CN112553291A - Rapid synchronous multiple detection method and kit for total number of shigella and bacteria - Google Patents
Rapid synchronous multiple detection method and kit for total number of shigella and bacteria Download PDFInfo
- Publication number
- CN112553291A CN112553291A CN202011464838.8A CN202011464838A CN112553291A CN 112553291 A CN112553291 A CN 112553291A CN 202011464838 A CN202011464838 A CN 202011464838A CN 112553291 A CN112553291 A CN 112553291A
- Authority
- CN
- China
- Prior art keywords
- shigella
- antibody
- bacteria
- sample
- fluorescence
- 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
- 241000607768 Shigella Species 0.000 title claims abstract description 199
- 241000894006 Bacteria Species 0.000 title claims abstract description 77
- 238000001514 detection method Methods 0.000 title claims abstract description 58
- 230000001360 synchronised effect Effects 0.000 title claims abstract description 8
- 238000000034 method Methods 0.000 claims abstract description 72
- 239000007850 fluorescent dye Substances 0.000 claims abstract description 47
- 239000000523 sample Substances 0.000 claims abstract description 33
- 108010043121 Green Fluorescent Proteins Proteins 0.000 claims abstract description 27
- 239000002245 particle Substances 0.000 claims abstract description 8
- 125000003636 chemical group Chemical group 0.000 claims abstract description 6
- 230000001580 bacterial effect Effects 0.000 claims description 30
- 239000000725 suspension Substances 0.000 claims description 17
- 108010052285 Membrane Proteins Proteins 0.000 claims description 12
- 239000007788 liquid Substances 0.000 claims description 12
- 239000004005 microsphere Substances 0.000 claims description 11
- 238000002189 fluorescence spectrum Methods 0.000 claims description 10
- 238000001215 fluorescent labelling Methods 0.000 claims description 9
- 239000000706 filtrate Substances 0.000 claims description 8
- 238000004458 analytical method Methods 0.000 claims description 7
- 239000002244 precipitate Substances 0.000 claims description 6
- 230000002163 immunogen Effects 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 238000001914 filtration Methods 0.000 claims description 4
- 238000002372 labelling Methods 0.000 claims description 4
- 239000012521 purified sample Substances 0.000 claims description 4
- 230000009977 dual effect Effects 0.000 claims description 3
- 239000012535 impurity Substances 0.000 claims description 3
- 239000011148 porous material Substances 0.000 claims description 3
- 238000000746 purification Methods 0.000 claims description 3
- 230000035945 sensitivity Effects 0.000 claims description 3
- 206010034960 Photophobia Diseases 0.000 claims description 2
- 230000004069 differentiation Effects 0.000 claims description 2
- 208000013469 light sensitivity Diseases 0.000 claims description 2
- 238000002474 experimental method Methods 0.000 description 14
- 235000013305 food Nutrition 0.000 description 10
- 239000000463 material Substances 0.000 description 9
- 239000000427 antigen Substances 0.000 description 7
- 102000036639 antigens Human genes 0.000 description 7
- 108091007433 antigens Proteins 0.000 description 7
- 238000005206 flow analysis Methods 0.000 description 7
- 239000012528 membrane Substances 0.000 description 6
- 102000018697 Membrane Proteins Human genes 0.000 description 5
- 238000003556 assay Methods 0.000 description 5
- 238000011156 evaluation Methods 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 244000063299 Bacillus subtilis Species 0.000 description 4
- 235000014469 Bacillus subtilis Nutrition 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 4
- 241000191967 Staphylococcus aureus Species 0.000 description 4
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 4
- 238000002965 ELISA Methods 0.000 description 3
- 241000607766 Shigella boydii Species 0.000 description 3
- 241000607764 Shigella dysenteriae Species 0.000 description 3
- 241000607762 Shigella flexneri Species 0.000 description 3
- 241000607760 Shigella sonnei Species 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 230000003053 immunization Effects 0.000 description 3
- 238000002649 immunization Methods 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 229940007046 shigella dysenteriae Drugs 0.000 description 3
- 229940115939 shigella sonnei Drugs 0.000 description 3
- 230000005526 G1 to G0 transition Effects 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000000717 retained effect Effects 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 208000004429 Bacillary Dysentery Diseases 0.000 description 1
- 241000194103 Bacillus pumilus Species 0.000 description 1
- 238000000018 DNA microarray Methods 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- 206010017915 Gastroenteritis shigella Diseases 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 241000607142 Salmonella Species 0.000 description 1
- 238000011091 antibody purification Methods 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 244000078673 foodborn pathogen Species 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 244000000074 intestinal pathogen Species 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000012009 microbiological test Methods 0.000 description 1
