CN113049826A - Rapid synchronous multiple detection method and kit for total number of vibrio parahaemolyticus and bacteria - Google Patents
Rapid synchronous multiple detection method and kit for total number of vibrio parahaemolyticus and bacteria Download PDFInfo
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Abstract
A method and a detection kit for rapidly and synchronously detecting the total number of vibrio parahaemolyticus and bacteria in a sample in a multiplex manner are disclosed, wherein the total number of the bacteria is marked by using a red fluorescent probe capable of marking all the bacteria, so that the bacteria in the sample can be distinguished from other background particles: meanwhile, a green fluorescent probe is used for distinguishing vibrio parahaemolyticus and non-vibrio parahaemolyticus in the sample through a vibrio parahaemolyticus antibody crosslinked by a chemical group; and simultaneously counting red and green fluorescent signals by a flow analyzer to realize synchronous detection of the total number of vibrio parahaemolyticus and bacteria. The method can simultaneously detect the total number of the vibrio parahaemolyticus and the total number of the bacteria, is simple and convenient to operate, consumes short time, and can finish detection in 0.5h for most samples.
Description
The technical field is as follows:
the invention belongs to the field of food microorganism detection, and particularly relates to rapid synchronous multiplex detection of two indexes of vibrio parahaemolyticus and total number of bacteria in a food sample and a kit.
Background art:
vibrio parahaemolyticus and total number of bacteria are the most basic and common detection items in food microbiological examination. The colony count generally refers to the total number of bacteria, and is used for determining the degree of bacterial contamination of food and the hygienic quality, and marks the quality of the hygienic quality of the food to a certain extent. Vibrio parahaemolyticus is a marine bacterium that can cause acute onset of illness, abdominal pain, vomiting, diarrhea in humans.
The traditional culture method wastes time and labor for measuring the vibrio parahaemolyticus, for example, the national standard GB 4789.7-2013 Vibrio parahaemolyticus test for food safety national standard food microbiology test requires a series of steps of bacterium increasing, culturing, biochemical identification and the like, and the time is over 60 hours.
The plate count method of total bacteria also has many disadvantages, such as the national standard GB 4789.2-2016 Total colony count, which requires a series of steps of sampling, diluting, pouring plates, culturing, and takes 48 hours.
Both methods require a large amount of manual operation, are time-consuming and labor-consuming, and the detection of the two indicators of the total number of Vibrio parahaemolyticus and bacteria must be performed separately.
Although many new techniques are currently used for rapid detection of the total number of Vibrio parahaemolyticus or bacteria, these techniques are difficult to use for multiplex detection of the total number of Vibrio parahaemolyticus and bacteria. For example, the existing biochip method cannot identify all kinds of bacteria; the PCR method can only realize multiple detection of different types of bacteria, and cannot realize the detection of the total number of the bacteria.
The flow analysis technology has the potential of realizing multiple detection of indexes of the total number of vibrio parahaemolyticus and bacteria, but because the development of a vibrio parahaemolyticus specific fluorescent probe has a plurality of technical difficulties, no relevant report exists at present.
In conclusion, the rapid detection, the synchronous multiple detection and the simultaneous detection of the total number of the vibrio parahaemolyticus and the total number of the bacteria are the actual needs in the field of food microorganism detection and the technical difficulties.
The invention content is as follows:
the first objective of the present invention is to provide a rapid and simultaneous multiple detection method for vibrio parahaemolyticus and total number of bacteria, especially to satisfy the following requirements: 1. must be able to distinguish between bacteria and impurities; 2. the total number of bacteria can be quantitatively measured; 3. can specifically identify and quantify vibrio parahaemolyticus from total bacteria; 4. and rapidly synchronizing to obtain 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 two technical key points: firstly, how to accurately distinguish bacterial from non-bacterial particles; secondly, how to specifically identify the vibrio parahaemolyticus.
