PCR detection method for human hsa-miR-124a-3p and application of PCR detection method to DS (Down's syndrome) pregnancy rick prediction

A detection method and risk prediction technology, applied in the field of molecular biology, can solve the problems of long inspection cycle, high condition requirements, complex technical operation, etc., and achieve the effects of wide detection linear range, high detection sensitivity and good repeatability

Inactive Publication Date: 2017-10-10
THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Down's screening is highly safe, but its specificity is poor. Although the prevalence of DS is as high as 84% ​​when the fetal nuchal translucency (NT) > 2.5 mm, it still cannot meet the requirements for diagnosis. Down's screening High-risk pregnant women still need follow-up examinations to confirm the diagnosis
Non-invasive DNA testing is a prenatal detection method for DS that has emerged in recent years, also known as non-invasive prenatal DNA testing or non-invasive fetal chromosome aneuploidy testing. And its sequence analysis is performed to obtain the genetic information of the fetus, so as to diagnose DS, Patau Syndrome (PS) and Edwards Syndrome (Edwards Syndrome, ES), its safety and sensitivity are high, but the test The cost is high, the technical operation is complicated, and the inspection cycle is long. At presen

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  • PCR detection method for human hsa-miR-124a-3p and application of PCR detection method to DS (Down's syndrome) pregnancy rick prediction
  • PCR detection method for human hsa-miR-124a-3p and application of PCR detection method to DS (Down's syndrome) pregnancy rick prediction
  • PCR detection method for human hsa-miR-124a-3p and application of PCR detection method to DS (Down's syndrome) pregnancy rick prediction

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Example 1, RT-qPCR detection of hsa-miR-124a-3p

[0032] 1. hsa-miR-124a-3p RT reaction

[0033] (1) Synthesis of hsa-miR-124a-3p standard product: log on to http: / / www.mirbase.org / website, obtain the base sequence of hsa-miR-124a-3p as 5′-UAAGGCACGCGGUGAAUGCC-3′ (such as shown in SEQ ID NO:1). The obtained hsa-miR-124a-3p sequence was sent to Dalian Bao Biological Engineering Co., Ltd. to synthesize hsa-miR-124a-3p standard (12.8nmols).

[0034] (2) Preparation of hsa-miR-124a-3p standard products with different concentrations: add 1.28ml RNase-free water to 12.8nmol hsa-miR-124a-3p standard products to prepare 10pmol / μL hsa-miR- 124a-3p standard solution, as a storage solution for the standard. Then continue to use RNase-free water to dilute the 10pmol / μL hsa-miR-124a-3p standard to 10-fold concentration gradient to two concentrations of 1 and 0.1pmol / μL.

[0035] (3) Design of hsa-miR-124a-3p RT primers: Replace all U in the hsa-miR-124a-3p sequence with T to ob...

Embodiment 2

[0054] Example 2. Clinical application of maternal plasma hsa-miR-124a-3p in prenatal screening for Down syndrome

[0055] 1. Extraction of total RNA in plasma

[0056] Collection and grouping of clinical specimens: 36 cases of plasma from pregnant women in the second trimester (13-27 weeks) of healthy fetuses aged 20-35 were collected from the Prenatal Diagnosis Center of the Department of Obstetrics and Gynecology, the First Affiliated Hospital of Third Military Medical University, and they were used as the normal control group; DS was diagnosed in pregnancy 11 cases of pregnant women's plasma in the second trimester of pregnancy belonged to the DS pregnancy group; the blood samples were anticoagulated with EDTA, centrifuged at 3000r / min at 25°C for 3min, and the upper layer of plasma was drawn, and stored at -80°C for later use. Total RNA was extracted from plasma.

[0057] 2. RT-qPCR reaction

[0058] According to the method in Example 1, RT-qPCR was performed on the ext...

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Abstract

The invention discloses a PCR detection method for human hsa-miR-124a-3p. The PCR detection method comprises the following steps: 1) extracting total RNA; 2) performing RT-qPCR detection: an RT primer is 5'-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACGGCATTC-3', an RTreaction procedure refers to 16DEG C for 30min, 42DEG C for 30min and 85DEG C for 5min; qPCR procedure refers to 50DEG Cfor 2min, 95DEG C for 10min, 95DEG Cfor 15s, 60DEG C for 60s, and 35cycles; 3)calculating concentration of to-be-detected hsa-miR-124a-3p. The invention further discloses an application of the PCR detection method to DS (Down's syndrome) pregnancy rick prediction.10<-5>-10<-3> pmol/mu L is determined as a DS high risk interval value of the detection method.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a human hsa-miR-124a-3p PCR detection method and its application in DS pregnancy risk prediction. Background technique [0002] Down's syndrome (Down's syndrome, DS) is the most common genetic disease in humans that causes mental retardation and developmental disorders in newborns, also known as trisomy 21 (trisomy 21, T21) or congenital stupidity Its incidence rate in newborn fetuses is about 1 / 700, while the incidence rate of DS in my country is about (1.53-2.40) / 10,000. Most fetuses with DS stop developing or cause miscarriage in utero. Therefore, the actual incidence of DS should be much higher than 1 / 700. And with the increase of women's pregnancy age, especially the change of my country's current population and childbearing policy, the increase of older or elderly pregnant women and the further promotion and application of assisted reproductive technol...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6883C12Q1/6851C12Q2600/178C12Q2545/114C12Q2525/207
Inventor 张波李胜蓝罗娟吴柳
Owner THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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