- 230000004001 molecular interaction Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 230000003716 rejuvenation Effects 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 201000005113 shigellosis Diseases 0.000 description 1
- 239000010802 sludge Substances 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Images
Classifications
-
- 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
- C12Q1/10—Enterobacteria
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Toxicology (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
A method and a detection kit for rapidly and synchronously detecting total number of shigella and bacteria in a sample in multiple ways are disclosed, wherein a red fluorescent probe capable of marking all bacteria is used for marking the total number of the bacteria, and the bacteria in the sample are distinguished from other background particles: meanwhile, a green fluorescent probe is used for distinguishing shigella from non-shigella in the sample through a shigella antibody cross-linked by chemical groups; and simultaneously counting red and green fluorescent signals by a flow analyzer to realize synchronous detection of the total number of the shigella and the bacteria. The method can simultaneously detect the total number of shigella and 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 total shigella and bacteria in a food sample and a kit.
Background art:
the total colony number and shigella indexes are the most basic and common detection items in food microorganism detection. Shigella is a gram-negative Bacillus pumilus, a common food-borne pathogen, which is mainly prevalent in developing countries, and is an intestinal pathogen of humans and primates, causing bacillary dysentery. 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.
The traditional culture method is time-consuming and labor-consuming in measuring the Shigella, for example, the national standard GB 4789.5-2016 (national food safety Standard food microbiology test for Shigella detection), the method needs a series of steps of enrichment, culture, biochemical identification and the like, takes more than 72 hours, needs XLD, MAC and a chromogenic medium, and needs a large amount of reagents for subsequent biochemical identification.
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 indexes of the total number of the salmonella and the bacteria must be carried out respectively.
Although many new techniques are currently used for rapid detection of shigella or total bacteria, these techniques are difficult to use for multiplex detection with shigella and total 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 total indexes of shigella and bacteria, but because the development of shigella specific fluorescent probes has many technical difficulties, no relevant report exists at present.
In conclusion, the rapid detection and the synchronous multiple detection can simultaneously detect the total number of the shigella and the bacteria, which is the actual requirement in the field of food microorganism detection and is a technical difficulty.
The invention content is as follows:
the first purpose of the invention is to provide a rapid synchronous multiple detection method for total shigella and bacteria, in particular to meet the following requirements: 1. must be able to distinguish between bacteria and impurities; 2. the total number of bacteria can be quantitatively measured; 3. shigella can be specifically identified and quantified 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 recognize shigella.
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 Shigella are selected as the representative strains of gram-negative bacteria, Staphylococcus aureus is selected as the representative strain of gram-positive bacteria, and Bacillus subtilis is selected as the 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 shigella
The method adopts an antibody of the shigella coupled with a green fluorescent probe, carries out specific fluorescent labeling on the shigella, and analyzes the fluorescence of the shigella by using a flow analyzer.
The shigella antibodies suitable for flow analyzers are very difficult to screen:
firstly, the shigella species are various and comprise shigella dysenteriae (group a), shigella flexneri (group B), shigella boydii (group C) and shigella sonnei (group D), and each species comprises a plurality of shigella species; and different shigella species have different serotypes. Thus, antibodies capable of specifically recognizing all shigella bacteria have been difficult to develop.
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 the Shigella structural antigen, and the Shigella antibody which is high in affinity, good in specificity and suitable for a flow analyzer is finally screened out and named Ab-1 (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 is preliminarily judged whether the antibody can react with Shigella in the mobile phase, and the specificity of the antibody is preliminarily evaluated (example 2).
