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

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...

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 extraction methods provided by these existing kits have many shortcomings: 1) The nucleic acid extraction process is complicated, and the sample processing takes a long time, and when processing the sample, it needs to go through multiple steps, and the DNA in the sample There is loss, especially for high-concentration samples, insufficient lysis and incomplete enrichment will cause a large loss of DNA, leading to sample loss verification, and low nucleic acid extraction efficiency; / ml; 3) There are generally no measures to prevent PCR product contamination; 4) There is no fluorescence normalization correction system, and the result judgment is greatly affected by subjective factors

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