In view of the above problems, the present inventors have worked as follows:
1. development of fluorescent probes capable of identifying total bacteria
As the bacteria are various, Escherichia coli and Vibrio parahaemolyticus are used as representative strains of gram-negative bacteria, Staphylococcus aureus is used as a representative strain of gram-positive bacteria, and Bacillus subtilis is used as a representative strain of spore-form bacteria. Four representative strains were studied and fluorescent probes capable of identifying total bacteria were developed and optimized.
2. Preparing specific antibody of suitable vibrio parahaemolyticus
The method adopts an antibody of the vibrio parahaemolyticus coupled with a green fluorescent probe, carries out specific fluorescent labeling on the vibrio parahaemolyticus, and analyzes the fluorescence of the vibrio parahaemolyticus by using a flow analyzer.
The screening of vibrio parahaemolyticus antibodies suitable for flow analyzers is very difficult:
first, the O antigen classification of Vibrio parahaemolyticus is more, including O1 to O13. Among them, all serotypes of Vibrio parahaemolyticus have been isolated from food, animal and human samples. Therefore, a suitable Vibrio parahaemolyticus antibody needs to be capable of specifically recognizing all the strains of the above 13 serotypes. However, at present, no antibody capable of recognizing all the Vibrio parahaemolyticus O antigens has been prepared.
Second, 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.
The invention prepares a plurality of batches of antibodies (example 1) aiming at vibrio parahaemolyticus, and finally screens out the vibrio parahaemolyticus antibody which has high affinity and good specificity and is suitable for a flow analyzer, is named Ab-3 (examples 3 and 4) and is currently stored in China institute of metrology science.
The screening of the antibodies is carried out by three steps:
first, it was preliminarily judged whether the antibody can react with Vibrio parahaemolyticus in the mobile phase, and the specificity of the antibody was preliminarily evaluated (example 2).
Second, the affinity between the Vibrio parahaemolyticus antibody and the antigen was analyzed, and a Vibrio parahaemolyticus antibody having high affinity was selected (example 3).
Thirdly, 12 strains of Vibrio parahaemolyticus (containing the four serotypes 1/2a, 1/2b, 1/2c and 4 b) and 24 other strains were selected, and whether the antibodies specifically recognized Vibrio parahaemolyticus and distinguished Vibrio parahaemolyticus from other bacteria was verified by using a flow analyzer (example 4).
The technical indexes for determining the vibrio parahaemolyticus antibody suitable for the purpose of the invention are as follows:
1. the immunogen used for preparing the antibody is a mixture of 4 vibrio parahaemolyticus specific outer membrane proteins, and the 4 outer membrane proteins are OmpU, OmpK, OmpC and OmpA respectively;
2. the antibody can react with vibrio parahaemolyticus in a mobile phase;
3. after the green fluorescent probe is crosslinked, the antibody can carry out fluorescent labeling on the vibrio parahaemolyticus, and the fluorescence can be observed by a fluorescent microscope and a flow analyzer;
4. the affinity KD of the antibody and the specific mycoprotein of the vibrio parahaemolyticus is less than 2 multiplied by 10-10；
5. The antibody is capable of recognizing all strains of Vibrio parahaemolyticus.
Antibodies meeting the above requirements may be used in the method of the invention.
Therefore, the invention establishes a method for rapidly and synchronously detecting the total number of vibrio parahaemolyticus and bacteria in multiple ways by adopting a flow analysis technology for the first time, which is characterized in that: 1) differentiation of bacteria from other background particles in the sample: marking the total number of bacteria by using a red fluorescent probe capable of marking all bacteria; 2) distinguishing between vibrio parahaemolyticus and non-vibrio parahaemolyticus in the sample: using a green fluorescent probe to specifically mark vibrio parahaemolyticus in the sample through a vibrio parahaemolyticus antibody crosslinked by a chemical group; 3) and simultaneously counting the red and green fluorescent signals generated by 1) and 2) by a flow analyzer to realize synchronous quantitative detection of the total number of the vibrio parahaemolyticus and the bacteria.