Secondly, the affinity of the shigella antibody and the antigen is analyzed, and the shigella antibody with high affinity is selected (example 3).
Thirdly, 24 shigella strains (including group a, group B, group C, and group D) and 24 other strains were selected, and it was verified whether the antibody specifically recognized the shigella and distinguished the shigella from other bacteria using a flow analyzer (example 4).
The technical indexes of Shigella antibodies determined to be suitable for the purpose of the invention are as follows:
1. the immunogen used for preparing the antibody is a mixture of specific membrane proteins of 4 strains of Shigella, wherein the 4 strains are respectively from Shigella A group, Shigella B group, Shigella C group and Shigella D group.
2. The antibody can react with Shigella in a mobile phase (unlike the antibodies used in ELISA, IHC-Fr, WB, etc. which react on a stationary phase).
3. After the antibody is crosslinked by the green fluorescent probe, the shigella can be subjected to fluorescent labeling, and fluorescence can be observed by a fluorescent microscope and a flow analyzer.
4. The affinity KD of the antibody and the shigella specific mycoprotein should be less than 1 x 10-9。
5. The antibodies are capable of recognizing all subgroups of Shigella strains.
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 shigella 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 shigella from non-shigella in a sample: specifically labeling shigella in a sample by using a green fluorescent probe through a shigella antibody crosslinked by a chemical group; 3) and simultaneously counting the red and green fluorescent signals generated by 1) and 2) by using a flow analyzer to realize synchronous quantitative detection of the total number of the shigella and the bacteria.
The technical indexes of the Shigella antibody are as follows: a) the immunogen used for preparing the antibody is a mixture of specific membrane proteins of 4 strains of Shigella, wherein the 4 strains are respectively from Shigella A group, Shigella B group, Shigella C group and Shigella D group; b) the antibody can react with Shigella in a mobile phase; c) after the antibody is crosslinked by the green fluorescent probe, shigella can be subjected to fluorescent labeling, and fluorescence can be observed by a fluorescent microscope and a flow analyzer; d) the affinity KD of the antibody and the shigella specific mycoprotein should be less than 1 x 10-9(ii) a e) The antibodies are capable of recognizing all subgroups of Shigella strains.
The method for judging the total number of shigella and 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 shigella; if two kinds of fluorescence, namely red fluorescence and green fluorescence are detected simultaneously, judging the shigella; if no fluorescence is detected, it is judged as an impurity particle which is not bacterial.
Counting by a flow analyzer, namely performing gate closing 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 count the total number of shigella and 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 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 shigella antibodies.
Example 2 is a preliminary screen for shigella 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 rapid detection of total shigella 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. And adding a shigella antibody (Gr-Ab) crosslinked by a green fluorescent probe with a final concentration of 1 mug/mL and a red fluorescent probe (Rd) with a 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. Shigella concentrations and total bacteria were calculated by analysis of events within the gates on dual fluorescence channels.
The inventor verifies the effect of the detection method:
mixed bacterial liquid of Shigella, Escherichia coli, Staphylococcus aureus and Bacillus subtilis is prepared, and mixed bacterial liquid with 10-fold serial dilution is obtained. Detecting the mixed bacterial liquid diluted by 10 times in series by adopting a shigella plate counting method and a total bacteria pouring plate method, and simultaneously detecting the total shigella 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 Shigella detection, the results obtained by the method and the flat plate counting method have good linear relation, which shows that the method has good accuracy, and the lower detection limit of the method is 89 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 shigella 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 green fluorescent probe is used for identifying shigella and non-shigella in the sample through the specific shigella antibody crosslinked by the chemical group; 3. calibration microspheres (500nm and 2500 nm).
