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A human cytomegalovirus gene PCR detection chip covering the whole genome

A human cytomegalovirus and detection chip technology, which is applied in the directions of microorganism-based methods, microorganism determination/inspection, combinatorial chemistry, etc., can solve the problems that PCR detection chips have not yet appeared and cannot be satisfied.

Active Publication Date: 2021-04-02
WENZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there are a considerable number of kits for HCMV gene detection in China, but DNA and cDNA PCR detection chips that can perform a large number of related cytomegalovirus genes at the same time have not yet appeared, which cannot meet the requirements for the detection of cytomegalovirus infection in human tissue and cell specimens and in vitro cultured cells. or expression required for genome-wide PCR detection

Method used

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  • A human cytomegalovirus gene PCR detection chip covering the whole genome
  • A human cytomegalovirus gene PCR detection chip covering the whole genome
  • A human cytomegalovirus gene PCR detection chip covering the whole genome

Examples

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Effect test

example 1

[0054] Example 1 Using the present invention to detect HCMV gene expression in human peripheral blood mononuclear cells, gastric cancer tissue and paracancerous tissues

[0055] Step 1: Isolation of PBMCs

[0056] 1. Required reagents and preparation methods

[0057] A: 10×PBS buffer solution: weigh NaCl, 8g; NaCl 2 HPO 4 , 1.42g; KH 2 PO 4 , 0.27g; KCl, 0.2g. After adding 800ml of deionized water, add concentrated hydrochloric acid dropwise to pH = 7.4, dilute to 1L with deionized water, and store at room temperature after autoclaving. Note: When washing peripheral blood mononuclear cells, 10×PBS must be diluted 10 times with deionized water before use.

[0058] B: 0.9% physiological saline.

[0059] C: Peripheral blood lymphocyte separation medium.

[0060] 2. Peripheral Blood Mononuclear Cell Isolation Steps

[0061] A. Take 2ml of EDTA anticoagulant blood taken intravenously on the same day, and centrifuge at 3000rpm at 4°C for 10min in 2 tubes.

[0062] B. Disca...

example 2

[0105] Example 2 Using the present invention to detect HCMV infection in human peripheral blood mononuclear cells, gastric cancer tissue and paracancerous tissues

[0106] Step 1: DNA Extraction Method

[0107] 1. Reagents and preparation

[0108] A. Electrophoresis 50×TAE buffer: weigh 242g Tris, Na2EDTA·2H 2 O 37.2g, add about 800mL of deionized water, add 57.1mL of acetic acid, and dilute to 1L. For electrophoresis or preparation of agarose gel, dilute 50×TAE 50 times into 1×TAE buffer.

[0109] B. Preparation of agarose gel

[0110] Prepare an appropriate amount of 1×TAE buffer for electrophoresis and gel preparation, accurately weigh the agarose according to the amount of gel preparation and gel concentration, add it to the Erlenmeyer flask, and heat and melt the agarose in a microwave oven. After the solution was cooled for several minutes, ethidium bromide solution (final concentration 0.5 mg / ml) was added and mixed well. Pour the agarose solution into the gel-maki...

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Abstract

The invention relates to a human cytomegalovirus gene PCR detection chip covering a whole genome. The detection chip is characterized in that 125 gene forward primer (5'-3') and reverse primer (5'-3') sequences of human cytomegalovirus are immobilized on the chip, and the primer sequences are Seq NO.1-Seq NO.250. Infection and expression of the cytomegalovirus genes can be rapidly detected, DNA samples can be detected, cDNA samples can also be detected, the PCR chip is used for detecting a human cytomegalovirus whole genome sample in China for the first time, and the chip has the advantages of being rapid, economic, capable of covering the cytomegalovirus whole genome and the like compared with a traditional cytomegalovirus whole genome detection method based on high-throughput sequencing.

Description

technical field [0001] The invention relates to the field of medical microbiology detection, in particular to a PCR detection chip that can be used for human cytomegalovirus (Human cytomegalovirus, HCMV) infection or human cytomegalovirus gene expression in human tissue and cell specimens and in vitro cultured cells, and can be used for Clinical testing and related academic research. Background technique [0002] Human cytomegalovirus (HCMV) is an opportunistic double-stranded linear DNA virus. The length of the genome is about 235kb, which is one of the currently known viruses with a large genome. The HCMV infection rate in the population is 60-100%, most of which are asymptomatic infections, but have extremely high morbidity and mortality in newborns and immunocompromised populations. It has the characteristics of lifelong latency and periodic activation, which makes HCMV more likely to cross-react with the body, thereby affecting the immune state of the body. [0003] ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/686C40B40/06C12R1/93
CPCC12Q1/701C40B40/06
Inventor 薛向阳郭刚强杨敏陈文静李宝青章慧娣沈贤
Owner WENZHOU MEDICAL UNIV