CN113293212A - Primer probe for detecting amplification of neuroblastoma recurrence transfer gene FZD2 and application thereof - Google Patents
Primer probe for detecting amplification of neuroblastoma recurrence transfer gene FZD2 and application thereof Download PDFInfo
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Abstract
The invention provides a primer probe for detecting amplification of a neuroblastoma relapse metastasis gene FZD2 and application thereof, wherein the sequences of the primer probe are respectively an upstream primer sequence FZD2-F of a target gene: 15 '-TTTCTGTCGGGCTGCTACAC-3' of SEQ ID No, a downstream primer sequence FZD2-R of a target gene: 25 '-GTAACCGTCCTCGGAGAAGC-3' of SEQ ID No, and a target gene probe sequence FZD 2-P: 35 '-FAM-GCTCCAGGAGCGCGTGGTGTGCA-BHQ 1-3' of SEQ ID No, and an upstream primer sequence EFTUD2-F of an internal reference gene: SEQ ID No. 45 '-CTCAAAGTGCGGGGACTGAT-3', reference gene downstream primer sequence EFTUD 2-R: 55 '-GGCATCAGGGTGACTCCAAA-3' of SEQ ID No, reference gene probe sequence EFTUD 2-P: SEQ ID No. 65 '-HEX-AGCCTGCTTCCTGGGAATGTGCTGCT-BHQ 1-3'. The primer probe is used for a digital PCR kit. The kit can detect FZD2 gene amplification quickly, specifically and sensitively, has simple one-step amplification method and low cost, is very suitable for the gene copy number variation conditions in various samples such as tumor tissues, circulating tumor cells, circulating tumor DNA and the like, and is suitable for wide popularization and application.
Description
Technical Field
The invention belongs to the technical field of tumor molecule detection, and particularly relates to a primer probe for detecting amplification of a neuroblastoma recurrence and metastasis gene FZD2 and application thereof.
Background
Neuroblastoma (GBM) has been designated by the world health organization as a grade IV cancer, the most common and fatal CNS tumor in adults. Currently, standard treatment for GBM patients involves maximal surgical resection followed by concurrent radiotherapy and chemotherapy. Temozolomide (Temodal) is a DNA alkylating agent and is the most commonly used chemotherapeutic agent. Despite these therapies, most patients eventually relapse. Therefore, there is an urgent clinical need to develop effective anti-GBM therapeutic agents.
The prognosis for GBM patients is generally poor. GBM tumors are highly heterogeneous. On the other hand, GBM also appears to have a cellular hierarchy, so that there is a sub-population of GBM cells that is enriched for the capacity of tumorigenesis and spread, which drive tumor growth and therapeutic resistance. Numerous studies have shown that WNT signaling is abnormally activated in GBM, and it promotes GBM growth and invasion by maintaining stem cell characteristics. WNT proteins are a highly conserved class of secretory signal molecules. Since the discovery of WNT signaling as an oncogene in the mouse breast cancer model in 1982, 30 WNT signals have emerged as key regulators of cell-cell interactions, cell fate determination and migration. Mutations in WNT pathway components lead to specific developmental defects, while aberrant WNT signaling often leads to cancer. WNT proteins bind to receptors of the Frizzled (FZD) and low density lipoprotein receptor-related proteins/alpha 2-macroglobulin receptor (LRP) family on the cell surface. After WNT ligand molecules bind to receptors (FZD2, etc.), downstream pathways can be activated, resulting in the stabilization and transformation of the transcription factor β -Catenin into the nucleus, ultimately regulating the expression of numerous target genes through transcriptional regulation. FZD2 is an important target of GBM, and its amplification may become an important indicator for GBM diagnosis and prognosis.
