述公式1、2、3如下所示：
公式1：ΔCt＝Ct目的基因-Ct参照基因
公式2：ΔΔCt＝ΔCt未知样本-ΔCt对照样本；
公式3：倍数变化＝2-ΔΔCt。
本实施例中以包含HER2与RPPH1基因片段比例为1：1的单一质粒作为对照样本，对所述照样本进行荧光定量PCR定量检测，根据HER2与RPPH1的平均Ct值获得平均ΔCt对照样本值；
在评估未知样本的HER2基因扩增情况时，分析步骤为：
第一、通过qPCR定量检测，获得目的基因及参照基因的Ct值后，根据公式1得到ΔCt未知样本、ΔCt对照样本；
第二、利用公式2及已有的ΔCt未知样本值和ΔCt对照样本值，算得ΔΔCt后，即可根据公式3得到倍数变化关系。
利用ΔΔCt方法对50例样本的HER2基因的扩增情况进行评估，具体检测分析结果见表10。
6、样本检测结果分析
本实施例对50例乳腺癌石蜡包埋组织样本进行了HER2基因的相对定量检测，并与荧光原位杂交(FISH)、免疫组化(IHC)的检测结果进行了比较。
本发明样本处理方法参照Qiagen公司的GeneRead DNA FFPE KIT使用说明，引物序列、扩增体系和扩增条件参照表7、8、9所述内容，在ABI7500荧光定量PCR仪上进行扩增，同时利用两种HER2基因相对定量检测方法对扩增结果进行评估。本实验中IHC检测抗体购自Abcam公司，FISH检测试剂购自雅培Vysis，实验操作参照说明书进行。本发明提供的两种HER2基因扩增检测方法与荧光原位杂交(FISH)、免疫组化(IHC)的检测结果，如表10所示。
表10、50例石蜡包埋组织样本的HER2与RPPH1基因拷贝数比例关系及其荧光免疫原位杂交与免疫组化的检测结果
Figure PCTCN2017073336-appb-000011
Figure PCTCN2017073336-appb-000012
Figure PCTCN2017073336-appb-000013
IHC是利用福尔马林固定的石蜡切片免疫显色反应在蛋白水平上进行HER2蛋白过表达检测，而FISH则是通过荧光标记的核酸探针与石蜡切片上固定的细胞核内的HER2基因的DNA靶序列杂交以检测其在染色体上的基因扩增情况。当IHC检测结果为2+时还需要进一步进行FISH检测确诊。
通过分析表10所得的结果后发现，对30例FISH阳性样本，本发明提供的两种HER2基因相对定量检测方法检测均判断为HER2扩增阳性；对20例FISH阴性样本，本发明提供的两种HER2基因相对定量检测方法检测均判断为HER2扩增阴性，表明，本发明提供的两种方法获得的结果与临床金标准FISH结果高度一致。
传统的IHC检测以及FISH检测方法在临床检测中不仅操作繁琐，价格昂贵，而且耗时较长(IHC检测需要1-2工作日，FISH检测方法则需要4-5个工作日)，无法进行标准化。与本发明所述方法相比，不仅灵敏度较低，而且还有非特异的假阳性。
本方法整个检测过程可在3-5小时内完成，可实现全自动化操作；另外本方 法采用双通道检测，操作简便，一次可实现40-50个样本的同时检测，而FISH完全是手工操作，一次检测样本难以超过10人份；最后本方法操作简便，因而稳定性好，其重复性远优于IHC及FISH检测。因此本方法完全可以成为临床HER2基因检测的新标准，具有巨大的经济价值。
应理解，在阅读了本发明的上述内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种基因拷贝数相对定量的实时荧光定量PCR检测方法，其特征在于，所述实时荧光定量PCR检测方法包括以下步骤：
    (1)将参照基因与待测目的基因片段等比例连接，克隆到同一个质粒载体上，构建一种可同时用于参照基因以及待测目的基因扩增检测的标准品，并将所得标准品按照浓度梯度稀释后同时用于绘制参照基因以及待测目的基因的标准曲线；
    (2)待测样本与步骤(1)所述标准品经同步进行实时荧光定量PCR扩增后，目的基因与参照基因按照各自的标准曲线计算各自的拷贝数，计算待测样本的目的基因与参照基因拷贝数比值，即得目的基因的拷贝数相对定量检测结果。
  2. 如权利要求1所述的实时荧光定量PCR检测方法，其特征在于，步骤(1)所述参照基因为RPPH1基因，所述待测目的基因为HER2基因，所述等比例为1:1；所述质粒载体为PCR克隆载体，所述标准品的核酸序列如序列表中SEQ ID NO:6所示。
  3. 如权利要求1所述的实时荧光定量PCR检测方法，其特征在于，步骤(2)所述荧光定量PCR扩增为双通道荧光定量PCR扩增。
  4. 一种用于基因拷贝数相对定量的实时荧光定量PCR检测的标准品，其特征在于，所述标准品是一种等比例包含参照基因与待测目的基因片段的质粒载体。
  5. 如权利要求4所述的标准品，其特征在于，所述参照基因为RPPH1基因，所述待测目的基因为HER2基因，所述等比例为1：1；所述质粒载体为PCR克隆载体，所述标准品的核酸序列如序列表中SEQ ID NO:6所示。
  6. 一种实时荧光定量PCR检测试剂盒，其特征在于，该实时荧光定量PCR检测试剂盒包括：PCR反应缓冲液、Taq酶、dNTP混合液、目的基因以及参照基因各自对应的特异性引物和荧光探针、标准品、阴性对照和阳性对照。
  7. 如权利要求6所述的实时荧光定量PCR检测试剂盒，其特征在于，所述目的基因为HER2基因，其对应的特异性上游引物序列如序列表中SEQ ID NO:7所示，其对应的特异性下游引物如序列表中SEQ ID NO:8所示，其对应的探针序列如列表中SEQ ID NO:11所示。
  8. 如权利要求6所述的实时荧光定量PCR检测试剂盒，其特征在于，所述参照基因为RPPH1基因，其对应的特异性上游引物序列如序列表中SEQ ID NO:9所示，其对应的特异性下游引物如序列表中SEQ ID NO:10所示，其对应的探针序列如序列表中SEQ ID NO:12所示。
  9. 如权利要求6所述的实时荧光定量PCR检测试剂盒，其特征在于，所述标准品为等比例包含参照基因RPPH1基因以及待测目的基因HER2基因的pMD18-T载体，其序列如序列表中SEQ ID NO:6所示。
  10. 如权利要求6所述的实时荧光定量PCR检测试剂盒，其特征在于，每个扩增反应中，所述目的基因、参照基因所对应的引物终浓度为0.14-0.18μM，所对应的探针终浓度为0.06-1.0μM，Taq酶的用量为0.02-0.06U。
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