Multiplex amplification detection kit for detecting 60 loca of autosomes and Y-chromosomes simultaneously

A detection kit and multiple amplification technology, applied in the biological field, can solve problems such as unsatisfactory, and achieve the effects of good balance, rapid amplification, and high sensitivity

Active Publication Date: 2019-03-08
江苏苏博生物医学科技南京有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A few such as Wuxi Zhongde Meilian (a six-color kit (patent 201510477236.9) that simultaneously detects 34 DNA loci (15 autosomal STR loci, 1 sex locus and 18 Y-STR loci), and Detect kits for simultaneous detection of 39 loci, including 18 A-STR, 20 Y-STR and gender (Amel) loci (patent 201810051679.5), developed by Ningbo Haiers Gene Technology Co., Ltd. The Compass Human DNA Identification Kit simultaneously detects 15 autosomal loci, 17 Y chromosome loci and 1 sex locus Amel. Can not meet the requirements of 20 STR loci for frequent dyeing and 35 STR loci for Y chromosome building

Method used

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  • Multiplex amplification detection kit for detecting 60 loca of autosomes and Y-chromosomes simultaneously
  • Multiplex amplification detection kit for detecting 60 loca of autosomes and Y-chromosomes simultaneously
  • Multiplex amplification detection kit for detecting 60 loca of autosomes and Y-chromosomes simultaneously

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1. often adds the preparation of Y composite amplification reagent

[0045] 1. Arrangement of sites in the multiplex amplification detection system

[0046] The present invention simultaneously amplifies 60 loci, divided into seven groups, the first group: rs771783753, DYS481, DYS389I / II, DYS635, DYS533, DYS627, DYS391, D7S820 and D1S1656; the second group: DYS576, DYS437, DYS439, DYS392 ,DYS448,DYS518,D12S391和D21S11;第三组:rs1759551978, DYS460,DYS459,DYS19,DYF387S1a / b,DYS456,DYS385a / b,CSF1PO和D5S818;第四组:DYS393,DYS570,DYS390,DYS438,Y_GATA_H4,DYS449 and PentaD; the fifth group rs199815934, D19S433, vWA, D8S1179, TPOX, FGA and D6S1043; the sixth group Amelogenin X / Y, D3S1358, TH01, D13S317, D16S539, D18S51, D2S1338 and PentaE; the seventh group DYS6445, DYS5 , DYS444, DYS643, DYS557, DYS596 and DYS527a / b. The first base at the 5' end of the primers in the first group is labeled with FAM; the first base at the 5' end of the primers in the second group is labeled w...

Embodiment 2

[0062] Example 2. Application of Multiple Amplification Verification Reagent

[0063] 1. Sample amplification

[0064] Add 24 μL of the DNA test reagent prepared in Example 1 to 1 μL of 0.5 ng / μL Male DNA Standard 9948, and perform PCR amplification.

[0065] The PCR amplification program is: 95°C for 1 minute; 94°C for 2 seconds, 60°C for 1 minute, 26 cycles; 60°C final extension for 5 minutes; 4-16°C hold.

[0066] 2. Detection of PCR products

[0067]After the amplification reaction was completed, the reaction tube was taken out, and electrophoresis and detection were performed with Nanjing Traceability Honor1816 Genetic Analyzer.

[0068] 1) Take (0.5 μL molecular weight internal standard + 10 μL deionized formamide) × (number of samples) to make a mixed solution, mix well and dispense, 10 μL per tube.

[0069] 2) Then add 1 μL of the amplification product or allele ladder (ladder), and centrifuge briefly to collect the liquid at the bottom of the centrifuge tube. The s...

Embodiment 3

[0071] Example 3. Amplification of male and female blood filter paper samples by composite amplification verification reagent

[0072] Add 24 μL of the DNA test reagent prepared in Example 1 to a 1.2 mm male (female) blood filter paper sample, configure the amplification system according to Example 2, and the PCR amplification program is: 95°C for 1 minute; 94°C for 2 seconds; 60°C for 1 minute; 28 cycles; 60°C final extension for 5 minutes; 4-16°C hold. Detection was performed according to the detection process of PCR products in Example 2. Its test results are as Figure 4 , as shown in 5, all loci were detected in male samples, Y loci were not detected in female samples, and all autosomal typing was detected. It shows that the specificity of male and female DNA in the kit is very good. The analysis results were compared with Jiangsu Subo YESU 21plex and Y45SE kits (operated according to the instructions), and the typing results of the same gene loci were consistent. The...

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Abstract

The invention discloses a multiplex amplification detection kit for detecting 60 loca of autosomes and Y-chromosomes simultaneously. The 60 loca are amplified in a multiplex manner through a polymerase chain reaction, and amplification products of the loca are detected by using a gene sequencer. The kit can be used for detecting the 60 loca of the autosomes and the Y-chromosomes simultaneously, which is a case that most STR loca can be detected by a primary reaction with a capillary electrophoresis method at present, and databases can be built for autosome STR and Y-chromosome STR simultaneously by the primary reaction. The kit has strong adaption to biomaterials, namely, one kit can be used for amplifying various biomaterial samples, wherein different biomaterial samples comprise a male genome DNA extracted by a Chelex100 method, a magnetic bead extracting method or an organic extracting method and male blood or oral cells of human, which is/are collected by any one carrier of filterpaper, an FTA card, a cotton bud, gauze and the like. The kit has higher amplification specificity and higher thermal tolerance.

Description

technical field [0001] The invention relates to a compound amplification detection kit for simultaneously detecting 60 autosome and Y chromosome loci, belonging to the field of biotechnology. Background technique [0002] Short tandem repeat sequence (STR for short), also called microsatellite DNA, is a kind of nucleotide repeat sequence widely present in prokaryotic and eukaryotic genomes. It generally consists of tens to more than one hundred nucleotide repeating sequences consisting of 2-6 nucleotides as repeating units. Different numbers of core sequences are arranged in tandem repeats, and the length is polymorphic. [0003] In recent years, the capacity of DNA databases of public security organs across the country has grown by leaps and bounds, and more and more cases have been solved directly using DNA databases. The conventional DNA database is a "one-to-one, point-to-point" comparison mode, which requires corresponding reference comparison samples to be identified ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/6858
CPCC12Q1/6858C12Q1/6888C12Q2563/107C12Q2565/125C12Q2531/113
Inventor 葛斌文黄春丽高智勇张明航赵晓芳苏雪峰王宇峰
Owner 江苏苏博生物医学科技南京有限公司
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