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Gene chip and kit for detecting common pathogenic bacteria in cosmetics

A gene chip and kit technology, which is applied in the field of gene chips and kits for detecting common pathogenic bacteria in cosmetics, can solve the problems of pathogenic bacteria harm, not high throughput, and unable to deal with samples, etc., to achieve strong repeatability, Easy operation and high accuracy

Active Publication Date: 2014-07-23
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this technique is not high-throughput and cannot cope with a large number of samples.
[0018] These detection techniques have their own advantages and disadvantages, and the pathogenic bacteria in cosmetics are also very harmful to humans, so it is very important to find a fast, sensitive and high-throughput method

Method used

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  • Gene chip and kit for detecting common pathogenic bacteria in cosmetics
  • Gene chip and kit for detecting common pathogenic bacteria in cosmetics
  • Gene chip and kit for detecting common pathogenic bacteria in cosmetics

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Probe design and preparation

[0060] 1. Sequence acquisition

[0061] (1) Acquisition of the 16S-23S rDNA region sequence: Staphylococcus aureus, Streptococcus pyogenes, Salmonella, Proteus mirabilis, Citrobacter freundii, Pseudomonas aeruginosa, Enterobacter aerogenes were downloaded from the GenBank public database , Providencia stutzeri, Shigella and their relatives all 16S-23S rDNA sequences.

[0062] (2) The sequence of the interval region of Micrococcus luteus is not available in the public database. We sequenced its ITS, amplified the ITS of Micrococcus luteus with universal primers for bacterial ITS, and purified the PCR product and connected it to the T vector Above, after electroporation into DH5α competent cells, the plasmids containing 500bp-1kbp were picked and sequenced with ABI 3700 sequencer. The measured sequences were spliced ​​with Staden Package software to obtain the sequence of the M. luteus region.

[0063] (3) Acquisition of the 18S-28S rDNA ...

Embodiment 2

[0076] Gene Chip Preparation: Chip Spotting

[0077] 1. Dissolving probes: Dissolve the probes synthesized in Example 1 in 50% DMSO solution, and dilute so that the final concentration of the probes reaches 1 μg / μl.

[0078] 2. Add plate: Add the dissolved probe to the corresponding position of the 384-well plate, 10 μl per well.

[0079] 3. Spotting: will be as figure 1 The shown 57.5mm×25.5mm×1mm (length×width×height) clean aldehydated glass slide (CEL Associates, Inc.) was placed on the stage of the chip spotter (Spotarray 72), using SpotArray Control software (Tele chem smp3 stealty pin), run the program, press figure 2 The arrangement shown is spotted on the aldehydeized glass slide in the spotting area of ​​4.5 mm×4.5 mm to form a medium-low density DNA micro-array, and the array arrangement rules in the six dot matrix areas on the glass slide are the same. The size of the dot matrix area is 3mm×2.25mm, the dot pitch in the dot matrix is ​​250 μm, the matrix: 12×9, ...

Embodiment 3

[0084] Rapid detection of common pathogenic bacteria in cosmetics using gene chips

[0085] 1. Sample handling:

[0086] (1) Water-soluble liquid samples: Use a sterilized measuring cylinder to measure 10mL of liquid samples in an ultra-clean bench, add them to 90mL of pre-prepared and sterilized medium, and make a 1:10 dilution. If it is less than 10mL, the dosage can be appropriately reduced, and all samples can be added to the corresponding medium.

[0087] (2) Insoluble oily liquid sample: Measure 10mL of the sample, add it to sterilized mineral oil and mix evenly, then add 10mL Tween-80, mix it in a 42 water bath for about 10 minutes, add sterilized Bacteria pre-enrichment medium 75mL, and then emulsified in a 42°C water bath to make a 1:10 dilution.

[0088] (3) Hydrophilic semi-solid sample: Weigh 10g of sample in a sterile environment, add it to 90mL of sterilized pre-enrichment medium, vortex and oscillate to mix, leave it at room temperature for about 20 minutes, ...

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Abstract

The invention provides a gene chip for detecting common pathogenic bacteria in cosmetics. The gene chip comprises a solid phase carrier and an oligonucleotide probe fixed on the solid phase carrier, wherein the oligonucleotide probe comprises DNA segments which are selected from 16S-23SrDNA intermediate zones of micrococcus luteus, staphylococcus aureus, streptococcus pyogenes, salmonella, proteus mirabilis, citrobacter freundii, pseudomonas aeruginosa, enterobacter aerogenes and providencia stuartii and 18S-28SrDNA intermediate zones of the ipaH gene of shigella, candida albicans and cryptococcus neoformans, or complementary DNA or RNA sequences of the DNA segments. The invention also provides a kit for detecting the common pathogenic bacteria in the cosmetics by use of the gene chip. The gene chip and the kit can be used for detecting the common pathogenic bacteria in the cosmetics, and are simple and convenient to operate, high in accuracy and excellent in repeatability.

Description

technical field [0001] The invention relates to a gene chip and a test kit for detection, in particular to a gene chip and a test kit for detecting common pathogenic bacteria in cosmetics. Background technique [0002] In recent years, in order to meet the needs of consumers, cosmetics manufacturers have added nutrients from plants and animals to cosmetics while improving efficacy and returning to nature. In addition, cosmetics also contain amino acids, proteins, fats, Nutrients such as vitamins, inorganic salts and placental fluid provide sufficient nutrients for the growth and reproduction of various pathogenic bacteria such as bacteria and fungi, so cosmetics are particularly susceptible to microbial contamination. In addition, pH 4-7 is also the most suitable condition for microbial growth. In addition, cosmetics are placed at room temperature during production, storage and daily use, and room temperature is also a suitable temperature for microbial growth and reproducti...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/14C12Q1/10C12Q1/04C40B40/06
CPCC12Q1/6837C12Q2565/501C12Q2545/101Y02A50/30
Inventor 王磊曹勃阳陈敏徐洋洋冯露
Owner NANKAI UNIV
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