Digital PCR is the third generation PCR technology that has been developed in the last decade, and the main principle is to place a single DNA molecule in an independent reaction chamber, where each reaction chamber either contains no nucleic acid target molecule to be detected or at least one nucleic acid target molecule to be detected, and perform PCR amplification on it, and detect a specific target sequence using TaqMan chemical reagents and dye-labeled probes. After PCR amplification, an analyzer detects each microdroplet one by one, the microdroplet with a fluorescent signal is judged as '1', the microdroplet without the fluorescent signal is judged as '0', and finally the copy number concentration of the target molecule to be detected is given according to the Poisson distribution principle and the proportion of positive microdroplets. The result of digital PCR quantification does not depend on Ct value any more and directly gives the initial copy number concentration of the target sequence, has the advantages of accuracy, hypersensitiveness, insusceptibility to PCR inhibitor and the like, can detect low-abundance tumor DNA variation, reduces false negative results, and is particularly suitable for the field of liquid biopsy (such as circulating tumor cell CTC or circulating tumor DNA). Therefore, the development of related digital PCR technology for amplification detection of the neuroblastoma recurrence and metastasis gene FZD2 is beneficial to dynamic disease course tracking and drug prognosis monitoring, greatly reduces health damage to patients caused by untimely or excessive drug administration and economic pressure and burden on individuals, families and national medical care systems, and has great benefits for economic, social and scientific development.
Disclosure of Invention
The invention aims to provide a primer probe for detecting amplification of a neuroblastoma recurrence transfer gene FZD2 based on a digital PCR technology and application thereof, and in order to realize the purpose of the invention, the following technical scheme is adopted:
a primer probe for detecting the amplification of a neuroblastoma recurrence transfer gene FZD2 based on a digital PCR technology comprises a primer probe with a nucleotide sequence shown in SEQ ID NO. 1-SEQ ID NO. 6, and the sequence is as follows:
the upstream primer sequence of the target gene FZD 2-F: 5'-TTTCTGTCGGGCTGCTACAC-3' SEQ ID NO 1,
The downstream primer sequence of the target gene FZD 2-R: 5'-GTAACCGTCCTCGGAGAAGC-3' SEQ ID NO:2,
The sequence of the target gene probe FZD 2-P: 5 '-FAM-GCTCCAGGAGCGCGTGGTGTGCA-BHQ 1-3' SEQ ID NO 3,
The upstream primer sequence of the internal reference gene EFTUD 2-F: 5'-CTCAAAGTGCGGGGACTGAT-3' SEQ ID NO. 4,
The downstream primer sequence EFTUD2-R of the internal reference gene: 5'-GGCATCAGGGTGACTCCAAA-3' SEQ ID NO 5,
Reference gene probe sequence EFTUD 2-P: 5 '-HEX-AGCCTGCTTCCTGGGAATGTGCTGCT-BHQ 1-3' SEQ ID NO 6.
Preferably, the target gene primer probe is designed according to a gene coding region of a human gene FZD2, the internal reference gene primer probe is designed according to a gene region of a human EFTUD2, the sizes of amplified target fragments are 102bp and 109bp respectively, and an amplification sequence (102bp) is as follows:
5’-TTTCTGTCGGGCTGCTACACCATGGTGTCGGTGGCCTACATCGCGGGCTTCGTGCTCCAGGAGCGCGTGGTGTGCAACGAGCGCTTCTCCGAGGACGGTTAC-3’、
the amplified sequence (109bp) was:
5’-CTCAAAGTGCGGGGACTGATTGTTAGCTCTAAAAGCTCTGGCACTATAGATAGTGATAGCCTGCTTCCTGGGAATGTGCTGCTAGAAGATTTGGAGTCACCCTGATGCC-3’。
preferably, the digital PCR primer probe is applied to the preparation of an amplification detection kit for detecting the neuroblastoma recurrence and metastasis gene FZD 2.
The invention also provides a digital PCR kit for detecting FZD2 gene amplification, which comprises a primer probe with a nucleotide sequence shown in SEQ ID NO. 1-SEQ ID NO. 6.
The use method of the kit comprises the following steps:
(1) preparing a digital PCR reaction solution; the formulation of 20. mu.l of digital PCR reaction solution is: 4 mul of one-step reaction buffer solution, 2 mul of enzyme mixed solution, 1.2 mul of each of 10 mul FZD2-F, FZD2-R, EFTUD2-F and EFTUD2-R primers, 0.6 mul of each of 10 mul MFZD2-P and EFTUD2-P, and 8 mul of DNA template of a sample to be detected;
(2) preparing microdroplets, transferring the microdroplets into a PCR plate, and carrying out amplification in a PCR instrument; reaction procedure: 15min at 45 ℃; 10min at 95 ℃; followed by 35 cycles, each cycle comprising 95 ℃ for 15s, 55 ℃ for 20s, 72 ℃ for 20 s; setting a temperature rise and fall speed of 2 ℃/sec for each step;
(3) and (3) putting the PCR plate subjected to the PCR amplification into a microdroplet analyzer, detecting microdroplets, analyzing data and displaying a detection result.
