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Diagnostic kit for recurrent spontaneous abortion and application of NLRC5 gene

A diagnostic kit and technology of spontaneous abortion, applied in the direction of DNA / RNA fragments, recombinant DNA technology, microbial measurement / inspection, etc., can solve problems such as missed diagnosis and incompleteness

Pending Publication Date: 2021-10-15
中国科学院大学深圳医院
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, it is not perfect to judge whether spontaneous abortion will occur only by detecting the abnormal expression of HLA-A / -B in peripheral blood alone, and there may be cases of missed diagnosis

Method used

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  • Diagnostic kit for recurrent spontaneous abortion and application of NLRC5 gene
  • Diagnostic kit for recurrent spontaneous abortion and application of NLRC5 gene
  • Diagnostic kit for recurrent spontaneous abortion and application of NLRC5 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1: Analysis of NLRC5 protein expression in tissue embryo trophoblast by immunohistochemical method

[0059] NLRC5 immunohistochemical detection kit composition: blocking solution (containing 10% goat serum), rabbit anti-NLRC5 polyclonal antibody (primary antibody), goat anti-rabbit IgG H&L-HRP antibody (secondary antibody), DAB chromogenic solution.

[0060] Specific steps of immunohistochemistry:

[0061] A) Preparation of specimens: Paraffin sections were dewaxed to water, rinsed with distilled water, placed in PBS buffer for 10 min, and then placed in 3% H 2 o 2 Incubate for 10 min, remove the slices and wash 3 times with PBS, 2 min each time.

[0062] B) Blocking plus antibody: block with 10% goat serum blocking solution, and incubate at room temperature for 10 min. Pour off the serum, add 1:1000 diluted primary antibody dropwise as the primary antibody working solution, and incubate overnight at 4°C. The slices were removed and washed 3 times with PBST,...

Embodiment 2

[0065] Example 2: Detection of NLRC5 mRNA expression in tissue embryo trophoblast by qPCR method

[0066] The composition of NLRC5 qPCR detection kit: Trizol solution, 75% ethanol, DEPC water, reverse transcriptase, SYBRGreen PCR Master Mix, NLRC5 forward primer and NLRC5 reverse primer.

[0067] qPCR specific steps:

[0068] A) Extraction of RNA: Grind the aborted tissue thoroughly in liquid nitrogen, add 1ml Trizol solution to 50g tissue sample, mix well and let stand at room temperature for 5min, add 0.2ml chloroform, shake vigorously for 30s, let stand at room temperature for 5min, centrifuge at 12000g at 4°C for 15min, Transfer the uppermost layer in the stratification to an RNase-free EP tube, add an equal volume of isopropanol to precipitate the RNA, mix gently, place at room temperature for 10 minutes, and centrifuge at 12000g at 4°C for 10 minutes to remove the supernatant. Add 1ml of 75% ethanol and centrifuge at 10,000g for 5min, discard the supernatant, wash twice...

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Abstract

The invention discloses a diagnostic kit for detecting recurrent spontaneous abortion based on abnormal expression of a trophoblast NLRC5 gene. Comprising an NLRC5 gene expression detection reagent and an NLRC5 protein expression detection reagent. The NLRC5 gene expression detection reagent is used for detecting the expression quantity of NLRC5 gene mRNA of trophoblast cells. The NLRC5 protein expression detection reagent is used for detecting the expression of the protein coded by the NLRC5 of the trophoblast. As one of NOD-like protein family members, NLRC5 is a key transcriptional regulatory factor of MHC-I type molecular gene expression. In combination with a part of results of the embodiment of the invention, the abnormal expression of the NLRC5 gene can be clearly detected in the RSA trophoblast, and the abnormal expression of the NLRC5 gene does not appear in normal tissues, so that the abnormal expression of the NLRC5 gene can be used as a target for detecting the recurrent spontaneous abortion. In addition, the invention also discloses application of the NLRC5 gene in preparation of diagnostic drugs and diagnostic reagents for recurrent spontaneous abortion.

Description

technical field [0001] The invention relates to the field of recurrent spontaneous abortion research, in particular to a diagnostic kit for detecting recurrent spontaneous abortion based on the abnormal expression of trophoblast NLRC5 gene and the application of NLRC5 gene. Background technique [0002] Recurrent spontaneous abortion (RSA) refers to 2 or more consecutive spontaneous abortions. It is a common complication during pregnancy, which plagues about 5% of women trying to conceive worldwide, and seriously affects family happiness and social harmony [1]. Factors such as chromosomal abnormalities, uterine malformations, endocrine dysfunction, and immune rejection are all potential causes of RSA [2]. In recent years, more and more evidence has shown that immune rejection is one of the important factors leading to RSA [3], but its pathogenic mechanism is still unclear. The immunosuppressive state of the utero-placental microenvironment is required for the establishment ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q2600/158
Inventor 张慧李宁麦细贤刘璐李艳
Owner 中国科学院大学深圳医院
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