CN111122689A - Tracing method for microbial pollution source - Google Patents
Tracing method for microbial pollution source Download PDFInfo
- Publication number
- CN111122689A CN111122689A CN201911346844.0A CN201911346844A CN111122689A CN 111122689 A CN111122689 A CN 111122689A CN 201911346844 A CN201911346844 A CN 201911346844A CN 111122689 A CN111122689 A CN 111122689A
- Authority
- CN
- China
- Prior art keywords
- single colony
- information
- microorganisms
- sample
- analyzed
- 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
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
- G01N27/626—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode using heat to ionise a gas
Landscapes
- Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Electrochemistry (AREA)
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a tracing method for a microbial pollution source, which comprises the following steps: determining each suspicious pollution link of a polluted product, and sampling respectively to obtain a plurality of samples to be analyzed; determining corresponding culture conditions according to the environment of each sample to be analyzed, and culturing microorganisms in each sample to be analyzed according to the determined culture conditions to obtain corresponding purified single colonies; performing mass spectrometry on each single colony by using a matrix-assisted laser desorption ionization-time-of-flight mass spectrometer to obtain spectrogram data of each single colony and identifying the species information of each single colony; determining species distribution information of microorganisms in each sample to be analyzed according to the species information of each single colony; and analyzing and comparing the species distribution information of the microorganisms of the polluted product with the species distribution information of the microorganisms in each suspected pollution link, and determining a pollution source according to an analysis result. The method can quickly and efficiently trace the source of the microbial pollution.
Description
Technical Field
The application relates to the technical field of tracing of pollution sources, in particular to a method for tracing a microbial pollution source.
Background
The analysis of the trace source of the microorganisms is to judge the relationship between the sample and the source of the contamination by comparing the presence or absence of a difference or biomarker indicative of the microorganism in the contaminated sample and the source of the contamination in order to determine the likely source of the contamination.
The determination of the pollution-indicating microorganisms is a key component of the tracing of the microorganisms, and usually, the presence or absence of the indicating microorganisms in a sample is directly detected by using a traditional microorganism isolation culture technology, or the presence of the indicating microorganisms is indirectly judged by using a molecular biology method (based on a nucleic acid probe and PCR, searching for similar but different gene maps, and reversely tracing a host where the gene maps are located) and a phenotypic method (comparing specific biochemical substances produced by bacteria under different environments to judge the source of the host).
However, the tracing analysis method in the prior art is generally difficult to be applied to tracing the pollution source in the food production process. For example, when the end product of a certain food production line is found to be contaminated, if the prior art traceability analysis method is used, since a one-to-one method is used, the selective culture medium and the biochemical characteristics of bacteria are used for identification, a prejudgment on the contaminating bacteria is needed before the experiment, and then a corresponding method is selected. However, since there is more than one process involved in the production process, the types and the number of the contaminating bacteria involved in each process are also large, and it is difficult to perform pre-determination, it is difficult to perform traceability analysis using the prior art.
Disclosure of Invention
In view of this, the invention provides a method for tracing the source of microbial contamination, so that the tracing of the source of microbial contamination can be realized quickly and efficiently.
The technical scheme of the invention is realized as follows:
a method for tracing a source of microbial contamination, the method comprising:
determining each suspicious pollution link of the polluted product, and respectively sampling the polluted product and each suspicious pollution link to obtain a plurality of samples to be analyzed;
determining corresponding culture conditions according to the environment of each sample to be analyzed, and culturing microorganisms in each sample to be analyzed according to the determined culture conditions to obtain corresponding purified single colonies;
performing mass spectrometry on each single colony by using a matrix-assisted laser desorption ionization-time-of-flight mass spectrometer to obtain spectrogram data of each single colony and identifying the species information of each single colony;
determining species distribution information of microorganisms in each sample to be analyzed according to the species information of each single colony;
analyzing and comparing the species distribution information of the microorganisms of the polluted product with the species distribution information of the microorganisms in each suspected pollution link, and determining a pollution source according to an analysis result.
Further, the spectrogram data is mass-to-charge ratio information and peak intensity information of the microbial protein.
