Fluorescence quantitative polymerase chain reaction (PCR) detection kit for alpha-thalassemia and application thereof

A technology of thalassemia and detection kits, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems of large influence, low detection sensitivity, and inability to see, and achieve good specificity and specificity Good, accurate measurement of the effect

Active Publication Date: 2014-05-14
SANSURE (SHANGHAI) GENE TECH LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The advantage is that it is easy to operate; the disadvantage is that it cannot effectively remove the factors that inhibit PCR in peripheral blood, and weak positives often have no amplification
[0006] (2) Concentrated boiling method: first concentrate the peripheral blood, add the lysate, boil, centrifuge at high speed, and use the supernatant as a template. This method is currently a common clinical method in China, and its advantage is that it can partially remove the "direct boiling method" Inhibitory factors that cannot be removed; the disadvantage is that different manufacturers have different concentration effects, some can see precipitation, and some cannot
The α-thalassemia e

Method used

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  • Fluorescence quantitative polymerase chain reaction (PCR) detection kit for alpha-thalassemia and application thereof
  • Fluorescence quantitative polymerase chain reaction (PCR) detection kit for alpha-thalassemia and application thereof
  • Fluorescence quantitative polymerase chain reaction (PCR) detection kit for alpha-thalassemia and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Embodiment 1: Provide a kind of α-thalassemia fluorescent quantitative PCR detection kit

[0088] A fluorescent quantitative PCR detection kit for α-thalassemia, which at least consists of the following independent components:

[0089] (1) PCR reaction solution Ⅰ: Contains primers and probes for detecting α-thalassemia SEA deletion derived from the conserved regions of the α1 and α2 genes on chromosome 16 of the human genome, and the probe α-SEA-P, amplified The sequences of the upstream primer α-SEA-F and the downstream primer α-SEA-R (synthesized by Invitrogen) are:

[0090] Upstream primer α-SEA-F: 5'-TCAGCCACCCGCAGACCAAGACCTA-3';

[0091] Downstream primer α-SEA-R: 5'-AGACAGCGTCACCCTCAGAGCCAT-3';

[0092] Probe α-SEA-P: 5'-AAATGGATGAGGACGGAGCGATCTG-3';

[0093] Preferably, the carboxyl end of the probe α-SEA-P is labeled with FAM, and the hydroxyl end is modified with a BHQ1 quenching group. In other embodiments of the present invention, fluorescein labels such as...

Embodiment 2

[0106] Embodiment 2: Provide a kind of α-thalassemia fluorescent quantitative PCR detection kit

[0107] A fluorescent quantitative PCR detection kit for α-thalassemia, which contains the following independent components in addition to each component that exists independently in Example 1:

[0108] (1) Enzyme mixture: DNA polymerase (Taq enzyme) (purchased from Promega);

[0109] (2) α-thalassemia negative control: inactivated negative peripheral blood that does not contain any of the three deletion types of α-thalassemia;

[0110] Among them, the enzyme mixture contains heat-resistant DNA polymerase (Taq enzyme) at a concentration of 1-5U / μl and uracil DNA glycosylase (UNG enzyme) at a concentration of 0.05-0.2U / μl, wherein UNG enzyme has the ability to degrade The function of the PCR product containing dU, the use of UNG enzyme and dUTP in the PCR reaction solution can prevent the contamination of the PCR product.

Embodiment 3

[0111] Embodiment 3: Provide a kind of α-thalassemia fluorescent quantitative PCR detection kit

[0112] A fluorescent quantitative PCR detection kit for α-thalassemia, which is composed of at least a dozen of the following independent components:

[0113] (1) PCR reaction solution Ⅰ: Contains primers and probes for detecting α-thalassemia SEA deletion derived from the conserved regions of the α1 and α2 genes on chromosome 16 of the human genome, and the probe α-SEA-P, amplified The sequences of the upstream primer α-SEA-F and the downstream primer α-SEA-R (synthesized by Invitrogen) are:

[0114] Upstream primer α-SEA-F: 5'-TCAGCCACCCGCAGACCAAGACCTA-3';

[0115] Downstream primer α-SEA-R: 5'-AGACAGCGTCACCCTCAGAGCCAT-3';

[0116] Probe α-SEA-P: 5'-AAATGGATGAGGACGGAGCGATCTG-3';

[0117] Preferably, the carboxyl end of the probe α-SEA-P is labeled with FAM, and the hydroxyl end is modified with a BHQ1 quenching group. In other embodiments of the present invention, fluorescei...

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Abstract

The invention relates to a fluorescence quantitative polymerase chain reaction (PCR) detection kit for alpha-thalassemia. The kit comprises probes for detecting SEA, 4.2 and 3.7 type deletion, a forward primer and a reverse primer for amplifying SEA, 4.2 and 3.7 type deletion, a PCR buffer and an enzyme mixed liquor. The kit is convenient and simple to operate, high in sensitivity, and good in specificity, repeatability and stability, and can prevent pollution of a PCR product. The fluorescence quantitative PCR detection of the alpha-thalassemia is carried out by using the kit, the deletion type of the alpha-thalassemia in a peripheral blood sample can be accurately determined, and a reliable experimental evidence is provided for sensitive and early diagnosis of the alpha-thalassemia deletion type.

Description

technical field [0001] The invention belongs to the technical field of thalassemia detection and relates to a fluorescent quantitative PCR detection kit for α-thalassemia. Background technique [0002] α-Thalassemia is a genetic disease with recessive chromosomal disorder, which is characterized mainly by anemia caused by red blood cell and hemoglobin deficiency, and the clinical symptoms also change from asymptomatic to fatal hemolytic anemia. The Gap-PCR method is now used by most of the hospitals that are qualified to detect α-thalassemia. This method requires operators to be very familiar with the whole process of ordinary PCR reactions, because each step has a great impact on the results, so it is very important for clinical practice. The experimental operation level of genetic professionals is very high. In addition, the pollution of carcinogens (EB) during the experiment, the timeliness of obtaining the experimental results (about 2 to 4 days), and the high cost of t...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/686C12Q2561/113C12Q2563/107
Inventor 戴立忠付亚成邓中平
Owner SANSURE (SHANGHAI) GENE TECH LTD
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