Deletion-type alpha-thalassemia detection kit based on Taqman probe and detection method of deletion-type alpha-thalassemia detection kit

Abstract

The invention provides a multiplicity multicolor real-time fluorescence PCR method and a detection kit regarding the defects of existing deletion-type alpha-thalassemia detection methods and detectionsites. By means of the method, deletion-type alpha-thalassemia (such as --SEA, -alpha 3.7, -alpha 4.2 and -THAI deletion which are common in China) can be detected, whether sites exist or not is determined by detecting genotypes, whether alleles of alpha-thalassemia of a to-be-examined person are wild type alleles, heterozygous-deletion-type alleles or homozygous-deletion-type alleles is judged,and thus the method is beneficial to clinical genetic counseling. Meanwhile, the method provided by the invention can effectively amplify long fragments with high GC content, and provides a real-timefluorescence PCR detection method and an amplification system suitable for the Taqman probe in combination with an optimized reaction program and reaction system. The method has the advantages of simplicity, convenience, stain resistance, high sensitivity, stability and accuracy, high specificity and the like, the time for clinical thalassemia screening and diagnosis can be greatly shortened, andthe clinical efficiency is improved.

Description

Technical field [0001] The present invention belongs to the field of molecular biology, and involves a lack type α-medlar anemia detection method based on Taqman probes, in particular, involving a multiple multicolor real-time fluorescent PCR method, while detecting a kit for deletion type α Mediterranean anemia. Background technique [0002] Mediterranean anemia is referred to as poverty, but also known as marine anemia, bead protein to generate disabilities of anemia, is due to defects of the bead protein gene, resulting in one or more of the bead protein chains that cannot be synthesized or synthesized, the chain structure is normal but the proportion is imbalance Hereditary hemolytic hemoglobin disease. [0003] Mediterranean anemia is one of the most common genetic diseases worldwide. It is mainly distributed in the Mediterranean region, the Middle East, India, Southeast Asia and Africa, Mediterranean anemia is also one of the most common and harmful genetic diseases in the ...

AI Technical Summary

Problems solved by technology

Limitations: 1. The concentration of DNA needs to be accurately measured, and the sample is easily contaminated; 2. It cannot be used for the detection of a single cell; 3. MLPA is used to detect gene deletion or duplication, and is not suitable for detecting unknown point mutation types; 4. Unable to detect balanced translocations of chromosomes

This method does not directly judge the detection result through the amplification curve, but requires complex calculations to obtain the detection result; at the same time, it requires high precision of instruments and reagents, which may easily lead to false negatives

[0017] The above-mentioned methods for the molecular diagnosis of α-thalassemia have high accuracy, but the electrophoresis detection step involved may cause contamination of the amplification product and lead to false positive results; at the same time, the operation is cumbersome, time-consuming and labor-intensive, the detection throughput is small, and the use of Reagents or instruments are expensive and other defects; in addition, most of the approved deletion α-thalassemia detection kits only detect three deletion types (--SEA, -α3.7 and -α4.2), and with the clinical In-depth research has found that there is also a certain proportion of --THAI deletion type distribution in the Chinese population. Therefore, in order to reduce missed diagnosis, it is necessary to detect --THAI deletion type

[0018] Of the various molecular diagnostic methods and approved kits for α-thalassemia mentioned above, there is no real-time fluorescent PCR detection method based on Taqman probes, which can determine the genotype of the sample to be tested in real time according to the amplification curve

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