Further, the mass spectrometry of the single colony by using the matrix-assisted laser desorption ionization-time-of-flight mass spectrometer to obtain the spectrogram data of the single colony comprises:
smearing the obtained single colony on a target plate, and adding a matrix solution to enable the single colony to be in a crystallization state; or, extracting protein from single colony, dripping the extracted protein onto a target plate, and adding matrix solution to form cocrystallization;
and putting the target plate into a matrix-assisted laser desorption ionization-time-of-flight mass spectrometer, ionizing the sample, and acquiring mass-to-charge ratio information and peak intensity information of microbial protein.
Further, the method further comprises:
and converting the mass-to-charge ratio information and the peak intensity information of the microbial protein into digital information.
Further, the method further comprises:
and comparing the spectrogram data of the single colony with data in a preset standard database to determine the species information of the microorganisms in each sample to be analyzed.
As can be seen from the above, in the method for tracing the source of the microbial contamination source in the present invention, since each suspected contamination link of the contaminated product is determined, the contaminated product and each suspected contamination link are sampled, the microbes of the multiple samples to be analyzed are cultured, mass spectrometry is performed on each single colony by MALDI-TOF MS, species distribution information of the microbes in each sample to be analyzed is determined, and finally, the species distribution information of the microbes of the contaminated product is compared with the species distribution information of the microbes of each suspected contamination link, so as to determine the contamination source according to the comparison result, thereby rapidly and efficiently tracing the source of the microbial contamination source.
Drawings
FIG. 1 is a flowchart of a tracing method of a source of microbial contamination according to an embodiment of the present invention.
Detailed Description
In order to make the technical scheme and advantages of the invention more apparent, the invention is further described in detail with reference to the accompanying drawings and specific embodiments.
FIG. 1 is a flowchart of a tracing method of a source of microbial contamination according to an embodiment of the present invention.
As shown in fig. 1, the method for tracing the source of microbial contamination in the embodiment of the present invention includes the following steps:
and 11, determining each suspicious pollution link of the polluted product, and respectively sampling the polluted product and each suspicious pollution link to obtain a plurality of samples to be analyzed.
According to the technical scheme, the general range of possible pollution can be defined, and then each suspicious pollution link is determined according to the production flow of the polluted product. Then, the contaminated product and each suspected contamination link are sampled respectively to obtain a sample to be analyzed.
For example, if the contaminated product is a certain food, each link that may possibly cause contamination may be determined from each link in the production flow as a suspected contamination link according to the whole production flow of the food. Then, sampling operation is carried out on the polluted food, and sampling operation is also carried out on each determined suspicious pollution link respectively, so that a plurality of samples to be analyzed can be obtained.
And step 12, determining corresponding culture conditions according to the environment of each sample to be analyzed, and culturing microorganisms in each sample to be analyzed according to the determined culture conditions to obtain corresponding purified single colonies.
In the technical scheme of the invention, the environment of each sample to be analyzed can be determined according to the actual application condition, so that the corresponding culture condition can be determined. For example, the culture conditions described above may include: temperature, humidity, gas conditions, and culture medium.
Then, culturing the microorganisms in each sample to be analyzed according to the determined culture conditions to obtain a corresponding purified single colony, and culturing to the amount required by the operation. If necessary, streak enrichment culture can be continued.
And step 13, performing mass spectrometry on each single colony by using a matrix-assisted laser desorption ionization-time-of-flight mass spectrometer to obtain spectrogram data of each single colony and identifying the species information of each single colony.
In the technical scheme of the invention, after the purified single colonies are obtained, Mass Spectrometry is carried out on each single colony by using a Matrix-Assisted Laser Desorption/Ionization Time of flight Mass Spectrometry (MALDI-TOF MS) instrument, so as to obtain spectrogram data of each single colony. Specifically, the spectrogram data can be mass-to-charge ratio information and peak intensity information of microbial protein.
For example, in a preferred embodiment of the present invention, the step 13 may specifically include the following steps:
step 31, smearing the obtained single colony on a target plate, and adding a matrix solution to enable the single colony to be in a crystalline state; or, extracting protein from single colony, dropping the extracted protein onto target plate, and adding matrix solution to form cocrystal.
