Primer probe combination and kit for detecting CAD gene D1371 mutation

Abstract

The invention discloses a primer probe combination and a kit for detecting mutation of CAD (computer aided design) gene D1371. The primer probe combination is shown in SEQ ID NO: 1 to SEQ ID NO: 11. The primer probe combination disclosed by the invention can be used for detecting CAD gene D1371 mutation.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a primer-probe combination and a kit for detecting the D1371 mutation of the CAD gene. Background Art

[0002] Mammalian cell proliferation requires de novo synthesis of pyrimidine nucleotides. This gene encodes a trifunctional protein that is associated with the enzymatic activities of the first three enzymes in six steps of the pyrimidine biosynthetic pathway: carbamoyl phosphate synthetase (CPSII), aspartate transcarbamylase, and dihydroorotase. That is, the metabolic flux of the de novo pyrimidine synthesis pathway is regulated by the first rate-limiting enzymes, Cytosolic carbamyl-phosphate synthetase II, asparate transcarbamylase, and Dihydroorotase (CAD). Summary of the Invention

[0003] One object of the present invention is to provide a primer-probe combination for detecting the D1371 mutation of the CAD gene, which includes:

[0004] CAD-GAT-F 5'TTGACTCCGGGTTGGCA 3'

[0005] CAD-GAT-R 5'CAGCTAGCTGCTCCAGGATG 3'

[0006] CAD-GAT-GAT-P TCACCATCCACAGCC

[0007] CAD-GAT-CAT-P TCACCATGCACAGCC

[0008] CAD-GAT-AAT-P TCACCATTCACAGCCT

[0009] CAD-GAT-TAT-P TCACCATACACAGCCT

[0010] CAD-GAT-GGT-P CACCACCCACAGCC

[0011] CAD-GAT-GCT-P CACCAGCCACAGCC

[0012] CAD-GAT-GTT-P TCACCAACCACAGCC

[0013] CAD-GAT-GAA-P TCACCTTCCACAGCC

[0014] CAD-GAT-GAG-P CACCCTCCACAGCC。

[0015] In the present invention, the mutation includes that the codon at D1371 is mutated from GAT to one of CAT, AAT, TAT, GGT, GCT, GTT, GAA, and GAG.

[0016] Further, the probe is a Taqman probe.

[0017] Further, the 5'-end of the Taqman probe is labeled with a fluorescent reporter group, and the 3'-end is labeled with a quenching group; the fluorescent reporter group includes FAM or VIC; the quenching group includes MGB or BHQ1.

[0018] Further, the fluorescence of the probe is:

[0019] CAD-GAT-GAT-P TCACCATCCACAGCC 5'-FAM 3'-MGB

[0020] CAD-GAT-CAT-P TCACCATGCACAGCC 5'-VIC 3'-MGB

[0021] CAD-GAT-AAT-P TCACCATTCACAGCCT 5'-VIC 3'-MGB

[0022] CAD-GAT-TAT-P TCACCATACACAGCCT 5'-VIC 3'-MGB

[0023] CAD-GAT-GGT-P CACCACCCACAGCC 5'-VIC 3'-MGB

[0024] CAD-GAT-GCT-P CACCAGCCACAGCC 5'-VIC 3'-MGB

[0025] CAD-GAT-GTT-P TCACCAACCACAGCC 5'-VIC 3'-MGB

[0026] CAD-GAT-GAA-P TCACCTTCCACAGCC 5'-VIC 3'-MGB

[0027] CAD-GAT-GAG-P CACCCTCCACAGCC 5'-VIC 3'-MGB。

[0028] Another object of the present invention is to provide the application of the primer-probe combination for detecting the D1371 mutation of the CAD gene in the preparation of a product for detecting the D1371 mutation of the CAD gene.

[0029] Still another object of the present invention is to provide a detection kit, which comprises the primer-probe combination for detecting the D1371 mutation of the CAD gene.

