Real-time fluorescence quantitative PCR(Polymerase Chain Reaction) detection primers and kit for thalassemia

A real-time fluorescence quantitative, thalassemia technology, applied in the field of medical detection, can solve the problems of affecting the resolution, distorted results, inability to achieve, etc., to achieve high detection sensitivity, accurate and reliable results, and meet the effect of rapid detection.

Inactive Publication Date: 2012-04-11
潮州市中心医院
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0034] 2.1 Gap PCR (gap-PCR), this method is the most commonly used detection method for deletion mutations, one of its principles is to design a pair of primers in the deletion region, due to the complete deletion of four α genes (Barts' edema fetus ), the primers have no complementary templates, so the corresponding PCR products cannot be amplified, but this method cannot identify heterozygous carriers
If you want to use the Sybr Green I melting curve to distinguish SNPs, it seems that it is currently impossible to achieve, which is related to the characteristics of this type of dye
Dyes such as Sybr Green I belong to unsaturated dyes. Due to the inhibitory effect of the dye on PCR, the concentration used in the experiment is very low, which is far lower than the concentration that saturates the minor groove in the DNA double helix structure. Saturation, coupled with the characteristics of the dye itself, in the process of DNA double-strand melting, Sybr Green I molecules rearrange, and those dye molecules that have been separated from the melted DNA fragments combine with the unmelted double-stranded DNA , causing the result to be distorted, unable to truly reflect the melting of DNA, and affecting the detection resolution

Method used

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  • Real-time fluorescence quantitative PCR(Polymerase Chain Reaction) detection primers and kit for thalassemia
  • Real-time fluorescence quantitative PCR(Polymerase Chain Reaction) detection primers and kit for thalassemia
  • Real-time fluorescence quantitative PCR(Polymerase Chain Reaction) detection primers and kit for thalassemia

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] The thalassemia gene diagnostic kit described in this embodiment,

[0083] Include primers from Table 2:

[0084] Table 2. Primers used for HBB gene in HRM mutation screening.

[0085]

[0086]

[0087] Fluorescent dye component: LC Green Plus.

[0088] Other dyes such as LC green, SYTO 9, Eva Green or other saturating fluorescent dyes can also be used in the present invention.

[0089] Experimental procedure

[0090] (1) DNA extraction: 2 ml of peripheral venous blood was collected using EDTA anticoagulant tubes. Genomic DNA was extracted using a DNA extraction kit, and diluted to 10-20 ng / μl after quantification.

[0091](2) Primer design: According to the gene mutation characteristics of HBB in southern my country, 6 pairs of primers were designed (Table 1, Figure 7 ), in the design, according to the inventor's rich experience and a large number of experiments, design primers that cover as many known β-thalassaemia mutation sites in the Chinese population...

Embodiment 2

[0111] Genetic diagnosis of non-deletion alpha-thalassemia.

[0112] The common non-deletion α-thalassemia genotypes in China are CS (constant spring), QS (Quong Sze) and WS (Westmead).

[0113] Experimental procedure

[0114] (1) DNA extraction: 2 ml of peripheral venous blood was collected using EDTA anticoagulant tubes. Genomic DNA was extracted using a DNA kit, and diluted to 10-20ng / μl after quantification.

[0115] (2) Primer Design: Primer Forward:

[0116] 5'-CACTGCCTGCTGGTGACC-3' (SEQ ID.NO.13)

[0117] Reverse: 5'-GCAGGAGGAACGGCTACC-3' (SEQ ID.NO.14)

[0118] (3) Use 96-well plates (for Roche480) for PCR amplification, the amplification system is 20 μl: 1 μl (about 10-20ng) of genomic DNA or 10 3 Copy / ml plasmid 1 μl, 5×reaction buffer, dNTP 250 μM, primers 0.2 μM, 1×LC Greens PLUS dye (Idaho Technology), Promega polymerase 1.0 U, add sterilized distilled water to 20 μL. Cycle conditions: 95°C for 3min; 98°C for 10s, 60°C for 10s, 72°C for 20s, 33 cycles.

[0...

Embodiment 3

[0125] Multiple samples of thalassemia of different genotypes were collected, and the detection methods of Examples 1 and 2 were used to detect α-thalassemia and β-thalassemia together. The PCR reaction system and HRM analysis were the same as above. At the same time, the samples were verified by DNA sequencing and RDH (Shenzhen Yaneng Reverse Dot Hybridization Kit).

[0126] Table 3.1. β-Thalassaemia The number of specimens detected by HRM analysis, gene sequencing and Shenzhen Yaneng reverse dot blot kit

[0127]

[0128]

[0129] Table 3.2. The number of specimens detected by HRM analysis, gene sequencing and Shenzhen Yaneng reverse dot blot kit for α-thalassemia

[0130]

[0131] From the above results, it can be seen that the thalassemia gene diagnosis kit, the primers and the test kit for thalassemia gene diagnosis of the present invention have good detection specificity and high sensitivity.

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Abstract

The invention discloses a real-time fluorescence quantitative PCR(Polymerase Chain Reaction) detection kit and related primers for thalassemia genes. The kit comprises more than one pair of the following primers: SEQ ID NO.1 and SEQ ID NO.2 aiming at TATA kits nt-28 (A->G) (-28M), -29 (A->G) (-29M) and CAP-40-43 (AACC) (CAPM) genotypes; SEQ ID NO.3 and SEQ ID NO.4 aiming at CD14-15 (+G) (14-15M), CD17 (A->T) (17M) and the initiation codon mutant ATG->AGG(IntM) genotypes; SEQ ID NO.5 and SEQ ID NO.6 aiming at CD26 (G->A)(betaEM) CDs27-28+C(27-28M), IVS1-1(G->T)(IVS1-1M), IVS1-5(G->C)(IVS1-5M) genotypes; SEQ ID NO.7 and SEQ ID NO.8 aiming at CDs41-42-TCTT (41-42M), CD43G->T(43M), 41-42M/41-42M genotypes; SEQ ID NO.9 and SEQ ID NO.10 aiming at CD71-72(+A) (71-72M) genotypes; SEQ ID NO.11 and SEQ ID NO.12 aiming at IVS2-654C->T(654M) and 654M/654M genotypes; and SEQ ID NO.13 and SEQ ID NO.14 aiming at constant spring (CS), Quong Sze (QS) and Westmead (WS) genotypes. The kit has the advantages of good detection specificity and high detection sensitivity.

Description

technical field [0001] The invention belongs to the field of medical detection, in particular to primers and a kit for real-time fluorescent quantitative PCR detection of thalassemia. Background technique [0002] Thalassemia is a group of hereditary hemolytic anemias, whose common feature is that the globin peptide chain synthesis in hemoglobin is reduced or cannot be synthesized due to globin gene defects, resulting in changes in the composition of hemoglobin. The clinical symptoms of this disease vary in severity, and most of them are chronic progressive hemolytic anemia. [0003] Thalassemia is more common in Mediterranean countries and Southeast Asian countries. It has been reported in all provinces south of the Yangtze River in my country. The incidence rate is relatively high in Guangxi, Guangdong, Hainan, Sichuan, Chongqing and other provinces, and it is relatively rare in the north. If the couple is a carrier of the thalassemia gene, each pregnancy, the child has a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 林敏杨立业郑佳坤吴教仁
Owner 潮州市中心医院
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