CN105486862A - Bacterium detection method - Google Patents
Bacterium detection method Download PDFInfo
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- CN105486862A CN105486862A CN201510837667.1A CN201510837667A CN105486862A CN 105486862 A CN105486862 A CN 105486862A CN 201510837667 A CN201510837667 A CN 201510837667A CN 105486862 A CN105486862 A CN 105486862A
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- bacterium
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
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Abstract
The invention discloses a bacterium detection method. The method comprises 1, carrying out sample treatment through separating bacteria from a sample under aseptic conditions, putting the bacteria in a constant temperature incubator, carrying out culture to obtain a single colony, collecting the bacterial liquid, respectively and repeatedly carrying out freezing-thawing on the bacterial liquid in liquid nitrogen at a temperature of 35-40 DEG C to crack the bacteria or carrying out supersonic wave fragmentation on the bacteria and then carrying out digestion treatment, 2, connecting the antibody of the object bacteria to surfaces of a magnetic nanosphere and a fluorescent nanosphere to obtain an immune magnetic ball and an immune fluorescent ball, 3, adding the immune magnetic ball and the immune fluorescent ball into a solution to be detected to obtain a magnetic ball-bacterium-fluorescent ball compound, and 4, detecting the magnetic ball-bacterium-fluorescent ball compound through a fluorescence microscope. The bacterium detection method can fast distinguish different bacteria, has sensitivity and accuracy better than those of the traditional detection method, has good specificity and high sensitivity, lays a technological base for bacterium fast diagnostic kit research and development, has universality and is convenient for promotion.
Description
Technical field
The present invention relates to detection field, be specifically related to the detection method of a kind of bacterium.
Background technology
Bacterium is distributed widely in soil and water, or outstanding and other biological symbiosis.Human body is with it also with considerable bacterium.According to estimates, in human body and supracutaneous bacterial cell sum be about ten times of human body cell sum.But the kind of bacterium is so many, scientist studied and the kind named only accounts for little part wherein.Micro biological Tests is the important content that medicine equipment, biologics etc. manufacture industry product quality control, and its content mainly comprises the pollution whether having the microorganism such as bacterium, mycoplasma with cultural method detection; Wherein bacterium inspection be microorganism detection be substantially the most also most important ingredient, the experiment of pure bacterium detects the viable bacteria amount of such as attenuated live vaccine self thalline, total bacterium amount and whether has the existence of other miscellaneous bacterias.
Summary of the invention
The object of this invention is to provide the detection method of a kind of bacterium, can realize the detection of bacterium fast, result is more accurate simultaneously, facilitates testing crew to adopt.
A detection method for bacterium, comprises the steps:
(1) sample preparation: under aseptic condition from sample separation of bacterial, streak inoculation, on nutrient agar panel, blood agar plate and Mai Kangkai flat board, is put in constant incubator, cultivate, obtain single bacterium colony; The single colony inoculation fluid nutrient medium of single bacterium colony is cultivated; Collect bacterium liquid, at liquid nitrogen and 35-40 DEG C, multigelation cracking bacterium or ultrasonic disruption bacterium, afterwards digestion process respectively;
(2) antibody of target bacteria to be connected to magnetic nano-balls and fluorescent nanosphere surface, and to close with the magnetic nano-balls of bovine serum albumin(BSA) antibody to coupling and fluorescent nanosphere, obtain biomolecular and immunofluorescence ball;
(3) biomolecular and immunofluorescence ball are joined in solution to be measured, hatch and biomolecular, immunofluorescence ball and target bacteria are fully combined, then Magneto separate washing, finally adds physiological saline and evenly resuspended, obtains magnetic ball-bacterium-fluorescent balls compound;
(4) fluorescent microscope or fluorescence spectrophotometer is utilized to detect magnetic ball-bacterium-fluorescent balls compound.
Further, in described step (1), the temperature of constant incubator is 38-42 DEG C, and incubation time is 25-36h.
Further, digestion process process is the ribonuclease adding 6 μ l in the bacterium of 1ml respectively in described step (1), and 10 × damping fluid, and 38 DEG C of digestion are after 1 hour, 85 DEG C, 3min stops digesting.