The technical indexes of the vibrio parahaemolyticus antibody are as follows: a) the immunogen used for preparing the antibody is a mixture of 4 vibrio parahaemolyticus specific outer membrane proteins, and the 4 outer membrane proteins are OmpU, OmpK, OmpC and OmpA respectively; b) the antibody can react with vibrio parahaemolyticus in a mobile phase; c) after the green fluorescent probe is crosslinked, the antibody can carry out fluorescent labeling on the vibrio parahaemolyticus, and the fluorescence can be observed by a fluorescent microscope and a flow analyzer; d) the affinity KD of the antibody and the specific mycoprotein of the vibrio parahaemolyticus is less than 2 multiplied by 10-10(ii) a e) The antibody is capable of recognizing all strains of Vibrio parahaemolyticus.
The method for judging the total number of the vibrio parahaemolyticus and the bacteria comprises the following steps: for an event produced by one particle, if only red fluorescence is detected, it is judged as a bacterium, but not as Vibrio parahaemolyticus; if two kinds of fluorescence, namely red fluorescence and green fluorescence are detected simultaneously, determining that the vibrio parahaemolyticus is vibrio parahaemolyticus; if no fluorescence is detected, it is judged as an impurity particle which is not bacterial.
The counting by the flow analyzer is realized by performing a gate circle by a scattered light channel of the flow analyzer and detecting the red and green fluorescent signals by a double fluorescent channel, so that the total number of vibrio parahaemolyticus and bacteria is counted; 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 a sample to be detected into a suspension in water, filtering and collecting filtrate, centrifuging the filtrate, removing upper-layer liquid, retaining the bottom precipitate, and adding PBS for heavy suspension to obtain a 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 the preparation of Vibrio parahaemolyticus antibodies.
Example 2 is a preliminary screening of Vibrio parahaemolyticus antibodies.
Example 3 affinity assay for antigen antibodies
Example 4 is the evaluation of the specificity of the antibody.
Examples 5-8 are evaluations of the assay methods established in this patent.
Example 9 is the components of the kit for rapidly detecting the total number of Vibrio parahaemolyticus and bacteria.
The following is a specific operation of one practical test of the invention:
1. and (3) purifying a sample to be detected:
adding 225mL of deionized water into 25mL or 25g of sample to be detected, filtering the sample to be detected by adopting a filter membrane with the aperture of 2.5-15 microns, and reserving the filtrate. The filtrate was centrifuged at 5000 Xg for 5 min. The upper layer of liquid was discarded, and the bottom precipitate was retained. Adding 2.5mL of PBS to resuspend the precipitate, transferring to a new centrifuge tube to obtain a purified sample bacterial suspension.
2. Adding a green fluorescent probe cross-linked vibrio parahaemolyticus antibody (Gr-Ab) with the final concentration of 1 mug/mL and a red fluorescent probe (Rd) with the concentration of 1 mug/mL into the purified bacterial suspension, uniformly mixing, and incubating for 5-15 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 concentration of Vibrio parahaemolyticus and the total number of bacteria were calculated by analyzing the events within the gates in the dual fluorescence channel.
The inventor verifies the effect of the detection method:
preparing the mixed bacterial liquid of vibrio parahaemolyticus, escherichia coli, staphylococcus aureus and bacillus subtilis, and obtaining the mixed bacterial liquid with 10-time serial dilution. And detecting the mixed bacterial liquid diluted by 10 times in series by adopting a vibrio parahaemolyticus plate counting method and a total bacteria pouring plate method, and simultaneously detecting the total vibrio parahaemolyticus and the total bacteria in the mixed bacterial liquid by using the method. Comparing the detection results of the plate counting method and the flow analysis method: for vibrio parahaemolyticus detection, the results obtained by the method and the plate counting method have good linear relation, which shows that the method has good accuracy, and the lower detection limit of the method is 49 CFU/mL; for the total number of bacteria detection, the results obtained by the method and the plate counting method have good linear relation, which shows that the method has good accuracy, and the lower detection limit of the method is 225 CFU/mL.
