Primer, probe and kit for detecting mutation of human JAK2 gene V617F

A V617F, detection probe technology, applied in the biological field, can solve the problems of low detection efficiency, high false positive rate, long detection time, etc., and achieve the effect of high sensitivity, good specificity and rapid detection

Inactive Publication Date: 2016-03-30
武汉海吉力生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In order to solve the problems of low sensitivity, long detection time, low detection efficiency, easy contamination of samples and high false positive rate in existing detection technologies, the present invention provides primers and probes for detecting the V617F mutation of human JAK2 gene, which have high sensitivity, Strong specificity, effectively avoiding false positives

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  • Primer, probe and kit for detecting mutation of human JAK2 gene V617F
  • Primer, probe and kit for detecting mutation of human JAK2 gene V617F
  • Primer, probe and kit for detecting mutation of human JAK2 gene V617F

Examples

Experimental program
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Effect test

Embodiment 1

[0054] 1. Mutation detection primers and probes, quality control primers and probe sequence design:

[0055] Based on the wild-type sequence of the JAK2 gene (NCBI Reference Sequence: NM_004972.3) published in the NCBI database, and with reference to the V617F mutation site of exon 14 (see Table 1), specific mutation primers and probes (see Table 2) were designed. Using the mutant plasmid constructed by genetic engineering (insert gene fragment >gi|568815589:5073601-5074000, and V617F base change) and wild-type plasmid (insert gene fragment>gi|568815589:5073601-5074000) as templates, establish real-time fluorescence The PCR mutation (Mutation) detection system realizes the high sensitivity and high specificity detection of the JAK2 gene V617F mutation.

[0056] Table 2: Specific primer and probe combinations for detection of human JAK2 gene V617F mutation

[0057] SEQ ID NO:

name

Sequence (5'→3')

end modification

1

JW1-F

TCTTTGAAGCAGCAAGTAT...

Embodiment 2

[0093] Use the human JAK2 gene V617F mutation detection kit of the present invention to detect clinically collected blood samples.

[0094] In this example, blood samples from patients diagnosed as myeloproliferative neoplasms (MPN) were collected and genomic DNA was extracted from them. Use the human JAK2 gene V617F mutation detection kit to detect whether there is a JAK2 gene V617F mutation in the test sample. Specific steps are as follows:

[0095] (1) Sample genomic DNA extraction

[0096] Blood samples were extracted using Ningbo Youcheng Blood Genomic DNA Extraction Kit (KR008F). Extract the genomic DNA of the sample to be tested according to the instructions, and use the ultraviolet spectrophotometer to detect the concentration and DNA quality of the extracted DNA sample. The DNA concentration is required to be ≥10ng / μL, and the DNA quality OD260 / 280 is between 1.8 and 2.0. Genomic DNA that meets the requirements was diluted to 2-10 ng / μL with TE (10 mmol / L, pH 8.0) ...

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Abstract

The invention discloses a primer, a probe and a kit for detecting a mutation of a human JAK2 gene V617F. The primer comprises a mutation forward primer JW1-F and a mutation reverse ARMS primer JM1-R, and the probe comprises a detection probe JW1-P and a blocking probe JW1-B, wherein the mutation forward primer JW1-F is combined with a JAK2 gene conserved sequence; the mutation reverse ARMS primer JM1-R is specifically combined with a V617F (1849G>T) site mutation sequence to selectively amplify the mutation sequence; a 5minute end of the detection probe JW1-P is marked with an FAM (carboxy fluorescein) signal; a 3minute end of the detection probe is marked with an MGB (Minor Groove Binder); the detection probe JW1-P can be combined with an amplified fragment; a 5minute end of the blocking probe JW1-B is double-deoxidized and modified; a 3minute end of the blocking probe JW1-B is marked with the MGB; the blocking probe JW1-B can be specifically combined with a V617 site wild-type sequence to inhibit wild-type nonspecific amplification. The primer and the probe which are disclosed by the invention are high in specificity and good in sensitivity, and have high detectability of 1 percent. The kit prepared from the primer and the probe is accurate in detection result and easy in reading of the detection result, is simple and rapid to operate, and is wide in application range.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to primers, probes and kits for detecting the V617F mutation of human JAK2 gene. Background technique [0002] The incidence of single disease of myeloproliferative neoplasms (MPN) is mostly less than 1 / 100,000, which is a rare disease. In the past 10 years, with the improvement of people's living standards and medical conditions, the total number of MPN patients found has increased by about three times. JAK2 gene has higher mutation rate in PV, ET, IMF (~90%, ~50%, ~50%). [0003] JAK2 is a type of tyrosine protein kinase, after binding to the hematopoietic growth factor receptor, it affects the transcriptional regulation of genes (JAK-STAT pathway) through signal transducers and activators of transcription (STAT). JAK2 gene V617F is a point mutation (G1849T) at position 617 of exon 14, and valine (valine, V) is replaced by phenylalanine (phenylalanine, F). Val617 is located in JH2 ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6886C12Q1/6858C12Q2600/156
Inventor 朱峰邹芳段卫涛赵平锋
Owner 武汉海吉力生物科技有限公司
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