And 32, putting the target plate into a MALDI-TOF MS instrument, ionizing the sample, acquiring mass-to-charge ratio information and peak intensity information of the microbial protein, and identifying the species information of the microbial protein.
In mass spectrometry using MALDI-TOF MS, a matrix solution is applied to a sample and crystallized. When laser irradiates a cocrystallized film formed by a sample and a matrix, the matrix absorbs energy from the laser and transfers the energy to biomolecules in the sample, the sample is desorbed and ionized, charge transfer occurs between the matrix and the sample to ionize the sample molecules, the ionized biomolecules in the sample fly through a vacuum flight tube under the action of an electric field and are detected according to different flight times of the arrival detectors, namely, the ions are analyzed by the mass-to-charge ratio (mass-to-charge ratio, m/z) of the ions and the flight time of the ions in direct proportion, and the molecular weight of the sample molecules is measured, so that the mass-to-charge ratio information and the peak intensity information of the microbial protein can be obtained; then, the species information thereof can be identified from the mass-to-charge ratio information and the peak intensity information described above.
After the mass-to-charge ratio information and the peak intensity information of the microbial protein are obtained, the mass-to-charge ratio information and the peak intensity information of the microbial protein can be further converted into digital information so as to facilitate subsequent processing.
For example, in a preferred embodiment of the present invention, the spectral data of a single colony can be compared with the data in a preset standard database to determine the species information of the microorganism in each sample to be analyzed.
In addition, in the technical scheme of the invention, the standard database can use a self-built spectrogram database or an identification system, and can also use other commercialized spectrogram databases or identification systems.
And step 14, determining the species distribution information of the microorganisms in each sample to be analyzed according to the species information of each single colony.
In the technical scheme of the invention, after the species information of each single colony is identified, the species distribution information of microorganisms in each sample to be analyzed, namely the species distribution information of the microorganisms of a polluted product, can be determined according to the species information of each single colony.
And step 15, analyzing and comparing the species distribution information of the microorganisms of the polluted product with the species distribution information of the microorganisms of each suspected pollution link, and determining a pollution source according to an analysis result.
After the species distribution information of the microorganisms in each sample to be analyzed is obtained through the steps 12 to 14, the species distribution information of the microorganisms of the contaminated product and the species distribution information of the microorganisms in each suspected contamination link can be analyzed and compared, and then the sampling position of the strain generating the contamination can be determined according to the analysis result, so that the source of the contamination can be determined.
For example, if 20 single colonies are isolated from the sample to be analyzed of the contaminated product, 15 single colonies among them are determined to be Staphylococcus epidermidis through the above-mentioned steps 12 to 14. At the moment, if staphylococcus epidermidis appears in sampling of the hands of the staff in each suspected pollution link, the pollution can be basically determined to be artificially introduced, so that the source of the pollution is determined, and the pollution can be stopped through staff disinfection treatment in the subsequent generation process.
Therefore, the tracing of the source of the microbial pollution can be realized through the steps 11 to 15.
Compared with the prior art, the method for tracing the source of the microbial pollution at least has the following advantages:
1) saving time, only needing to culture pure bacteria, one bacterium can be identified in ten seconds after the machine is operated. The target plate can spot 48, 96 or 384 samples at a time, high-throughput detection can be realized, and the whole identification process can be completed within hours. Whereas, if the methods of the prior art are used, it takes 2-7 days to identify a bacterial conservation estimate.
2) The cost is saved, the required reagent consumables are low in price, and the dosage of a single experiment is very small, so that the cost is very low.
3) The method can be carried out only by obtaining pure culture of bacteria or fungi, the pretreatment and operation are very simple and quick, the technical requirement is low, the detection personnel can operate on the machine without long-time special training, the requirement on the experimenter is low, and the detection is convenient.
4) In addition, the method can also adopt a cloud identification mode, so that the memory and a larger memory of a user computer are not required to be occupied, and the cost is reduced for customers.
