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Primer probe combination for detecting human leukemia FLT3 gene internal tandem repeat mutation, kit and application

A detection kit and internal tandem technology, applied in the field of primer-probe combination for the detection of internal tandem repeat mutations of the human leukemia FLT3 gene, can solve problems such as complex mutation types, uncertain insertions, and uncertain sequence lengths of mutant sequences, and achieve simplified Detection steps, high sensitivity and accuracy, simple and efficient detection process

Pending Publication Date: 2021-11-30
广东永诺医疗科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, the type of FLT3-ITD mutation is complicated, and the specific upstream and downstream sites of the mutation sequence insertion and the length of the inserted sequence are uncertain. Uncertain insertions can be made in multiple regions on exon 14, and the length of the inserted sequence can range from several bp To a few hundred bp, primers designed specifically for individuals are required according to the length of the FLT3-ITD gene inserted into different fragments

Method used

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  • Primer probe combination for detecting human leukemia FLT3 gene internal tandem repeat mutation, kit and application
  • Primer probe combination for detecting human leukemia FLT3 gene internal tandem repeat mutation, kit and application
  • Primer probe combination for detecting human leukemia FLT3 gene internal tandem repeat mutation, kit and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] First, one human leukemia FLT3 internal tandem duplication (ITD) mutations in the droplet digital PCR detection kit, the kit comprising:

[0059] (1) using droplet digital PCR reaction mix: including buffer, dNTPs (0.2mM), taq enzyme (2U), UDG enzyme (0.2U) (Guangdong Wing Connaught Medical Products Ltd., catalog S02000301, and MicroDrop- digital droplet 100 supporting the use of PCR system).

[0060] (2) Detection of FLT3 mixture A: a probe and detected by the wild-type FLT3 gene of a pair of primers, the detection of the reference gene RPP30 a pair of primers and a probe consisting of; deionized water concentration was adjusted to 900nM primer , concentration of the probe was adjusted to 250 nM;

[0061] FLT3 wild type primer, probe sequences as shown in SEQ ID NO.1-3:

[0062] FLT3-Exon14-CF: 5'-GTACGTGCATTTTAAAGATTTTCCAA-3 '(SEQ ID N O.1);

[0063] FLT3-Exon15-R1: 5'-GCAATTTAGGTATGAAAGCCAGC-3 '(SEQ ID NO.2);

[0064] FLT3-Intron14-Probe: 5'-FAM-CTGCAGAAACATTTGGCACATTCCA...

Embodiment 2

[0113] Example 2 verification kit detecting method, AS-PCR mutagenic primer validity embodiment

[0114] First, using the human in Example 1 leukemia FLT3 internal tandem droplet digital PCR test kit and method for detecting duplicate (ITD) mutation to the actual mutation clinical samples for the object, test kit and method for detecting, mutagenic primer the effectiveness of specific testing process:

[0115] (1) clinical samples: 301 provided by a hospital patient carries three DNA samples FLT3-ITD mutation has been detected, the hospital these DNA samples FLT3-ITD mutation by capillary electrophoresis levels (Table 4).

[0116] Table 4

[0117] Patient sample Sample 1 Sample 2 Sample 3 FLT3-ITD mutation Qiangyang Qiangyang Qiangyang

[0118] (2) First, the patient's DNA samples Sanger DNA sequencing of exon regions 15 to 14 of FLT3 outside, in order to obtain a mutated sequence, using the following primers Double Ended Sequencing:

[0119] Upstream sequen...

Embodiment 3

[0134] Example 3 A kit for detecting actual clinical samples

[0135] The present embodiment employs the actual clinical samples for testing digital droplet PCR Kit and method for detecting a human leukemia internal tandem duplication (ITD) mutations of the embodiment, the specific detection method:

[0136] (1) collected clinical samples: by 301 hospital carried FLT3-ITD mutations in six patients were extracted blood DNA samples treatment at different times, the hospital has been detected, these DNA samples FLT3-ITD mutation level by capillary electrophoresis, see table 6 below.

[0137] Table 6

[0138]

[0139] (2) First, DNA samples six different treatment period of the patient's condition for Sanger DNA sequencing of exon regions 15 to 14 of FLT3 outside, mutated sequences obtained, a total of 18. Using the following primers Double Ended Sequencing:

[0140] Upstream sequencing primer FLT3-Exon14-F: 5'-GCAATTTAGGTATGAAAGCCAGC-3 ';

[0141] Downstream sequencing primer FLT3-...

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Abstract

The invention discloses a primer probe combination for detecting human leukemia FLT3 gene internal tandem repeat mutation, a kit and application. In the primer probe combination, primer sequences are shown as SEQ ID NO.1 and SEQ ID NO. 2; and a probe sequence is shown as SEQ ID NO. 3. The kit is a microdroplet type digital PCR detection kit and comprises detection mixed liquid, reaction premixed liquid, a negative quality control material and blank control, wherein the detection mixed liquid is prepared from the primer probe combination of an FLT3 gene and a primer probe of a reference gene RPP 30. The primer probe combination and the kit disclosed by the invention are used for detecting the human leukemia FLT3 gene internal tandem repeat mutation, have the advantages of simple and efficient detection process, absolute quantification, high sensitivity and accuracy and the like and have potential application value for continuously monitoring a treatment effect, guiding medication and predicting disease progression of an AML patient carrying FLT3-ITD mutation.

Description

Technical field [0001] The present invention relates to the field of biotechnology, in particular, to one human leukemia FLT3 internal tandem duplication mutation primer probe combination, and application of a test kit. Background technique [0002] FLT3 gene is located on chromosome 13q12.2, with 24 exons encoding a protein belonging to one of the type III receptor tyrosine kinase family, the full name of FMS-like tyrosine kinase 3 (FMS-like tyrosine kinase 3), hematopoietic cell survival, proliferation, differentiation, and plays an important role. According to statistics reported tumor in 2015 China, China leukemia is the number 75300 / year, the death toll was 53400 / year, of which adult acute myeloid leukemia (AML) accounts for about 65% of adult leukemia, AML accounts for about 25% of children of childhood leukemia . The presence of FLT3 internal tandem duplication (ITD) mutations in AML patients about one third of the mutation in the FLT3 gene occurs primarily exon region...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q2545/101C12Q2545/113C12Q2563/159C12Q2537/16C12Q2563/107
Inventor 李静芳许少飞罗景燕赖炳权
Owner 广东永诺医疗科技有限公司
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