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Fluorescent in situ hybridization detecting method for nitrate bacteria 16SrDNA

A fluorescent in situ hybridization and nitrifying bacteria technology, applied in the field of fluorescent in situ hybridization technology, can solve the problems of inconvenient separation and detection work, easy adhesion of nitrifying bacteria growth, inability to determine the optimum temperature, pH and hybridization time of nitrifying bacteria, and achieve Improve accuracy and timeliness, improve the effect of resolution

Inactive Publication Date: 2009-01-21
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Since the growth of nitrifying bacteria is easily inhibited by environmental factors such as temperature, pH, dissolved oxygen and some chemical factors, and they often attach to insoluble solid particles or the surface of the vessel wall during growth, the adhesion together brings inconvenience to the separation and detection work
However, the current general-purpose fluorescence in situ hybridization technology cannot determine the optimum temperature, pH and hybridization time for the detection of nitrifying bacteria, and fails to solve the problem of easy adhesion of nitrifying bacteria, so it cannot accurately determine the temporal and spatial distribution of nitrifying bacteria. position

Method used

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  • Fluorescent in situ hybridization detecting method for nitrate bacteria 16SrDNA

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Embodiment 1

[0022] 1. Slide processing

[0023] (1) Slide cleaning: Scrub with hot soapy water, rinse with tap water 5 times, soak in 96% alcohol, burn, then soak in 1% (mass) hydrochloric acid for 24 hours, rinse with tap water 4 times, and wash with deionized water 3-5 times.

[0024] (2) Siliconization treatment: Boil slides and coverslips in 1% (mass) hydrochloric acid for 10 minutes, wash with deionized water three times, dry at 50°C, wrap in tin foil and store at 4°C for later use. After the glass slide is siliconized, it is water-resistant, which can effectively prevent cell loss.

[0025] (3) Preparation of gelatin smears: put the slides into the gelatin solution for 10 minutes, and then dry them overnight at 60°C for later use.

[0026] Preparation of gelatin solution: Weigh 1.0g of gelatin and dissolve it in 500-800mlddH 2 O, heat and stir to help dissolve. After the gelatin is completely dissolved, add 0.5g of formazan alum, dissolve and dilute to 1000ml before use.

[0027]...

Embodiment 2

[0044] 1. Slide processing

[0045] (1) Slides are cleaned by scrubbing with hot soapy water, rinsed three times with tap water, soaked in 96% alcohol, burned, then soaked in 1% (mass fraction) hydrochloric acid for 24 hours, rinsed five times with tap water, and washed three to five times with deionized water.

[0046] (2) Siliconized slides and coverslips were boiled in 1% (mass fraction) hydrochloric acid for 10 minutes, washed three times with deionized water, dried at 50°C, wrapped in tin foil and stored at 4°C for later use. After the glass slide is siliconized, it is water-resistant, which can effectively prevent cell loss.

[0047] (3) Preparation of gelatin smear Put the glass slide in gelatin solution for 10 minutes, and then dry it at 60°C overnight for later use.

[0048] Preparation of gelatin solution: Weigh 1.0g of gelatin and dissolve it in 500-800mlddH 2 O, heat and stir to help dissolve. After the gelatin is completely dissolved, add 0.5g of formazan alum, ...

Embodiment 3

[0065] 1. Slide processing

[0066] (1) Cleaning slides with hot soapy water, scrubbing with tap water several times, soaking in 96% alcohol, burning, then soaking in 1% (mass fraction) hydrochloric acid for 24 hours, rinsing with tap water several times, and deionized water for 3-5 times.

[0067] (2) Siliconized slides and coverslips were boiled in 1% (mass fraction) hydrochloric acid for 10 minutes, washed three times with deionized water, dried at 50°C, wrapped in tin foil and stored at 4°C for later use. After the glass slide is siliconized, it is water-resistant, which can effectively prevent cell loss.

[0068] (3) Preparation of gelatin smear Put the glass slide in gelatin solution for 10 minutes, and then dry it at 60°C overnight for later use. Preparation of gelatin solution: Weigh 1.0g of gelatin and dissolve it in 500-800mlddH 2 O, heat and stir to help dissolve. After the gelatin is completely dissolved, add 0.5g of formazan alum, dissolve and dilute to 1000ml b...

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Abstract

The invention discloses a fluorescence in situ hybridization method for detecting nitrobacteria 16SrDNA. The method comprises the following steps that the method carries out treatment for a glass slide and then completes collection and pretreatment of a sample; then, hybridization is carried out; and finally, observation is carried out by means of a fluorescence microscope so as to count the number of nitrobacteria thalli. Moreover, the hybridization comprises the following steps that a NIT3 probe is marked by HEX, and a CNIT3 probe, the fluorescence-labeled NIT3 probe and a hybridization solution are mixed on the glass slide to carry out hybridization reaction inside a hybridization box at a temperature controlled between 46 and 48 DEG C for between 2 and 3 hours; and the probes are cleaned by an eluent after hybridization. The fluorescence in situ hybridization method can be directly used for detecting the number distribution and spatial distribution of nitrobacteria inside a nitration system, and directly guides the determination for community diversity of nitrobacteria in nature and under artificial condition; moreover, the method greatly increases the accuracy and timeliness of monitoring community distribution of nitrobacteria, and is particularly suitable for fluorescence in situ hybridization of nitrobacteria.

Description

Technical field: [0001] The invention relates to the fields of molecular ecology and environmental microorganisms, in particular to the fluorescent in situ hybridization technique of 16srDNA of nitrifying bacteria. Background technique: [0002] FISH technology enables simultaneous visualization, identification, counting, and fixation of individual microbial cells. Its outstanding feature is that it can overcome the technical limitations of pure culture, and can detect and analyze the composition and succession of microbial communities in the biological treatment process system at the in-situ level, as well as the ecological changes such as the spatial connection of different populations. Since the 1990s, FISH technology has been widely used in the rapid detection of nitrifying bacteria abroad. Using specific target oligonucleotide probes for nitrifying bacteria, when detecting nitrifying bacteria in water environment or biofilm in situ, it can not only detect them qualitat...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/06G01N21/64
Inventor 林炜铁朱雅楠张星王周
Owner SOUTH CHINA UNIV OF TECH