The method for judging the detection result of the kit comprises the following steps: (1) positive control: the copy number of the target gene/the copy number of the internal reference gene is more than 2.2; (2) negative control: the copy number of the target gene/the copy number of the internal reference gene is less than 1.8; and (3) judging the result of the sample to be detected: (1) positive: the copy number of the target gene/the copy number of the internal reference gene is more than 2.2; (2) negative: the copy number of the target gene/the copy number of the internal reference gene is less than 1.8; (3) suspected positive: 1.8< copy number of target gene/copy number of internal reference gene < 2.2.
The invention designs a pair of specific primers and probes according to the FZD2 gene region, and designs a pair of primers and probes by taking the human EFTUD2 gene region as an internal reference, establishes a detection method based on the digital PCR technology, and has the advantages that:
(1) high sensitivity: the detection sensitivity reaches one FZD2 amplified molecule in 10 ten thousand normal DNA molecules, and the FZD2 amplified molecule is suitable for liquid biopsy samples (such as circulating tumor cells CTC or circulating tumor DNA).
(2) High specificity: the specific primer probe designed aiming at the FZD2 gene region can specifically detect the copy number change of the FZD2 gene, and does not interfere with other homologous genes.
(3) The interference of complex samples on the experimental results can be eliminated: is not influenced by PCR inhibitors, can avoid PCR false negative caused by sample complexity, and is suitable for body fluid samples.
Drawings
FIG. 1 is a schematic diagram of an experimental result of the effectiveness of the primer provided by the present invention, which is a result of detecting a gene of FZD2 destination, wherein the positions of the holes in the diagram are, from left to right, 3 positive samples of FZD2 gene copy number variation and 3 negative samples of FZD2 gene copy number variation, and channel 1 represents a FAM channel;
FIG. 2 is a schematic diagram showing the experimental results of the effectiveness of the primers provided by the present invention, which is the detection result of the reference gene in human EFTUD2, wherein the positions of the holes in the diagram are 3 FZD2 gene copy number variation positive samples, 3 FZD2 gene copy number variation negative samples, and channel 2 represents the HEX channel;
FIG. 3 is a schematic diagram of the sensitivity test results of the primers provided by the present invention, which is the detection result of FZD2 target gene, the detection results of the critical concentration positive samples at the positions G01-A02, the detection results of the non-template control at the positions B02-D02, and the channel 1 represents the FAM channel;
FIG. 4 is a schematic diagram of the sensitivity test results of the primers provided by the present invention, which is the detection results of reference genes in human EFTUD2, wherein the positions G01-A02 are the detection results of positive samples with critical concentration, the positions B02-D02 are the detection results of no template control, and channel 2 represents the HEX channel.
Detailed Description
Hereinafter, specific embodiments of the present invention will be described in detail. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation. The technical scheme of the invention is a conventional scheme in the field if not specifically stated, and the used reagents or raw materials are purchased from commercial sources or published if not specifically stated.
Example 1 design of specific primers and probes for detecting amplification of neuroblastoma recurrence metastasis gene FZD2
The design of specific primers and probes suitable for ddPCR is carried out by taking human FZD2 as a target gene and human EFTUD2 as an internal reference gene, and the sequences of the primers and the probes are as follows:
the upstream primer sequence of the target gene FZD 2-F: 5'-TTTCTGTCGGGCTGCTACAC-3' SEQ ID NO 1,
The downstream primer sequence of the target gene FZD 2-R: 5'-GTAACCGTCCTCGGAGAAGC-3' SEQ ID NO:2,
The sequence of the target gene probe FZD 2-P: 5 '-FAM-GCTCCAGGAGCGCGTGGTGTGCA-BHQ 1-3' SEQ ID NO:3, reference gene upstream primer sequence EFTUD 2-F: 5'-CTCAAAGTGCGGGGACTGAT-3' SEQ ID NO. 4,
The downstream primer sequence EFTUD2-R of the internal reference gene: 5'-GGCATCAGGGTGACTCCAAA-3' SEQ ID NO 5,
Reference gene probe sequence EFTUD 2-P: 5 '-HEX-AGCCTGCTTCCTGGGAATGTGCTGCT-BHQ 1-3' SEQ ID NO 6.