The technical indexes of the Shigella antibody are as follows: a) the immunogen used for preparing the antibody is the specificity of 4 strains of ShigellaA mixture of membrane proteins, said 4 strains being from shigella group a, group B, group C, group D, respectively; b) the antibody can react with Shigella in a mobile phase; c) after the antibody is crosslinked by the green fluorescent probe, shigella can be subjected to fluorescent labeling, and fluorescence can be observed by a fluorescent microscope and a flow analyzer; d) the affinity KD of the antibody and the shigella specific mycoprotein should be less than 1 x 10-9(ii) a e) The antibodies are capable of recognizing all subgroups of Shigella strains.
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 green fluorescent probe is used for identifying shigella and non-shigella in the sample through the specific shigella antibody crosslinked by the chemical group; 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 Shigella 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. Shigella concentrations and total bacteria were calculated by analysis of events within the gates on dual fluorescence channels.
The invention has the following innovations and advantages:
1. simultaneous detection of shigella and total bacteria
The shigella antibody and the red fluorescent probe which are crosslinked by the green fluorescent probe are adopted for double-fluorescence labeling, so that the total number of shigella and bacteria can be accurately and quantitatively detected at the same time.
The red fluorescent probe was stained for all bacteria and shigella was specifically labeled by immunofluorescent antibodies with green fluorescence. If one bacterium is marked by green and red simultaneously, the bacterium is Shigella; if a bacterium is only red-marked, it is a bacterium, but not shigella.
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 Shigella antibodies in example 1;
FIG. 2 is fluorescence microscopy of FITC-labeled Shigella in example 2;
FIG. 3 shows the results of detecting double fluorescently labeled Shigella in example 6 using a flow analyzer;
FIG. 4 shows the results of the flow analyzer in example 6 for detecting Shigella and infectious microbe at different concentration ratios;
FIG. 5 is a linear relationship between the results of the method of example 7 and Shigella measurements by plate count.
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 consumables of the experimental reagents used in the following examples are all conventional biochemical reagents unless otherwise specified. The experimental method not specifying the specific conditions is usually performed under the conditions in the conventional conditions or the conditions recommended by the manufacturer. 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 of Shigella antibodies
Materials and methods
1. In total, 2 rabbits were tested, with an initial weight of 2.1-2.4 kg. The immunization mode comprises the following steps: back intradermal multiple injection. The immunogen is Shigella specific membrane protein. 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 Shigella antibodies were obtained.
2. The titer of the obtained 2 shigella antibodies was detected by ELISA endpoint method. The antibody concentration was determined by SDS-PAGE.
Second, experimental results
2 shigella are prepared preliminarily, and the titer of the antibody 1 is 1:5 × 106 Antibody 2 titer 1: 2X 107. The electrophoresis results are shown in FIG. 1.
TABLE 1 antibody potency assay
Third, conclusion of experiment
The antibody 2 is an antibody prepared from the Shigella antibody at the time 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 Shigella antibodies
Materials and methods
1. The prepared 6 Shigella antibodies (Ab-1 to Ab-6) were labeled with fluorescein FITC using a FITC labeling kit.
2. Shigella flexneri, Shigella sonnei, Shigella boydii and Shigella dysenteriae. Prepared separately at a concentration of about 1X 107cells/mL of the above Shigella bacterial solution.
3. FITC-labeled antibodies (Ab-1 to Ab-6) at a concentration of 1. mu.g/mL were added to the bacterial solutions of the four Shigella strains, respectively, and incubated for 15min in the dark. Detection was performed with a fluorescence microscope and a flow analyzer, respectively.
Second, experimental results
When Ab-1, Ab-2 and Ab-6 are used for marking, green fluorescence is detected in 4 Shigella strains (figure 2). When Ab-4 and Ab-5 are used for marking, green fluorescence is not detected by 4 Shigella strains, which indicates that the antibodies Ab-4 and Ab-5 can not react with antigen in a mobile phase. When Ab-3 is used for labeling, only part of Shigella detects green fluorescence, which indicates that the Ab-3 antibody cannot specifically recognize all subgroups of Shigella strains.
Third, conclusion of experiment
Three Shigella antibodies (Ab-1, Ab-2 and Ab-6) are successfully screened out, can react with Shigella in a mobile phase, and can identify Shigella strains from all subgroups through preliminary verification.