[0030] Compared with the background art, the present technical solution has the following advantages:

[0031] In the preliminary research of the inventors, it was found that the multifunctional enzyme CAD is the physiological substrate of Caspase-3 in the endogenous apoptosis pathway induced by chemotherapeutic drugs. Subsequently, the research results of the inventors showed that the overexpression of CAD or gain-of-function (GOF) mutations at the aspartic acid cleavage site codon 1371 enhanced chemoresistance, which was confirmed in clinical samples. In addition, the inventors demonstrated that the pharmacological inhibition of anti-cleavage CAD sensitized tumor cells to clinically relevant chemotherapeutic drugs. Therefore, CAD has become a key inhibitor of chemotherapy-induced cell death and a promising molecular target for treatment resistance. The primer-probe combination of the present invention can completely cover eight possible mutation combinations of CAD at its cleavage site D1371, and whether there is a mutation can be detected at one time. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The present invention will be further described below with reference to the drawings and embodiments.

[0033] Figure 1 For the single-sample detection result of the kit, the WT-CAD is labeled in the blue fluorescence channel.

[0034] Figure 2 For the single-sample detection result of the kit, the MUT-CAD is labeled in the green fluorescence channel. DETAILED DESCRIPTION OF THE INVENTION

[0035] Example 1

[0036] In this kit, the codon of wild-type CAD at its cleavage site D1371 is GAT. In theory, each nucleotide mutates separately, excluding the case of synonymous mutations. A total of eight possible mutations are considered, which are

[0037] CAT, AAT, TAT, GGT, GCT, GTT, GAA, GAG, and the 5' ends of the mutant nucleic acid probes all carry the VIC fluorescent group, and the 5' end of the wild-type CAD nucleic acid probe carries the FAM fluorescent group.

[0038] Composition of Reagents

[0039]

[0040] GATATCTTTCCTGCCAAGAAATACACTTTGCCAGTATCAAATAGAGGCAGCCCTCAGTGCCCACCCTAT

[0041] GGGTCCTCAGTCTCCTCATCATGGGCTCCTGGGCCAGCTCCTCTCCCTTAAGGCTAGCCTTCCTGACC

[0042] GCTGCCAGACAGCTTGGCCCTGACCTTGACTCCGGGTTGGCAGGTAACAGCTGTGGACTGGCACTTTGAGGAGGCTGTGGAT(Asp / D)GGTGAGTGCCCACCACAGCGGAGCATCCTGGAGCAGCTAGCTGAGA AAAACTTTGAGCTGGTGATTAACCTGTCAATGCGTGGAGCGGGGGCCGGCGTCTCTCTTCCTTTGTCACCAAGGGCTACCGCACCCGACGCTTGGCCGCTGACTTCTCCGTGCCCTGTGGAGGTAACTGAGACCCATGTGCTGGGAGGGAGACTGCCAGTGCTAATCATCGATATCAAGTGCACCAAACTCTT

[0043] The above is the CAD gene fragment, including the mutation site "GAT(ASP / D)".

[0044] Composed of 20×multiplex primers / probes

[0045] Primer and probe sets, where F represents the forward primer, R represents the reverse primer, and P represents the probe.

[0046] CAD-GAT-F 5'TTGACTCCGGGTTGGCA 3'

[0047] CAD-GAT-R 5'CAGCTAGCTGCTCCAGGATG 3'

[0048] CAD-GAT-GAT-P TCACC ATC CACAGCC 5’-FAM 3’-MGB

[0049] CAD-GAT-CAT-P TCACC ATG CACAGCC 5’-VIC 3’-MGB

[0050] CAD-GAT-AAT-P TCACC ATT CACAGCCT 5’-VIC 3’-MGB

[0051] CAD-GAT-TAT-P TCACC ATA CACAGCCT 5’-VIC 3’-MGB

[0052] CAD-GAT-GGT-P CACC ACC CACAGCC 5’-VIC 3’-MGB

[0053] CAD-GAT-GCT-P CACC AGC CACAGCC 5’-VIC 3’-MGB

[0054] CAD-GAT-GTT-P TCACC AAC CACAGCC 5’-VIC 3’-MGB

[0055] CAD-GAT-GAA-P TCACC TTC CACAGCC 5’-VIC 3’-MGB

[0056] CAD-GAT-GAG-P CACC CTC CACAGCC 5’-VIC 3’-MGB

[0057] Reaction steps

[0058]

