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Human PAH gene SNP typing rapid test paper strip detection method with characteristics of no extraction and direct amplification, and kit

A detection method and test strip technology, applied in the field of SNP detection, can solve the problems of being unsuitable for promotion and use in clinical hospitals, increasing cross-contamination of samples, and long detection time, saving the cost of DNA purification reagents, and improving detection sensitivity and accuracy. , the effect of reducing the cost of testing

Active Publication Date: 2018-06-05
XIAN GOLDMAG NANOBIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods generally have limitations such as complicated operation, long detection time, or the need for expensive large-scale instruments and equipment, and these methods need to purify DNA from samples. The complicated and cumbersome purification steps not only consume a lot of time, but also increase the cost. The risk of cross-contamination between samples, so it is not suitable for most clinical hospitals to promote the use in routine clinical tests

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  • Human PAH gene SNP typing rapid test paper strip detection method with characteristics of no extraction and direct amplification, and kit
  • Human PAH gene SNP typing rapid test paper strip detection method with characteristics of no extraction and direct amplification, and kit
  • Human PAH gene SNP typing rapid test paper strip detection method with characteristics of no extraction and direct amplification, and kit

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Experimental program
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Effect test

Embodiment 1

[0049] The design and optimization of embodiment 1 primer

[0050] Design primers for the seven mutation sites of PAH gene: confirm the flanking sequence of the SNP site through the dbSNP database of NCBI, and design synthetic primers accordingly. For each SNP site, a wild-type specific primer was designed, whose 3' end was complementary to the wild-type base of the SNP site, and a mutant-specific primer, whose 3' end was complementary to the mutant base of the SNP site and a Common primers for identification and amplification. In order to facilitate subsequent chromatographic detection techniques, the designed primers were labeled, among which the common primers were labeled with biotin, and the specific primers were labeled with digoxigenin. In order to increase amplification specificity and detection resolution, multiple sets of primers were designed for each locus for test screening.

[0051] The optimal primers were screened according to the following objectives: (1) Hi...

Embodiment 2

[0057] Embodiment 2, the detection of whole blood sample

[0058] The whole blood samples of 10 people were taken respectively, and the mutation types were detected respectively for the seven mutation sites involved in the present invention. The specific operation steps were as follows: take a small amount of whole blood samples and add them to PCR centrifuge tubes, and draw the samples with a pipette Add 10 μL of the treatment solution into a centrifuge tube, and let it stand for 5 minutes for later use; take two PCR centrifuge tubes, A and B, and add the following substances (mutant primers are added to tube A, and wild-type primers are added to tube B):

[0059]

[0060] Put the two tubes A and B into the PCR machine, and proceed according to the optimized amplification program as follows:

[0061] 50°C UNG enzyme action for 2min, 95°C pre-denaturation for 5min, 94°C denaturation for 30s, annealing at a temperature 5°C lower than the Tm value for 30s, 65°C extension for ...

Embodiment 3

[0066] Embodiment 3: the detection of oral swab sample

[0067] Using the same sample source individuals as in Example 2, the mutation types were detected for the seven mutation sites involved in the present invention. Draw 10 μL of the sample treatment solution into the centrifuge tube, and let it stand for 5 minutes for later use; take two PCR centrifuge tubes, A and B, and add the following substances respectively:

[0068]

[0069]

[0070] Put the two tubes A and B into the PCR machine, carry out the amplification reaction and test strip detection and judgment according to the same procedure as in Example 2. The results were the same as in Example 2, and were consistent with the sequencing results. It is shown that the method is also reliable when applied to buccal swab samples.

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Abstract

The invention discloses a rapid test paper strip detection method for directly amplifying seven hot spot mutation sites (R111X, IVS4-1, Y204C, R243Q, W326X, Y356X and R413P) of a human PAH gene without extraction, and a kit. The rapid test paper strip detection method comprises: directly adding a treatment liquid to a collected sample to prepare a sample liquid; carrying out identification and amplification on SNP allele sites based on an AS-PCR method; and achieving rapid genotyping by using the interaction between amplification fragment labeled digoxin and digoxin monoclonal antibody on thesurface of nanogold magnetic microparticles and by combining with a lateral flow chromatography technology. According to the present invention, with the rapid test paper strip detection method, the DNA separation extraction process is not performed, and the surface modified magnetic gold microparticles are used as the hybrid carrier, such that the problems of high cost, time saving and labor saving due to nucleic acid purification and product treatment can be eliminated, and high sensitivity and high accuracy are achieved.

Description

technical field [0001] The invention belongs to the technical field of SNP detection, in particular to a rapid test strip detection method for human PAH gene SNP typing without extraction and direct amplification. Background technique [0002] Phenylketonuria (PKU) is a common disorder of amino acid metabolism, caused by a defect in the phenylalanine hydroxylase gene (PAH) in the phenylalanine metabolic pathway, resulting in the conversion of phenylalanine into tyrosine. The acid pathway is blocked, resulting in the accumulation of phenylalanine and phenylpyruvate, and affecting the synthesis of neurotransmitters in the brain. PKU is an autosomal recessive inheritance, which mainly causes abnormal development of the nervous system in newborns. The main clinical features are mental retardation, neuropsychiatric symptoms, abnormal EEG, eczema, skin scratch signs, depigmentation, and rat odor. The incidence of phenylalanine varies from country to country, with an average of ab...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6804
CPCC12Q1/6804C12Q2531/113C12Q2563/131C12Q2563/143C12Q2563/149C12Q2565/625
Inventor 刘枭男崔亚丽张学张朝朱娟莉惠文利刘克武
Owner XIAN GOLDMAG NANOBIOTECH
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