Example 2 detection of amplification of neuroblastoma relapse metastasis Gene FZD2 Using digital PCR detection primers and probes
1. Plasma free DNA extraction
Nucleic acid extraction of the Plasma sample was carried out using a NucleoSpin Plasma XS kit (cat # 740901.50) manufactured by MNG. Mu.l of plasma was taken, nucleic acid extraction was performed according to the extraction kit instructions, and finally resuspended with 20. mu.l of eluent.
2. Digital PCR amplification reaction
(1) Preparing a digital PCR reaction solution; the formulation of 20. mu.l of digital PCR reaction solution is: 4 mul of one-step reaction buffer solution, 2 mul of enzyme mixed solution, 1.2 mul of each of 10 mul FZD2-F, FZD2-R, EFTUD2-F and EFTUD2-R primers, 0.6 mul of each of 10 mul MFZD2-P and EFTUD2-P, and 8 mul of DNA template of a sample to be detected;
(2) on the production chip of the droplet PCR, 20. mu.L of the droplet PCR reaction system of step 1) was added, and 40. mu.L of the droplet-producing oil was added to carry out the droplet-producing step, and after completion, the reaction system formed into droplets was transferred to a 96-well plate and sealed.
(3) Putting the 96-well plate after the membrane sealing into a PCR instrument for amplification; reaction procedure: 15min at 45 ℃; 10min at 95 ℃; followed by 35 cycles, each cycle comprising 95 ℃ for 15s, 55 ℃ for 20s, 72 ℃ for 20 s; setting a temperature rise and fall speed of 2 ℃/sec for each step;
(4) the PCR plate with completed PCR amplification is put into a microdroplet analyzer, microdroplets are detected, data are analyzed, and the detection result is displayed (FIG. 1-FIG. 2).
(5) The determination method of the detection result comprises the following steps: (1) positive control: the copy number of the target gene/the copy number of the internal reference gene is more than 2.2; (2) negative control: the copy number of the target gene/the copy number of the internal reference gene is less than 1.8; and (3) judging the result of the sample to be detected: (1) positive: the copy number of the target gene/the copy number of the internal reference gene is more than 2.2; (2) negative: the copy number of the target gene/the copy number of the internal reference gene is less than 1.8; (3) suspected positive: 1.8< copy number of target gene/copy number of internal reference gene < 2.2.
TABLE 1 results of detection of FZD2 gene and EFTUD2 gene in different samples.
Example 3 digital PCR primer Probe sensitivity assay for neuroblastoma samples
This experiment was performed using a neuroblastoma positive sample for gradient dilution. The detection was carried out by the detection method given in example 2. The results show that the ontology can detect the number of copies that differ from the no template control at 0.001% template amount (FIGS. 3-4).
Table 2 shows the results of the detection of FZD2 gene and EFTUD2 gene in positive samples at the critical concentration.
Sequence listing
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Claims (3)
1. A primer probe for detecting amplification of a neuroblastoma recurrence transfer gene FZD2, characterized in that the sequences of the primer probe are respectively:
the upstream primer sequence of the target gene FZD 2-F: 15 '-TTTCTGTCGGGCTGCTACAC-3' of SEQ ID No,
The downstream primer sequence of the target gene FZD 2-R: SEQ ID Nos 25 '-GTAACCGTCCTCGGAGAAGC-3',
the sequence of the target gene probe FZD 2-P: 35 '-FAM-GCTCCAGGAGCGCGTGGTGTGCA-BHQ 1-3' of SEQ ID No,
The upstream primer sequence of the internal reference gene EFTUD 2-F: SEQ ID Nos 45 '-CTCAAAGTGCGGGGACTGAT-3',
the downstream primer sequence EFTUD2-R of the internal reference gene: SEQ ID Nos 55 '-GGCATCAGGGTGACTCCAAA-3',
reference gene probe sequence EFTUD 2-P: SEQ ID No. 65 '-HEX-AGCCTGCTTCCTGGGAATGTGCTGCT-BHQ 1-3'.
2. The primer probe of claim 1, wherein the primer probe sequences SEQ ID NO 1-SEQ ID NO 3 are designed according to the coding region of human FZD2 gene, the amplified target fragment is 102bp in size, SEQ ID NO 4-SEQ ID NO 6 are designed according to the region of human EFTUD2 gene, and the amplified target fragment is 109bp in size.
3. Use of the primer probe of claims 1-2 in the preparation of a digital PCR kit for detecting amplification of FZD2 gene.
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