[0059] Sample detection

[0060] Samples were obtained from the First Affiliated Hospital of China Medical University. Paraffin-embedded tissue (formalin-fixed, paraffin-embedded (FFPE)) samples were taken from two gastric cancer patients who received neoadjuvant chemotherapy in the Department of General Surgery of the First Affiliated Hospital of China Medical University and had an outcome of "Progressive Disease" (PD).

[0061] Genomic DNA was extracted from formalin-fixed paraffin-embedded (FFPE) tissues using the following kit: The DNA FFPE Tissue Kit (#56404; QIAGEN, Hilden, Germany, QIAamp DNA FFPE Tissue Kit (qiagen.com)). Instruction manuals in both Chinese and English are provided.

[0062] Detection steps:

[0063] The CAD (reference sequence NM_004341.5) mutations of interest in ctDNA were detected on the Naica digital PCR Naica Crystal digital PCR system (Stilla Technologies, France). The primers and probes for detecting exon 26 are as follows:

[0064] CAD-GAT-F: 5'-TTGACTCCGGGTTGGCA-3'

[0065] CAD-GAT-R: 5'-CAGCTAGCTGCTCCAGGATG-3'

[0066] CAD-GAT-GAT-P: 5’-VIC-TCACCATCCACAGCC-MGB-3’

[0067] CAD-GAT-TAT-P: 5’-FAM-TCACCATACACAGCCT-MGB-3’

[0068] CAD-GAT-GAA-P: 5’-FAM-TCACCTTCCACAGCC-MGB-3’

[0069] The digital PCR reaction was assembled using PerFecTa Multiplex qPCR ToughMix (Quanta Biosciences, Gaithersburg, MD, USA), 40 nM FITC (Saint Louis, MO, USA), 1 μl of the primer and probe multiplex mixture, and 3 μl of the DNA template. The total volume of the PCR reaction was 20 μl. The cycling conditions were 95 °C for 10 minutes, followed by 45 cycles of 95 °C for 10 seconds and 62 °C for 15 seconds. After the sample plate was successfully set up on the computer, it was placed in the QX200 Droplet Digital PCR System for droplet analysis and detection. The data could be uploaded to the computer for final analysis. After the reaction program was completed, the threshold line was adjusted to an appropriate position according to the specific reaction conditions for the next result interpretation. Standard wild-type DNA and standard mutant DNA were used as negative and positive controls respectively.

[0070] This detection was based on two samples selected according to the positive results of the previous whole exome sequencing (WES). Corresponding fluorescent probes were designed for ddPCR according to the mutation conditions detected by WES. If the fluorescent signals corresponding to the mutations were detected, it could confirm the existence of positive mutations, which was determined by the characteristics of the fluorescent probes, that is, there was no fluorescent signal for non-specific mutations. Both of the two selected samples had signal detections.

[0071] Samples with the CAD-D1371 mutation obtained according to the sequencing results can all detect positive results using the kit designed by the present invention, indicating the accuracy and high feasibility of the kit.

[0072] As mentioned above, it is only a preferred embodiment of the present invention, so the scope of implementation of the present invention cannot be limited thereby. That is, equivalent changes and modifications made according to the scope of the present invention patent and the content of the specification should still fall within the scope covered by the present invention.

