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Reagent and kit for vascular calcification diagnosis, and application

A diagnostic kit and vascular calcification technology, applied in the field of biomedicine, can solve the problems of limited accuracy, long time and high cost of PCR detection

Inactive Publication Date: 2021-12-17
THE FIRST AFFILIATED HOSPITAL HENGYANG MEDICAL SCHOOL UNIV OF SOUTH CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the reagents used for detection are conventional, and the time for extraction, reverse transcription, and PCR detection is long, the cost is high, and the accuracy is limited.

Method used

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  • Reagent and kit for vascular calcification diagnosis, and application
  • Reagent and kit for vascular calcification diagnosis, and application
  • Reagent and kit for vascular calcification diagnosis, and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] 1. Research object

[0046] A total of 66 patients diagnosed with coronary artery calcification by coronary angiography and dual-source CTC were selected from June 2013 to May 2014 in the First Affiliated Hospital of Nanhua University. Those with a history of myocardial infarction, history of coronary or peripheral arterial revascularization, hepatic and renal insufficiency, thyroid or adrenal dysfunction, abnormal calcium metabolism, chronic infection (active tuberculosis, COPD protracted stage), connective tissue disease, and tumor were excluded. , end-stage of immune system disease or mental illness and other diseases, etc. According to the Agatston method score (CACS) of coronary artery CTA, they were divided into coronary artery calcification group and control group, in which 8 patients with coronary artery calcification score equal to 0 points were in the non-coronary artery calcification group, and 8 patients with coronary artery calcification score greater than ...

Embodiment 3

[0093] The research objects, plasma collection and template preparation were the same as in Example 1.

[0094] 1. Configure the qPCR reaction system (Kangwei):

[0095]

[0096]

[0097] Wherein primer and internal reference are with embodiment 1.

[0098] 2. The amplification reaction program (three-step method) is set as follows:

[0099] Stage 1: Reverse transcription at 45°C for 10 minutes

[0100] Stage 2: Pre-denaturation at 95°C for 5 minutes

[0101] Stage 3: PCR reaction (40 cycles)

[0102] Denaturation 95℃ 15sec

[0103] Annealing 60℃ 30sec

[0104] Extend 72℃ 30ses

[0105] Stage 4: Melting curve analysis

[0106]

[0107] 3. Result Analysis

[0108] as attached Figure 6-9 As shown, the amplification curve of the internal reference is not clear, but the amplification curve of miR-32 is messy. It can be seen from the dissolution curves that the dissolution curves of both are non-unimodal and chaotic. It shows that this amplification system has ...

Embodiment 4

[0110] The research objects, plasma collection and template preparation were the same as in Example 1.

[0111] 1. Configure the qPCR reaction system (Kangwei):

[0112]

[0113] Wherein primer and internal reference are with embodiment 1.

[0114] 2. The amplification reaction program (three-step method) is set as follows:

[0115] Stage 1: Reverse transcription at 45°C for 10 minutes

[0116] Stage 2: Pre-denaturation at 95°C for 5 minutes

[0117] Stage 3: PCR reaction (40 cycles)

[0118] Denaturation 95℃ 15sec

[0119] Annealing 60℃ 30sec

[0120] Extend 72℃ 30ses

[0121] Stage 4: Melting curve analysis

[0122]

[0123] 3. Result analysis

[0124] as attached Figure 10-11 As shown, the amplification curves of internal references with different amounts of serum mixture added are acceptable, but there is no gradient difference. The amplification curve of miR-32 was messy, and the amplification value was high.

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Abstract

The invention belongs to the field of biomedicine, and particularly relates to a reagent and kit for vascular calcification diagnosis, and an application. The reagent or kit for vascular calcification diagnosis comprises a No.1 reagent, a No.2 reagent and a No.3 reagent, wherein the No.1 reagent is an rRNA PCR amplification reagent; the No.2 reagent is a cDNA PCR amplification reagent; and the No.3 reagent is a sample releasing agent. The reagent and the kit have obvious advantages in the aspect of detection cost. An old method needs three steps to complete detection, and the time is about 6 hours. The optimized detection method only comprises one step, and the detection time is about 1.5 h. The method provided by the invention has obvious advantages in detection time efficiency.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a reagent, kit and application for vascular calcification diagnosis. Background technique [0002] Vascular calcification is a common pathological change in diseases such as diabetes, hypertension, atherosclerotic vascular disease, and other vascular injuries. It is one of the important factors for the high morbidity and mortality of cardiovascular and cerebrovascular diseases. It is considered an accurate predictor of adverse cardiovascular events. In the past, vascular calcification was considered to be the passive deposition of calcium salts in cells and extracellular matrix. In recent years, however, vascular calcification has been found to be an active, highly tunable biological process similar to bone formation. As the main constituent cells of the cardiovascular system, vascular smooth muscle cells change from a contractile phenotype to an osteoblast-like phenotype...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/686
CPCC12Q1/6883C12Q1/686C12Q2600/158C12Q2600/166C12Q2600/178C12Q2527/125
Inventor 刘江华祖旭宇曹劲松
Owner THE FIRST AFFILIATED HOSPITAL HENGYANG MEDICAL SCHOOL UNIV OF SOUTH CHINA
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