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Detection method of symbiotic bacteria in sponge cell based on quantum dot fluorescence in-situ hybridization

A technology of fluorescence in situ hybridization and symbiotic bacteria, applied in biochemical equipment and methods, fluorescence/phosphorescence, microbial measurement/inspection, etc., can solve the problems of difficult antigen-antibody reactions, low antigenicity, and complex components. Achieve the effect of avoiding the interference of autofluorescence

Inactive Publication Date: 2011-02-16
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the detection of this prior art often relies on the antigen-antibody reaction. For environmental samples (marine organisms, geological samples) with complex components, low antigenicity, and low content of detection objects, the antigen-antibody reaction is difficult to meet the requirements.

Method used

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  • Detection method of symbiotic bacteria in sponge cell based on quantum dot fluorescence in-situ hybridization
  • Detection method of symbiotic bacteria in sponge cell based on quantum dot fluorescence in-situ hybridization

Examples

Experimental program
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Effect test

Embodiment 1

[0029] The specific implementation steps of this embodiment are as follows:

[0030] 1. Isolation of sponge cells:

[0031] (1) The following operations are performed under sterile conditions. Spongy tissue (1-2g) was thawed at 4°C and cut into pieces no larger than 0.5cm 3 of small pieces. Add 15-20 times the volume of artificial seawater (1.1g CaCl 2 , 10.2 g MgCl 2 ·6H 2 O, 31.6g NaCl, 0.75g KCl, 1.0g NaCl 2 SO 4 , 2.4g Tris / HCl, 0.02g NaHCO 3 , 1L deionized water, pH 7.6) in an Erlenmeyer flask, 110rpm, 20°C gentle shaking for 40min-60min.

[0032] (2) Gently rubbing the sponge fragments washed in the previous step on a 200-mesh stainless steel cell sieve for mechanical dissociation, allowing the sponge fragments to become a paste, while using 20-25 times the volume of calcium-magnesium-free artificial seawater ( 31.6g NaCl, 0.75g KCl, 1.0gNa 2 SO 4 , 2.4g Tris / HCl, 0.02g NaHCO 3 , 7.2g EDTA, 1L deionized water, pH 7.6) wash, transfer to Erlenmeyer flask at 110...

Embodiment 2

[0040] The specific implementation steps of this embodiment are as follows:

[0041] 1. Isolation of sponge cells:

[0042] (1) The following operations are performed under sterile conditions. Spongy tissue (1-2g) was thawed at 4°C and cut into pieces no larger than 0.5cm 3 of small pieces. Add 15-20 times the volume of artificial seawater (1.1g CaCl 2 , 10.2 g MgCl 2 ·6H 2 O, 31.6g NaCl, 0.75g KCl, 1.0g NaCl 2 SO 4 , 2.4g Tris / HCl, 0.02g NaHCO 3 , 1L deionized water, pH 7.6) in an Erlenmeyer flask, 110rpm, 20°C gentle shaking for 40min-60min.

[0043] (2) Gently rubbing the sponge fragments washed in the previous step on a 200-mesh stainless steel cell sieve for mechanical dissociation, allowing the sponge fragments to become a paste, while using 20-25 times the volume of calcium-magnesium-free artificial seawater ( 31.6g NaCl, 0.75g KCl, 1.0gNa 2 SO 4 , 2.4g Tris / HCl, 0.02g NaHCO 3 , 7.2g EDTA, 1L deionized water, pH 7.6) wash, transfer to Erlenmeyer flask at 110...

Embodiment 3

[0051] The specific implementation steps of this embodiment are as follows:

[0052] 1. Isolation of sponge cells:

[0053] (1) The following operations are performed under sterile conditions. Spongy tissue (1-2g) was thawed at 4°C and cut into pieces no larger than 0.5cm 3 of small pieces. Add 15-20 times the volume of artificial seawater (1.1g CaCl 2 , 10.2 g MgCl 2 ·6H 2 O, 31.6g NaCl, 0.75g KCl, 1.0g NaCl 2 SO 4 , 2.4g Tris / HCl, 0.02g NaHCO 3 , 1L deionized water, pH 7.6) in an Erlenmeyer flask, 110rpm, 20°C gentle shaking for 40min-60min.

[0054] (2) Gently rubbing the sponge fragments washed in the previous step on a 200-mesh stainless steel cell sieve for mechanical dissociation, allowing the sponge fragments to become a paste, while using 20-25 times the volume of calcium-magnesium-free artificial seawater ( 31.6g NaCl, 0.75g KCl, 1.0gNa 2 SO 4 , 2.4g Tris / HCl, 0.02g NaHCO 3 , 7.2g EDTA, 1L deionized water, pH 7.6) wash, transfer to Erlenmeyer flask at 110...

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Abstract

The invention provides a detection method of symbiotic bacteria in sponge cells based on quantum dot fluorescence in-situ hybridization, which relates to the technical field of bacterial detection. The detection method comprises the following steps of: obtaining a sponge single cell; according to a classical fluorescence in-situ hybridization method, adopting an oligonucleotide probe marked by biotin to hybridize microbial genome DNA (deoxyribonucleic acid) in the sponge single cell; finally, adopting a quantum dot marked by streptavidin to carry out incubation treatment; and obtaining blue sponge cells in a fluorescence microscope with exciting light as an ultraviolet band and red bacteria. The invention combines a mechanical method and a chemical method, can quickly obtain sponge cells, avoids the autofluorescence interference of the sponge cells and successfully detects the bacteria in the sponge cells.

Description

technical field [0001] The invention relates to a method in the technical field of bacteria detection, in particular to a method for detecting symbiotic bacteria in sponge cells based on quantum dot fluorescence in situ hybridization. Background technique [0002] As the oldest metazoan, sponges have a unique cavity-like structure and live by filtering microorganisms in seawater. There are a large number of symbiotic microorganisms in the body, which can generally reach more than 40% of the sponge's volume. A lot of evidence shows that the medicinal natural products derived from sponges may be produced by their symbiotic microorganisms, and sponge symbiotic microorganisms have therefore become one of the focuses of marine drug research and development. Analyzing the diversity of these microorganisms and identifying host-specific microorganisms are the prerequisites for the development and utilization of sponge symbiotic microbial resources. Fluorescence in situ hybridizatio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/04C12Q1/68G01N21/64
Inventor 刘放李志勇张风丽韩敏奇杨振亚
Owner SHANGHAI JIAO TONG UNIV
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