DNA probe and gene chip for detecting cryptococus neoformans and application thereof

A technology of Cryptococcus neoformans and DNA probes, which is applied in the direction of DNA/RNA fragments, recombinant DNA technology, microbial measurement/inspection, etc. It can solve the problems of not being able to detect fungi at the same time, not being able to really apply clinically, and not having high throughput. , to achieve the effect of high sensitivity, small sample volume and low reagent consumption

Active Publication Date: 2011-05-04
TARGETINGONE CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] (4) There is contamination when the operator handles the specimen
[0014] Including in situ hybridization, polymerase chain reaction, determination of G+C mol% content in DNA, random amplified polymorphic DNA (RAPD) analysis, restriction endonuclease polymorphism analysis (RFLP), etc. It is time-consuming and labor-intensive, does not have the characteristics of high-throughput, and cannot quickly and simultaneously detect a variety of possible fungi with a small amount of samples, so it is mostly limited to laboratory research and cannot be truly applied to clinical practice.

Method used

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  • DNA probe and gene chip for detecting cryptococus neoformans and application thereof
  • DNA probe and gene chip for detecting cryptococus neoformans and application thereof
  • DNA probe and gene chip for detecting cryptococus neoformans and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Extraction of DNA

[0044] Extraction can be performed according to conventional methods in the art, and samples can be fresh tissue, blood, urine, cerebrospinal fluid, secretions (sputum, pleural effusion, ascites, alveolar lavage fluid, etc.) and the like. In this embodiment, Qiagen micro DNA extraction kit is used, taking fresh tissue as an example:

[0045] (1) Put the fresh tissue into a high-temperature sterilized mortar, pour liquid nitrogen into it and grind it, and transfer the ground tissue into a 1.5ml EP tube;

[0046] (2) Add 1ml of normal saline to rinse, centrifuge at 3000rpm for 5min, and discard the supernatant;

[0047] (3) Add 200μl NaOH (50mM), incubate at 95°C for 10min, centrifuge at 5000rpm for 10min, and discard the supernatant;

[0048] (4) Add 500 μl Lyticase solution and incubate at 37°C for 30 minutes;

[0049] (5) Centrifuge at 14000rpm for 10min, discard the supernatant;

[0050] (6) Quickly add 180 μl ATL and 20 μl proteinas...

Embodiment 2

[0061] Embodiment 2: the PCR amplification of sample

[0062] PCR reaction system: The total volume of PCR reaction is 25 μl, containing template DNA (Cryptococcus neoformans, Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Aspergillus terreus, Aspergillus nidulans, Candida kefir, Candida rugosa, Trichospore asahi Rhizopus aureus, Rhizopus microsporum, Rhizomucor micromus, Absidia umbelliferum, Candida glabrata, Candida albicans, Candida krusei, Candida tropicalis, Candida parapsilosis, Candida quaternidae or Candida Dublin DNA) 5μl, 10×Buffer 2.5μl, 2.5mM dNTP 1.25μl, 25mM MgCl 2 3 μl, 0.25 μl each of primers ITS1 and ITS4 50 pmol, 0.1 μl of Hotstart Taq enzyme (5U / μl), and supplemented with ultrapure water to 25 μl.

[0063] PCR reaction conditions: 95°C for 15 minutes, amplification denaturation, annealing, and extension conditions were denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 1 minute, 35 cycles, and final...

Embodiment 3

[0064] Embodiment 3: the labeling of probe and the preparation of gene chip

[0065] (1) with 0.5M NaHCO 3 (PH 8.4) Dilute the probe of the present invention to an appropriate concentration, take 4ul of the probe solution into the microwell plate, add 4ul of sample solution to each well, mix well, and the blank control is an equivalent amount of the above-mentioned NaHCO 3 Mixture with sample solution.

[0066] (2) Cut the BiodyneC nylon membrane to a suitable size.

[0067] (3) Biodyne C nylon membrane was incubated with 16% w / v EDAC for 10 minutes at room temperature.

[0068] (4) Rinse the Biodyne C nylon membrane with deionized water for 1 minute.

[0069] (5) Dry the nylon membrane with filter paper.

[0070] (6) Spot the probe solution and blank control solution on the prepared nylon membrane with a spotting instrument.

[0071] (7) Incubate at room temperature for 5 minutes.

[0072] (8) Remove the liquid on the surface of the nylon membrane, and put the nylon mem...

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Abstract

The invention relates to the field of biotechnology, and discloses a deoxyribonucleic acid (DNA) probe for detecting cryptococus neoformans. The DNA probe for detecting the cryptococus neoformans has a nucleotide sequence shown as SEQ ID No. 1 in a sequence table, and has high specificity and sensitivity of 0.04ng for detecting the cryptococus neoformans. The invention also provides a gene chip of the DNA probe. By using the gene chip, high-flux, automation and quick detection of a sample which possibly contains the cryptococus neoformans can be realized, the specificity is high, the accuracy of a detection result can be guaranteed, and the DNA probe and the gene chip have broad application prospect.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a DNA probe for detecting cryptococcus neoformans, a gene chip and applications thereof. Background technique [0002] Invasive fungal infection is a serious infection in which fungi invade the human body and cause blood infection, infection of various organs, or systemic dissemination. In the past 20 years, the incidence of invasive fungal infection has shown an obvious increasing trend, which is related to the extensive use of broad-spectrum antibiotics, anticancer radiotherapy, chemotherapy and other immunosuppressants, steroid drugs, organ transplantation, intravenous catheterization and various invasive operations. [0003] The main pathogenic fungi of invasive fungal infections include opportunistic fungi such as Candida, Aspergillus, and Cryptococcus, as well as endemic pathogenic fungi such as Histoplasma, Blastomyces dermatitidis, and Sporothrix. In different environments, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
Inventor 李国辉
Owner TARGETINGONE CORP
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