CN113403364A - Rapid quantitative detection method and kit for total number of bacteria in cow milk - Google Patents
Rapid quantitative detection method and kit for total number of bacteria in cow milk Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/06—Quantitative determination
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
- G01N15/1434—Electro-optical investigation, e.g. flow cytometers using an analyser being characterised by its optical arrangement
- G01N15/1436—Electro-optical investigation, e.g. flow cytometers using an analyser being characterised by its optical arrangement the optical arrangement forming an integrated apparatus with the sample container, e.g. a flow cell
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
- G01N2015/1486—Counting the particles
Abstract
The invention relates to a rapid quantitative detection method and a kit for the total number of bacteria in cow milk, wherein a green fluorescent probe is used for respectively marking gram-negative bacteria and gram-positive bacteria in a sample through an antibody of gram-negative bacteria and an antibody of gram-positive bacteria which are cross-linked by chemical groups; the total number of dead bacteria was labeled with a red fluorescent nucleic acid probe that can be membrane-blocked. And simultaneously counting red and green fluorescent signals by a flow analyzer to realize the rapid quantitative detection of the total number of bacteria. The kit has the advantages of accurate quantification, short detection time and capability of distinguishing dead bacteria from live bacteria.
Description
The technical field is as follows:
the invention belongs to the field of rapid detection of food microorganisms, relates to rapid quantitative detection of total bacteria in cow milk, and particularly relates to a method for rapidly and quantitatively detecting total viable bacteria by using an antibody labeling and flow analysis technology based on chemical group crosslinking of a fluorescent probe and a detection kit.
Background art:
with the improvement of domestic living standard, the consumption of cow milk is on the trend of increasing year by year, but at the same time, the cow milk also faces the threat of food safety. The total number of bacterial colonies is one of the important indexes of national standard GB 19301 raw milk for food safety and GB 19645 pasteurized milk for food safety national standard.
At present, the detection of microorganisms in cow milk mainly depends on a conventional bacteriological culture method, but the culture method is time-consuming and labor-consuming, the detection result is delayed, and the requirement of large-scale screening detection cannot be met. Great inconvenience and even economic loss are brought to enterprise production and quality supervision. Therefore, the traditional microbiological culture method is increasingly difficult to meet the requirement of fast and accurate detection of microorganisms in cow milk, so that the research and establishment of a fast, accurate and sensitive detection method for the total number of bacterial colonies plays an important role in food safety monitoring.
The flow analysis technology has the advantages of rapidness and high efficiency in the field of bacteria detection. The research on the application of the compound in pathogenic bacteria detection is also increasingly emphasized. However, the current methods for specifically detecting the total number of bacteria in cow milk based on flow analysis technology are few.
The invention content is as follows:
the first purpose of the invention is to provide a method for rapidly and quantitatively detecting the total number of bacteria in cow milk, which particularly meets the following requirements: 1. must be able to distinguish between bacteria and impurities; 2. the total number of bacteria can be quantitatively measured; 3. must be able to distinguish between dead and live bacteria; 4. and (5) rapidly obtaining a detection result.
The second object of the present invention is to provide a detection kit which can achieve the above object.
The method has a technical key point that: how to specifically distinguish all bacteria from somatic cells and other impurities in cow's milk.
It is well known that bacteria are divided into gram-negative and gram-positive bacteria. Therefore, the invention specially prepares an antigen lipoid A antibody aiming at the lipopolysaccharide characteristic structure on the surface of gram-negative bacteria and an antigen teichoic acid antibody aiming at the surface characteristic structure of gram-positive bacteria, and couples green fluorescent probes on the lipoid A antibody and the teichoic acid antibody respectively to prepare the bacteria specific fluorescent probe.
Meanwhile, the invention also screens out the membrane-blocking red fluorescent nucleic acid probe to distinguish dead bacteria from live bacteria. The total number of live bacteria in the cow milk is rapidly and quantitatively analyzed by fluorescence labeling of the bacteria in the cow milk and then multi-parameter detection of the bacteria by adopting a flow analysis technology.
In view of the above problems, the present inventors have worked as follows:
1. preparation of antibodies recognizing all gram-negative bacteria
The antibody of gram-negative bacteria coupled with the green fluorescent probe is adopted to carry out specific fluorescence labeling on the gram-negative bacteria, and the fluorescence of the gram-negative bacteria is analyzed by using a flow analyzer.