Claims

1. A primer-probe combination for detecting the D1371 mutation of the CAD gene, characterized in that: It includes the following sequences: CAD-GAT-F: 5'-TTGACTCCGGGTTGGCA-3' CAD-GAT-R: 5'-CAGCTAGCTGCTCCAGGATG-3' CAD-GAT-GAT-P: 5’-VIC-TCACCATCCACAGCC-MGB-3’ CAD-GAT-TAT-P: 5’-FAM-TCACCATACACAGCCT-MGB-3’ CAD-GAT-GAA-P: 5’-FAM-TCACCTTCCACAGCC-MGB-3’.

2. A primer-probe combination for detecting the D1371 mutation of the CAD gene, characterized in that: It includes the following sequences: CAD-GAT-F 5'TTGACTCCGGGTTGGCA 3' CAD-GAT-R 5'CAGCTAGCTGCTCCAGGATG 3' CAD-GAT-GAT-P TCACCATCCACAGCC CAD-GAT-CAT-P TCACCATGCACAGCC CAD-GAT-AAT-P TCACCATTCACAGCCT CAD-GAT-TAT-P TCACCATACACAGCCT CAD-GAT-GGT-P CACCACCCACAGCC CAD-GAT-GCT-P CACCAGCCACAGCC CAD-GAT-GTT-P TCACCAACCACAGCC CAD-GAT-GAA-P TCACCTTCCACAGCC CAD-GAT-GAG-P CACCCTCCACAGCC.

3. The primer-probe combination for detecting the D1371 mutation of the CAD gene according to claim 2, characterized in that: The mutation described above includes that the codon at D1371 is mutated from GAT to one of CAT, AAT, TAT, GGT, GCT, GTT, GAA, GAG.

4. The primer-probe combination for detecting the D1371 mutation of the CAD gene according to claim 2, wherein The probe is a Taqman probe.

5. The primer-probe combination for detecting the D1371 mutation of the CAD gene according to claim 2, characterized in that, The 5' end of the Taqman probe is labeled with a fluorescent reporter group, and the 3' end is labeled with a quenching group; the fluorescent reporter group includes FAM or VIC; the quenching group includes MGB or BHQ1.

6. The primer-probe combination for detecting the D1371 mutation of the CAD gene according to claim 2, characterized in that The fluorescence of the probe is: CAD-GAT-GAT-P TCACCATCCACAGCC 5’-FAM 3’-MGB CAD-GAT-CAT-P TCACCATGCACAGCC 5’-VIC 3’-MGB CAD-GAT-AAT-P TCACCATTCACAGCCT 5’-VIC 3’-MGB CAD-GAT-TAT-P TCACCATACACAGCCT 5’-VIC 3’-MGB CAD-GAT-GGT-P CACCACCCACAGCC 5’-VIC 3’-MGB CAD-GAT-GCT-P CACCAGCCACAGCC 5’-VIC 3’-MGB CAD-GAT-GTT-P TCACCAACCACAGCC 5’-VIC 3’-MGB CAD-GAT-GAA-P TCACCTTCCACAGCC 5’-VIC 3’-MGB CAD-GAT-GAG-P CACCCTCCACAGCC 5’-VIC 3’-MGB。 7. Use of a primer-probe combination for detecting the D1371 mutation of the CAD gene according to claim 1 or 2 in the preparation of a product for detecting the D1371 mutation of the CAD gene.

8. Detection kit, characterized in that: Comprising the primer-probe combination for detecting the D1371 mutation of the CAD gene according to claim 1 or 2.

9. Detection system for CAD gene D1371 mutation, characterized in that: Comprising: The primer and the probe are the primer-probe combination for detecting the D-1371 mutation of the CAD gene described in claim 1 or 2.

10. The detection system for the CAD gene D1371 mutation according to claim 9, characterized in that: The reaction steps are as follows: Enzyme activation: 95 °C, 10 min; then 40 cycles, with each cycle having denaturation at 94 °C for 30 s; annealing: 55 °C, 1 min; enzyme inactivation at 98 °C, 10 min.

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