In summary, in the technical solution of the present invention, because each suspected contamination link of the contaminated product is determined, the contaminated product and each suspected contamination link are sampled, a plurality of samples to be analyzed are cultured, mass spectrometry is performed on each single colony by MALDI-TOF MS to determine species distribution information of microorganisms in each sample to be analyzed, and finally the species distribution information of the microorganisms in the contaminated product is compared with the species distribution information of the microorganisms in each suspected contamination link, so as to determine the contamination source according to the comparison result, thereby rapidly and efficiently tracing the contamination source of the microorganisms, providing an accurate, simple, fast, high-throughput, low-cost method combination for the tracing analysis of the contamination source of the microorganisms, and being widely applied to food, clinical, environment, animal, food, and the like, Tracing the source of microbial pollution in the fields of plants and the like.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (5)
1. A method for tracing a source of microbial contamination, comprising:
determining each suspicious pollution link of the polluted product, and respectively sampling the polluted product and each suspicious pollution link to obtain a plurality of samples to be analyzed;
determining corresponding culture conditions according to the environment of each sample to be analyzed, and culturing microorganisms in each sample to be analyzed according to the determined culture conditions to obtain corresponding purified single colonies;
performing mass spectrometry on each single colony by using a matrix-assisted laser desorption ionization-time-of-flight mass spectrometer to obtain spectrogram data of each single colony and identifying the species information of each single colony;
determining species distribution information of microorganisms in each sample to be analyzed according to the species information of each single colony;
and analyzing and comparing the species distribution information of the microorganisms of the polluted product with the species distribution information of the microorganisms in each suspected pollution link, and determining a pollution source according to an analysis result.
2. The method of claim 1, wherein:
the spectrogram data is mass-to-charge ratio information and peak intensity information of the microbial protein.
3. The method of claim 2, wherein performing mass spectrometry on the single colony using a matrix-assisted laser desorption ionization-time-of-flight mass spectrometer to obtain spectral data of the single colony comprises:
smearing the obtained single colony on a target plate, and adding a matrix solution to enable the single colony to be in a crystallization state; or, extracting protein from single colony, dripping the extracted protein onto a target plate, and adding matrix solution to form cocrystallization;
and putting the target plate into a matrix-assisted laser desorption ionization-time-of-flight mass spectrometer, ionizing the sample, and acquiring mass-to-charge ratio information and peak intensity information of microbial protein.
4. The method of claim 3, further comprising:
and converting the mass-to-charge ratio information and the peak intensity information of the microbial protein into digital information.
5. The method of claim 1, further comprising:
and comparing the spectrogram data of the single colony with data in a preset standard database to determine the species information of the microorganisms in each sample to be analyzed.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911346844.0A CN111122689A (en) | 2019-12-24 | 2019-12-24 | Tracing method for microbial pollution source |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911346844.0A CN111122689A (en) | 2019-12-24 | 2019-12-24 | Tracing method for microbial pollution source |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111122689A true CN111122689A (en) | 2020-05-08 |
Family
ID=70501741
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911346844.0A Pending CN111122689A (en) | 2019-12-24 | 2019-12-24 | Tracing method for microbial pollution source |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111122689A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113406245A (en) * | 2021-03-31 | 2021-09-17 | 广州海关技术中心 | Method for tracing and identifying origin of soybean based on combination of MALDI-TOF/TOF and IRMS technologies |