2. Preparation of antibodies recognizing all gram-positive bacteria
The method adopts an antibody of gram-positive bacteria coupled with a green fluorescent probe, carries out specific fluorescence labeling on the gram-positive bacteria, and uses a flow analyzer to analyze the fluorescence of the gram-positive bacteria.
Gram-negative and gram-positive bacterial antibodies suitable for flow analyzers are difficult to screen: first, gram-negative bacteria are of a wide variety of species. Suitable antibodies need to be able to specifically recognize all gram-negative bacterial strains. However, at present, no human has screened for such antibodies. Second, there are a wide variety of gram-positive bacteria. Suitable antibodies need to be able to specifically recognize all gram-positive bacterial strains. However, at present, no human has screened for such antibodies. Third, commercially available antibodies are mainly used in ELISA, IHC-Fr, WB and other experiments. In these experiments, the antigen-antibody reaction is mainly carried out on a stationary phase, such as a reaction plate. In the flow analysis technique, the reaction of antigen and antibody is carried out in a mobile phase. Therefore, a flow assay technique requires an antibody having good affinity.
According to the invention, a large amount of literature research and experiments are carried out at the early stage, and a lipopolysaccharide characteristic structure, namely lipoid A, on the surface of gram-negative bacteria is selected as a specific marker of the gram-negative bacteria; the surface characteristic structure of gram-positive bacteria thallus, teichoic acid, is selected as the specific marker of gram-positive bacteria.
A plurality of batches of antibodies are prepared aiming at the lipoid a, and then the gram-negative bacterial antibody which has high affinity and good specificity and is suitable for a flow analyzer is finally screened out and named Ab-G (-), and is currently stored in China institute of metrology science (example 1). A plurality of batches of antibodies are prepared aiming at teichoic acid, and the gram-positive bacterial antibody which has high affinity and good specificity and is suitable for a flow analyzer is finally screened out and named Ab-G (+), and is stored in China institute of metrology science (example 2).
The screening of the antibodies is carried out by two steps:
first, the affinity between the lipid a antibody and the lipid a was analyzed, and gram-negative bacterial antibodies having high affinity were selected (example 1). The affinity of teichoic acid antibodies and teichoic acid was analyzed and gram-positive bacterial antibodies with high affinity were selected (example 2).
Secondly, it was judged whether the gram-negative bacterial antibody reacted with gram-negative bacteria in the mobile phase (example 3). Antibodies to gram-positive bacteria were judged to react with gram-positive bacteria in the mobile phase (example 4).
The technical indexes for determining the gram-negative bacteria antibody suitable for the purpose of the invention are as follows: a) the immunogen used for preparing the antibody is lipoid A in lipopolysaccharide on the surface of gram-negative bacteria; b) the antibody may react with gram-negative bacteria within the mobile phase; c) after the green fluorescent probe is crosslinked, the antibody can perform fluorescent labeling on gram-negative bacteria, and fluorescence can be observed by a fluorescent microscope and a flow analyzer; d) the antibody should have an affinity for lipid a of less than 2 x 10-10; e) the antibody is capable of recognizing all gram-negative bacteria.
The technical indexes of the gram-positive bacteria antibody determined by the invention are as follows: a) the immunogen used for preparing the antibody is teichoic acid on the surface of gram-positive bacteria thallus; b) the antibody may react with gram positive bacteria within the mobile phase; c) the antibody can perform fluorescence labeling on gram-positive bacteria after being crosslinked by a green fluorescent probe, and fluorescence can be observed by a fluorescent microscope and a flow analyzer; d) the affinity KD of the antibody and teichoic acid should be less than 2 x 10-10; e) the antibody is capable of recognizing all gram-positive bacteria.
Both gram-negative and gram-positive antibodies meeting the above requirements can be used in the method of the invention.
3. Screening out suitable membrane-resistant red fluorescent nucleic acid probe
The dead bacteria are marked by adopting a membrane barrier red fluorescent nucleic acid probe, and fluorescence analysis is carried out by using a flow analyzer, so that the dead bacteria and the live bacteria are distinguished.
