General atherosclerosis detection primer group for persons and monkey, detection chip and detection method

A technology for detection of atherosclerosis and primers, applied in biochemical equipment and methods, recombinant DNA technology, microbiological determination/testing, etc. Chips, waste, etc.

Inactive Publication Date: 2013-03-20
广东蓝岛生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] 1. The atherosclerosis PCR chips mentioned above are not clearly applicable to that kind of tissue. Considering the spatiotemporal nature of gene expression, there will be a lot of waste when we use these chips for high-throughput detection;
[0007] 2. At present, there is still no atherosclerosis PCR chip suitable for monkeys, which brings inconvenience for us to use monkeys, especially model monkeys, as experimental animals for atherosclerosis gene diagnosis research

Method used

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  • General atherosclerosis detection primer group for persons and monkey, detection chip and detection method
  • General atherosclerosis detection primer group for persons and monkey, detection chip and detection method
  • General atherosclerosis detection primer group for persons and monkey, detection chip and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Example 1. Extraction of white blood cells.

[0079] Collect venous blood into anticoagulant tubes and label them separately; 7 cases in each of healthy cynomolgus monkeys, cynomolgus monkeys with atherosclerosis, healthy people, and patients with atherosclerosis. Samples 1-7 are samples of healthy cynomolgus monkeys; samples 8-14 are samples of cynomolgus monkeys with atherosclerosis; samples 15-21 are samples of healthy people; samples 22-28 are samples of patients with atherosclerosis. The atherosclerotic cynomolgus monkey mentioned above is a model monkey of atherosclerosis obtained by inducing a normal cynomolgus monkey with a high-energy diet. The relevant indicators of the obtained model monkeys were detected as follows: C-reactive protein > 10㎎ / L, cholesterol > 7.97mmol / L, low-density lipoprotein cholesterol > 6.57mmol / L, high-density lipoprotein cholesterol 1.58mmol / L, fasting blood glucose >4.62mmol / L, blood pressure ≥140 / 90mmHg. In addition, the test resul...

Embodiment 2

[0082] Example 2. Extraction of RNA.

[0083] ①Take out the leukocytes obtained in Example 1 from the liquid nitrogen, let stand at room temperature, flick the wall of the tube to disperse the leukocytes, add 1 mL TRIZOL to each cryovial, and mix well.

[0084] ②Layering: After adding TRIZOL to the sample, place it at room temperature for 5 minutes to fully lyse the sample. Then add 200 μL of chloroform, vibrate vigorously and mix well, then place at room temperature for 3-5min.

[0085] ③ RNA precipitation: centrifuge at 10000g for 15min at 4°C. The sample will be divided into three layers: the yellow organic phase in the lower layer, the colorless aqueous phase in the middle and upper layers, and the RNA is mainly in the aqueous phase. Transfer the aqueous phase (usually 550 μL can be pipetted) to a new tube. Pipette the aqueous phase carefully and never pipette the intermediate interface, as this will result in DNA contamination in the RNA sample. Add an equal volume o...

Embodiment 3

[0090] Example 3. Reverse transcription of cDNA.

[0091] Use Transgene's cDNA reverse transcription kit——EasyScript First-Strand cDNA Synthesis SuperMix (TransGen Biotech, AE301) to reverse-transcribe the RNA extracted in Example 2 into cDNA. The specific method can be carried out by referring to the instructions, and the prepared cDNA is placed in Store at -20°C until use.

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PUM

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Abstract

The invention relates to a detection primer group, a polymerase chain reaction (PCR) detection chip and a detection method for analyzing atherosclerosis related gene expression conditions of human beings and machin. The general atherosclerosis detection primer group for persons and machin comprises amplification primer pairs for respectively amplifying atherosclerosis related genes ACE, APOA1, APOC1, APOF, CCL2, CD40LG, DBP, EDN1, FAS, FASLG, FGG, GLG1, GRN, ICAM1, IL18, IL1beta, IL1RN, IL6, IL6ST, IL8, ITGalpha2beta, ITGalpha4, ITGalpha6, ITGalphaM, ITGalphaV, ITGbeta1, ITGbeta2, LTA, LTB, MTHFR, NFKbeta1, OLR1, PLAUR, PLTP, PON2, SELL, SELP, SELPG, SERPINE1, TIMP1, TNF and TNFRSF1B, and the sequences of the amplification primer pairs are nucleotide sequences shown as SEQ ID NO:1-84. The invention also provides the PCR detection chip containing the primer group and the detection method. The invention can accurately quantify and detect the expression condition of the atherosclerosis related genes of the persons and the machin, and has significance for promotion of basic research of atherosclerosis, prevention detection and clinical treatment.

Description

technical field [0001] The invention relates to a detection primer group, a fluorescence quantitative PCR detection chip and a detection method which can be used for analyzing the expression of atherosclerosis-related genes in the peripheral venous blood leukocytes of human beings and cynomolgus monkeys. Background technique [0002] Gene diagnosis was born in the late 1970s. Gene diagnosis is an example of the combination of modern molecular biology theory and technology with medicine. Genetic diagnosis is the diagnosis of etiology, which is both specific and sensitive. Its advantages are mainly reflected in the following aspects: It can reveal the genetic status related to the disease before symptoms appear, so that it can be used to diagnose the carriers with normal phenotype and the susceptibility of a certain disease. It can also be combined with conventional clinical diagnostic indicators to play an important role in clinical diagnosis and prognosis evaluation; in addi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 刘晓明彭白露夏机良郝香芬季芳
Owner 广东蓝岛生物技术有限公司
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