According to the detection method established by the invention, the invention also provides a kit for rapidly and synchronously detecting the total number of vibrio parahaemolyticus and bacteria in a sample in multiple ways, which is characterized by comprising the following components: 1. red fluorescent probes that can label all bacteria in the sample; 2. the specific vibrio parahaemolyticus antibody cross-linked by a chemical group by using the green fluorescent probe can distinguish vibrio parahaemolyticus and non-vibrio parahaemolyticus in a sample; 3. calibration microspheres (500nm and 2500 nm).
The technical indexes of the vibrio parahaemolyticus antibody are as follows: a) the immunogen used for preparing the antibody is a mixture of 4 vibrio parahaemolyticus specific outer membrane proteins, and the 4 outer membrane proteins are OmpU, OmpK, OmpC and OmpA respectively; b) the antibody can react with vibrio parahaemolyticus in a mobile phase; c) after the green fluorescent probe is crosslinked, the antibody can carry out fluorescent labeling on the vibrio parahaemolyticus, and the fluorescence can be observed by a fluorescent microscope and a flow analyzer; d) the affinity KD of the antibody and the specific mycoprotein of the vibrio parahaemolyticus is less than 2 multiplied by 10-10(ii) a e) The antibody is capable of recognizing all strains of Vibrio parahaemolyticus.
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 kit also comprises a filter membrane with the pore diameter of 2.5-15 mu m.
In one example of the invention, the kit comprises the following components:
1. the red fluorescent probe can mark all bacteria in a sample, and the fluorescence emission spectrum of the red fluorescent probe is 601 nm-640 nm;
2. the specific vibrio parahaemolyticus antibody cross-linked by a chemical group by using the green fluorescent probe can distinguish vibrio parahaemolyticus and non-vibrio parahaemolyticus in a sample; wherein the fluorescence emission spectrum of the green fluorescent probe is 501 nm-540 nm;
3. calibration microspheres (500nm and 2500 nm).
4. Filter membrane (2.7-15 μm pore size).
The operation method for detecting the vibrio parahaemolyticus by using the kit is characterized by comprising the following steps:
1. and (3) purifying a sample to be detected:
adding 225mL of deionized water into 25mL or 25g of sample to be detected, filtering the sample to be detected by adopting a filter membrane with the aperture of 2.5-15 microns, and reserving the filtrate. The filtrate was centrifuged at 5000 Xg for 5 min. The upper layer of liquid was discarded, and the bottom precipitate was retained. Adding 2.5mL of PBS to resuspend the precipitate, transferring to a new centrifuge tube to obtain a purified sample bacterial suspension.
2. Adding Gr-Ab with the final concentration of 1 mu g/mL and Rd with the final concentration of 1 mu g/mL into the purified bacterial suspension, uniformly mixing, and incubating for 5-15 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 concentration of Vibrio parahaemolyticus and the total number of bacteria were calculated by analyzing the events within the gates in the dual fluorescence channel.
The invention has the following innovations and advantages:
1. simultaneous detection of Vibrio parahaemolyticus and total number of bacteria
By adopting the green fluorescent probe to cross-link the vibrio parahaemolyticus antibody and the red fluorescent probe to carry out double-fluorescence labeling, the total number of the vibrio parahaemolyticus and the bacteria can be simultaneously and accurately and quantitatively detected.
The red fluorescent probe was stained for all bacteria and specifically labeled with green fluorescent immunofluorescent antibody for Vibrio parahaemolyticus. If a bacterium is marked green and red at the same time, it is vibrio parahaemolyticus; if a bacterium is only red-labeled, it is a bacterium, but not Vibrio parahaemolyticus.
2. The method is simple and convenient to operate, short in time consumption, and most of samples can be detected within 0.5 h.
3. 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 is the result of electrophoresis of the Vibrio parahaemolyticus antibody in example 1;
FIG. 2 is a fluorescent microscope observation of FITC-labeled Vibrio parahaemolyticus in example 2;
FIG. 3 shows the results of detecting Bifluorescence-labeled Vibrio parahaemolyticus by the flow analyzer in example 6;
FIG. 4 is the results of flow analysis in example 6 for detecting Vibrio parahaemolyticus and miscellaneous bacteria at different concentration ratios;
FIG. 5 is a linear relationship between the results of the measurement of Vibrio parahaemolyticus by the present method and the plate counting method in example 7.