Through experimental verification and screening, the membrane-resistant red fluorescent nucleic acid probe suitable for the purpose of the invention has two characteristics: first, it cannot penetrate the cell membrane; second, it is capable of labeling bacterial nucleic acids, with red fluorescence. The cell membrane of the viable bacteria is complete, and the probe cannot enter the bacteria; the cell membrane of the dead bacteria is damaged, and the probe can enter the bacteria to mark nucleic acid. Furthermore, the probe fluoresces red and the emission spectrum does not overlap with that of the green immunofluorescent antibody. Therefore, the membrane-blocking red fluorescent nucleic acid probe can distinguish dead bacteria from live bacteria by combining with a flow analysis technology.
Therefore, the invention establishes a method for rapidly, sensitively and quantitatively detecting the total number of bacteria in milk by adopting a flow analysis technology for the first time, which is characterized by comprising the following steps: 1) identifying gram-negative bacteria in the sample: labeling gram-negative bacteria in the sample with an antibody to the gram-negative bacteria crosslinked by a chemical group using a green fluorescent probe; 2) identifying gram-positive bacteria in the sample: labeling gram positive bacteria in the sample with an antibody to the gram positive bacteria crosslinked by a chemical group using a green fluorescent probe; 3) distinguishing dead bacteria from live bacteria in the sample: marking the total number of dead bacteria by using a red fluorescent nucleic acid probe capable of blocking the membrane; 4) the red and green fluorescence signals generated by 1), 2) and 3) are counted simultaneously by a flow analyzer, so that the total number of bacteria can be detected quickly and quantitatively.
The technical indexes of the gram-negative bacteria antibody are as follows: a) the immunogen used for preparing the antibody is lipoid A in lipopolysaccharide on the surface of gram-negative bacteria; b) the antibody may react with gram-negative bacteria within the mobile phase; c) after the green fluorescent probe is crosslinked, the antibody can perform fluorescent labeling on gram-negative bacteria, and fluorescence can be observed by a fluorescent microscope and a flow analyzer; d) the antibody should have an affinity for lipid a of less than 2 x 10-10; e) the antibody is capable of recognizing all gram-negative bacteria.
The technical indexes of the gram-positive bacteria antibody are as follows: a) the immunogen used for preparing the antibody is teichoic acid on the surface of gram-positive bacteria thallus; b) the antibody may react with gram positive bacteria within the mobile phase; c) the antibody can perform fluorescence labeling on gram-positive bacteria after being crosslinked by a green fluorescent probe, and fluorescence can be observed by a fluorescent microscope and a flow analyzer; d) the affinity KD of the antibody and teichoic acid should be less than 2 x 10-10; e) the antibody is capable of recognizing all gram-positive bacteria.
The method for determining the total number of bacteria is as follows: for an event generated by one particle, if only green fluorescence is detected, determining that the particle is a viable bacterium; if two kinds of fluorescence of red and green are detected simultaneously, determining dead bacteria; if no fluorescence is detected, the particle is judged to be an impurity particle.
Counting by a flow analyzer, namely performing gating by a scattered light channel of the flow analyzer, and detecting the red and green fluorescent signals by a double fluorescent channel so as to detect the total number of bacteria; wherein the circular gate of the forward angle scattered light channel is between 500nm and 2500 nm. The method of the ring door comprises the following steps: and measuring 500nm standard microspheres and 2500nm standard microspheres by using a flow analyzer, and performing gate looping according to the signal positions of the microspheres on a histogram of a forward angle scattered light channel, wherein the lower limit is 500nm, and the upper limit is 2500 nm.
The fluorescence emission spectrum of the red fluorescent probe is 601 nm-640 nm; the fluorescence emission spectrum of the green fluorescent probe is 501 nm-540 nm.
The technical indexes and detection parameters of the flow analyzer are as follows: the fluorescence sensitivity is less than 10MESF, the scattered light sensitivity is less than 50nm, the fluorescence resolution RSD is less than 3%, and the scattered light resolution is less than 3%; the analysis speed is 1-30 mu L/min, and the detection time is 15-300 s.
In the detection method, a sample to be detected needs to be purified before detection; and the purification method comprises the steps of adding protease and surfactant into cow milk, and removing protein and fat in the filtrate to obtain purified sample bacterial suspension.
The invention clearly discloses the method of the invention by a plurality of experiments, which are detailed in the examples. Wherein:
example 1 is an affinity assay for gram negative bacterial antibodies and lipid a.
