Single-stranded DNA aptamer specifically bonded with (1,3)-beta-D-glucose in candida albicans and screening method of single-stranded DNA aptamer and reagent

A technology of Candida albicans and dextran, which is applied in the fields of molecular microbiology and infection immunity, can solve the problems of difficult antibody preparation and detection, and achieve good application prospects

Active Publication Date: 2017-02-01
阜阳职业技术学院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The (1,3)-β-D-glucan in the fungal cell wall is a polysaccharide substance with poor water solubility, and the immunogenicity of the carbohydrate antigen is poor, so it is difficult to prepare antibodies, and it is difficult to use conventional immunological methods. detection

Method used

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  • Single-stranded DNA aptamer specifically bonded with (1,3)-beta-D-glucose in candida albicans and screening method of single-stranded DNA aptamer and reagent
  • Single-stranded DNA aptamer specifically bonded with (1,3)-beta-D-glucose in candida albicans and screening method of single-stranded DNA aptamer and reagent
  • Single-stranded DNA aptamer specifically bonded with (1,3)-beta-D-glucose in candida albicans and screening method of single-stranded DNA aptamer and reagent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Selection of Materials

[0033] Reagents and Instruments:

[0034] Candida albicans ATCC10231 was purchased from Wuhan University Culture Collection Center; Candida albicans culture medium was purchased from Qingdao Haibo Biotechnology Co.; I. T4 DNA ligase was purchased from Fermentas Company; protein marker (Cat: SM1861) was purchased from Baoke Biological Company; DNA marker was purchased from Dongsheng Biological Company; AxyPrep Plasmid DNA Mini Kit was purchased from AXYGEN Company; DNA gel recovery reagent The cassette was purchased from AxyPrep Company; the PCR mix was purchased from TIANGEN Company; Wavelength Scanner (Beckmann-Coulter); Nucleic Acid Protein Detector (German EPPENDORF Company); PCR instrument (German EPPENDORF Company); single-stranded DNA library and primers were synthesized by Invitrogen Company; chemical reagents were all analytically pure, purchased from Sinopharm Chemical.

[0035] Construction of random single-stranded DNA (s...

Embodiment 2

[0039] Example 2: Extraction method

[0040] Extraction and identification of (1,3)-β-D-glucan:

[0041] Collect Candida albicans cells on the solid medium, add a small amount of PBS to wash, centrifuge and freeze-dry, take 5 grams of dry cells and add 100 mL of 1M sodium hydroxide to resuspend them in a 90°C water bath for 3 hours; after centrifugation, wash the precipitate with pure water 3 times, and then treated with 4% acetic acid solution at 37°C for 3 hours. After centrifugation, the precipitate was washed 3 times with pure water, and then washed 3 times with absolute ethanol. After dehydration with ether, the product was dried with nitrogen to obtain the product, which was passed through gas chromatography. After identification, cryopreservation for future use. Enzymatic hydrolysis with (1,3)-β-glucanase was used to increase its water solubility, and the water-soluble (1,3)-β-D-glucan with a molecular weight of about 1.4kD was recovered by cutting the gel. Sugars as ...

Embodiment 3

[0050] Example 3: Results

[0051] Extraction, purification and identification of (1,3)-β-D-glucan:

[0052] (1,3)-β-D-glucan was extracted from Candida albicans, treated with (1,3)-β-D-glucanase, the supernatant was subjected to SDS-PAGE electrophoresis and then stained with glycogen , found mainly concentrated in three regions, figure 1 As shown in A, the arrow shows the target substance of this study; in this experiment, the low-polymerization degree (1,3)-β-D-glucan with a molecular weight figure 1 As shown in B, the arrow shows the highest absorption peak at 196 nm; figure 1 The arrows in C are from left to right: rhamnose, glucose, arabinose, mannose, galactose and xylose standard, figure 1 The arrow in D shows the monosaccharide after hydrolysis. The recovered material is detected by gas chromatography after acid hydrolysis, reduction and acylation derivatization, and there is only a single peak at the retention time of 5.896min (the rest are miscellaneous peaks), suc...

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Abstract

The invention relates to a single-stranded DNA aptamer specifically bonded with (1,3)-beta-D-glucose in candida albicans and a screening method of the single-stranded DNA aptamer and a reagent. The nucleotide sequence of the single-stranded DNA aptamer specifically bonded with (1,3)-beta-D-glucose in the candida albicans is shown as SEQ ID NO.1 or SEQ ID NO.2. According to the single-stranded DNA aptamer specifically bonded with (1,3)-beta-D-glucose in the candida albicans, invasive fungal infection can be differentiated and diagnosed, a new idea is provided for differentiation and diagnosis of invasive fungal infection, and a good application prospect is achieved.

Description

technical field [0001] The invention relates to the fields of molecular microbiology and infection immunity, in particular to a single-stranded DNA aptamer that specifically binds to (1,3)-β-D-glucan in Candida albicans and a screening method and reagent thereof. Background technique [0002] Invasive fungal infection (IFI) is a fungal infection caused by pathogenic fungi invading subcutaneous tissue, mucous membranes and organs. At present, due to the irregular use of antibiotics and immunosuppressants and the widespread use of invasive medical methods, the infection rate of deep fungus in hospitals is on the rise, and its high fatality rate has also attracted the attention of medical staff. Early diagnosis and treatment are effective means to reduce the lethality of deep fungus. [0003] Fungal culture is still the gold standard for diagnosing deep fungal infections. However, factors such as the difficulty in obtaining deep fungal materials and the long fungal culture cyc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115C12Q1/68
CPCC12N15/1048C12N15/115C12N2310/16C12Q2531/107C12Q2521/301
Inventor 华影唐晓磊
Owner 阜阳职业技术学院
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