FIG. 6 is a linear relationship between the results of the method of example 8 and the total number of bacteria measured by plate counting.
Detailed Description
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, may be applied to quantitative terms indicating that a change in quantity may be permissible without departing from 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 of Vibrio parahaemolyticus antibody
Materials and methods
1. In total, 2 rabbits were tested, with an initial weight of 2.2-2.5 kg. The immunization mode comprises the following steps: back intradermal multiple injection. Mixing the immunogen and the specific membrane protein of the vibrio parahaemolyticus. The process comprises the following steps: three immunizations were performed in total, half a month apart. Then titer detection is performed. The fourth immunization was then performed, and serum was collected after appropriate titer detection and antibody purification was performed. 2 Vibrio parahaemolyticus antibodies were obtained.
2. The titer of the obtained 2 antibodies to vibrio parahaemolyticus was determined by ELISA endpoint assay. The antibody concentration was determined by SDS-PAGE.
Second, experimental results
2 pieces of vibrio parahaemolyticus with 1 valence of antibody 1:1 × 10 are prepared preliminarily6 Antibody 2 titer 1: 1.5X 106. The electrophoresis results are shown in FIG. 1.
TABLE 1 antibody potency assay
Third, conclusion of experiment
The antibody 2 is the antibody prepared from the vibrio parahaemolyticus antibody and is numbered Ab-1.
A total of 6 antibodies (Ab-1 to Ab-6) were prepared according to the same protocol.
Example 2 evaluation of Vibrio parahaemolyticus antibody
Materials and methods
1. The prepared 6 Vibrio parahaemolyticus antibodies (Ab-1 to Ab-6) were labeled with fluorescein FITC using a FITC labeling kit.
2. Four vibrio parahaemolyticus standard strains are numbered as follows: CICC 21617, CICC10552, CICC 23924 and CMCC 20001. Prepared separately at a concentration of about 1X 107cells/mL of the above-mentioned Vibrio parahaemolyticus bacterial liquid.
3. FITC-labeled antibodies (Ab-1 to Ab-6) were added to the bacterial solutions of the four Vibrio parahaemolyticus strains at a concentration of 1. mu.g/mL, respectively, and incubated for 15min in the absence of light. Detection was performed with a fluorescence microscope and a flow analyzer, respectively.
Second, experimental results
When Ab-1, Ab-3, Ab-4 and Ab-6 were used for labeling, green fluorescence was detected from 4 strains of Vibrio parahaemolyticus (FIG. 2). When Ab-2 was used for labeling, no green fluorescence was detected from any of the 4 strains of Vibrio parahaemolyticus, indicating that the antibody Ab-2 could not react with the antigen in the mobile phase. When Ab-5 is used for marking, only part of the vibrio parahaemolyticus detects green fluorescence, which indicates that the antibody Ab-5 cannot specifically recognize 4 serotype vibrio parahaemolyticus strains.
Third, conclusion of experiment
Three antibodies (Ab-1, Ab-3, Ab-4 and Ab-6) of the vibrio parahaemolyticus are successfully screened out, can react with the vibrio parahaemolyticus in a mobile phase, and can identify 4 serotype vibrio parahaemolyticus strains through preliminary verification.
TABLE 2 evaluation of Vibrio parahaemolyticus antibody
Example 3 affinity analysis of Vibrio parahaemolyticus antibody and Vibrio parahaemolyticus Membrane protein
Materials and methods
1. The 3 Vibrio parahaemolyticus antibodies (Ab-1, Ab-3, Ab-4, Ab-6) obtained by the primary screening and 4 commercially available Vibrio parahaemolyticus antibodies (Nos. Ab-7 to Ab-10) were diluted to the same initial concentration and serially diluted 2-fold respectively.
