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Fetal chromosome library construction kit and method and application thereof

A technology for library construction and kits, applied in chemical libraries, biochemical equipment and methods, and microbial measurement/inspection, etc., can solve the problems of cumbersome steps, complicated operation steps, complicated rinsing, etc., and achieve the effect of simple operation steps

Active Publication Date: 2016-06-01
北京爱普益医学检验中心有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In this method, DNA extraction and DNA high-throughput sequencing database construction are two independent processes, and the steps are cumbersome and rinsing is complicated, which easily affects the detection results.
[0006] In the current non-invasive prenatal screening and diagnosis process, the extraction of cell-free DNA is generally carried out by using a commercially available cell-free DNA extraction kit alone, and library construction is carried out as two independent links. There are many kits involved, and the operation steps are complicated, which may easily cause loss or contamination of DNA samples, thus affecting the results of high-throughput sequencing
At the same time, the purification process of the free DNA library is not complete, and it is easy to leave the problem of self-ligation of the adapter, which will pollute the sequencing library and affect the detection results.

Method used

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  • Fetal chromosome library construction kit and method and application thereof
  • Fetal chromosome library construction kit and method and application thereof
  • Fetal chromosome library construction kit and method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0112] Example 1 Preparation of kit for non-invasive prenatal screening for fetal chromosomal aneuploidy

[0113] Free DNA extraction reagent: the final concentration of Tris-HCl buffer is 0.1M, proteinase K is 500μg / mL, EDTA is 0.02M, Triton-100 is 0.05%, mixed and stored in 25mL / cartridge.

[0114] Free DNA Precipitation Reagent: Isopropanol, stored in 25mL / cartridge.

[0115] Free DNA washing reagent: the final concentration of Tris-HCl buffer is 0.1M, guanidine isothiocyanate is 8M, and NaCl is 1.5M. After mixing, store in 50mL / cartridge.

[0116] DNA end repair reagent: the final concentration of Tris-HCl buffer is 0.2M, TaqDNA polymerase is 0.2U / μL, MgCl 2 20mM, KCl 100mM, mix and store in 450μL / cartridge.

[0117] Adapter for repairing DNA: the final concentration of Tris-HCl buffer is 100mM, T4DNAligase is 0.05mM, adapter is 0.5μM, mixed and stored in 1mL / cartridge.

[0118] PCR amplification primers: Index primers and universal primers, used at a final concentratio...

Embodiment 2

[0135] The construction of embodiment 2 high-throughput library

[0136] 1) Extract the DNA of the sample to be tested:

[0137] (1) Take 300-500 μL of peripheral blood plasma from pregnant women, add 500 μL of free DNA extraction reagent, and keep warm in a 56°C water bath for 10 minutes;

[0138] (2) Add 500 μL of free DNA precipitation reagent, incubate at room temperature for 5 minutes, and then transfer to the spin column;

[0139] (3) Centrifuge at 12000r / min for 1min to discard the waste liquid, add 500 μL of free DNA washing reagent, centrifuge at 12000r / min for 1min to discard the waste liquid, repeat this step once;

[0140] (4) Take out the spin column, transfer it to a clean collection tube, open the cap of the spin column tube, and dry at room temperature for 5 minutes;

[0141] (5) Add 40 μL of ultrapure water to the spin column, and centrifuge at 12000 r / min for 1 min to collect free DNA;

[0142] 2) Perform PCR on the DNA obtained in step (1) to construct a ...

Embodiment 3

[0150] Example 3 library quality inspection

[0151] Real-time fluorescence quantitative PCR was used to further detect whether there was adapter self-ligation contamination in the library, and 1 μL of the solution was taken out of the library, and the library was detected using the high-throughput sequencing library quantification kit from KAPABIOSYSTEMS according to the instructions. See the test results image 3 and Figure 4 . image 3 It shows that the repeatability of the library amplification curve is very good, and the Ct values ​​of the three repeated detections are 11.98, 11.94 and 11.95, respectively, indicating that the library was successfully constructed and the qPCR operation did not cause contamination. Figure 4 is the melting curve graph of the library, from Figure 4 It can be seen that there is only a single peak in the melting curve of the library, and there are no miscellaneous peaks, indicating that the library constructed by the kit and method involv...

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Abstract

The invention relates to the field of noninvasive prenatal screening detection, in particular to a fetal chromosome library construction kit and method and application thereof.The kit includes a reagent for DNA extraction and a reagent for DNA library construction.Compared with other free DNA library construction kits in the markets at home and abroad, the fetal chromosome library construction kit can directly extract free DNA from the plasma of a pregnant woman to perform sequencing library construction, can completely and systematically achieve sample free DNA extraction and library construction, can be used for detecting whether fetal chromosomes are aneuploid or not through high-throughput noninvasive screening, is convenient to operate, simple, rapid, efficient and accurate in detection result and high in sensitivity and has an important value.

Description

technical field [0001] The invention relates to the field of non-invasive prenatal screening and detection, in particular to a kit, method and application for constructing a fetal chromosome library. Background technique [0002] Down syndrome, also known as trisomy 21 or congenital stupidity, is a congenital disease caused by chromosomal abnormalities in newborns. The incidence rate of Down's syndrome is about 1 / 800~1 / 600, and it accounts for the highest proportion among trisomy chromosomal diseases in children, about 70%~80%. increase and rise. Prenatal diagnosis and screening is an important and practical part of screening for Down syndrome. The well-known prenatal diagnosis and screening methods are mainly traditional screening methods, such as amniocentesis to extract amniotic fluid, chorionic villi Membrane sampling and puncture to draw umbilical cord blood and other methods. These methods are invasive to a certain extent and cause certain dangers to pregnant women ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/06C12Q1/68
Inventor 陈勇张梦玲谢文龙王玲
Owner 北京爱普益医学检验中心有限公司
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