Primer group and kit for single-tube detection of human spinal muscular atrophy

A spinal muscular atrophy, human detection technology, applied in recombinant DNA technology, microbial determination/examination, biochemical equipment and methods, etc. Application requirements, etc.

Pending Publication Date: 2020-03-17
GUANGZHOU DARUI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0014] The purpose of the present invention is to overcome the shortcomings of the prior art that the current human motor neuron gene detection technology and products cannot simultaneously meet the multiple clinical application requirements of patient diagnosis, phenotype typing, and carrier screening, and provide a simple-to-operate, Low-cost, fast and reliable primers and reagents for the single-tube detection based on the combination of QF-PCR technology and CE technology to simultaneousl...

Method used

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  • Primer group and kit for single-tube detection of human spinal muscular atrophy
  • Primer group and kit for single-tube detection of human spinal muscular atrophy
  • Primer group and kit for single-tube detection of human spinal muscular atrophy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0152] The selection of the primer design position of embodiment 1 highly homologous SMN1 and SMN2

[0153] Amplification primers include universal sequences (or specific sequences) and recognition sequences, among which the universal primers are used to realize the simultaneous amplification of different amplicons, and can bind and extend the amplification products of different recognition sequences, finally in the amplification program The upper Tm value is higher and the second cycle amplification; specific sequence, similar to the tag sequence on the next generation sequencing method, the designed primers for different targets include different nucleotide lengths, which are used to distinguish different SMN amplifications in length sub-length; recognition sequence, the recognition sequence on different primers can specifically bind to exon 7 of SMN1 and SMN2, exon 4 and exon 5 of NIAP gene, and exon 10 of GTF2H2 gene respectively , and extend and amplify. The following ma...

Embodiment 2

[0161] Example 2 Amplification blocking primer design of SNP site difference and amplification blocking primer design for copy number quantification of SMN1 / 2 and "2+0" carrier

[0162] For the difference base between SMN1 and SMN2, the SNP mutation site on the SMN1 gene, etc., the 3' end of the sequence adopts the amplification block design, which is different from the general amplification block design, which can only match the template at the 3' end, while the There is a mismatch with the homologous template, and the present invention introduces an additional 1 to 3 mismatched bases in the 2 to 5 bases at the 3' end. Based on the strength of base matching, while ensuring the quality of primers (try to avoid primer dimers, mismatches, hairpin structures, etc.), additional mismatched bases are introduced.

[0163] Primer design for different positions:

[0164] (1) Design primers for the differential bases in the 7th intron (+100A) of SMN1, except that the 3' end of the prim...

Embodiment 3

[0174] Example 3 Single-tube human spinal muscular atrophy-related gene copy number and point mutation detection, carrier typing kit

[0175] 1. Composition

[0176] The kits of the present invention are designed according to 50 people, including: 5 × PCR reaction mixture, 1 tube (275 μL / tube); primer mixture, 1 tube (138 μL / tube); PCR enzyme mixture, 1 tube (28 μL / tube); tube); SMN calibrator, 1 tube (275 μL / tube); SMN quality control product, 1 tube (275 μL / tube); diluent, 1 tube (1000 μL / tube).

[0177] Among them, the 5×PCR reaction mixture includes Tris-Hcl buffer 175mmol / L, potassium chloride 200mmol / L, ammonium sulfate 83mmol / L, calf serum albumin 500μg / mL, gelatin 0.05%, Tween-200.5%, di Thiothreitol 25mmol / L, dATP 1000nM, dGTP 1000nM, dCTP 1000nM, dTTP 500nM, dUTP 1000nM.

[0178] This kit designs primers based on the base difference between Exon 7 and Intron 7, and the PCR product is generally "Exon 7 of SMN1 + Intron 7 partial sequence" or "Exon 7 of SMN2 No. exo...

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Abstract

The invention discloses a primer group and a kit for detecting copy number and point mutation of a single-tube human spinal muscular atrophy related gene and typing a carrier. The nucleotide sequencesof the primer group are shown as SEQ ID NO. 1 to 38. The primer group is reasonable in design and high in specificity. The amplification bias is reduced, and the quantification accuracy is ensured. An identification sequence is introduced to realize identification of the conversion condition of the seventh exon of the SMN1 and the SMN2. UDG enzyme is introduced, and amplification products are cut, so as to avoid pollution. Clinical applications such as SMN1/2 gene copy number detection of human amyotrophy, SNP site detection of SMN1/2 Chinese population, NAIP and GTF2H2 gene copy number detection, typing of patients (I- VI types) and carriers ('1 + 0' and '2 + 0') and the like are simultaneously realized by the single tube. The method and the kit are rapid in detection, accurate in result, appropriate in cost, high in clinical occupancy of applicable instruments and suitable for clinical application.

Description

technical field [0001] The invention belongs to the field of in vitro diagnosis, in particular to a primer set and a kit for single-tube detection of human spinal muscular atrophy. Background technique [0002] Spinal muscular atrophy (SMA) is a recessive genetic disease characterized by progressive degeneration and loss of spinal cord anterior horn cells (ie, lower motor neurons) and brain stem cell nuclei, resulting in muscle weakness and atrophy. According to multi-ethnic studies on SMA, the overall carrier frequency of SMA is 1 / 40-1 / 100, the incidence rate is about 1 / 11000, and the carrier rate of the Chinese population is about 1 / 50. [0003] Survival motor neuron genes (SMN, NCBI #6606) are located at the 5q13 position of human chromosome 5, including survival motor neuron 1 (SMN1) on the telomeric side and survival motor neuron 2 (SMN2) on the centromere side . SMN1 and SMN2 are highly homologous, both contain 9 exons, and there are only 5 base differences. [0004...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858C12Q1/6851C12Q1/6883C12N15/11
CPCC12Q1/6858C12Q1/6851C12Q1/6883C12Q2600/156C12Q2600/172C12Q2531/113C12Q2547/101C12Q2537/16C12Q2563/107C12Q2545/101
Inventor 吴英松周其伟康小龙李明杨学习梁志坤
Owner GUANGZHOU DARUI BIOTECH
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