2. Mixing specific membrane proteins OmpU, OmpK, OmpC and OmpA of vibrio parahaemolyticus.
3. The specific membrane protein of Vibrio parahaemolyticus was linked to the carboxyl chip using an EDC/NHS reaction. And the carboxyl chip was loaded into an analytical interaction analyzer.
4. For a Vibrio parahaemolyticus antibody, 2 serially diluted antibodies were loaded onto the molecular interaction instrument, data were collected and fitted to a series of reaction curves using TraceDrawer, and the affinity KD of the antibody and the membrane protein was calculated.
Second, experimental results
The affinity of 8 Vibrio parahaemolyticus antibodies and membrane proteins was analyzed using an analytical interaction analyzer, and the results are shown in Table 1. As a result, the affinity between the antibody Ab-3 and the membrane protein of Parahemolytic vibrio was found to be 4.89X 10-11。
Third, conclusion of experiment
The invention screens out the vibrio parahaemolyticus antibody Ab-3 with the best affinity.
TABLE 3 affinity of different Vibrio parahaemolyticus antibodies and antigens
Example 4 evaluation of specificity of Vibrio parahaemolyticus antibody Ab-3
Materials and methods
1. Vibrio parahaemolyticus strain 12 (Table 4), and Vibrio non-parahaemolyticus strain 24 (Table 5). 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 vibrio parahaemolyticus antibody Ab-1 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 vibrio parahaemolyticus antibody Ab-1 and bacterial suspensions of 36 different strains are reacted, and then the specificity of the vibrio parahaemolyticus antibody Ab-1 is detected by a flow analyzer, and the results are shown in tables 4 and 5: green fluorescence was detected in all 12 Vibrio parahaemolyticus, and no green fluorescence was detected in all 24 non-Vibrio parahaemolyticus.
TABLE 4 results of the method specificity experiments (12 parahaemolytic arc strains)
TABLE 5 results of the method specificity experiments (24 non-parahemolytic arc strains)
Third, conclusion of experiment
The vibrio parahaemolyticus antibody Ab-3 can be used for a flow analysis technology, has good specificity, and can accurately identify and distinguish vibrio parahaemolyticus.
Example 5 method for measuring the universality of the total number of bacteria
Materials and methods
1. The standard strains of different genera, 16 strains in total (Table 6).
2. Membrane permeable nucleic acid fluorescent material SYTO 62.
3. Suspensions of the 16 strains were prepared and adjusted to a turbidity of 0.5 McF. The bacterial solutions were stained with SYTO 62 (final concentration 1. mu.g/mL) for 15 min.
5. The stained sample was tested using a flow analyzer.
Second, experimental results
The results showed that red fluorescence was detected in 16 fluorescently labeled bacterial cells by the flow analyzer. For 16 fluorescently unlabeled bacterial fluids, no red fluorescence was detected by the flow analyzer. SYTO 62 was shown to stain 16 strains tested.
TABLE 6 fluorescent labeling of different strains
Third, conclusion of experiment
The method can identify all kinds of bacteria and has universality.
EXAMPLE 6 detection of samples of Vibrio parahaemolyticus and miscellaneous bacteria at different concentration ratios
Materials and methods
1. Prepared separately at a concentration of about 1X 107cells/mL of Vibrio parahaemolyticus bacterial liquid and 1X 107cells/mL of mixed bacteria liquid (containing escherichia coli, staphylococcus aureus and bacillus subtilis).
2. The vibrio parahaemolyticus suspension and the mixed bacteria suspension are mixed in different volume ratios (0/10, 1/9, 5/5, 9/1, 10/0). The sample was then examined with a flow analyzer.
Second, experimental results
Vibrio parahaemolyticus not fluorescently labeled as shown in FIG. 3A, without any fluorescence; vibrio parahaemolyticus labeled with a green fluorescent antibody fluoresces green as shown in FIG. 3B; vibrio parahaemolyticus labeled with a green fluorescent antibody and a nucleic acid fluorescein fluoresces both green and red as shown in FIG. 3C.
