Oligonucleotide microarray technique for detecting pathogen contamination in seawater

An oligonucleotide and microarray technology, applied in the field of seawater pollution monitoring, can solve the problem that the overall pollution status of seawater pathogens cannot be effectively given.

Inactive Publication Date: 2009-11-18
NANKAI UNIV
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Problems solved by technology

However, the detection of two or three kinds of fungal diseases cannot effectively give the overall pollution status of

Method used

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  • Oligonucleotide microarray technique for detecting pathogen contamination in seawater
  • Oligonucleotide microarray technique for detecting pathogen contamination in seawater
  • Oligonucleotide microarray technique for detecting pathogen contamination in seawater

Examples

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Embodiment 1

[0071] Embodiment 1: seawater sample 1

[0072] 1 Oligonucleotide Microarray Fabrication:

[0073] 1) Preparation of probe dilution: 1× salmon sperm DNA solution (1g / L)

[0074] 2) Prepare probe solution: use probe diluent to prepare 0.1ug / μL probe solution

[0075] 3) Immobilizing the probe on the nylon membrane substrate: spot the probe solution on the 5cm×5cm nylon membrane substrate, and keep warm at 80°C for 2 hours.

[0076] 2 Seawater sample processing and DNA extraction

[0077] 1) Sample processing

[0078] ①Take 20L of surface seawater and pack it into sterilized plastic buckets.

[0079] ②Filter with a 0.15mm pore size sieve to remove suspended matter, and then use a Mini-pellicon system (Millipore, USA) to carry out pressure filtration to collect the bacteria. The filter membrane has a pore size of 0.22 μm (Milipo, USA). Every 2L of seawater filtered corresponds to a filter membrane, that is, every 2L of seawater is taken as a sample, and each sampling point c...

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Abstract

The invention relates to an oligonucleotide microarray technique for detecting pathogen contamination in seawater, belonging to the field of seawater contamination monitoring. The technique comprises the main technical schemes that a 16S-23S rRNA gene transcription interval sequence is used as a detection target and is amplified by a one-step polymerase chain reaction, a digoxin mark is obtained simultaneously, and then oligonucleotide hybridization is carried out; and the obtained monitoring result is interpreted in a manner that an enzyme-labeled antibody catalyzes the substrate colour development. Compared with the traditional product for detecting seawater contamination, the invention utilizes microarray detection to obtain the distribution situation of large numbers of pathogens and contamination index bacteria, and has the advantage of high flux; the invention can directly utilize seawater as a sample and truly obtain the contamination situation information of target bacteria under a condition of keeping the natural proportion of the flora number in the seawater; however, most existing detection techniques need the step of enrichment culture, destroy the original proportion of a flora composition, have lower reliability of the result and have longer detection procedure; and the oligonucleotide microarray detection operation has short procedure and is comparatively sensitive and fast.

Description

technical field [0001] The invention belongs to the field of seawater pollution monitoring, and the main content is an oligonucleotide microarray technology, which can monitor the pollution of pathogenic bacteria in seawater with high throughput. It can simultaneously detect a variety of pathogenic bacteria, including Vibrio riverine, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio cholerae, Vibrio mimicus, Vibrio furnisis, Vibrio Harvey, Vibrio vulnificus, Enterococcus faecalis, Listeria, Pseudomonas aeruginosa, Salmonella, Staphylococcus aureus, Clostridium perfringens. And this microarray technology can directly detect a variety of indicator bacteria from seawater without enrichment culture, including bacteria that cannot be cultured artificially, without being affected by seasons. In addition to being able to assess the pollution degree of pathogenic bacteria in the corresponding sea area, it can also indicate whether the sea area is polluted by domestic wastewater....

Claims

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Application Information

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IPC IPC(8): C12Q1/68C40B40/06C12R1/63C12R1/01C12R1/385C12R1/42C12R1/445C12R1/145
Inventor 黄熙泰许晶晶李永君田雪莹孙谦
Owner NANKAI UNIV
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