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Neuronal intranuclear inclusion disease NOTCH2NLC gene GGC repeat amplification detection kit

A kit and neuron technology are applied in the field of detection of GGC repeat expansion of NOTCH2NLC gene in neuron nuclear inclusion body disease, which can solve the problems of easy misdiagnosis or missed diagnosis, difficulty in amplification of high GC sequences, and difficult detection, etc. The effect of small amount, overcoming the limitation of amplification length, and low price

Pending Publication Date: 2020-12-18
杭州方夏生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the difficulty of high GC sequence amplification, it is difficult for laboratories to perform this test, and no company has launched a corresponding product. If the operation is not performed properly, misdiagnosis or missed diagnosis will easily occur.

Method used

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  • Neuronal intranuclear inclusion disease NOTCH2NLC gene GGC repeat amplification detection kit
  • Neuronal intranuclear inclusion disease NOTCH2NLC gene GGC repeat amplification detection kit
  • Neuronal intranuclear inclusion disease NOTCH2NLC gene GGC repeat amplification detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Preparation of NOTCH2NLC gene GGC repeat rapid screening kit

[0027] 1. Primer design

[0028] A pair of PCR system I specific upstream primer NOTCH2NLC-F and downstream primer NOTCH2NLC-R were designed within 200 bp of the upstream 5' end and downstream 3' end of the GGC repeat sequence of the NOTCH2NLC gene to amplify the target genome including the GGC repeat region, 5' flanking region and 3' flanking region. The 5' flanking region is 80bp, the 3' flanking region is 158bp, and the length of the amplified fragment is (238+3xn)bp, where n represents the number of GGC repeats. The formula for calculating the GGC repeat number in the normal range is: GGC repeat number=(PCR product length-238) / 3. When the length of the PCR product is 358bp, it is the upper limit of the normal value (40 repeats). When the number of repeats exceeds 100 GGCs, the resolution of capillary electrophoresis will continue to decrease, so the number of repeats needs to be corrected b...

Embodiment 2

[0037] Example 2: PCR system 1 screening of abnormally amplified GGC samples

[0038]Use a commercially available genomic DNA extraction kit to extract genomic DNA from the peripheral blood of female samples with a concentration of at least 50ng / μL, 260 / 230 value greater than 2.0, and 260 / 280 value greater than 1.8.

[0039] PCR system I reaction system prepared using the kit of Example 1: prepare 25 μL PCR system in a 200 μL PCR thin-walled tube. The sequences of primers NOTCH2NLC-F and NOTCH2NLC-R are: SEQ ID NO:1 and SEQ ID NO:2, respectively. PCR amplification program: pre-denaturation at 98°C for 10 minutes; 30 cycles of 97°C for 35 seconds→65°C for 35 seconds→68°C for 5 minutes; extension at 72°C for 10 minutes. After the reaction, 1 μL PCR product was taken, mixed with 10 μL deionized formamide and 1 μL DNA internal standard ROX1000, denatured at 95°C for 5 minutes, and cooled on ice. The mixture was subjected to capillary electrophoresis with ABI 3730, the injection ...

Embodiment 3

[0040] Example 3: PCR system 2 to screen abnormally amplified samples of GGC

[0041] Use the kit of Example 1 to prepare PCR system 2 reaction system: prepare 25 μL PCR system in a 200 μL PCR thin-walled tube.

[0042] The sequence of primer NOTCH2NLC-R is SEQ ID NO:2, the sequence of NOTCH2NLC-T is SEQ ID NO:3; the sequence of NOTCH2NLC-GGC4 is SEQ ID NO:4. PCR amplification program: pre-denaturation at 98°C for 10 minutes; 98°C for 45 seconds → 64°C for 45 seconds → 68°C for 6 minutes, 10 cycles; 98°C for 45 seconds → 68°C for 5 minutes, 20 cycles; extension at 72°C for 10 minutes . After the reaction, 1 μL of PCR product was taken, mixed with 10 μL of deionized formamide and 1 μL of DNA internal standard ROX1000, denatured at 95° C. for 5 minutes, and cooled on ice. The mixture was subjected to capillary electrophoresis with ABI 3730, the injection voltage was 2.5 kV, 20 seconds, and the electrophoresis time was 4000 seconds. The capillary electrophoresis diagram of PCR...

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Abstract

The invention provides a method and kit for rapidly detecting abnormal GGC repeat amplification of NOTCH2NLC genes. The components of the kit include a reaction solution, a reinforcing agent, a specific primer, dNTPs and DNA polymerase. Through a high GC content PCR system, GGC can be repeatedly amplified through the DNA polymerase of high thermal stability and high GC content templates, so that the number of GGC repeat of to-be-detected samples can be calculated through fluorescence scanning, and whether amplification occurs can be judged as well. The kit can detect the repeat number of NOTCH2NLC gene GGC through the PCR system 1, determine whether the repeat number is in a normal range or a mutation range, and screen whether subjects carry GGC pathogenic mutation; and whether amplification exists can be judged through the trapezoidal peak of a PCR system 2, so that missed detection can be avoided. The method can be applied to neuronal intranuclear inclusion diseases and the gene detection in some dementia patients. The kit has the characteristics of being fast, accurate and high insensitivity, and can prevent missed detection.

Description

technical field [0001] The invention relates to the field of molecular biology diagnosis, in particular to a method and a kit for detecting the GGC repeat amplification of the NOTCH2NLC gene of neuron intranuclear inclusion body disease. Background technique [0002] Neuronal Intranuclear Inclusion Disease (NIID) is a rare multisystem chronically progressive neurodegenerative disease characterized by eosinophilic hyaline inclusions in the central and peripheral nervous systems and internal organs. The clinical presentation of NIID is highly heterogeneous depending on the primary site of neuronal loss and includes mainly pyramidal and extrapyramidal symptoms, cerebellar ataxia, dementia, seizures, peripheral neuropathy, and autonomic dysfunction Wait. Most of the onset is juvenile, a few are middle-aged, and familial is common. Usually 10-20 years after the onset of death. [0003] The first case of NIID was reported by Lindenderg et al. in 1968. In a male patient with men...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/6858
CPCC12Q1/6883C12Q1/6858C12Q2600/156C12Q2531/113C12Q2525/151
Inventor 段然慧唐北沙金赟懿
Owner 杭州方夏生物科技有限公司
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