Detection kit and detection method for folate metabolism related molecular marker gene mutation

A technology of molecular markers and detection kits, applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., can solve the cost of only one gene detection and cannot be applied to large-scale clinical samples Research and other issues to achieve the effects of improved detection sensitivity and specificity, fast detection speed, and low cost

Pending Publication Date: 2021-03-19
江苏博嘉生物医学科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, patients are generally tested by quantitative real-time polymerase chain reaction (Quantitative real timepolymerase chain reaction, Q-PCR), non-radioactive in situ hybridization (fluorescence in situ hybridization, Fish), multiplex primer PCR (multiplex PCR), etc. Gene mutation detection of folic acid metabolism-related loci, however, these detection technologies have problems such as only one gene can be detected at a time or the detection cost is too high, which makes it impossible to apply to large-scale clinical sample research

Method used

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  • Detection kit and detection method for folate metabolism related molecular marker gene mutation
  • Detection kit and detection method for folate metabolism related molecular marker gene mutation
  • Detection kit and detection method for folate metabolism related molecular marker gene mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Design and acquisition of crRNA targeting gene mutation site

[0040] 1. Discovery of folic acid metabolism detection sites based on CRISPR-Cpf1 system

[0041] According to the gene sequence and a large amount of clinical test data, the common mutation site sequence of the folic acid generation-related mutation gene was determined, and crRNA was designed for these different regions and the CRISPR-Cpf1 system was constructed for research. The results show that the sequence of the region shown in SEQ ID NO.1-3 in Table 1 is used as the detection site for the mutation of folic acid metabolism based on the CRISPR-Cpf1 system, which has a good detection effect.

[0042] Table 1 Detection sites for mutations in folic acid metabolism

[0043]

[0044]Among them, the MTHFR-667 mutation site, that is, there is a C / T polymorphism at the 677th position of the MTHFR gene, corresponding to the C→T mutation at the 25th bp in SEQID NO.1; the MTHFR-1298 mutation site, th...

Embodiment 2

[0061] Example 2 Detection Kit and Detection Method for Gene Mutation of Molecular Markers Related to Folic Acid Metabolism

[0062] 1. The composition of the kit

[0063] This kit includes 6 crRNAs for folic acid metabolism detection (crRNAs corresponding to 3 mutation sites are obtained as shown in Example 1) or crDNA of 6 mutation sites (when crDNA is used in the kit, the operator needs to first The crDNA fragments were generated RNA under the action of T7 RNA polymerase, recovered and purified to obtain crRNA, see Example 1), a specific fluorescent probe (as shown in Table 3), and Cpf1 protein (this example uses FnCpf1) , enzyme-free water, DNase inhibitors;

[0064] Table 3 Fluorescent probe sequences

[0065] fluorescent probe Sequence (5'-3') Probe 1 FAM-TTTTTTTT-BHQ1 Probe 2 FAM-TTTTTTTTTT-BHQ1 Probe 3 FAM-TTTTTTTTTTTT-BHQ1

[0066] Further, the kit may also include an amplification system, the amplification system includes a pai...

Embodiment 3

[0079] Example 3 crRNA specific detection of wild-type and mutant sequences

[0080] Synthesize the target sequences of the wild strain (WT) and the mutant strain (MUT), and use the 6 crRNAs designed in Example 1 to construct the detection system for detection and screening, and the detection results are as follows figure 1 Shown, wherein 667-crRNA1 is the sequence shown in SEQ ID NO.11, 667-crRNA2 is the sequence shown in SEQ ID NO.12; 1298-crRNA1 is the sequence shown in SEQ ID NO.13, 1298-crRNA2 is the sequence shown in SEQ ID NO.13 The sequence shown in .14; 66-crRNA1 is the sequence shown in SEQ ID NO.15, and 66-crRNA2 is the sequence shown in SEQ ID NO.16. According to the detection results, compared with the wild strain, the fluorescence value changes more significantly in the mutant strain, that is, the crRNA with higher sensitivity and better effect is selected, which are 667-crRNA2 shown in SEQ ID NO.12 and SEQ ID NO.13 respectively. 1298-crRNA1 shown and 66-crRNA1 ...

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Abstract

The invention discloses crRNA, a kit and a method for detecting folate metabolism related molecular marker gene mutation. The crRNA comprises crRNA of a mutation site of MTHFR-667 with a sequence as shown in any one of SEQ ID NO.11-12; crRNA of a mutation site of MTHFR-1298 with a sequence as shown in any one of SEQ ID NO.13-14; and crRNA of a mutation site of MTRR-66 gene with a sequence as shownin any one of SEQ ID NO. 15-16. By screening crRNA and combining with a CRISPR-Cpf1 system, whether folate metabolism related molecular marker gene mutation exists in a to-be-detected sample or not can be detected in a short time, the kit and the detection method are easy to operate, high in detection speed and low in cost, repeated detection can be achieved, and meanwhile detection sensitivity and detection specificity are remarkably improved.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a detection kit and a detection method for gene mutations of molecular markers related to folic acid metabolism. Background technique [0002] Folic acid, also called pteroyl glutamic acid, belongs to the B vitamins and is an important nutrient needed by the human body. Folic acid acts in the form of tetrahydrofolate in the body, and as a donor of one-carbon units, participates in DNA oxidative damage repair, cell proliferation and tissue growth, helps protein metabolism, and promotes the production and maturation of red blood cells together with vitamin B12. A variety of enzymes in the folic acid metabolic pathway participate in the metabolism and transport of folic acid, and methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (methionine synthase reductase, MTRR) are the two most critical enzymes. A large number of studies hav...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6858C12N15/113
CPCC12Q1/6858C12Q2531/119C12Q2537/1376C12Q2563/107C12Q2527/127C12Q2521/327C12Q2565/625
Inventor 姚杰赵洪友程诚
Owner 江苏博嘉生物医学科技有限公司
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