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A kind of npm1 mutation detection kit

A mutation detection and kit technology, applied in the biological field, can solve the problems of easy error in results, low sensitivity of the kit, false positives, etc., and achieve the effects of high accuracy, enhanced sensitivity, and strong specificity

Active Publication Date: 2021-03-19
宁波胤瑞生物医学仪器有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the sensitivity of the kit is relatively low, and it is prone to false positives or false negatives
[0005] Aiming at the problems of high cost and error-prone results in NPM1 mutation detection, the present invention provides a NPM1 mutation detection kit, which has high sensitivity and accuracy, strong specificity, and can quickly detect NPM1 A type mutation

Method used

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  • A kind of npm1 mutation detection kit
  • A kind of npm1 mutation detection kit
  • A kind of npm1 mutation detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] A NPM1 mutation detection kit, including upstream primers, downstream primers, specific probes, RT-PCR reaction enzyme system, RT-PCR master mix, DEPC H 2 O, dNTPs and quality controls;

[0038] Among them, the RT-PCR master mix includes: 14mmol / LMgCl 2 , 110mmol / LKCl, 7mmol / L tricalcium phosphate, 3.2mmol / L gentiobiose, 0.68mmol / L parathyroid hormone and 35mmol / L glycine-MOPS-HCl buffer system; the pH of the RT-PCR master mix is ​​8 ;

[0039] The upstream primer is: 5'-ATTGCTTCCGGATGACTG-3';

[0040] The downstream primer is: 5'-CTGTTACAGAAATGAAATAAGACG-3';

[0041] The specific probe is: 5'-FAM-CCTCCAGATCTCTGTCTGGCAGTGGAGG-BHQ1-3'.

[0042] Wherein, the concentrations of the upstream primer and the downstream primer are both 0.35 μmol / L, and the concentration of the probe is 0.18 μmol / L;

[0043] The RT-PCR reaction enzyme system includes: Taq enzyme, reverse transcriptase and phosphatidylserine, the weight ratio of the three is 1:2:2;

[0044] The quality contro...

Embodiment 2

[0054] The only difference from Example 1 is that the RT-PCR master mix includes: 12mmol / LMgCl 2 , 100mmol / L KCl, 5mmol / L tricalcium phosphate, 3mmol / L gentiobiose, 0.5mmol / L parathyroid hormone and 30mmol / L glycine-MOPS-HCl buffer system, the pH of the RT-PCR master mix is ​​7.5; The concentration of upstream primer and downstream primer is 0.12 μmol / L, the concentration of probe is 0.12 μmol / L; the molar ratio of glycine, MOPS and HCl in the glycine-MOPS-HCl buffer system is 5:30:15. The RT-PCR reaction enzyme system includes: Taq enzyme, reverse transcriptase and phosphatidylserine, and the weight ratio of the three is 1:2:1.6. In addition, the cycle conditions in the detection method of using the above kit for non-disease diagnosis purposes are: 50°C for 35min, 1 cycle; 94°C for 2min, 1 cycle; 94°C for 30s, 60°C for 40s, 3 cycles; 94°C 15s, 50°C 40s, 63°C 35s, 40 cycles.

Embodiment 3

[0056] The only difference from Example 1 is that the RT-PCR master mix includes: 15mmol / LMgCl 2 , 120mmol / L KCl, 8mmol / L tricalcium phosphate, 5mmol / L gentiobiose, 0.8mmol / L parathyroid hormone and 40mmol / L glycine-MOPS-HCl buffer system, the pH of the RT-PCR master mix is ​​8.8; The concentration of upstream primer and downstream primer was 0.45 μmol / L, and the concentration of probe was 0.2 μmol / L; the molar ratio of glycine, MOPS and HCl in the glycine-MOPS-HCl buffer system was 10:50:25. The RT-PCR reaction enzyme system includes: Taq enzyme, reverse transcriptase and phosphatidylserine, and the weight ratio of the three is 1:2:2.4. In addition, the cycle conditions in the detection method of using the above kit for non-disease diagnosis purposes are: 50°C for 35min, 2 cycles; 95°C for 2min, 1 cycle; 95°C for 15s, 65°C for 30s, 8 cycles; 95°C 10s, 60°C for 30s, 65°C for 20s, 40 cycles.

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Abstract

The invention provides an NPM1 mutation detection kit, and relates to the field of biotechnology. The NPM1 mutation detection kit comprises an upstream primer, a downstream primer, a specific probe, an RT-PCR reaction enzyme system, an RT-PCR premix solution, DEPC H2O, dNTPs and quality control products, wherein the upstream primer is 5'-ATTGCTTCCGGATGACTG-3', the downstream primer is 5'-CTGTTACAGAAATGAAATAAGACG-3', and the specific probe is 5'-FAM-CCTCCAGATCTCTGTCTGGCAGTGGAGG-BHQ1-3'. The kit has the advantages of high sensitivity, good accuracy and high specificity. Through a detection method utilizing the kit, namely sample extraction, PCR amplification and PCR fluorescence detection, NPM1-A type mutation can be quickly detected.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a NPM1 mutation detection kit. Background technique [0002] NPM1 is a protein widely expressed in the nucleolus. It can shuttle between the nucleus and the cytoplasm, participate in the assembly and transport of ribosomal proteins, regulate the duplication of centrosomes and regulate the expression of the tumor suppressor ARF. Usually, the mutation of NPM1 This leads to the translocation of the protein from the nucleus to the cytoplasm, causing acute myeloid leukemia. About 55 variants of NPM1 mutations have been found so far, most of which involve insertion mutations on exon 12, especially the insertion of 4 bases. Among them, the most frequent type A mutation (insertion of TCTG), about Accounts for 77%-80% of mutations. The frequency of NPM1 mutations is high, and patients with NPM1 mutations usually have a high remission rate, long survival period and stable condition, so NPM1 ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6886C12Q1/686
CPCC12Q1/686C12Q1/6886C12Q2600/156C12Q2521/107C12Q2563/107
Inventor 隋硕任鲁风金鑫浩刘一博张未来俞育德于军
Owner 宁波胤瑞生物医学仪器有限责任公司