Example 2 is an affinity assay for gram negative bacterial antibodies and lipid a.
Example 3 detection of gram negative bacteria.
Example 4 detection of gram positive bacteria.
Example 5 is the quantitative determination of dead and viable bacteria.
Example 6 is the detection of total bacterial count in artificially contaminated milk samples.
Example 7 is a kit component for rapid detection of total bacteria in cow's milk.
The following is a specific operation of one practical test of the invention:
1. purification of bacteria in milk samples: 1mL of milk sample was digested with protease (1-3 mg) and nonionic surfactant (500. mu.L, 1%) at 37 ℃ for 30 min. Centrifuging at 8000 Xg-12000 Xg for 5-10 min, discarding the upper fat and middle liquid, resuspending the bottom bacterial sludge with 1mL PBS, and washing for 2-5 times.
2. Adding a green fluorescent probe-crosslinked gram-negative bacterial antibody (Gr-Ab-G (-), with the final concentration of 1 mu G/mL) and a gram-positive bacterial antibody (Gr-Ab-G (+), with the final concentration of 1 mu G/mL) and a membrane-blocking red fluorescent nucleic acid probe (Rd) (with the final concentration of 1 mu G/mL) into the purified bacterial liquid, uniformly mixing, and incubating for 10-20 min in a dark place.
3. Detecting by a flow analyzer: the analysis speed is 1-30 mu L/min, the detection time is 15-300 s, and the ring gate of the forward angle scattering light channel is 500 nm-2500 nm. The total number of viable bacteria in the milk was calculated by analysis of events within the clan in a dual fluorescence channel.
The inventor verifies the effect of the detection method:
nine gram-negative bacterial standard strains of different genera and nine gram-positive bacterial standard strains of different genera were selected and the ability of the method to detect G + and G-bacteria was evaluated. The results show that the G + bacteria antibody can specifically recognize all gram-positive bacteria, and the G-bacteria antibody can specifically recognize all gram-negative bacteria.
The method is used for measuring 11 dead and live bacteria suspensions of common food-borne pathogenic bacteria, and the result shows that the dead and live ratio measured by all the strains is close to 5:5, so that the method can be used for detecting the number of the dead and live bacteria of all the bacteria.
The method and the plate counting method are adopted to simultaneously detect three representative bacterial liquids of 10-time serial dilution strains, and the results of the two representative bacterial liquids are compared. As a result, it was found that: the method has good linear relation with the result obtained by the flat plate counting method, and the method has good accuracy.
According to the detection method established by the invention, the invention also provides a kit for rapidly and quantitatively detecting the total number of bacteria in cow's milk, which comprises the following components in addition to the common reagents and tools of the common kit: 1. the green fluorescent probe is crosslinked with an antibody of gram-negative bacteria, so that the gram-negative bacteria in the sample can be identified; 2. the green fluorescent probe is crosslinked with an antibody of gram-positive bacteria, so that the gram-positive bacteria in the sample can be identified; 3. a membrane-blocking red fluorescent nucleic acid probe; 4. calibration microspheres (500nm and 2500 nm).
The technical indexes of the gram-negative bacteria antibody are as follows: a) the immunogen used for preparing the antibody is lipoid A in lipopolysaccharide on the surface of gram-negative bacteria; b) the antibody may react with gram-negative bacteria within the mobile phase; c) after the green fluorescent probe is crosslinked, the antibody can perform fluorescent labeling on gram-negative bacteria, and fluorescence can be observed by a fluorescent microscope and a flow analyzer; d) the antibody should have an affinity for lipid a of less than 2 x 10-10; e) the antibody is capable of recognizing all gram-negative bacteria.
The technical indexes of the gram-positive bacteria antibody are as follows: a) the immunogen used for preparing the antibody is teichoic acid on the surface of gram-positive bacteria thallus; b) the antibody may react with gram positive bacteria within the mobile phase; c) the antibody can perform fluorescence labeling on gram-positive bacteria after being crosslinked by a green fluorescent probe, and fluorescence can be observed by a fluorescent microscope and a flow analyzer; d) the affinity KD of the antibody and teichoic acid should be less than 2 x 10-10; e) the antibody is capable of recognizing all gram-positive bacteria.
The fluorescence emission spectrum of the red fluorescent probe is 601 nm-640 nm; the fluorescence emission spectrum of the green fluorescent probe is 501 nm-540 nm.
