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Method for detecting mutation rate of JAK2 V617F as well as special primer and probe thereof

A technology of JAK2V617F and detection probes, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of inability to completely eliminate non-specific amplification reactions, detection sensitivity limitations, etc., and achieve result interpretation Simple and accurate, high sensitivity, avoid pollution effect

Pending Publication Date: 2020-04-03
济南金域医学检验中心有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the limitations of primer design in the common AS-PCR method, non-specific amplification will still occur.
Although the non-specific amplification reaction cannot be completely eliminated by strictly controlling the lower template DNA concentration (typically 10-15 ng / μL), this requires the wild and mutant δC T Value ranges define negative and positive possible
Due to the above-mentioned technical limitations, the detection sensitivity of ordinary AS-PCR method is obviously limited, and generally only reaches about 1%.

Method used

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  • Method for detecting mutation rate of JAK2 V617F as well as special primer and probe thereof
  • Method for detecting mutation rate of JAK2 V617F as well as special primer and probe thereof
  • Method for detecting mutation rate of JAK2 V617F as well as special primer and probe thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1: Primer probe design and screening

[0058] In the present invention, by designing specific primers and Taqman fluorescent probes for detecting JAK2 gene wild and V617F mutation sites, real-time fluorescent PCR method is used to quantitatively detect JAK2 gene wild and V617F mutations in human peripheral blood or bone marrow genomic DNA, and calculate Percentage of wild type and mutant type. The present invention firstly designs the upstream mutation of JAK2 and the wild primer, and mutates the base before the mutation site (G>T) to obtain better specific amplification. The combination of different mutation bases at the 3' end will affect the amplification. Therefore, the previous base of the mutation site was mutated to T, C, and G in sequence to obtain three pairs of primer-probe combinations. Add these two primers and the 3'-end common primer to perform two parallel PCRs. Only the primers that are completely complementary to the wild or mutant site (G / T) D...

Embodiment 2

[0064] Embodiment 2: Construction of mutant plasmid reference product and wild plasmid reference product

[0065] According to the upstream and downstream primers designed in Example 1, select the amplified fragment and about 100 bp upstream and downstream of the amplified fragment to construct the mutant plasmid reference product and the wild plasmid reference product respectively. Plasmids were constructed by Shanghai Jierui Bioengineering Co., Ltd. The sequence of the JAK2 gene fragment (>gi|568815589:5073611-5074001) inserted in the wild plasmid reference product is shown in SEQID NO.14; the sequence of the JAK2 gene fragment (>gi|568815589:5073611-5074001) inserted in the mutant plasmid reference product As shown in SEQ ID NO.15.

[0066] 5’-GCATCTTTATTATGGCAGAGAGAATTTTCTGAACTATTTATGGACAACAGTCAAACAACAATTCTTTGTACTTTTTTTTTTCCTTAGTCTTTCTTTGAAGCAGCAAGTATGATGAGCAAGCTTTCTCACAAGCATTTGGTTTTAAATTATGGAGTATGTGTCTGTGGAGACGAGAGTAAGTAAAACTACAGGCTTTCTAATGCCTTTCTCAGAGCATCTGTTTTTGTTTATAT...

Embodiment 3

[0072] Example 3: A kit for detecting the mutation rate of JAK2 V617F

[0073] 1. Kit composition:

[0074] (1) PCR reaction tube 1, comprising:

[0075] name sequence modify JAK2 upstream wild primer 5'-AGCATTTGGTTTTAAAATTATGGAGTATGAG-3' — Common primers downstream of JAK2 5'-CTAGCTGTGATCCTGAAACTGAATT-3' — JAK2 detection probe TGCCTTTTCTCAGAGCATCTGT 5'-FAM; 3'-MGB JAK2 mutation blocking probe TGGAGTATGTTTCTGTGGAGA 3'-MGB GAPDH upstream primer 5'-CCCACTCCTCCCACCTTTGAC-3' — GAPDH downstream primer 5'-CTGGCCCCAGCCACATAC-3' — GAPDH probe CATTGCCCTCAACGACCACTTTGTCAAGC 5'-HEX; 3'-TAMRA

[0076] (2) PCR reaction tube 2, comprising:

[0077] name sequence modify JAK2 upstream mutation primers 5'-AGCATTTGGTTTTAAAATTATGGAGTATGAT-3' — Common primers downstream of JAK2 5'-CTAGCTGTGATCCTGAAACTGAATT-3' — JAK2 detection probe TGCCTTTTCTCAGAGCATCTGT 5'-FAM; 3'-MGB JAK2 wi...

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Abstract

The invention relates to the technical field of gene mutation detection, in particular to a method for detecting mutation rate of JAK2V617F and a special primer and probe thereof. The special primer and probe comprise a JAK2 upstream wild primer, a JAK2 upstream mutation primer, a JAK2 shared downstream primer, a JAK2 detection probe, a JAK2 wild closed probe and a JAK2 mutation closed probe. According to the invention, a closed probe is introduced into a detection system, so that non-specific amplification can be effectively avoided under high sample DNA concentration, and the detection sensitivity is improved; mGB markers are selected for the design of the detection probe, the wild closed probe and the mutation closed probe, so that the Tm value difference between paired and non-paired templates is improved, the signal to noise ratio is increased, the detection result is more accurate, and the resolution ratio is higher.

Description

technical field [0001] The invention relates to the technical field of gene mutation detection, in particular to a method for detecting the mutation rate of JAK2 V617F and special primers and probes thereof. Background technique [0002] In 2005, a study found that the JAK2 V617F mutation exists in polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The mutation of JAK2 exon14 from valine to phenylalanine changes the JH2 domain of the JAK2 protein, and JH2 loses its negative regulatory effect on the downstream and continues to activate the JAK2 pathway. In 2008, the World Health Organization (WHO) regarded positive JAK2 V617F gene mutation as one of the diagnostic conditions for PV, ET, and PMF. JAK2 V617F gene mutation can occur in 93%-95% of PV patients, so JAK2V617F is a sensitive indicator for the diagnosis of PV. 53%-64% of ET patients carry JAK2 V617F gene mutation, and 58%-65% of PMF patients carry JAK2 V617F mutation. In additi...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q2600/156
Inventor 祁德波郭心玮姜玲波王丙维彭德志王佳佳张陈祎
Owner 济南金域医学检验中心有限公司
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