The invention has the beneficial effects as follows:
Bacterium of the present invention leads to detection method, can differentiate different bacterium fast, and susceptibility, accuracy are better than traditional detection method; There is good specificity and higher susceptibility, simultaneously for technical foundation has been established in the research and development of bacterium quick diagnosis reagent kit.There is universality, be convenient to promote.
Embodiment
Embodiment 1
A detection method for bacterium, is characterized in that, comprises the steps:
(1) sample preparation: under aseptic condition from sample separation of bacterial, streak inoculation, on nutrient agar panel, blood agar plate and Mai Kangkai flat board, is put in constant incubator, cultivate, obtain single bacterium colony; The single colony inoculation fluid nutrient medium of single bacterium colony is cultivated; Collect bacterium liquid, at liquid nitrogen and 35 DEG C, multigelation cracking bacterium or ultrasonic disruption bacterium, afterwards digestion process respectively;
(2) antibody of target bacteria to be connected to magnetic nano-balls and fluorescent nanosphere surface, and to close with the magnetic nano-balls of bovine serum albumin(BSA) antibody to coupling and fluorescent nanosphere, obtain biomolecular and immunofluorescence ball;
(3) biomolecular and immunofluorescence ball are joined in solution to be measured, hatch and biomolecular, immunofluorescence ball and target bacteria are fully combined, then Magneto separate washing, finally adds physiological saline and evenly resuspended, obtains magnetic ball-bacterium-fluorescent balls compound;
(4) fluorescent microscope or fluorescence spectrophotometer is utilized to detect magnetic ball-bacterium-fluorescent balls compound.
Preferably, in described step 1), the temperature of constant incubator is 38 DEG C, and incubation time is 25h.
Preferably, in described step 1), digestion process process is the ribonuclease adding 6 μ l in the bacterium of 1ml respectively, and 10 × damping fluid, 38 DEG C digestion 1 hour after, 85 DEG C, 3min stops digest.
Embodiment 2
A detection method for bacterium, is characterized in that, comprises the steps:
(1) sample preparation: under aseptic condition from sample separation of bacterial, streak inoculation, on nutrient agar panel, blood agar plate and Mai Kangkai flat board, is put in constant incubator, cultivate, obtain single bacterium colony; The single colony inoculation fluid nutrient medium of single bacterium colony is cultivated; Collect bacterium liquid, at liquid nitrogen and 38 DEG C, multigelation cracking bacterium or ultrasonic disruption bacterium, afterwards digestion process respectively;
(2) antibody of target bacteria to be connected to magnetic nano-balls and fluorescent nanosphere surface, and to close with the magnetic nano-balls of bovine serum albumin(BSA) antibody to coupling and fluorescent nanosphere, obtain biomolecular and immunofluorescence ball;
(3) biomolecular and immunofluorescence ball are joined in solution to be measured, hatch and biomolecular, immunofluorescence ball and target bacteria are fully combined, then Magneto separate washing, finally adds physiological saline and evenly resuspended, obtains magnetic ball-bacterium-fluorescent balls compound;
(4) fluorescent microscope or fluorescence spectrophotometer is utilized to detect magnetic ball-bacterium-fluorescent balls compound.
Preferably, in described step 1), the temperature of constant incubator is 40 DEG C, and incubation time is 30h.
Preferably, in described step 1), digestion process process is the ribonuclease adding 6 μ l in the bacterium of 1ml respectively, and 10 × damping fluid, 38 DEG C digestion 1 hour after, 85 DEG C, 3min stops digest.
