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Fluorescence quantitative PCR (polymerase chain reaction) detection method of aspergillus fumigatus

A fluorescence quantification and detection method technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems of unsuitability for the growth of desiccated fungi, easy inactivation of bioaerosols, and inability to form colonies, etc. The effect of shortening the detection cycle, high speed and high sensitivity

Inactive Publication Date: 2012-01-11
SHANDONG AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the culture counting method depends on the cultivability of microorganisms. First, not all microorganisms can grow on the sampling medium, and different microorganisms have different growth requirements. For example, MEA is not suitable for the growth of xerophilic fungi; secondly, biogas The sol is easily inactivated during the sampling process, so that it cannot form colonies on the medium; again, many microorganisms in the air have died, but their cellular components can still cause allergies or poisoning, and this part of the aerosol cannot be cultured. determination

Method used

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  • Fluorescence quantitative PCR (polymerase chain reaction) detection method of aspergillus fumigatus
  • Fluorescence quantitative PCR (polymerase chain reaction) detection method of aspergillus fumigatus
  • Fluorescence quantitative PCR (polymerase chain reaction) detection method of aspergillus fumigatus

Examples

Experimental program
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Effect test

Embodiment 1

[0032] Example 1: Aspergillus fumigatus strain was used as the detection material, and ultrapure water was used as the negative control.

[0033] 1. DNA extraction from Aspergillus fumigatus

[0034] The purchased Aspergillus fumigatus standard strain was inoculated on potato dextrose agar medium, and cultured at 28°C for 7 days for DNA extraction. Under sterile conditions, scrape a small amount of mycelium with an inoculation loop and put it into a 1.5ml centrifuge tube that has been added with the extract in advance, mash the mycelium with the grinding pestle attached to the kit, and then press the kit (Tiangen Company, fast DNA extraction and amplification set, catalog number: KG201, instructions see the website of Tiangen Company) to perform DNA extraction.

[0035] 2. Fluorescent quantitative PCR detection

[0036] The above-mentioned DNA was taken as a template for fluorescence quantitative PCR detection, and a standard curve was prepared at the same time. The amplific...

Embodiment 2

[0038] Example 2: Fluorescent quantitative PCR detection of Aspergillus fumigatus in disease materials to be tested (rabbit throat swabs and cloacal swabs with respiratory symptoms).

[0039] 1. Sample preparation:

[0040] After the rabbit throat swab and cloacal swab samples were fully mixed on the mixer, the liquid in the swab was squeezed out with sterilized tweezers, left at room temperature for 30min, and the supernatant was transferred to a sterile 1.5mL Eppendorf tube. No. spare.

[0041] 2. DNA extraction from Aspergillus fumigatus

[0042] The above liquid was subjected to DNA extraction according to the instructions of the kit (Tiangen Company, Rapid DNA Extraction and Amplification Kit, catalog number: KG201, instructions on the website of Tiangen Company).

[0043] 3. Fluorescent quantitative PCR detection

[0044] Take the above DNA as a template, carry out fluorescent quantitative PCR detection, and formulate a standard curve at the same time, the amplification...

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Abstract

The invention relates to a fluorescence quantitative PCR (polymerase chain reaction) detection method of aspergillus fumigatus. In the detection method, the mitochondrial translation optimization protein (Mtol) of aspergillus fumigatus is used as a reference sequence to design specific primers and probes, and the quantitative analysis is carried out on aspergillus fumigatus by virtue of fluorescence quantitative PCR. The fluorescence quantitative PCR detection method provided by the invention has the advantages of high sensitivity, strong specificity, simple operation, high speed, and high flux, greatly shortens the detecting cycle, provides guarantee for the disease treatment and epidemic control, and avoids the possible biological pollution during the detecting process.

Description

(1) Technical field [0001] The invention relates to a fluorescent quantitative PCR detection method for Aspergillus fumigatus. (2) Background of the invention [0002] Aspergillus fumigatus is a common air fungus, and it is also a fungus that can produce toxic substances. Its pathogenic factors can be divided into toxins and enzymes, which work together in the pathogenicity of Aspergillus fumigatus: first, the protease produced by Aspergillus fumigatus may not directly participate in the invasion of the host by Aspergillus fumigatus, but it can promote the adhesion of the fungus to the host tissue. In addition to penetration, because the respiratory epithelium constitutes an internal and external environmental barrier, microorganisms must first penetrate the epithelial tissue to enter the lung parenchymal cells to form lung infection, and Aspergillus fumigatus can produce certain proteases, especially serine proteases, which can promote the shedding of alveolar epithelial ce...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/06
Inventor 柴同杰苗增民
Owner SHANDONG AGRICULTURAL UNIVERSITY