The Vibrio parahaemolyticus and the miscellaneous bacteria were mixed at different concentration ratios and detected by a flow analyzer, and the results are shown in FIG. 4. The result shows that the ratio of the vibrio parahaemolyticus and the infectious microbe detected by the flow type is close to the actual value.
Third, conclusion of experiment
The detection method of the invention can accurately and quantitatively detect the total number of the vibrio parahaemolyticus and the bacteria in the liquid.
Example 7 comparison of the method of the present invention and the plate counting method for detecting Vibrio parahaemolyticus
Materials and methods
1. The cultured parahemolytic vibrio suspension is centrifuged at 12000 Xg for 5min, the supernatant is discarded, and the bacterial sludge is resuspended by PBS.
2. The suspension of the vibrio parahaemolyticus is serially diluted by 10 times by using the sterilized purified water, and the sample is quantitatively detected by respectively adopting the detection method and the plate counting method, so that the linearity and the sensitivity of the method are analyzed.
Second, experimental results
When the concentration of Vibrio parahaemolyticus is 102～108At CFU/mL, the detection results of the plate counting method and the flow analysis method are close to each other, and the linearity is good (R)20.9991) (fig. 5).
Third, conclusion of experiment
The detection method can accurately and sensitively quantitatively detect the vibrio parahaemolyticus, and has good linearity with a plate counting method.
Example 8 comparison of the Total number of bacteria detected by the method of the invention and by plate counting
Materials and methods
1. Standard strain of Escherichia coli, number CMCC 44102; a standard strain of staphylococcus aureus, No. ATCC 6538P; bacillus subtilis standard strain, No. ATCC 6633.
2. The bacterial liquid of the three cultured representative strains is respectively diluted by 10 times in series. Quantitative detection is carried out by adopting a flow detection method and a plate counting method in national standard GB4789.2 determination of total number of bacterial colonies for food safety national standard food microbiological test, and each concentration is respectively repeated for 3 times by using the flow detection method and the plate counting method.
Second, experimental results
FIG. 6 is a linear relationship between the flow assay results and plate count results for the three strains. The results showed that the concentration of bacteria was 103～108The flow results at CFU/mL were essentially identical to the plate count method with good linearity.
Third, conclusion of experiment
The method for detecting the total number of bacteria by using the flow analysis technology in the method has good accuracy, and the lower detection limit is 225 CFU/mL. Example 9 Rapid detection kit for Vibrio parahaemolyticus and Total bacteria count
The kit is internally provided with:
red fluorescent probes that can label all bacteria in the sample;
a green fluorescent probe-crosslinked vibrio parahaemolyticus antibody;
a filter membrane (aperture of 2.5-15 μm);
calibration microspheres (500nm and 2500 nm).
Claims (10)
1. A method for rapidly and synchronously detecting the total number of vibrio parahaemolyticus and bacteria in a sample in a multiplex manner is characterized by comprising the following steps:
1) differentiation of bacteria from other background particles in the sample: marking the total number of bacteria by using a red fluorescent probe capable of marking all bacteria;
2) distinguishing between vibrio parahaemolyticus and non-vibrio parahaemolyticus in the sample: using a green fluorescent probe to specifically mark vibrio parahaemolyticus in the sample through a vibrio parahaemolyticus antibody crosslinked by a chemical group;
3) and simultaneously counting the red and green fluorescent signals generated by 1) and 2) by a flow analyzer to realize synchronous quantitative detection of the total number of the vibrio parahaemolyticus and the bacteria.
In the above 2), the vibrio parahaemolyticus antibody is prepared from a mixture of immunogens of 4 vibrio parahaemolyticus specific outer membrane proteins, the 4 outer membrane proteins are OmpU, OmpK, OmpC and OmpA, respectively;
in the above 3), the method for determining the total number of Vibrio parahaemolyticus and bacteria is as follows: for an event produced by one particle, if only red fluorescence is detected, it is judged as a bacterium, but not as Vibrio parahaemolyticus; if two kinds of fluorescence, namely red fluorescence and green fluorescence are detected simultaneously, determining that the vibrio parahaemolyticus is vibrio parahaemolyticus; if no fluorescence is detected, it is judged as an impurity particle which is not bacterial.
