Primers for detecting BCR/ABL fusion genes by ddPCR technology and detection method thereof
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A fusion gene and technical detection technology, which is applied in ddPCR technology to detect BCR/ABL fusion gene primers and its detection field, can solve the problem of low sensitivity of RT-PCR, achieve strong design specificity, reduce recurrence, and avoid false negative results Effect
Inactive Publication Date: 2018-06-01
PRIMBIO GENES BIOTECH WUHAN CO LTD
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[0012] In order to overcome the above-mentioned deficiencies of the prior art, the purpose of the present invention is aimed at the low defect of RT-PCR sensitivity, proposes a kind of ddPCR technology to detect the primer of BCR/ABL fusion gene and detection method thereof, this pri
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Embodiment 1
[0037] Embodiment 1: Design and synthesis of primers and probes for detection of BCR / ABL fusion gene by ddPCR
[0038] For BCR / ABLP210, BCR / ABL P190 and BCR / ABL P2303 types of fusion genes, using the full-length cDNA of BCR / ABL fusion genes e13 / 14a2, e1a2, and e19a2 as templates, use Oligo software to analyze TaqMan primers and probes site, and according to the consideration of the two genomic DNA sequences of BCR and ABL, the best combination is selected as follows:
[0039] BCR / ABL fusion gene upstream and downstream primers:
[0040] BCR / ABLP210 upstream primer SEQ NO:1: 5'-CATTCCGCTGACCATCAAT-3'
[0041] BCR / ABLP190 upstream primer SEQ NO:2: 5'-ACTGCCCGGTTGTCGTG-3'
[0043] BCR / ABLP230 upstream primer SEQ NO:3: 5'-GGTCCAAGGTGCCCTACATC-3'
[0044] BCR / ABLP230 downstream primer SEQ NO:5: 5'-GTCCAGCGAGAAGGTTTTCC-3'
[0045] The BCR-ABL fusion gene detection probe is: SEQ NO:6: 5'-...
Embodiment 2
[0051] Embodiment 2: Detection of BCR / ABL fusion gene in tissue
[0052] 1. Prepare the samples to be tested: RNA containing the internal reference BCR gene, BCR / ABL integrated fusion-positive RNA, and a mixed sample of the internal reference gene and fusion RNA, where the content ratios of the fusion type and the internal reference gene are 1 / 100 and 1 / 1000, respectively.
[0053] 2. Extraction of RNA: Biomiga Blood RNA miniprep kit (Cat#R6411) was used to extract the mRNA of white blood cells in whole blood. For specific operations, refer to the instructions of the RNABlood Mini Kit kit from QIAGEN.
[0054] 3. Reverse transcription of RNA into cDNA: Refer to the instructions of the High Capacity cDNA ReverseTranscription Kit kit from ThermoFisher Company, and reverse transcribe RNA into cDNA.
[0055] 4. Prepare the PCR reaction solution in the PCR plate according to the following ratio: 2× digital PCR master mix (Biorad, #1863010) and BCR / ABL fusion gene upstream and downs...
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Abstract
The invention discloses primers for detecting BCR/ABL fusion genes by a ddPCR technology and a detection method thereof. With digital PCR as a detection platform, in allusion to three types of BCR/ABLfusion genes, upstream and downstream primers for detecting the BCR/ABL fusion genes, a BCR/ABL fusion gene detection probe, upstream and downstream primers for detecting an internal reference gene and an internal reference gene detection probe are designed and synthesized, cDNA templates of a sample to be detected, the upstream and downstream primers for detecting the BCR/ABL fusion genes, the BCR/ABL fusion gene detection probe, the upstream and downstream primers for detecting an internal reference gene, the internal reference gene detection probe and a PCR premixed solution are mixed forpreparing ddPCR microreaction liquid drops for a PCR amplification reaction, and whether fusion gene templates are contained in the sample to be detected and the number and the content thereof are judged according to the types of fluorescence signals. The design specificity of the primers and the probes are strong, the detection sensitivity is higher through combination with the digital PCR technology, and a false negative result is avoided by automatically reading the result by software, so that the primers and the probes can be applied to detection of a small number of leukemia cells and thepossibility of relapse of a patient is reduced.
Description
technical field [0001] The invention relates to the molecular biological detection of genes in the field of biotechnology, in particular to a ddPCR technique for detecting a primer for BCR / ABL fusion gene and a detection method thereof. Background technique [0002] abl is a proto-oncogene, located on chromosome 9 q34, the gene product is a non-receptor tyrosine protein kinase; bcr gene is located on chromosome 22 q11, the normal bcr gene product is a 160kD cytoplasmic phosphoprotein, due to Translocation of t(9;22)(q34;q11) to form a bcr / abl fusion gene, the translocation produces a bcr / abl chimeric gene, the gene product is a fusion protein of 210 kD, its expression activates the tyrosine protein Kinase, which changes the protein tyrosine level and actin binding ability of cells, disrupts the normal signal transduction pathway, and inhibits the occurrence of apoptosis. [0003] The bcr gene breakpoints were concentrated in three regions: major (majorbcr, M-bcr), minor (mi...
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