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Multi-QF-PCR STR detection system for performing fast diagnosis on numerical abnormality of chromosomes

A QF-PCR STR and chromosome number technology, applied in the field of QF-PCR rapid diagnosis, can solve the problems of low heterozygosity rate of STR loci, poor reproducibility, insufficient STR loci, etc.

Inactive Publication Date: 2010-09-22
GUANGZHOU WOMEN AND CHILDRENS MEDICAL CENTER
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

[0006] In view of the shortcomings of the existing QF-PCR system, such as insufficient STR sites, poor relative quantitative repeatability, and low heterozygosity rate of the selected STR sites in the Chinese population, the purpose of the present invention is to establish a set of STR sites containing enough STR sites. A multiplex QF-PCR STR rapid detection system for diagnosing abnormalities in the number of chromosomes 21, 18, and 13, and each STR has a high heterozygosity rate in the Chinese population, and a combination of relative quantification and increasing STR loci to establish a A relatively complete QF-PCR STR detection system for detecting abnormalities in the number of sex chromosomes, that is, fluorescent quantitative PCR detection technology, its operation and implementation steps are as follows:

Method used

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  • Multi-QF-PCR STR detection system for performing fast diagnosis on numerical abnormality of chromosomes
  • Multi-QF-PCR STR detection system for performing fast diagnosis on numerical abnormality of chromosomes
  • Multi-QF-PCR STR detection system for performing fast diagnosis on numerical abnormality of chromosomes

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Embodiment 1

[0035] Embodiment one: detection method of the present invention

[0036] Specimens for prenatal diagnosis include amniotic fluid, villi, fetal blood, etc. Whole blood samples need ACD anticoagulation, amniotic fluid samples need to be centrifuged at 3000rpm to collect cells, and the villi are soaked in normal saline. Various specimens were stored at 4°C, and DNA was extracted within 48 hours. DNA was extracted using a kit, such as QIAamp DNA Mini kit (QiaGen), and operated strictly according to the instructions. Finally, the DNA product was dissolved in double-distilled water, and the concentration and purity were measured on the N.D1000V3.7 DNA analyzer, and the DNA concentration was adjusted to 10-50ng / μL. OD260 / 280 ratio between 1.6-2.1 is a qualified DNA sample.

[0037] PCR reaction: DNA polymerase using Takara Taq TM For Hot Start Version, the PCR reaction uses a 25μL system, and the amount of DNA template is 15-70ng. Three sets of reactions A, B, and C were perfo...

Embodiment 2

[0046] Embodiment 2: QF-PCR detection of a healthy male

[0047] refer to figure 1 As shown, using the method of Example 1, DNA was extracted from 200 μL of a peripheral blood sample of a healthy adult male with a normal karyotype confirmed by karyotype analysis, and a total of 45 μL of DNA with a concentration of 23.5 ng / μL was obtained, and the OD260 / 280 ratio was 1.82. For qualified DNA samples. Take 2 μL of DNA each for PCR reactions and fragment analysis in groups A, B, and C. The analysis results of the collected fluorescence data by GeneMapper software are shown in Table 4:

[0048] Table 4 GeneMapper software to the data analysis results of the QF-PCR fluorescence signal of a healthy male

[0049]

[0050]

[0051] The fluorescence signal intensity of all allele peaks in this sample is between 500-8000, and the fluorescence signal intensity is suitable for karyotype judgment; the gender determination gene AMXY shows a 1:1 (AMX:AMY) double peak, and the male-sp...

Embodiment 3

[0052] Embodiment three: the QF-PCR detection of a healthy female

[0053] refer to figure 2 As shown, using the method of Example 1, DNA was extracted from 200 μL of a peripheral blood sample of a healthy adult female confirmed by karyotype analysis as a normal karyotype, and a total of 45 μL of DNA with a concentration of 26 ng / μL was obtained, and the OD260 / 280 ratio was 1.91, which was Qualified DNA samples. Take 2 μL of DNA each for PCR reactions and fragment analysis in groups A, B, and C. The analysis results of the collected fluorescence data by GeneMapper software are shown in Table 5:

[0054] Table 5 GeneMapper software to the data analysis results of the QF-PCR fluorescence signal of a healthy female

[0055]

[0056]

[0057] *DXS1053 site PCR amplified fragment has a simple repetition of two bases "CA" in the middle, resulting in slippage in the PCR process, so the product doublet ratio is not strictly close to 1:1 (<1.6), which is still normal.

[005...

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Abstract

The invention provides a multi-QF-PCR STR detection system for performing fast diagnosis on the numerical abnormality of chromosomes, which comprises A, B and C three groups of PCR systems, wherein detection loci of the group A comprise AMXY, D21S1433, D21S1411, D13S258, D21S1414, D21S1412, D21S1445 and 21q11.2; the detection loci of the group B comprise D18S535, D18S391, SRY, D18S51, D18S386, D13S742, D13S634 and D13S303; and the detection loci of the group C comprise AMXY, DXS1187, DXS8377, SRY, DXS6809, DXS981, DXS1053, D13S305 and D21S11. When used in prenatal diagnosis, the fast detection system with enough STR loci and high heterozygosis rate of the STR loci can greatly shorten diagnosis time and buy precious time for timely treatment for clinicians and is prenatal diagnosis means with extremely high practicability.

Description

technical field [0001] The present invention relates to a QF-PCR rapid diagnosis technology for 21, 18, 13 and sex chromosome abnormalities, in particular to three multiplex PCR amplification systems for detecting 21, 18, 13 and sex chromosome-specific STRs and its primers. Background technique [0002] There is no effective treatment for neonatal genetic diseases, and prenatal diagnosis is the most effective means to prevent the birth of newborns with congenital defects. Chromosomal disease is the most common type of genetic disease in newborns, with Down syndrome (trisomy 21) and Turner syndrome (45, X) being the most common, followed by trisomy 18, and trisomy 13 occasionally, Klinefelter Syndrome and XYY Syndrome etc. At present, the most important technical method in the prenatal diagnosis of chromosomal diseases is karyotype analysis, but due to its complicated operation and long time-consuming (about 14 working days), it will not only miss the best time for clinicia...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 廖灿梁巧仪杨昕黄以宁
Owner GUANGZHOU WOMEN AND CHILDRENS MEDICAL CENTER
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