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Kit for detecting aneuploidy of five human chromosomes through monotube multiple amplification

A technology of multiple amplification and aneuploidy, applied in the field of molecular biology, can solve the problems of cumbersome operation, difficulty in unifying the expression of test results, and low detection throughput of QF-PCR kits, so as to avoid maternal and fetal risks and reduce waiting time. Anxiety, high sensitivity effect

Active Publication Date: 2014-02-05
AGCU SCIENTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, QF-PCR commercial kits for the detection of 5 kinds of chromosome numbers, such as Aneuploidy and Elucigene QSTR from the UK, Chromo Quent and Deryser from Sweden, and TrueScience TM Aneuploidy STR Kits from ABI in the United States, etc., the selected STR loci are mainly Group samples based on Caucasians may not be applicable to Chinese populations; domestic self-developed QF-PCR kits (or patents) have a relatively low detection throughput, and generally require multiple reaction systems to detect five chromosomes, and the operation is cumbersome
At the same time, the existing QF-PCR kits or patents for chromosome number diagnosis at home and abroad lack the standardization of STR genotype analysis, and the expression of test results is difficult to unify, which is not conducive to the routine development and quality control of clinical testing.

Method used

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  • Kit for detecting aneuploidy of five human chromosomes through monotube multiple amplification
  • Kit for detecting aneuploidy of five human chromosomes through monotube multiple amplification
  • Kit for detecting aneuploidy of five human chromosomes through monotube multiple amplification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1: Determination of 23 loci

[0060] The kit provided by the invention selects the following 23 loci to detect altogether, based on:

[0061] The selected loci are all loci with high polymorphism in the Chinese Han population. In the previous study, through the genotype detection and analysis of 175 unrelated individuals of the Han nationality in China, 21 STRs with good polymorphism and high clinical diagnostic value were selected from the loci with high polymorphism reported in the literature. loci. See Table 3 for details:

[0062] Table 3175 unrelated individuals of the Chinese Han population polymorphic information content (PIC) analysis at 21 STR loci

[0063] loci

[0064] The 23 STR loci screened are all 4-5 nucleotide repeats, have good polymorphism, high heterozygosity, and can be amplified and detected in the same tube. The STR loci with 4-5 nucleotide repeats have a low ratio of replication slip peaks (ie stutter peaks) and fewer miscel...

Embodiment 2

[0065] The determination of the design of embodiment 2 primers and their concentration, kit composition

[0066] In the case of the above 23 preferred loci, the present invention has designed primers and concentrations corresponding to each site through a large number of experiments, and the sequences and concentrations of the primers are shown in Table 1.

[0067] The primer mixture includes the above 23 pairs of primers, which are used in the same amplification system. With the increase of the number of loci in the multiplex amplification system, due to the influence of amplification competition, the amplification efficiency of different primers varies, and multiple pairs of primers will affect each other, making it more difficult to control the relative balance of each locus , it is difficult to carry out quantitative analysis. By repeatedly optimizing the primer sequence, balancing the concentration of primers at different loci, and controlling the parameters of the compl...

Embodiment 3

[0097] Example 3: Rapid prenatal diagnosis of target chromosomal abnormality syndrome using this kit

[0098] 558 remaining amniotic fluid, villi, and blood samples were analyzed by clinical routine karyotype, among which 0.5-1ml of amniotic fluid was sampled, a few villi (0.1-0.5g) were sampled, and 0.2ml of blood sample was sampled. The reagents finally determined according to Example 2 The optimal composition of the box and the detection method were used for blind detection analysis. This kit detected 5 cases of trisomy 21, 3 cases of trisomy 18, 10 cases of 45X, 1 case of 47XXY, 1 case of 47XYY and 2 cases of 47XXX in 558 samples (all were complete, no chimerism and translocation ), at the same time, karyotype diagnosis was used to verify the results. The detection accuracy of this method is 100%, no missed detection, no amplification failure; each sample can obtain the detection result within 24 hours.

[0099] For the results, please refer to accompanying drawings 4, 5,...

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Abstract

The invention relates to a kit for detecting the STR (Short Tandem Repeat) genetype of human chromosomes 21, 18 and 13 and sex chromosomes, and particularly relates to a QF-PCR (Quantitative Fluorescence-Polymerase Chain Reaction) kit for detecting the number of the chromosomes 21, 18 and 13 and the sex chromosomes by adopting five-color fluorescence labeling monotube fast multiple amplification and mainly for diagnosing 21 trisomy syndrome, 18 trisomy syndrome, 13 trisomy syndrome and the aneuploid abnormality of the sex chromosomes. The kit comprises a primer mixture, a hot start C-Taq enzyme, an amplification reaction solution, a positive quality control product, a negative reference product, a fluorescence interior label Siz-500 and an allelic gene typing standard substance. Compared with the traditional antenatal diagnosis method, the kit disclosed by the invention can realize high-flux, fast, reliable and standardized detection.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to fluorescent quantitative PCR technology (QF-PCR), in particular to a rapid and high-throughput method for detecting STR genotypes of human autosomes No. 21, No. 18, No. 13 and sex chromosomes X and Y The kit is used for the detection of aneuploidy of the above five chromosomes. Background technique [0002] Chromosomal disease is a common type of genetic disease that causes birth defects, accounting for about 0.6% of live births. It mainly manifests as mental retardation, developmental delay, and multiple organ deformities, which brings heavy mental and physical stress to society and families. economic burden. Chromosomal abnormalities in live births mainly include trisomy 21, trisomy 18, trisomy 13, and abnormal numbers of sex chromosomes. [0003] Trisomy 21, also known as Down syndrome and congenital stupidity, has an incidence rate of 1 / 800-1 / 600 in newborns. It is a serious ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6883C12Q2600/156
Inventor 吕时铭朱宇宁薛佳陈雁张民郑卫国葛海鹏
Owner AGCU SCIENTECH
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