In one example of the invention, the kit comprises the following components:
1. the green fluorescent probe is crosslinked with an antibody of gram-negative bacteria, so that the gram-negative bacteria in the sample can be identified; wherein the fluorescence emission spectrum of the green fluorescent probe is between 501nm and 540 nm.
2. The green fluorescent probe is crosslinked with an antibody of gram-positive bacteria, so that the gram-positive bacteria in the sample can be identified; wherein the fluorescence emission spectrum of the green fluorescent probe is between 501nm and 540 nm.
3. The membrane barrier red fluorescent nucleic acid probe has a fluorescence emission spectrum of 601-640 nm;
4. calibration microspheres (500nm and 2500 nm).
The operation method for detecting the total number of bacteria in milk by using the kit is characterized by comprising the following steps:
1. purification of bacteria in milk samples: 1mL of milk sample was digested with protease (1-3 mg) and nonionic surfactant (500. mu.L, 1%) at 37 ℃ for 30 min. Centrifuging at 8000 Xg-12000 Xg for 5-10 min, discarding the upper fat and middle liquid, resuspending the bottom bacterial sludge with 1mL PBS, and washing for 2-5 times.
2. Adding a green fluorescent probe-crosslinked gram-negative bacterial antibody (Gr-Ab-G (-), with the final concentration of 1 mu G/mL) and a gram-positive bacterial antibody (Gr-Ab-G (+), with the final concentration of 1 mu G/mL) and a membrane-blocking red fluorescent nucleic acid probe (Rd) (with the final concentration of 1 mu G/mL) into the purified bacterial liquid, uniformly mixing, and incubating for 10-20 min in a dark place.
3. Detecting by a flow analyzer: the analysis speed is 1-30 mu L/min, the detection time is 15-300 s, and the ring gate of the forward angle scattering light channel is 500 nm-2500 nm. The total number of viable bacteria in the milk was calculated by analysis of events within the clan in a dual fluorescence channel.
The invention has the following innovations and advantages:
1. capable of specifically recognizing all kinds of bacteria
The invention selects the lipopolysaccharide characteristic structure-lipoid A on the surface of gram-negative bacteria as the specificity marker of the gram-negative bacteria; the surface characteristic structure of gram-positive bacteria thallus, teichoic acid, is selected as the specific marker of gram-positive bacteria. The gram-negative bacterial antibodies and gram-negative bacterial antibodies thus prepared were able to recognize all kinds of bacteria.
2. Can accurately identify live bacteria
Fluorescence labeling of all bacteria can distinguish bacteria from somatic cells and foreign particles in cow milk, and detect the total number of live bacteria.
3. The method is simple and convenient to operate, short in consumed time and capable of completing detection within 0.5 h.
4. The kit is additionally provided with the flow analyzer calibration microspheres, so that the accuracy and the precision of a detection result can be ensured to the greatest extent.
Drawings
FIG. 1 shows the results of the flow analyzer in example 4, in which the ratio of viable and dead bacteria in E.coli was 5: 5;
FIGS. 2 to 4 are results of flow analysis of the percentage and concentration of dead bacteria of three representative strains in example 4; wherein: FIG. 2 E.coli; FIG. 3 Staphylococcus aureus; FIG. 4 Bacillus subtilis.
Detailed Description
The invention is further illustrated below with reference to specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Unless defined otherwise, technical and scientific terms used in the following examples have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The consumables of the experimental reagents used in the following examples are all conventional biochemical reagents unless otherwise specified. The experimental methods in the following examples, in which specific conditions are not specified, are generally performed under the conditions in the conventional conditions or the conditions recommended by the manufacturers. The strains referred to in the examples are well known in the art and are readily available from open commercial sources to those skilled in the art.
Approximating language, as used herein in the following examples, may be applied to identify quantitative indicators that could vary from one another without necessarily altering the basic function. Accordingly, a numerical value modified by a language such as "about", "left or right" is not limited to the precise numerical value itself. In some cases, the approximating language may be related to the precision of a measuring instrument.
