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: 2012-08-29
SHANGHAI JIAOTONG 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
Comparison scheme
Effect test

Embodiment 1

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

[0030] 1. Separation of sponge cells:

[0031] (1) The following operations are all performed under aseptic conditions. Sponge tissue (1-2g) is thawed at 4℃ and cut into less than 0.5cm 3 Of small pieces. Add 15-20 times the volume of artificial seawater (1.1g CaCl 2 , 10.2g MgCl 2 ·6H 2 O, 31.6g NaCl, 0.75gKCl, 1.0g Na 2 SO 4 , 2.4g Tris / HCl, 0.02g NaHCO 3 , 1L deionized water, pH 7.6) in an Erlenmeyer flask, 110rpm, 20℃, gentle shaking for 40min-60min.

[0032] (2) The sponge pieces washed in the previous step are gently rubbed on a 200-mesh stainless steel cell sieve to mechanically dissociate, so that the sponge pieces become pureed, and at the same time use 20-25 times the volume of calcium and 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) rinse, transfer to an Erlenmeyer flask 110rpm, 20°C shaking, di...

Embodiment 2

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

[0041] 1. Separation of sponge cells:

[0042] (1) The following operations are all performed under aseptic conditions. Sponge tissue (1-2g) is thawed at 4℃ and cut into less than 0.5cm 3 Of small pieces. Add 15-20 times the volume of artificial seawater (1.1g CaCl 2 , 10.2g MgCl 2 ·6H 2 O, 31.6g NaCl, 0.75gKCl, 1.0g Na 2 SO 4 , 2.4g Tris / HCl, 0.02g NaHCO 3 , 1L deionized water, pH 7.6) in an Erlenmeyer flask, 110rpm, 20℃, gentle shaking for 40min-60min.

[0043] (2) The sponge pieces washed in the previous step are gently rubbed on a 200-mesh stainless steel cell sieve to mechanically dissociate, so that the sponge pieces become pureed, and at the same time use 20-25 times the volume of calcium and 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) rinse, transfer to an Erlenmeyer flask 110rpm, 20°C shaking, di...

Embodiment 3

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

[0052] 1. Separation of sponge cells:

[0053] (1) The following operations are all performed under aseptic conditions. Sponge tissue (1-2g) is thawed at 4℃ and cut into less than 0.5cm 3 Of small pieces. Add 15-20 times the volume of artificial seawater (1.1g CaCl 2 , 10.2g MgCl 2 ·6H 2 O, 31.6g NaCl, 0.75gKCl, 1.0g Na 2 SO 4 , 2.4g Tris / HCl, 0.02g NaHCO 3 , 1L deionized water, pH 7.6) in an Erlenmeyer flask, 110rpm, 20℃, gentle shaking for 40min-60min.

[0054] (2) The sponge pieces washed in the previous step are gently rubbed on a 200-mesh stainless steel cell sieve to mechanically dissociate, so that the sponge pieces become pureed, and at the same time use 20-25 times the volume of calcium and 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) rinse, transfer to an Erlenmeyer flask 110rpm, 20°C shaking, di...

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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 bacterial 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, the sponge has a unique cavity-like structure. It feeds on microorganisms in the seawater. A large number of symbiotic microorganisms accumulate in the body, which can generally reach more than 40% of the sponge volume. Many evidences indicate 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 is a prerequisite for the development and utilization of sponge-associated microorganism resources. Fluorescence in situ hybridization techn...

Claims

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Application Information

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