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MyD88 gene L265P mutation detection kit and detection method based on ARMS-PCR

A mutation detection and kit technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems of low detection rate and poor detection sensitivity, and achieve improved specificity and sensitivity. and low specificity, high detection sensitivity and specificity

Inactive Publication Date: 2020-04-10
苏州艾可瑞斯生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The Sanger sequencing method currently used for this mutation detection has the characteristics of low detection rate and poor detection sensitivity

Method used

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  • MyD88 gene L265P mutation detection kit and detection method based on ARMS-PCR
  • MyD88 gene L265P mutation detection kit and detection method based on ARMS-PCR
  • MyD88 gene L265P mutation detection kit and detection method based on ARMS-PCR

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Such as figure 1 As shown, a MyD88 gene L265P mutation detection kit based on ARMS-PCR, comprising: 1. Peripheral blood mononuclear cell enrichment reagent; 2. Proteinase K; 3. DNA extraction column; 4. DNA extraction sleeve 5. Cleaning solution A; 6. Cleaning solution B; 7. DNA eluent; 8. ARMS-PCR specific primer Fw; 9. ARMS-PCR specific primer Fm; 10. ARMS-PCR specific primer R0; 11. Reagents for ARMS-PCR reaction system.

[0045] Wherein, the ARMS-PCR specific primers are respectively:

[0046] Fw: 5'-gtgcccatcagaagcgcct-3';

[0047] Fm: 5'-gtgcccatcagaagcgccc-3';

[0048] R0: 5'-GACGTGTCTGTGAAGTTGGCATCTC-3'.

[0049] Wherein, the ARMS-PCR reaction system reagents include: 2 μl of DNA template, 10 μl of Power SYBR Green premixed enzyme solution, 1 μl of 1 μM upstream and downstream primers (wild-type and mutant upstream primers were added to different systems), deionized 6 μl of enzyme-free water, 20 μl in total.

[0050] The method for detecting gene L265P mut...

Embodiment 2

[0057] An ARMS-PCR-based MyD88 gene L265P mutation detection kit and detection method, the composition of which is the same as that of Example 1.

[0058] Using the above method, Sanger sequencing and ARMS-PCR detection were performed on the mixed plasmids with mutation ratios of 0%, 25%, 75% and 100%, respectively. The results showed that the consistency of the two methods was 100% (Table 1), indicating that this study The established ARMS-PCR method has good consistency with the clinical "gold standard" method.

[0059] Table 1, ARMS-PCR method accuracy verification table

[0060]

Embodiment 3

[0062] An ARMS-PCR-based MyD88 gene L265P mutation detection kit and detection method, the composition of which is the same as that of Example 1.

[0063] Using the above method to adjust the concentration to 10ng / μl, the results of the detection of mixed plasmids with a mutation ratio of 0%, 1%, 2%, 5%, 10%, 20% and 100% showed that the mixed plasmid with a mutation ratio of 1% The proportion of plasmids with Ct values ​​> 32 was relatively high, and for 2% of the mixed plasmids, the proportion of "mutation detected" was > 95%; while for the mixed plasmids with a mutation ratio of 5% to 100%, the coincidence degree was 100%. Therefore, 2% was defined as the detection limit of the method.

[0064] 21. ARMS-PCR Method Analysis Degree Verification Form

[0065]

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Abstract

The invention provides a MyD88 gene L265P mutation detection kit based on ARMS-PCR. The MyD88 gene L265P mutation detection kit comprises (1) a peripheral blood mononuclear cell enrichment reagent, (2) protease K, (3) a DNA extraction column, (4) a DNA extraction sleeve, (5) cleaning liquid A, (6) cleaning liquid B, (7) a DNA eluent, (8) an ARMS-PCR specific primer Fw, (9) an ARMS-PCR specific primer Fm, (10) an ARMS-PCR specific primer R0, and (11) an ARMS-PCR reaction system reagent. A mismatch is artificially introduced to the 3'end of the specific primers to improve the specificity of thespecific primers. The concentrations of a DNA template and the primers in the reaction are controlled at a low level to ensure the specificity of the reaction.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an ARMS-PCR-based MyD88 gene L265P mutation detection kit and detection method. Background technique [0002] Lymphoplasmacytic lymphoma (LPL) / Wahrenheit macroglobulinemia ( Macroglobulinemia (WM) is a hematological neoplastic disease, the incidence of which is positively correlated with age, and has a certain familial tendency. MyD88 gene L265P mutation is one of the most common somatic mutations in LPL / WM patients, and its detection rate can reach more than 90%, and this mutation is related to the growth and survival of tumor cells. Although the MyD88 L265P mutation can also be detected in some other hematological diseases such as diffuse large B-cell lymphoma and IgM monoclonal gammopathy of undetermined significance, in 2016 this mutation is still an important The auxiliary diagnostic index of lymphoplasmacytic lymphoma / Wahrenheit's macroglobulin has been written ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/686C12N15/11
CPCC12Q1/6886C12Q1/686C12Q2600/156C12Q2535/137
Inventor 袁嘉扬
Owner 苏州艾可瑞斯生物科技有限公司
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