Kit for detecting thalassemia gene mutations

A kit and thalassemia technology are applied in the field of kits for detecting thalassemia gene mutations, which can solve the problems of incomplete detection sites, inability to meet methodological requirements, and high costs, and achieve low labor costs and detection costs, and flexibility. The effect of good sensitivity combined with scalability

Active Publication Date: 2019-12-17
SOUTHERN MEDICAL UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Commercial kits based on these two methods have been used clinically for more than 20 years. However, there are problems such as low throughput, long time-consuming, cumbersome operation, high cost, and incomplete detection sites, especially after PCR amplification. Serious consequences of false positives or false negatives caused by laboratory carry-over contamination caused by opening tubes for product analysis have obviously failed to meet the current methodological requirements for large-scale population molecular screening and routine molecular diagnosis

Method used

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  • Kit for detecting thalassemia gene mutations
  • Kit for detecting thalassemia gene mutations
  • Kit for detecting thalassemia gene mutations

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1. Primer Design

[0066] Amplification primers and extension primers were designed based on the deletion mutation truncating the target sequence contained in the thalassemia gene mutation hotspot, and the point mutation physical position target gene sequence.

[0067] Wherein, amplification primers include:

[0068] (1) Among a pair of primers that specifically amplify the Z-box region of the α2 globin gene,

[0069] The upstream primer sequence is: 5'-CTTTCCCTACCCCAGAGCCAAG-3' (SEQ ID NO:29),

[0070] The downstream primer sequence is: 5'-CGAGGCTCCAGCTTAACGGTATTT 3' (SEQ ID NO: 30);

[0071] (2) Among a pair of primers for specific amplification-SEA thalassemia gene deletion truncated sequence,

[0072] The upstream primer sequence is: 5'-TTCCCATATCGCACAAAGATTG-3' (SEQ ID NO:31),

[0073] The downstream primer sequence is: 5'-GGGCATAAAATTGTATGTG-3' (SEQ ID NO: 32);

[0074] (3) Among a pair of primers for specific amplification-α4.2 thalassaemia gene dele...

Embodiment 2

[0142] Example 2. Assembly of Thalassaemia Gene Mutation Detection Kit

[0143] 1. The kit for detecting gene mutation of the present invention comprises the following components:

[0144] (1) primer set, as shown in embodiment 1;

[0145] (2) PCR amplification system is as shown in embodiment 1;

[0146] (3) The dNTPs removal reaction system is shown in Table 2:

[0147] Table 2 dNTPs removal reaction system

[0148]

[0149] (4) The single base extension reaction system is shown in Table 3:

[0150] Table 3 The single base extension reaction system of the kit of the present invention

[0151]

[0152] Among them, the composition of each 100.00 μl extension primer mixture is shown in Table 4

[0153] Table 4 Extension Primer Mixture System

[0154]

[0155]

[0156] 2. How to use the above kit

[0157](1) According to the number of samples to be detected, according to Table 1, add each component of the overall PCR reaction system (except the template gDNA) ...

Embodiment 3

[0173] Example 3. Sensitivity and accuracy small sample evaluation of the kit

[0174] (1) Source and type of samples: select gDNA specimens of confirmed deletion α-thalassemia and β-thalassemia, and the genotypes are -α 3.7 / αα, -α 4.2 / αα, -- SEA / αα, -- SEA / -α 4.2 、-- SEA / -- SEA 、- SEA / -α 3.7 , -α 3.7 / -α 3.7 , -α 4.2 / -α 4.2 , αα / α WS α, αα / α QS α, αα / α CS α,, α WS α / α WS α, α CS α / α CS α, α QS α / α QS α, β N / β CD41-42 , β CD41-42 / β CD41-42 , β N / β IVS-II-654 , β IVS-II-654 / β IVS-II-654 , β N / β -28 , β -28 / β -28 , β N / β CD71-72(+A) , β CD71-72(+A) / β CD71-72(+A) , β N / β CD17 , β CD17 / β CD17 , β N / β CD26 , β CD26 / β CD26 , β N / β IVS-I-1 , β IVS-I-1 / β IVS-I-1 , β N / β CD43 , β CD43 / β CD43 , β N / β -32 , β -32 / β -32 , β N / β -29 , β -29 / β -29 , β N / β Cap+1 , β Cap+1 / β Cap+1 , β N / β Int , β Int / β Int 1 part each (38 parts in total), dilute gDNA samples to 15-200ng / μl with sterilized double ...

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Abstract

The invention belongs to the technical field of nucleic acid detection, and particularly relates to a kit for detecting thalassaemia gene mutations. The kit includes amplification primers shown as SEQID NO:1-SEQ ID NO:10 and extension primers shown as SEQ ID NO:11-SEQ ID NO:28. The kit realizes the genotyping of various types of mutation hotspots of thalassaemia genes in the same reaction system,has both flexibility and scalability, is simple in operation and high in throughput and low in cost, and is of great significance for the screening, prenatal diagnosis and the like on people with thalassaemia.

Description

technical field [0001] The invention belongs to the technical field of nucleic acid detection, and in particular relates to a kit for detecting thalassemia gene mutation. Background technique [0002] Thalassemia ("thalassemia" for short) is a group of hereditary hemolytic diseases that have the greatest impact on human health. The molecular mechanism is derived from the α-globin gene cluster (16p13.3) or the β-globin gene cluster (11p15.5) gene deletion or mutation. Fetal death caused by severe α-thalassemia caused serious complications to pregnant women; children with severe β-thalassemia developed chronic progressive anemia 3 to 6 months after birth, and depended on blood transfusion and iron removal therapy for life, more than adolescents Death, although bone marrow transplantation can be used, but factors such as tissue matching, bone marrow source, and treatment costs determine that this treatment is only suitable for a very small number of patients. The disease is i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/6858
CPCC12Q1/6858C12Q1/6883C12Q2600/156C12Q2531/113C12Q2533/101C12Q2521/525C12Q2565/627
Inventor 周万军杨学习朱安娜李明李亮许明丽
Owner SOUTHERN MEDICAL UNIVERSITY
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