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Thalassemia gene detection method based on fluorescence labeling quantitation PCR (Polymerase Chain Reaction) technology
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A fluorescent labeling quantitative, thalassemia technology, applied in the field of medical detection
Inactive Publication Date: 2014-09-10
钦州市妇幼保健院
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Problems solved by technology
At present, there is no combination of AS-PCR method and Gap-PCR method with fluorescence-labeled quantitative PCR technology to realize the effect of single-tube multiplex PCR technology on α-thalassemia-- SEA type (Southeast Asian type), -α 3.7 ,-α 4.2 ,-- THAI (Thai type), α CS alpha, alpha QS α and α WS α, and β thalassemia HPFH-SEA (abnormal hemoglobin combined with hereditary persistent fetal hemoglobin), Southeast Asian deletion type (HPFH-SEA) and DBT (Chinese type) thalassemia genotype detection reports
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Embodiment 1
[0083] Embodiment 1: detection method of the present invention
[0084] (1) Primer design
[0085] (1) Through the Blast function of NCBI, design a DNA fragment with a length of 20 bp and less homology with human DNA sequences as a universal primer. The sequence of the universal primer is: 5'-CTCGACACGCATCTGCTCAG-3' (SEQ ID NO: 1) , using 6-FAM fluorescent labeling at the 5' end to obtain universal fluorescent labeling primers;
[0086] In addition, 5'-CTCGACACGCATCTGACGTT-3' (SEQ ID NO: 2) labeled with VIC, 5'-CTCGACACGCATCTGGCGAA-3' (SEQ ID NO: 3) labeled with NED, and 5'- CTCGACACGCATCTGCTACC-3' (SEQ ID NO: 4) has also been designed and can be used in subsequent multicolor fluorescent labeling systems;
[0087] (2) For α-thalassemia -- SEA Type, -α 3.7 ,-α 4.2 ,-- THAI , α CS alpha, alpha QS α and α WS α, and β thalassemia HPFH-SEA and DBT thalassemia genotype sequences, design amplification primer pairs, primer sequences and pairings are as follows:
[0088] 2.1)...
Embodiment 2
[0189] Repeat the method of embodiment 1, difference is, replace sample 1 with sample 2, the result is as follows Figure 6 shown.
[0190] Figure 6 Among them, 2Y1=Y2, CSM, QSN, WSN have peaks, HPFH-W has peaks, and the rest have no peaks.
[0191] Combining with existing test results Figure 1~4 , it can be independently judged that the genotype of sample 2 is -α 3.7 / α CS α genotype thalassemia; and the result graph independently made by the system of the present invention can also directly determine that the genotype of sample 2 is -α 3.7 / α CS Alpha genotype thalassemia. The two are mutually verified, which proves the simplicity of the present invention and the consistency of the results.
Embodiment 3
[0193] Repeat the method of embodiment 1, difference is, replace sample 1 with sample 3, the result is as follows Figure 7 shown.
[0194] Figure 7 Among them, Y1=2Y2, CSN, QSN, and WSW have peaks, HPFH-W has peaks, and the rest have no peaks.
[0195] Combining with existing test results Figure 1~4 , it can be independently judged that the genotype of sample 3 is -α 4.2 / α WS α genotype thalassemia; and the result graph independently made by the system of the present invention can also directly determine that the genotype of sample 3 is -α 4.2 / α WS Alpha genotype thalassemia. The two are mutually verified, which proves the simplicity of the present invention and the consistency of the results.
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Abstract
The invention discloses a thalassemia gene detection method based on a fluorescence labeling quantitation PCR (Polymerase Chain Reaction) technology, and the detection method mainly comprises the following steps of: firstly designing a general fluorescence labeling primer; then designing an amplification primer aiming at a detection site; when the amplification primer is designed, tailing one primer of an amplification primer pair, wherein the tailing sequence is consistent to the sequence of the general fluorescence labeling primer; when PCR reaction is carried out, mixing a tailed primer group and the general fluorescence labeling primer to realize the fluorescence labeling of an amplification product; and after the PCR reaction is finished, placing PCR products into a sequencer, and confirming the lengths of different PCR products through a series of internal references of known molecular weight of a result by utilizing the Genemapper function of the sequencer to judge the genetype of a detected sample. The detection method disclosed by the invention can realize the detection of different genotypes of alpha thalassemia through a single tube.
Description
technical field [0001] The invention relates to the technical field of medical detection, in particular to a thalassemia gene detection method based on fluorescent marker quantitative PCR technology. Background technique [0002] Thalassemia (Mediterranean Anemia), also known as Thalassemia (Thalassemia), Cooley (Cooley) anemia, globin synthesis disorder anemia, referred to as thalassemia. Thalassemia is one of the most common monogenic genetic diseases. Common types of thalassemia include α-thalassemia and β-thalassemia, which are caused by abnormal expression of α-protein gene cluster and β-globin gene cluster, respectively. The disease occurs frequently in the Mediterranean coast, Africa, Latin America, Southeast Asia, East Asia, South Asia, and other tropical and subtropical regions. In my country, Guangxi, Guangdong and Hainan are provinces with high incidence of thalassemia, among which the incidence of α-thalassemia in Guangxi is 15.5%, and the prevalence of α-thalas...
Claims
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