TABLE 2 evaluation of Shigella antibodies
Example 3 affinity analysis of Shigella antibodies and Shigella membrane proteins
Materials and methods
1. Shigella flexneri, Shigella sonnei, Shigella boydii and Shigella dysenteriae.
2. The 3 shigella antibodies (Ab-1, Ab-2, Ab-6) obtained by the primary screening and 4 shigella antibodies (accession No. Ab-7 to Ab-10) purchased on the market were diluted to the same initial concentration and serially diluted 2-fold respectively.
3. Prepared separately at a concentration of about 1X 107cells/mL of the shigella bacterial solution is heated and killed, cell membranes of the shigella bacterial solution are cracked, then an antibody of a shigella structural antigen is used for carrying out fishing reaction, and shigella specific membrane protein is captured.
4. The Shigella specific membrane protein was linked to the carboxyl chip using an EDC/NHS reaction. And the carboxyl chip was loaded into an analytical interaction analyzer.
5. For a shigella antibody, 2 serially diluted antibodies were loaded on the molecular interaction instrument separately, data were collected and fitted to a series of reaction curves using TraceDrawer, and affinity KD for the antibody and membrane protein was calculated.
Second, experimental results
The affinity of 7 shigella antibodies and membrane proteins 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-1 and Shigella membrane protein was the best, 7.81X 10-10。
Third, conclusion of experiment
The shigella antibody Ab-1 with the best affinity is screened out.
TABLE 3 affinity of different Shigella antibodies and antigens
Example 4 Shigella antibody Ab-1 specificity evaluation
Materials and methods
1. Shigella strain 24 (table 4), non-shigella strain 24 (table 5). All the above strains were rejuvenated and expanded and diluted to the appropriate concentration (about 10)6CFU/mL)。
2. And (3) respectively adding the green fluorescence-labeled Shigella 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
Shigella antibody Ab-1 is reacted with bacterial suspensions of 48 different strains, and then the specificity of the shigella antibody Ab-1 is detected by a flow analyzer, and the results are shown in tables 4 and 5: green fluorescence was detected for 24 shigella, while no green fluorescence was detected for 24 non-shigella.
TABLE 4 results of the method specificity experiments (24 Shigella strains)
TABLE 5 results of the method specificity experiments (24 non-Shigella strains)
Third, conclusion of experiment
The Shigella antibody Ab-1 can be used for a flow analysis technology, has good specificity, and can accurately identify and distinguish Shigella.
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 Shigella and miscellaneous bacteria samples at different concentration ratios
Materials and methods
1. Prepared separately at a concentration of about 1X 107cells/mL shigella bacterial liquid and 1 × 107cells/mL of mixed bacteria liquid (containing escherichia coli, staphylococcus aureus and bacillus subtilis).
2. Shigella suspensions and mixed suspensions at 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
Shigella without fluorescent labeling is shown in fig. 3A without any fluorescence; shigella labeled with green fluorescent antibody fluoresces green as shown in fig. 3B; shigella labeled with green fluorescent antibody and nucleic acid fluorescein fluoresces both green and red as shown in fig. 3C.
Shigella and sundry bacteria at different concentration ratios were mixed and detected by a flow analyzer, and the results are shown in fig. 4. The result shows that the ratio of shigella to mixed bacteria detected by flow detection is close to the actual value.
Third, conclusion of experiment
The detection method can accurately and quantitatively detect the total number of shigella and bacteria in the liquid.
Example 7 comparison of the method of the present invention with Shigella detection by plate count
Materials and methods
1. The cultured shigella bacterial suspension is centrifuged at 12000 Xg for 5min, the supernatant is discarded, and the bacterial sludge is resuspended by PBS.
2. The shigella bacterial suspension is serially diluted by 10 times with sterilized purified water, and the sample is quantitatively detected by the detection method and the plate counting method respectively, so that the linearity and the sensitivity of the method are analyzed.
Second, experimental results
When the concentration of Shigella 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)20.9999) (fig. 5).