Embodiment 3
A detection method for bacterium, is characterized in that, comprises the steps:
(1) sample preparation: under aseptic condition from sample separation of bacterial, streak inoculation, on nutrient agar panel, blood agar plate and Mai Kangkai flat board, is put in constant incubator, cultivate, obtain single bacterium colony; The single colony inoculation fluid nutrient medium of single bacterium colony is cultivated; Collect bacterium liquid, at liquid nitrogen and 40 DEG C, multigelation cracking bacterium or ultrasonic disruption bacterium, afterwards digestion process respectively;
(2) antibody of target bacteria to be connected to magnetic nano-balls and fluorescent nanosphere surface, and to close with the magnetic nano-balls of bovine serum albumin(BSA) antibody to coupling and fluorescent nanosphere, obtain biomolecular and immunofluorescence ball;
(3) biomolecular and immunofluorescence ball are joined in solution to be measured, hatch and biomolecular, immunofluorescence ball and target bacteria are fully combined, then Magneto separate washing, finally adds physiological saline and evenly resuspended, obtains magnetic ball-bacterium-fluorescent balls compound;
(4) fluorescent microscope or fluorescence spectrophotometer is utilized to detect magnetic ball-bacterium-fluorescent balls compound.
Preferably, in described step 1), the temperature of constant incubator is 42 DEG C, and incubation time is 36h.
Preferably, in described step 1), digestion process process is the ribonuclease adding 6 μ l in the bacterium of 1ml respectively, and 10 × damping fluid, 38 DEG C digestion 1 hour after, 85 DEG C, 3min stops digest.
Above-mentioned explanation is not limitation of the present invention, and the present invention is also not limited in above-mentioned citing, and the change that those skilled in the art make in essential scope of the present invention, remodeling, interpolation or replacement also should belong to protection scope of the present invention.
Claims (3)
1. a detection method for bacterium, is characterized in that, comprises the steps:
(1) sample preparation: under aseptic condition from sample separation of bacterial, streak inoculation, on nutrient agar panel, blood agar plate and Mai Kangkai flat board, is put in constant incubator, cultivate, obtain single bacterium colony; The single colony inoculation fluid nutrient medium of single bacterium colony is cultivated; Collect bacterium liquid, at liquid nitrogen and 35-40 DEG C, multigelation cracking bacterium or ultrasonic disruption bacterium, afterwards digestion process respectively;
(2) antibody of target bacteria to be connected to magnetic nano-balls and fluorescent nanosphere surface, and to close with the magnetic nano-balls of bovine serum albumin(BSA) antibody to coupling and fluorescent nanosphere, obtain biomolecular and immunofluorescence ball;
(3) biomolecular and immunofluorescence ball are joined in solution to be measured, hatch and biomolecular, immunofluorescence ball and target bacteria are fully combined, then Magneto separate washing, finally adds physiological saline and evenly resuspended, obtains magnetic ball-bacterium-fluorescent balls compound;
(4) fluorescent microscope or fluorescence spectrophotometer is utilized to detect magnetic ball-bacterium-fluorescent balls compound.
2. according to claim 1 tell the detection method of a kind of bacterium, it is characterized in that, in described step 1), the temperature of constant incubator is 38-42 DEG C, and incubation time is 25-36h.
3. according to claim 1 tell the detection method of a kind of bacterium, it is characterized in that, in described step 1), digestion process process is the ribonuclease adding 6 μ l in the bacterium of 1ml respectively, and 10 × damping fluid, 38 DEG C digestion 1 hour after, 85 DEG C, 3min stops digest.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510837667.1A CN105486862A (en) | 2015-11-26 | 2015-11-26 | Bacterium detection method |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510837667.1A CN105486862A (en) | 2015-11-26 | 2015-11-26 | Bacterium detection method |
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| CN105486862A true CN105486862A (en) | 2016-04-13 |
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| CN201510837667.1A Pending CN105486862A (en) | 2015-11-26 | 2015-11-26 | Bacterium detection method |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112462056A (en) * | 2020-11-19 | 2021-03-09 | 武汉大学 | Urine detection platform for detecting bacteria in urine on site and use method thereof |
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2015
- 2015-11-26 CN CN201510837667.1A patent/CN105486862A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112462056A (en) * | 2020-11-19 | 2021-03-09 | 武汉大学 | Urine detection platform for detecting bacteria in urine on site and use method thereof |
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Application publication date: 20160413 |
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| WD01 | Invention patent application deemed withdrawn after publication |