EXAMPLE 1 preparation and screening of antibodies against gram-negative bacteria
Materials and methods
1. The immunogen is a specific marker lipid A of gram-negative bacteria, 6 monoclonal antibodies of the lipid A are prepared, and the numbers Ab-1 to Ab-6 are numbered. From the market, 4 lipoid A monoclonal antibodies are purchased, and the numbers Ab-7 to Ab-10
2. Lipid A was linked to a carboxyl chip and the carboxyl chip was loaded into an analytical interaction analyzer.
3. For a lipid a monoclonal antibody, 2 serially diluted antibodies were loaded separately into the molecular interactor, data were collected and a series of response curves were fitted with TraceDrawer and the affinity KD between the antibody and lipid a was calculated.
Second, experimental results
The affinity of 10 gram-negative bacterial antibodies and lipid A was analyzed using an analytical interaction analyzer, and the results are shown in Table 1. The results showed that the affinity between the antibody Ab-6 and lipid A was the best at 7.03X 10-11。
Third, conclusion of experiment
The antibody Ab-6 of gram-negative bacteria with the best affinity is selected and named Ab-G (-).
TABLE 1 affinity of different gram-negative bacterial antibodies to lipid A
Example 2 preparation and screening of gram-Positive bacterial antibodies
Materials and methods
1. The immunogen is the specific marker teichoic acid of gram-positive bacteria, and 6 teichoic acid monoclonal antibodies are prepared, and are numbered Ab-1 to Ab-6. 4 monoclonal antibodies of teichoic acid, numbered Ab-7 to Ab-10, were purchased from the market
2. The teichoic acid is linked to the carboxyl chip and the carboxyl chip is loaded into an analytical interaction analyzer.
3. For a teichoic acid monoclonal antibody, 2 serially diluted antibodies were loaded onto the molecular interactor, data were collected and fitted to a series of reaction curves using TraceDrawer and affinity KD between the antibody and teichoic acid was calculated.
Second, experimental results
The affinity of 10 gram-negative bacterial antibodies and teichoic acid was analyzed using an analytical interaction analyzer, and the results are shown in table 2. The results showed that the affinity of antibody Ab-2 with teichoic acid was best, 4.46X 10-11。
Third, conclusion of experiment
The antibody Ab-2 of gram-negative bacteria with the best affinity is selected and named Ab-G (+).
TABLE 2 affinity of different gram-negative bacterial antibodies and teichoic acid
Example 3 detection of gram-negative bacteria
Materials and methods
1. Nine gram-negative bacterial standard strains of different genera and nine gram-positive bacterial standard strains of different genera. All the above strains were rejuvenated and expanded and diluted to the appropriate concentration (about 10)6CFU/mL)。
2. And respectively adding the green fluorescence labeled gram-negative bacterial antibody Ab-G (-) with the concentration of 1 mug/mL into the bacterial suspensions, and incubating for 5-15 min in a dark place. And then detected with a flow analyzer.
Second, experimental results
The gram-negative bacterial antibody Ab-G (-) was reacted with bacterial suspensions of 18 different species of strains, and then fluorescence was detected by flow analysis, with the results shown in Table 3: green fluorescence was detected for all 9 different species of gram negative bacteria, while no green fluorescence was detected for all 9 gram positive bacteria.
Third, conclusion of experiment
The gram-negative bacterium antibody Ab-G (-) can be used in flow analysis technology, and can accurately identify and distinguish gram-negative bacteria.
TABLE 3 detection of gram-negative bacteria by the method
Example 4 detection of gram-Positive bacteria
Materials and methods
1. Nine gram-negative bacterial standard strains of different genera and nine gram-positive bacterial standard strains of different genera. All the above strains were rejuvenated and expanded and diluted to the appropriate concentration (about 10)6CFU/mL)。
2. And respectively adding a green fluorescence labeled gram-positive bacterial antibody Ab-G (+) with the concentration of 1 mu G/mL into the bacterial suspensions, and incubating for 5-15 min in a dark place. And then detected with a flow analyzer.
Second, experimental results
The gram-positive bacterial antibody Ab-G (+) was reacted with bacterial suspensions of 18 different species of strains, followed by fluorescence detection using a flow analyzer, and the results are shown in Table 4: green fluorescence was detected for all 9 different species of gram-positive bacteria, while no green fluorescence was detected for all 9 gram-negative bacteria.
Third, conclusion of experiment
The gram-positive bacterium antibody Ab-G (+) can be used in flow analysis technology and can accurately identify and distinguish gram-positive bacteria.