Third, conclusion of experiment
The detection method can accurately and sensitively detect the shigella quantitatively, 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 Shigella and Total bacteria
The kit is internally provided with:
red fluorescent probes that can label all bacteria in the sample;
a shigella antibody Ab-1 crosslinked by a green fluorescent probe;
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 total number of shigella and bacteria in a sample in multiple ways 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 shigella from non-shigella in a sample: specifically labeling shigella in a sample by using a green fluorescent probe through a shigella antibody crosslinked by a chemical group;
3) and simultaneously counting the red and green fluorescent signals generated by 1) and 2) by using a flow analyzer to realize synchronous quantitative detection of the total number of the shigella and the bacteria.
In the above 2), the shigella antibody is prepared by using a mixture of specific membrane proteins of 4 strains of the genus shigella as immunogens, wherein the 4 strains are respectively from a group a, a group B, a group C and a group D of the genus shigella;
in the above 3), the method for determining the total number of shigella 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 shigella; if two kinds of fluorescence, namely red fluorescence and green fluorescence are detected simultaneously, judging the shigella; if no fluorescence is detected, it is judged as an impurity particle which is not bacterial.
2. The method of claim 1, the shigella antibody further having the following technical indicators:
a the antibody can react with Shigella in a mobile phase;
b, after the antibody is crosslinked by a green fluorescent probe, the antibody can carry out fluorescent labeling on Shigella, and fluorescence can be observed by a fluorescent microscope and a flow analyzer;
c the affinity KD of the antibody and the shigella specific mycoprotein is less than 1 x 10-9；
d the antibody is capable of recognizing all subgroups of Shigella strains.
3. The method of claim 1, wherein the counting by the flow analyzer is performed by gating a scattered light channel of the flow analyzer and detecting the red and green fluorescent signals by a dual fluorescent channel to count shigella and total 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 601nm to 640nm, and the fluorescence emission spectrum of the green fluorescent probe is 501nm to 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 shigella antibody for rapidly and synchronously detecting total shigella and bacteria in a sample in multiple ways is prepared by using a mixture of specific membrane proteins of 4 strains of Shigella as immunogen, wherein the 4 strains are respectively from Shigella A group, Shigella B group, Shigella C group and Shigella D group;
the shigella antibody should also meet the following technical criteria:
a the antibody can react with Shigella in a mobile phase;
b, after the antibody is crosslinked by a green fluorescent probe, the antibody can carry out fluorescent labeling on Shigella, and fluorescence can be observed by a fluorescent microscope and a flow analyzer;
c the affinity KD of the antibody and the shigella specific mycoprotein is less than 1 x 10-9；
d the antibody is capable of recognizing all subgroups of Shigella strains.
9. A kit for rapidly and synchronously detecting total number of shigella and bacteria in multiple samples is characterized by comprising:
red fluorescent probes that can label all bacteria in the sample;
the green fluorescent probe is used for identifying shigella and non-shigella in the sample through the specific shigella antibody crosslinked by the chemical group;
calibration microspheres (500nm and 2500 nm);
the shigella antibody of claim 8.