2. The method of claim 1, wherein the vibrio parahaemolyticus antibody further has the following technical indicators:
a the antibody can react with vibrio parahaemolyticus in a mobile phase;
b, after the antibody is crosslinked by a green fluorescent probe, the antibody can carry out fluorescent labeling on the vibrio parahaemolyticus, and the fluorescence can be observed by a fluorescent microscope and a flow analyzer;
c the affinity KD of the antibody and the specific mycoprotein of the vibrio parahaemolyticus is less than 2 multiplied by 10-10；
d the antibody is capable of recognizing all strains of Vibrio parahaemolyticus.
3. The method of claim 1, wherein the counting by the flow analyzer is performed by performing a gate-rounding through a scattered light channel of the flow analyzer and detecting the red and green fluorescent signals through a dual fluorescent channel, thereby counting the total number of Vibrio parahaemolyticus and 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 1, 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.
6. The method of claim 1, 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.
7. The method according to any one of claims 1 to 6, wherein the sample to be tested is subjected to purification treatment before detection; and the purification method comprises the steps of adding a sample to be detected into a suspension in water, filtering and collecting filtrate, centrifuging the filtrate, removing upper-layer liquid, retaining the bottom precipitate, and adding PBS for heavy suspension to obtain a purified sample bacterial suspension.
8. A is used for detecting the vibrio parahaemolyticus antibody of vibrio parahaemolyticus and total number of bacterium in the sample in step multiplex, it is prepared for 4 pieces of outer membrane protein mixture of vibrio parahaemolyticus specificity for immunogen, said 4 pieces of outer membrane protein are OmpU, OmpK, OmpC and OmpA separately;
the vibrio parahaemolyticus antibody also has the following technical indexes:
a the antibody can react with vibrio parahaemolyticus in a mobile phase;
b, after the antibody is crosslinked by a green fluorescent probe, the antibody can carry out fluorescent labeling on the vibrio parahaemolyticus, and the fluorescence can be observed by a fluorescent microscope and a flow analyzer;
c the affinity KD of the antibody and the specific mycoprotein of the vibrio parahaemolyticus is less than 2 multiplied by 10-10；
d the antibody is capable of recognizing all strains of Vibrio parahaemolyticus.
9. A kit for rapidly and synchronously detecting the total number of vibrio parahaemolyticus and bacteria in a sample in a multiplex manner is characterized by comprising the following components:
red fluorescent probes that can label all bacteria in the sample;
the specific vibrio parahaemolyticus antibody cross-linked by a chemical group by using the green fluorescent probe can distinguish vibrio parahaemolyticus and non-vibrio parahaemolyticus in a sample;
calibration microspheres (500nm and 2500 nm);
the technical indexes of the vibrio parahaemolyticus antibody are as follows:
a, preparing the antibody by using immunogen of a mixture of 4 vibrio parahaemolyticus specific outer membrane proteins, wherein the 4 outer membrane proteins are OmpU, OmpK, OmpC and OmpA respectively;
b the antibody can react with vibrio parahaemolyticus in a mobile phase;
c, after the antibody is crosslinked by the green fluorescent probe, the antibody can carry out fluorescent labeling on the vibrio parahaemolyticus, and the fluorescence can be observed by a fluorescent microscope and a flow analyzer;
d the affinity KD of the antibody and the specific mycoprotein of the vibrio parahaemolyticus is less than 2 multiplied by 10-10；
e the antibody is capable of recognizing all strains of Vibrio parahaemolyticus.
10. The kit of claim 9, further comprising a filter with a pore size of 2.5 μm to 15 μm, and the fluorescence emission spectrum of the red fluorescent probe is 601nm to 640 nm; the fluorescence emission spectrum of the green fluorescent probe is 501 nm-540 nm.
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