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Fluorescent quantitative PCR kit for detecting alpha-globin gene deletion

A fluorescent quantitative and globin technology, applied in the field of biochemistry, can solve the problems of operational feasibility, unsatisfactory detection throughput, false positives, false negatives, etc., and achieve good sensitivity, good specificity, repeatability, and good sensitivity and the effect of specificity

Inactive Publication Date: 2011-08-10
SOUTHERN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In general, some new techniques in molecular biology have been applied to the molecular diagnosis of deletional α-thalassaemia. Although there has been some progress in methodology, they basically belong to the qualitative analysis of various deletion types. Methodological improvements, but there are still technical bottlenecks such as false negatives and false positives, and its operational feasibility and detection throughput cannot meet the current needs of large-scale population screening and routine detection

Method used

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  • Fluorescent quantitative PCR kit for detecting alpha-globin gene deletion
  • Fluorescent quantitative PCR kit for detecting alpha-globin gene deletion
  • Fluorescent quantitative PCR kit for detecting alpha-globin gene deletion

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1: Small sample evaluation of the sensitivity and accuracy of the kit

[0055] 1. The composition of the kit:

[0056] (1) The upstream primers for specific amplification of α1 globin are:

[0057] 5'-CGTCGTGTACTTGTGTGATGGTTAGA-3' (SEQ NO.1),

[0058] The downstream primers for specific amplification of α1 globin are:

[0059] 5'-CGTCGTAAACAGGTAAACAAAGCAATAG-3' (SEQ NO.2),

[0060] (2) The upstream primer sequence for specific amplification of α2 globin is:

[0061] 5'-CGTCGTGTACAACTTCCTATTCTCAGTG-3' (SEQ NO.3),

[0062] The downstream primers for specific amplification of α2 globin are:

[0063] 5'-CGTCGGGAAGACTTGCTAGGTAAATACT-3' (SEQ NO.4),

[0064] (3) The upstream primers for specific amplification of ζ globin are:

[0065] 5'-CCGTCCAGGAGGCTGCTTACT-3' (SEQ NO.5),

[0066] Downstream primers that specifically amplify zeta globin are listed as:

[0067] 5'-GGTGCTCCTTATTCATTTCAGAATCAC-3' (SEQ NO.6),

[0068] (4) The upstream primers for specifically a...

Embodiment 2

[0103] Embodiment 2: Sensitivity and specificity evaluation of the kit of the present invention

[0104] 1. Specimen collection:

[0105] From the sample bank of our laboratory, 218 gDNA samples of known genotypes detected by gap-PCR were collected. This batch of samples contained αα / αα, -α 3.7 / αα, -α 4.2 / αα, -- SEA / αα, -- SEA / -α 4.2 、-- SEA / -- SEA 、- SEA / -α 3.7 , -α 3.7 / -α 3.7 , -α 4.2 / -α 4.2 , -α THAI For the 10 genotypes / αα, dilute the gDNA samples to 100-300ng / μl with sterilized double distilled water for later use. The gap-PCR detection system used is a detection system that has been verified by Southern Blot, and the relative copy numbers of ζ, α1, and α2 genes are accurately deduced according to the type of deletion.

[0106] 2. Specimen testing:

[0107] (1) Kit composition:

[0108] With embodiment 1.

[0109] (2) Sample detection: the above-mentioned gDNA specimens to be tested, 2 normal control gDNA samples with 2 copies of ζ, α1 and α2 glo...

Embodiment 3

[0118] Example 3: Small-scale crowd screening detection evaluation

[0119] 1. Specimen collection:

[0120] 104 peripheral blood samples were randomly collected from the Biochemical Genetics Department of the Prenatal Diagnosis Center of Nanfang Hospital, and routine blood analysis was performed on the 104 peripheral blood samples with F-820 blood analyzer to obtain phenotype data. Then use Tiangen column-type peripheral blood genomic DNA column extraction reagent (Beijing Tiangen Biotechnology Company) to extract gDNA, and dilute the gDNA sample to 100-300 ng / μl with sterilized double-distilled water for use.

[0121] 2. Specimen testing:

[0122] (1) Kit composition:

[0123] Same as Example 1

[0124] (2) Sample detection: The above 104 gDNA samples to be tested were double-blind, and the deletion α-thalassaemia gap-PCR method and this kit were used for parallel detection. The gap-PCR method used is the detection system and detection conditions that have been verified ...

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Abstract

The invention belongs to the field of biochemistry, and provides a fluorescent quantitative polymerase chain reaction (PCR) kit for detecting alpha-globin gene deletion. The kit consists of a pair of specific amplified xi globin primers, a pair of specific amplified alpha1 globin primers, a pair of specific amplified alpha2 globin primers, a pair of specific amplified beta-Actin gene primers, a fluorescent probe for specifically detecting xi globin, a fluorescent probe for specifically detecting alpha1 globin, a fluorescent probe for specifically detecting alpha2 globin, a fluorescent probe for specifically detecting beta-Actin genes, a DNA polymerase and the like. The kit has good sensitivity and accuracy for detecting depletion alpha-thalassemia, has good repeatability and stability, and is totally suitable for clinical detection of alpha-thalassemia.

Description

technical field [0001] The invention relates to the field of biochemistry, in particular to reagents for measuring or testing methods comprising nucleic acids. Background technique [0002] The human α-globin gene cluster is located on chromosome 16. There are three functional genes ζ, α1, and α2, which express the corresponding ζ and α globin chains, and then combine with the corresponding β globin chains to form ζ2γ2 (embryonic hemoglobin Hb Portland) and α2β2 (adult hemoglobin HbA). Normally, the human globin gene expresses an appropriate ratio of α- and β-globin chains to form a functional hemoglobin tetramer. When the α-globin gene is defective, the α-chain synthesis is reduced and the β-chain is relatively excess, which can lead to α-thalassemia disease. Thalassemia (referred to as "thalassemia") is one of the most common single-gene genetic diseases that have the greatest impact on human health in the world. It is mainly concentrated in countries along the Mediterra...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N21/64
Inventor 周万军熊符徐湘民赵镇
Owner SOUTHERN MEDICAL UNIVERSITY
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