Probe, kit and method thereof for detecting MAPT gene mutation

A kit and probe technology, applied in the field of biomedicine, can solve the problems of requiring a large number of manual operations, large human errors, and low degree of automation, and achieve the effects of reducing the probability of cross-contamination, being easy to automate, and improving accuracy

Pending Publication Date: 2017-03-22
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But there are many deficiencies in this method: first, this method relies on the activity of restriction endonucleases, and it is easy to produce false positive and false negative results in the process of enzyme digestion; secondly, the method of electrophoresis is likely to cause contamination of PCR products, resulting in erroneous results. Again, this method is time-consuming (about 5-6 hours) and labor-intensive, including at least four steps of PCR amplification, enzyme digestion, electrophoresis, and gel imaging analysis, requiring a lot of manual operations, low degree of automation, and large human errors
At present, there is no report on the use of real-time fluorescent PCR to study the mutations of MAPT genes R5L, A152T, L284R, S285R, N296del and G303V

Method used

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  • Probe, kit and method thereof for detecting MAPT gene mutation
  • Probe, kit and method thereof for detecting MAPT gene mutation
  • Probe, kit and method thereof for detecting MAPT gene mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Real-time fluorescent PCR detection of the genotype of the MAPT gene encoding protein R5L mutation site

[0044] 1. Materials

[0045] 1. Instrument:

[0046] Real-time fluorescent PCR instrument, pipette, centrifuge.

[0047] 2. Primer and probe design:

[0048] The present invention designs primers capable of specifically amplifying the R5 site of the protein encoded by the MAPT gene, and probes that specifically recognize the wild type and the mutant type of the R5 site of the protein encoded by the MAPT gene. The 5' end of the wild-type probe is labeled FAM, and the 3' end is labeled BHQ1; the 5' end of the wild-type probe is labeled HEX, and the 3' end is labeled BHQ1.

[0049] The primers and probes used are listed in Table 1.

[0050] Table 1 Primer and probe information list

[0051]

[0052]

[0053] 3. Reagents:

[0054] 1×PCR buffer: 10mmol / L Tris-HCl (pH 8.3), 50mmol / L KCl; MgCl 2 ; Hot start Taq enzyme; dATP, dCTP, dGTP, dTTP.

[005...

Embodiment 2

[0069] Example 2 Real-time fluorescent PCR detection of the genotype of the MAPT gene-encoded protein A152T mutation site

[0070] According to the method and steps of Example 1, real-time fluorescent PCR amplification was performed.

[0071] The primers and probes used are listed in Table 1.

[0072] Result analysis:

[0073] Genotype determination of the A152 locus of the MAPT gene-encoded protein: samples with amplification signals only in the FAM channel are homozygous for WT / WT; samples with amplification signals only in the HEX channel are homozygous for the A152T / A152T mutation; Samples that produced amplified signals in both the FAM and HEX channels were WT / A152T heterozygotes.

[0074] from figure 2 It can be seen that (A) is the positive standard 1 of the known MAPT genotype, which only has an amplification signal in the FAM channel, and is WT / WT homozygous, which is consistent with the actual situation; (B) is the known MAPT gene Genotype positive standard 2, o...

Embodiment 3

[0075] Example 3 Real-time fluorescent PCR detection of the genotype of the L284R mutation site of the protein encoded by the MAPT gene

[0076] According to the method and steps of Example 1, real-time fluorescent PCR amplification was performed.

[0077] The primers and probes used are listed in Table 1.

[0078] Result analysis:

[0079] Genotype determination of the L284 locus of the MAPT gene-encoded protein: samples with amplification signals only in the FAM channel are homozygous for WT / WT; samples with amplification signals only in the HEX channel are homozygous for the L284R / L284R mutation; Samples that produced amplified signals in both the FAM and HEX channels were WT / L284R heterozygotes.

[0080] from image 3 It can be seen that (A) is the positive standard 1 of the known MAPT genotype, which only has an amplification signal in the FAM channel, and is WT / WT homozygous, which is consistent with the actual situation; (B) is the known MAPT gene The positive stand...

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Abstract

The invention discloses a probe, a kit and a method thereof for detecting MAPT gene mutation, and relates to the field of bio-medicine. The probe for detecting the MAPT gene mutation includes, but not limited in, at least one of a TaqMan probe, a TaqMan-MGB probe, a molecular beacon, a modified molecular beacon, a double-stranded fluorescent displacing probe, a LightCycler probe, a double-loop probe and the like. The kit for detecting the MAPT gene mutation consists of an amplification primer, a fluorescent probe, an amplification reagent and a reference reagent. The method for detecting the MAPT gene mutation comprises the following steps: 1) selecting a sample; 2) extracting genome DNA; 3) conducting real-time fluorescent PCR amplification and detection; and 4) conducting result analysis. Experimental results can be observed in real time. Meanwhile, the probe, the kit and the method are more sensitive and rapid in detection, more convenient to operate and easy for automation, and the probability of cross pollution can be greatly reduced and the accuracy of detection can be improved. Cost is saved, and meanwhile flux is improved.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a probe, a kit and a method for detecting MAPT gene mutation. Background technique [0002] Progressive supranuclear palsy (PSP), also known as Steele-Richardson-Olszewski syndrome, is a clinically rare degenerative disease of the central nervous system. PSP is the most common degenerative parkinsonian syndrome after Parkinson's disease (PD), and its incidence accounts for about 6%-10% of PD. It was first discovered and described by Posey in 1904. In 1964, Steele et al. (Steele JC, Richardson JC, et al. .Arch Neurol,1964,10:333-359.) This disease is listed as an independent nervous system disease. The main clinical features of PSP are postural instability, vertical supranuclear palsy, pseudobulbar palsy, supranuclear oculomotor disturbance, cervical dystonia, dysarthria, pseudobulbar palsy, mild dementia and paresthesia. Similar symptoms of Kinson syndrome, etc. [0003] The onset ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6883C12Q1/6858C12Q2600/156C12Q2531/113C12Q2561/101C12Q2563/107
Inventor 张云武孟健张慕娴谢勇壮许华曦卜国军
Owner XIAMEN UNIV
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