10. The kit of claim 9, further comprising a filter having a pore size of 2.5 μm to 15 μm; 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.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2019112955961 | 2019-12-16 | ||
| CN201911295596 | 2019-12-16 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112553291A true CN112553291A (en) | 2021-03-26 |
Family
ID=75064180
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011464838.8A Pending CN112553291A (en) | 2019-12-16 | 2020-12-14 | Rapid synchronous multiple detection method and kit for total number of shigella and bacteria |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112553291A (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103323597A (en) * | 2012-11-21 | 2013-09-25 | 北京农学院 | Colloidal gold rapid detecting card for shigella detection and preparation method thereof |
| US20140273022A1 (en) * | 2013-03-14 | 2014-09-18 | Silver Lake Research Corporation | Biomarkers for detecting the presence of bacteria |
| KR20170105997A (en) * | 2016-03-11 | 2017-09-20 | 대한민국(관리부서 질병관리본부장) | Composition and method for detecting Shigella spp. belonging to 4 serotype groups |
| CN110218763A (en) * | 2019-05-27 | 2019-09-10 | 厦门大学 | A method of in edible raw egg pathogenic bacteria and total bacteria count carry out quantitative detection |
-
2020
- 2020-12-14 CN CN202011464838.8A patent/CN112553291A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103323597A (en) * | 2012-11-21 | 2013-09-25 | 北京农学院 | Colloidal gold rapid detecting card for shigella detection and preparation method thereof |
| US20140273022A1 (en) * | 2013-03-14 | 2014-09-18 | Silver Lake Research Corporation | Biomarkers for detecting the presence of bacteria |
| KR20170105997A (en) * | 2016-03-11 | 2017-09-20 | 대한민국(관리부서 질병관리본부장) | Composition and method for detecting Shigella spp. belonging to 4 serotype groups |
| CN110218763A (en) * | 2019-05-27 | 2019-09-10 | 厦门大学 | A method of in edible raw egg pathogenic bacteria and total bacteria count carry out quantitative detection |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Feng | Commercial assay systems for detecting foodborne Salmonella: a review | |
| US6225046B1 (en) | Method for detecting microorganisms | |
| CA2741008C (en) | Methods for separation and characterization of microorganisms using identifier agents | |
| CN102928590A (en) | Kit adopting fluorescent quantum dots to quickly screen, separate and detect salmonella | |
| Wuthiekanun et al. | a rapid method for the differentiation of Burkholderia pseudomallei and Burkholderia thailandensis. | |
| US20060129327A1 (en) | Ultrasensitive sensor and rapid detection of analytes | |
| CN108362629B (en) | Rapid detection method and kit for single viable bacteria of Escherichia coli O157H 7 | |
| CN102590506A (en) | Method for rapidly detecting and screening staphylococcus aureus | |
| Shrestha et al. | Rapid differentiation of methicillin-resistant and methicillin-susceptible Staphylococcus aureus by flow cytometry after brief antibiotic exposure | |
| CN101432739A (en) | Ultrasensitive sensor and rapid detection of analytes | |
| US20210405033A1 (en) | Analyte detection and methods therefor | |
| JP6563654B2 (en) | A method to detect and directly identify microorganisms in biological samples by light pathway | |
| CN108287236A (en) | A kind of biological sensor and its application based on High-gradient Magnetic separation and quantum dot | |
| US20030022270A1 (en) | Automated epifluorescence microscopy for detection of bacterial contamination in platelets | |
| Taha et al. | Rapid detection of Salmonella in chicken meat using immunomagnetic separation, CHROMagar, ELISA and real-time polymerase chain reaction (RT-PCR) | |
| CN112575054B (en) | Rapid synchronous multiple detection method and kit for total number of listeria monocytogenes and bacteria | |
| JP2021521462A (en) | Antibodies or combinations of antibodies and their use for the detection of mycobacterium-related antigens in subject urine samples | |
| CN112553291A (en) | Rapid synchronous multiple detection method and kit for total number of shigella and bacteria | |
| AU2014348685A1 (en) | Bacterial diagnosis | |
| JP3773633B2 (en) | Analysis method and reagent for E. coli O157 | |
| CN113049826A (en) | Rapid synchronous multiple detection method and kit for total number of vibrio parahaemolyticus and bacteria | |
| CN112553292A (en) | Rapid synchronous multiple detection method and kit for total number of salmonella and bacteria | |
| CN112577884A (en) | Rapid synchronous multiple detection method and kit for total number of enterobacter sakazakii and bacteria | |
| CN108982848A (en) | A kind of methicillin-resistant staphylococcus aureus fluorescence detection method based on aptamers | |
| Okada et al. | Cross‐Reactivity and Sensitivity of Two Legionella Urinary Antigen Kits, Biotest EIA and Binax NOW, to Extracted Antigens from Various Serogroups of L. pneumophila and Other Legionella Species |
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 | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20210326 |
|
| RJ01 | Rejection of invention patent application after publication |