TABLE 4 detection of gram-positive bacteria by the method
Example 5 detection of dead bacteria
Materials and methods
1. 11 common food-borne pathogenic bacteria of different genera are obtained. For all the strains mentioned aboveRejuvenating and amplifying, diluting to appropriate concentration, and preparing into viable bacteria liquid (about 10)6CFU/mL)。
2. And respectively treating the live bacteria liquid with isopropanol for 30min to obtain dead bacteria liquid with the same concentration.
3. And mixing the dead and live bacteria liquid of the bacteria in equal volume to respectively obtain the bacteria liquid with the dead and live bacteria ratio of about 1: 1.
4. And respectively adding a green fluorescent probe-crosslinked gram-negative bacterium antibody (Gr-Ab-G (-), with the final concentration of 1 mu G/mL) and a gram-positive bacterium antibody (Gr-Ab-G (+), with the final concentration of 1 mu G/mL) and a membrane barrier red fluorescent nucleic acid probe (Rd) (with the final concentration of 1 mu G/mL) into the obtained dead and live bacterium solution, uniformly mixing, and incubating for 10-20 min in a dark place.
5. The stained sample was tested using a flow analyzer.
Second, experimental results
FIG. 1 is a graph showing the results of flow-type detection of E.coli, in which live bacteria and dead bacteria were successfully distinguished. All experimental strains have the same experimental result, namely dead bacteria and live bacteria of different strains can be distinguished by using a flow analyzer, and the circle gates of the live bacteria in a result graph are close. The ratio of dead and live bacteria for all the experimental strains is shown in Table 5. The results show that the ratio of dead and live bacteria of 11 strains is very close to the theoretical value.
FIG. 2 is a representation of the total number of bacteria for the strains: and (3) flow detection results of dead bacteria samples of escherichia coli, staphylococcus aureus and bacillus subtilis in different proportions. For 3 representative strains, the ratio of dead bacteria to added bacteria was close to that of flow-detection.
TABLE 5 ratio of dead and live bacteria of the strains
Third, conclusion of experiment
The method can identify dead and living bacteria of all kinds of bacteria, and has universality. And the method can accurately and quantitatively detect dead bacteria and live bacteria.
Example 6 detection of three representative bacteria of Artificial contamination
Materials and methods
1. Escherichia coli standard strain, staphylococcus aureus standard strain and bacillus subtilis standard strain
2. Artificially inoculating three representative strains into cow milk respectively to prepare artificially contaminated cow milk sample with concentration of about 101～104CFU/mL. Plate counting was used and E.coli was plate counted.
3. According to the method and the flow of the patent, the milk sample with artificial pollution is detected.
Second, experimental results
Tables 6 to 8 show the results of the detection of milk samples artificially contaminated with Escherichia coli, Staphylococcus aureus and Bacillus subtilis, respectively. The results showed that the concentration of bacteria was 101In CFU/mL, the deviation of the flow detection result and the plate counting method is large; the concentration of bacteria is 102～104The flow detection result is basically consistent with the plate counting method at CFU/mL.
TABLE 6 detection of Escherichia coli in cow's milk by flow detection and plate counting
TABLE 7 detection of Staphylococcus aureus in cow's milk by flow assay and plate counting method
TABLE 8 detection of Bacillus subtilis in cow's milk by flow detection and plate counting
Third, conclusion of experiment
The method for detecting the total number of bacteria in the cow milk has good accuracy and sensitivity of 102CFU/mL。
Example 7 Rapid quantitative detection kit for total number of bacteria in cow's milk
The kit is internally provided with:
an antibody of a green fluorescent probe-crosslinked gram-negative bacterium;
an antibody of a green fluorescent probe-crosslinked gram-positive bacterium;
a membrane-blocking red fluorescent nucleic acid probe;
calibration microspheres (500nm and 2500 nm).
Claims (10)
1. The application of the following gram-negative bacteria antibodies and gram-positive bacteria antibodies in detecting the total number of bacteria in cow milk;
the technical indexes of the gram-negative bacteria are as follows:
a, preparing the antibody by using an immunogen which is lipoid A in lipopolysaccharide on the surface of gram-negative bacteria;
b the antibody can react with gram-negative bacteria in the mobile phase;
c, after the green fluorescent probe is crosslinked, the antibody can carry out fluorescent labeling on gram-negative bacteria, and fluorescence can be observed by a fluorescent microscope and a flow analyzer;
d the affinity KD of the antibody and the lipoid A is less than 2 x 10-10；
e the antibody is capable of recognizing all gram-negative bacteria.