| CN113419014A (en) * | 2021-03-31 | 2021-09-21 | 广州海关技术中心 | Method for tracing producing area of soybean and soybean oil based on characterization of triglyceride by MALDI-TOF/TOF |
| CN113743822A (en) * | 2021-09-16 | 2021-12-03 | 上海诺狄生物科技有限公司 | Environmental microorganism monitoring and tracing system and method |
| CN113755613A (en) * | 2021-08-20 | 2021-12-07 | 浙江省海洋水产养殖研究所 | Technology for high-throughput analysis of bacterial symbiosis, microbial pollution and strain identification |
| CN114291417A (en) * | 2021-12-31 | 2022-04-08 | 成都市食品检验研究院 | Microorganism and biotoxin tracing system |
| CN117054479A (en) * | 2023-08-16 | 2023-11-14 | 湖南工商大学 | Method and device for tracing watershed water pollutants |
-
2019
- 2019-12-24 CN CN201911346844.0A patent/CN111122689A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113406245A (en) * | 2021-03-31 | 2021-09-17 | 广州海关技术中心 | Method for tracing and identifying origin of soybean based on combination of MALDI-TOF/TOF and IRMS technologies |
| CN113419014A (en) * | 2021-03-31 | 2021-09-21 | 广州海关技术中心 | Method for tracing producing area of soybean and soybean oil based on characterization of triglyceride by MALDI-TOF/TOF |
| CN113755613A (en) * | 2021-08-20 | 2021-12-07 | 浙江省海洋水产养殖研究所 | Technology for high-throughput analysis of bacterial symbiosis, microbial pollution and strain identification |
| CN113743822A (en) * | 2021-09-16 | 2021-12-03 | 上海诺狄生物科技有限公司 | Environmental microorganism monitoring and tracing system and method |
| CN114291417A (en) * | 2021-12-31 | 2022-04-08 | 成都市食品检验研究院 | Microorganism and biotoxin tracing system |
| CN117054479A (en) * | 2023-08-16 | 2023-11-14 | 湖南工商大学 | Method and device for tracing watershed water pollutants |
| CN117054479B (en) * | 2023-08-16 | 2024-05-14 | 湖南工商大学 | Method and device for tracing watershed water pollutants |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111122689A (en) | Tracing method for microbial pollution source | |
| Angeletti | Matrix assisted laser desorption time of flight mass spectrometry (MALDI-TOF MS) in clinical microbiology | |
| Carbonnelle et al. | Robustness of two MALDI-TOF mass spectrometry systems for bacterial identification | |
| Sauer et al. | Mass spectrometry tools for the classification and identification of bacteria | |
| Krásný et al. | Identification of bacteria using mass spectrometry techniques | |
| Bunge et al. | On-line monitoring of microbial volatile metabolites by proton transfer reaction-mass spectrometry | |
| Patel | MALDI-TOF MS for the diagnosis of infectious diseases | |
| Keys et al. | Compilation of a MALDI-TOF mass spectral database for the rapid screening and characterisation of bacteria implicated in human infectious diseases | |
| JP5808398B2 (en) | System and method for determining drug resistance of microorganisms | |
| Dekker et al. | MALDI-TOF mass spectrometry in the clinical microbiology laboratory | |
| KR20000035935A (en) | Microorganism identification | |
| Reich et al. | Species identification of bacteria and fungi from solid and liquid culture media by MALDI-TOF mass spectrometry | |
| Arsova et al. | The use of heavy nitrogen in quantitative proteomics experiments in plants | |
| EP3083981B2 (en) | Microbe identification by mass spectrometry and infrared spectrometry | |
| US9695457B2 (en) | Rapid detection of bacteria using mass spectrometric analysis | |
| Rodriguez‐Temporal et al. | Impact of updating the MALDI‐TOF MS database on the identification of nontuberculous mycobacteria | |
| Wiesner et al. | Monitoring of bacterial growth and rapid evaluation of antibiotic susceptibility by headspace gas analysis | |
| US11754572B2 (en) | Mass spectrophotometric detection of microbes | |
| Rosenthal et al. | Volatile atmospheric pressure chemical ionisation mass spectrometry headspace analysis of E. coli and S. aureus | |
| Hang et al. | Fast detection of volatile organic compounds from Staphylococcal blood cultures by CDI-MS | |
| Mestas et al. | Direct Identification of Aerobic Bacteria by Matrix‐Assisted Laser Desorption Ionization Time‐of‐Flight Mass Spectrometry Is Accurate and Robust | |
| KR20170040078A (en) | Method for rapid identification of food poisoning bacteria | |
| Chac et al. | Sample Preparation for Detection of Different Bacterial Strains by Matrix‐Assisted Laser Desorption/Ionization Time‐of‐Flight (MALDI‐TOF) Mass Spectrometry | |
| Velichko et al. | Classification and identification tasks in microbiology: Mass spectrometric methods coming to the aid | |
| Uwakweh et al. | Nanospray desorption electrospray ionization mass spectrometry of untreated and treated probiotic Lactobacillus reuteri cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |