CN1828303A - Method for quick detection of microbe - Google Patents
Method for quick detection of microbe Download PDFInfo
- Publication number
- CN1828303A CN1828303A CN 200610025505 CN200610025505A CN1828303A CN 1828303 A CN1828303 A CN 1828303A CN 200610025505 CN200610025505 CN 200610025505 CN 200610025505 A CN200610025505 A CN 200610025505A CN 1828303 A CN1828303 A CN 1828303A
- Authority
- CN
- China
- Prior art keywords
- microorganism
- sample
- atriphos
- detecting method
- fast detecting
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000000034 method Methods 0.000 title claims abstract description 21
- 238000001514 detection method Methods 0.000 title claims description 11
- 238000005415 bioluminescence Methods 0.000 claims abstract description 10
- 230000029918 bioluminescence Effects 0.000 claims abstract description 10
- 239000005089 Luciferase Substances 0.000 claims abstract description 7
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 6
- 244000005700 microbiome Species 0.000 claims description 19
- ZKHQWZAMYRWXGA-KQYNXXCUSA-N Adenosine triphosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-N 0.000 claims description 17
- 239000011324 bead Substances 0.000 claims description 15
- 241000894006 Bacteria Species 0.000 claims description 10
- 238000006243 chemical reaction Methods 0.000 claims description 5
- 238000000354 decomposition reaction Methods 0.000 claims description 5
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 claims description 5
- 230000036039 immunity Effects 0.000 claims description 4
- 239000000725 suspension Substances 0.000 claims description 4
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 claims description 3
- 239000003153 chemical reaction reagent Substances 0.000 claims description 2
- 239000012467 final product Substances 0.000 claims description 2
- 238000012113 quantitative test Methods 0.000 claims description 2
- 210000000170 cell membrane Anatomy 0.000 claims 1
- 230000003834 intracellular effect Effects 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 2
- 238000005336 cracking Methods 0.000 abstract 1
- 235000013305 food Nutrition 0.000 description 8
- 241001646719 Escherichia coli O157:H7 Species 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 241000305071 Enterobacterales Species 0.000 description 1
- 108060001084 Luciferase Proteins 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 238000013016 damping Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 239000013558 reference substance Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The provided method comprises: with specific immune magnetic pearl to capture the special microbe in sample; releasing the ATP in microbe cell with cracking agent; finally, detecting the ATP to decide the target and opposite amount with the fluorescein-luciferase bioluminescence system. Compared with prior art, this invention has high sensitivity and strong specificity to complete whole process within 8h.
Description
Technical field:
The present invention relates to a kind of fast detecting method of microorganism, particularly immunomagnetic beads separates and adds ATP bioluminescence technique fast detecting method of microorganism, belongs to the microorganism detection field.
Background technology:
In recent years, the food origin disease that is caused by invasive organism happens occasionally, serious harm human health.The food origin disease that these are caused by invasive organism in the food is more and more higher to the evaluation requirement of pathogen.Effective detection at objective microbe needs fast, responsive and special method.Though the tradition cultural method has higher susceptibility and specificity, need a couple of days even above time in a week could report testing result usually.Therefore the foundation of microbial rapid detection technology and improve and have more and more important practical sense to ensuring food safety in microorganism, the especially food.
Summary of the invention:
The objective of the invention is to: a kind of fast detecting method of microorganism is provided, the present invention is directed to the deficiencies in the prior art, adopt the immunomagnetic beads separation to add atriphos (ATP) bioluminescence technique and detect, the method that is provided is quick, responsive, is no more than 8 hours detection time.
The present invention be achieved in that by the specific immunity magnetic bead come enrichment catch bacteria suspension or sample in microorganisms such as bacterium, add decomposition agent then in the immunomagnetic beads enrichment or in bacteria suspension after catching or the sample and discharge atriphos (ATP) in the microbial cell such as bacterium, detect ATP by the fluorescein-luciferase bio-luminescence system at last and measure and judge and have or not specified microorganisms and quantity thereof in the sample.
Wherein, described specific immunity magnetic bead is meant and constitutes directly or indirectly being coated on the magnetic bead at the monoclonal of the specified microorganisms that will detect or polyclonal antibody.
Used decomposition agent is the reagent that destroys microorganism walls such as bacterium, contains trichloroacetic acid TCA.
The detailed process that described fluorescein-luciferase bio-luminescence system detects ATP is: bioluminescence reaction needs ATP, fluorescein and luciferase, fluorescein is oxidized and send fluorescence in the course of reaction, this light intensity is directly proportional with ATP content in the sample, measures light intensity by the high-sensitivity detection instrument and carries out quantitative test and get final product.
Compared with prior art, the present invention combines the specific reaction of antigen-antibody and quick, responsive ATP bioluminescence technique, make it have characteristics such as quick, highly sensitive and high specificity simultaneously, all testing processes can be finished in 8 hours, can be used for the microorganism detection in the several samples such as clinical, food.
Embodiment:
Specific embodiments of the invention: immunomagnetic beads separates and adds Escherichia coli O 157: H7 in the ATP bioluminescence technique fast detecting food:
Sample collecting, increase bacterium and immunomagnetic beads enrichment: the food samples gathered such as beef 25 grams are waited increase the bacterium cultivation after national standard method is handled routinely, the immunomagnetic beads 15 μ l that then will be coated with Chinese People's Anti-Japanese Military and Political College's enterobacteria O157:H7 monoclonal or polyclonal antibody join and contain in the test tube that 0.9ml increases the bacterium culture, are placed in the magnetic concentrator after the sample mix device mixes and remove suspending liquid; The immunomagnetic beads that is combined with Escherichia coli O 157: H7 that to collect at last suspends again in damping fluid and detects to carry out ATP.
2.ATP detect: will there be the decomposition agent of trichloroacetic acid (TCA) to join in the suspending liquid of the immunomagnetic beads that is combined with Escherichia coli O 157: H7 after suspending again by equal-volume, the reaction back adds luciferin-luciferase solution, adopt high sensitivity luminous detection instrument to measure light intensity afterwards immediately and carry out assay determination, the numerical value that determines can relative light unit (RLU) expression.The numerical value that different luminous detection instrument determine may be incomplete same, and the therefore final numerical value that detects should be determined normal and exceptional value scope according to the instrument that is adopted and standard items, reference substance.
Claims (4)
1. fast detecting method of microorganism, it is characterized in that: by the specific immunity magnetic bead come enrichment catch bacteria suspension or sample in microorganism, add decomposition agent with the intracellular atriphos of releasing microbe in the immunomagnetic beads enrichment or in bacteria suspension after catching or the sample then, judge by fluorescein-luciferase bio-luminescence system detection atriphos amount at last to have or not specified microorganisms and quantity thereof in the sample.
2. according to the described fast detecting method of microorganism of claim 1, it is characterized in that: described specific immunity magnetic bead is meant and constitutes directly or indirectly being coated on the magnetic bead at the monoclonal of the specified microorganisms that will detect or polyclonal antibody.
3. according to the described fast detecting method of microorganism of claim 1, it is characterized in that: used decomposition agent is the reagent of destroy microorganisms cell membrane, contains trichloroacetic acid TCA.
4. according to the described fast detecting method of microorganism of claim 1, it is characterized in that: the detailed process that described fluorescein-luciferase bio-luminescence system detects atriphos is: fluorescein is oxidized and send fluorescence in the reaction, this light intensity is directly proportional with atriphos content in the sample, measures light intensity by the high-sensitivity detection instrument and carries out quantitative test and get final product.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200610025505 CN1828303A (en) | 2006-04-07 | 2006-04-07 | Method for quick detection of microbe |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200610025505 CN1828303A (en) | 2006-04-07 | 2006-04-07 | Method for quick detection of microbe |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1828303A true CN1828303A (en) | 2006-09-06 |
Family
ID=36946797
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200610025505 Pending CN1828303A (en) | 2006-04-07 | 2006-04-07 | Method for quick detection of microbe |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1828303A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101639479A (en) * | 2008-07-31 | 2010-02-03 | 上海美全生物科技有限公司 | Method for rapidly detecting mycobacterium |
| CN101024854B (en) * | 2006-02-24 | 2010-06-09 | 米利波尔公司 | Fast microbiological analysis method |
| CN101215598B (en) * | 2008-01-11 | 2010-11-17 | 中华人民共和国吉林出入境检验检疫局 | Method for detecting bacteria by using immunity enrichment thread, and immunity enrichment brush |
| CN102183648A (en) * | 2011-01-26 | 2011-09-14 | 中国科学院上海微系统与信息技术研究所 | Detection method and detection kit for detecting special pathogenic bacteria by bioluminescence |
| CN103776807A (en) * | 2014-01-22 | 2014-05-07 | 中国食品发酵工业研究院 | Method for detecting residual bacteria in flushed water of CIP (cleaning in place) cleaning system |
| EP4148419A4 (en) * | 2020-05-08 | 2023-10-04 | Tangshan Food and Drug Intergration Examination and Detection Center | Method for detecting pesticide residue, and kit |
-
2006
- 2006-04-07 CN CN 200610025505 patent/CN1828303A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101024854B (en) * | 2006-02-24 | 2010-06-09 | 米利波尔公司 | Fast microbiological analysis method |
| CN101215598B (en) * | 2008-01-11 | 2010-11-17 | 中华人民共和国吉林出入境检验检疫局 | Method for detecting bacteria by using immunity enrichment thread, and immunity enrichment brush |
| CN101639479A (en) * | 2008-07-31 | 2010-02-03 | 上海美全生物科技有限公司 | Method for rapidly detecting mycobacterium |
| CN102183648A (en) * | 2011-01-26 | 2011-09-14 | 中国科学院上海微系统与信息技术研究所 | Detection method and detection kit for detecting special pathogenic bacteria by bioluminescence |
| CN103776807A (en) * | 2014-01-22 | 2014-05-07 | 中国食品发酵工业研究院 | Method for detecting residual bacteria in flushed water of CIP (cleaning in place) cleaning system |
| CN103776807B (en) * | 2014-01-22 | 2016-01-13 | 中国食品发酵工业研究院 | A kind of method detecting remaining bacteria in CIP purging system wash-down water |
| EP4148419A4 (en) * | 2020-05-08 | 2023-10-04 | Tangshan Food and Drug Intergration Examination and Detection Center | Method for detecting pesticide residue, and kit |
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| PB01 | Publication | ||
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| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Open date: 20060906 |