The technical indexes of the gram-positive bacteria antibody are as follows:
a, preparing the antibody by using an immunogen which is teichoic acid on the surface of gram-positive bacteria thallus;
b the antibody can react with gram-positive bacteria in the mobile phase;
c, after the green fluorescent probe is crosslinked, the antibody can carry out fluorescent labeling on gram-positive bacteria, and fluorescence can be observed by a fluorescent microscope and a flow analyzer;
d the affinity KD of the antibody and teichoic acid should be less than 2 x 10-10；
e the antibody is capable of recognizing all gram-positive bacteria.
2. A method for rapidly and quantitatively detecting the total number of bacteria in cow milk is characterized by comprising the following steps:
1) identifying gram-negative bacteria in the sample: labeling gram-negative bacteria in the sample with an antibody to the gram-negative bacteria crosslinked by a chemical group using a green fluorescent probe;
2) identifying gram-positive bacteria in the sample: labeling gram positive bacteria in the sample with an antibody to the gram positive bacteria crosslinked by a chemical group using a green fluorescent probe;
3) distinguishing dead bacteria from live bacteria in the sample: marking the total number of dead bacteria by using a red fluorescent nucleic acid probe capable of blocking the membrane;
4) simultaneously counting green and red fluorescent signals generated by 1), 2) and 3) by a flow analyzer to realize the rapid quantitative detection of the total number of bacteria;
the above 1) and 2), wherein the antibody against the gram-positive bacterium and the antibody against the gram-negative bacterium are as defined in claim 1;
in the above 4), the method for determining the total number of bacteria is as follows: for an event generated by one particle, if only green fluorescence is detected, determining that the particle is a viable bacterium; if two kinds of fluorescence of red and green are detected simultaneously, determining dead bacteria; if no fluorescence is detected, the particle is judged to be an impurity particle.
3. The method of claim 2, wherein counting by the flow analyzer is by gating through a scattered light channel of the flow analyzer and detecting the red and green fluorescent signals by a dual fluorescent channel to detect total number of bacteria; wherein the circular gate of the forward angle scattered light channel is between 500nm and 2500 nm.
4. The method of claim 3, wherein the method comprises: and measuring 500nm standard microspheres and 2500nm standard microspheres by using a flow analyzer, and performing gate looping according to the signal positions of the microspheres on a histogram of a forward angle scattered light channel, wherein the lower limit is 500nm, and the upper limit is 2500 nm.
5. The method of claim 2, wherein the green fluorescent probe has a fluorescence emission spectrum of 501nm to 540 nm.
6. The method of claim 2, wherein the fluorescence emission spectrum of the red fluorescent probe is 601nm to 640 nm.
7. The method of claim 2, wherein the technical indicators and detection parameters of the flow analyzer are: the fluorescence sensitivity is less than 10MESF, the scattered light sensitivity is less than 50nm, the fluorescence resolution RSD is less than 3%, and the scattered light resolution is less than 3%; the analysis speed is 1-30 mu L/min, and the detection time is 15-300 s.
8. The method according to any one of claims 2 to 7, wherein the sample to be tested is purified before being tested; the purification method comprises the steps of adding protease and surfactant into cow milk, and removing protein and fat to obtain purified sample bacterial suspension.
9. A total number of bacteria rapid quantitative determination kit in cow's milk, characterized by having:
the green fluorescent probe is crosslinked with an antibody of gram-negative bacteria, so that the gram-negative bacteria in the sample can be identified;
the green fluorescent probe is crosslinked with an antibody of gram-positive bacteria, so that the gram-positive bacteria in the sample can be identified;
a membrane-blocking red fluorescent nucleic acid probe;
calibration microspheres (500nm and 2500 nm).
The antibody of the gram-negative bacterium and the antibody of the gram-negative bacterium are as defined in claim 1.
10. The kit of claim 9, wherein the fluorescence emission spectrum of the red fluorescent probe is between 601nm and 640 nm; the fluorescence emission spectrum of the green fluorescent probe is 501 nm-540 nm.
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