Serum/plasma miRNA marker related to assisted diagnosis of intrahepatic cholestasis of pregnancy and application of serum/plasma miRNA marker

A technique for cholestasis and auxiliary diagnosis, applied in the fields of genetic engineering and reproductive medicine

Active Publication Date: 2018-08-24
WUXI MATERNAL & CHILD HEALTH HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, there are currently no reports of relatively stable biomarkers for ICP-assisted diagnosis. If ICP-specific or abnormally expressed serum / plasma miRNAs can be screened as biomarkers and corresponding auxiliary diagnostic kits can be developed, it will be extremely important. Greatly improve the diagnosis status of ICP in my country

Method used

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  • Serum/plasma miRNA marker related to assisted diagnosis of intrahepatic cholestasis of pregnancy and application of serum/plasma miRNA marker
  • Serum/plasma miRNA marker related to assisted diagnosis of intrahepatic cholestasis of pregnancy and application of serum/plasma miRNA marker
  • Serum/plasma miRNA marker related to assisted diagnosis of intrahepatic cholestasis of pregnancy and application of serum/plasma miRNA marker

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] The collection of embodiment 1 sample and the arrangement of sample data

[0063] The inventor collected a large number of peripheral blood samples of ICP patients and healthy control pregnant women from October 2016 to March 2017 from the Wuxi Maternal and Child Health Hospital affiliated to Nanjing Medical University (the samples used for research were collected during the same period, sampled, sub-packaged, and stored) The conditions are uniform), and by sorting out the sample data, the inventor selected 88 samples that meet the following criteria as the experimental samples for Agilent miRNA chip detection and a series of subsequent qRT-PCR verifications:

[0064] 1. The above research objects are second trimester Pregnant women who were confirmed to have ICP during ICP screening (refer to the guidelines for diagnosis and treatment of ICP patients (first edition)) were defined as cases.

[0065] 2. The above research objects are in second trimester No ICP occurr...

Embodiment 2

[0067] Agilent miRNA chip detection of miRNA in embodiment 2 serum / plasma

[0068] The above-mentioned 4 eligible ICP cases and 4 healthy controls were detected by Agilent miRNA chip to obtain relevant results. The specific steps are:

[0069] 1. Take 600ul of serum from the "intrahepatic cholestasis of pregnancy" group and the "healthy female control" group, and add 3 times the volume of Trizol reagent;

[0070] 2. Phase separation: place it at 37°C for 15 minutes after shaking for 1 minute, and add the internal reference gene hsa-mir-16 (primers are SEQ ID No.7 and SEQ ID No.8) to control the difference between samples before resting, so that The final concentration is 10 -4 pmol / μl. Add an equal volume of chloroform to the turbid solution, shake for 50 s, and keep at room temperature for 15 min. After resting, centrifuge at 14,000rpm at 4°C for 15min;

[0071] 3. RNA precipitation: transfer the water phase to a new 15ml centrifuge tube, add 1.5 times the volume of the ...

Embodiment 3

[0079] qRT-PCR experiment of miRNA in embodiment 3 serum / plasma

[0080] In the Agilent miRNA chip, the CT values ​​of the two groups of research subjects were not greater than 35 and the miRNAs whose expression signals were relatively uniform among the samples of each group were selected for further verification by qRT-PCR method to improve the detection efficiency.

[0081] miRNAs meeting the above conditions include: miR-371a-5p, miR-6865-5p, miR-1182, miR-6803-5p.

[0082] According to the above Agilent miRNA results, select miR-371a-5p, miR-6865-5p, miR-1182, miR-6803-5p to design primers for reverse transcription and qRT-PCR. The qRT-PCR detection of miRNA was performed on individual individuals in the serum of the "ICP case" group and the "healthy control" group, see Table 1.

[0083] Table 1: Differential expression of miRNA detected by miR array in ICP group and control group

[0084]

[0085]

[0086] More than 2.0-fold up- or down-regulated miRNAs in ICP (P)...

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Abstract

The invention discloses a serum/plasma miRNA marker related to assisted diagnosis of intrahepatic cholestasis of pregnancy and an application of the serum/plasma miRNA marker. The serum/plasma miRNA marker related to ICP assisted diagnosis consists of miR-371a-5p, miR-6865-5p and miR-1182. The invention also discloses a primer of the serum/plasma miRNA marker and an application of the primer in preparing an ICP assisted diagnosis kit. The inventor, by separating and researching ICP cases of first pregnancy and singleton pregnancy and by comparing miRNAs in serum/plasma between the cases and healthy pregnant women matched in age, finds out a group of high-specificity and high-sensitivity miRNAs highly related to ICP attack; and an ICP assisted diagnosis kit convenient for clinical application is researched out, so that laboratory support is provided for screening and diagnosis & treatment of the ICP.

Description

field of invention [0001] The invention belongs to the fields of genetic engineering and reproductive medicine, and relates to a serum / plasma miRNA marker related to the auxiliary diagnosis of intrahepatic cholestasis during pregnancy and an application thereof. Background technique [0002] Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy-related syndrome characterized by pruritus, elevated serum liver enzymes and bile acids, and is a complication that seriously endangers the health of mother and child during pregnancy. Its incidence rate can be as high as 12.0%, which can cause adverse pregnancy outcomes such as fetal distress, spontaneous premature birth, and stillbirth, and increase perinatal morbidity and mortality. The greatest harm of ICP to pregnancy is the unpredictable sudden death of the fetus, and the perinatal mortality rate is about 6-10 times that of normal pregnancy. Although it has been reported that various genes and proteins of ICP will change, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q2600/118C12Q2600/158C12Q2600/178
Inventor 张婷邹萍王晶李娜董蕊锐王瑶
Owner WUXI MATERNAL & CHILD HEALTH HOSPITAL
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