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Method for detecting microbial population in sediment by fluorescent in situ hybridization technique

A fluorescence in situ hybridization and technical detection technology, which is applied in the field of detection of microbial populations in sediments by fluorescence in situ hybridization technology, can solve the problems of incomplete hybridization, increased non-specific coloring, non-specific hybridization, etc., to weaken autofluorescence and eliminate The effect of non-specific hybridization

Inactive Publication Date: 2009-03-11
NANJING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Not only that, but during the FISH experiment, due to the fixation method or incomplete elution, it is easy to cause non-specific hybridization and false positives; the time of the hybridization reaction is also an important factor, and the hybridization time is too short to cause incomplete hybridization, hybridization Excessive time will increase non-specific staining

Method used

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  • Method for detecting microbial population in sediment by fluorescent in situ hybridization technique
  • Method for detecting microbial population in sediment by fluorescent in situ hybridization technique
  • Method for detecting microbial population in sediment by fluorescent in situ hybridization technique

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1: Collection of sediment samples.

[0066] Collected in April 2006 from one sampling point (No. 2: N31°30.306', E120°13.370') of Taihu Lake Meiliang Bay and one sampling point (No. 5: N31°23.767', E120° 19.471'). On-site use a columnar sampler to collect 30cm of surface sediment, one sample per 1cm for the first 10cm, one sample per 2cm for the first 10-20cm, and one sample for every 5cm below 20cm, put them into sealed bags and label them, and store them in the refrigerator.

Embodiment 2

[0067] Example 2: Cleaning and coating of slides.

[0068] The slide cleaning procedure is as follows:

[0069] (1) Soak the slide in soapy water overnight;

[0070] (2) After rinsing with tap water, rinse with distilled water and soak;

[0071] (3) Rinse with distilled water after soaking in 1% HCl for 24 hours;

[0072] (4) After high-temperature sterilization for 20 minutes, store in the refrigerator at 4°C until use;

[0073] The slide coating steps are as follows:

[0074] (1) Soak the glass slides vertically placed on the slide rack in a mixture of APEs and acetone at a volume ratio of 1:50 for 1 min;

[0075] (2) Move the frame to acetone and drain it back and forth several times;

[0076] (3) Oven-dried at 37°C, stored in a slide box, and can be stored for 1 year.

Embodiment 3

[0077] Embodiment 3: Detection of samples.

[0078] For all the sediment samples collected in Example 1 at the two points, fluorescence in situ hybridization (FISH) technology was used to detect specific microorganisms. The specific operation is as follows:

[0079] (1) Pretreatment of sediment samples:

[0080] (1a) Take 0.2g sediment sample in a centrifuge tube, centrifuge (14000rpm refrigerated centrifugation for 10min), and remove the supernatant;

[0081] (1b) Add 1mL PBS buffer solution, mix thoroughly, centrifuge (14000rpm refrigerated centrifugation for 10min), and remove the supernatant;

[0082] (1c) repeat step (1b) 3 times;

[0083] (1d) Add 1mL of 4% paraformaldehyde fixative solution, mix well, then add 1mL of 0.2mol / L HCl, mix well, after overnight at 4°C, centrifuge (14000rpm refrigerated centrifugation for 10min), and remove the supernatant;

[0084] (1e) Add 1mL PBS buffer solution, mix well, centrifuge (14000rpm refrigerated centrifugation for 10min), an...

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Abstract

The invention discloses a method for detecting microbial communities in sediment by a fluorescence in situ hybridization (FISH) technique. The method comprises the steps of pretreatment of a sediment sample, spread coating and dehydration of the sample, hybridization and washing, and observation with a fluorescence microscope, and detects the microbial communities in the sediment through the fluorescence responses evoked by a different detection probes. By optimizing the analysis conditions of the fluorescence in situ hybridization technique, the method reduces autofluorescence in the sample, eliminates nonspecific hybridization, and determines the hybridization times and the concentration of an eluent, thereby applying the FISH technique to detection of microbial communities in the sediment sample.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a method for detecting microbial populations in sediments by fluorescence in situ hybridization (FISH). Background technique [0002] Traditionally, investigation methods of microbial community structure have been based on isolation and culture methods. However, because only a part of environmental microorganisms can be cultured, the results of microbial diversity analysis are often limited and cannot accurately reflect the composition and diversity of mixed flora. Bacteria often do not have the desired effect. Therefore, the survey results obtained by traditional culture methods cannot accurately reflect the composition of microbial communities. It is very necessary to establish and develop a method that does not rely on microbial culture to study the structure of microbial communities. [0003] In recent years, with the development of molecular biology techniques such as mole...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04G01N21/00
Inventor 宋炜段婧婧肖琳杨柳燕
Owner NANJING UNIV
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