CN111876343A - Mass spectrometry method for detecting Klebsiella pneumoniae in instant fresh fruits and vegetables - Google Patents
Mass spectrometry method for detecting Klebsiella pneumoniae in instant fresh fruits and vegetables Download PDFInfo
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Abstract
The invention relates to a method for detecting Klebsiella bacteria of an instant fruit and vegetable agricultural product by a mass spectrum, which comprises the following steps: s1, specifically selecting and culturing Klebsiella pneumoniae in the instant fruits and vegetables; s2, establishing a separation method for separating klebsiella pneumoniae from fruits and vegetables; s3, extracting suspected Klebsiella pneumoniae protein components from fruits and vegetables; and S4, collecting the protein map of the Klebsiella pneumoniae by matrix analysis ionization time-of-flight mass spectrometry, and identifying by adopting a characteristic peak fitting method. The core technology is research and development of components of a Klebsiella pneumoniae selective culture medium on the surface of the instant fruits and vegetables, establishment of a separation method, establishment of a pretreatment method of microbial mass spectrometry identification and establishment of microbial mass spectrometry conditions. The invention relates to a method for detecting and identifying klebsiella pneumoniae of instant fruits and vegetables of nonstandard agricultural products, which adopts a biochemical selective culture and mass spectrometry method to detect and identify klebsiella pneumoniae in the instant fruits and vegetables, thereby accurately identifying the klebsiella pneumoniae in the fruits and vegetables of agricultural products, obtaining more fruit and vegetable microbial protein data information and providing data for tracing and pathogenic analysis of the klebsiella pneumoniae in the fruits and vegetables.
Description
Technical Field
The invention relates to the technical field of agricultural product microorganism detection and mass spectrometry microorganism identification, in particular to food-borne pathogenic microorganism detection of instant fresh fruits and vegetables of agricultural products.
Background
At present, food-borne microorganism detection methods mainly comprise four types, one is a traditional conventional detection method, the detection method mainly comprises enrichment, separation, biochemical identification and the like, the detection steps are relatively complex, the period is long, and the detection period is as long as 5-10 days, but the traditional method is mature in technology and simple in required equipment, and the traditional method is a mainstream method for microorganism detection at present, but the detection method for klebsiella pneumoniae in food is not mature and standard in China. The second is a molecular biology method which comprises the following technologies: the method has the characteristics of high sensitivity and quick detection, and is generally used for clinical microbial detection. Thirdly, an immunological detection method, and the principle of the immunoassay method is as follows: the specific antibody of the microorganism to be detected is prepared, and the microorganism is identified by the specific reaction of the antibody, and the specific antibody mainly comprises an enzyme linked immunosorbent assay kit and a colloidal gold detection card. The method has very good sensitivity and specificity, the existing development ELISA kit of the antibody of the Klebsiella exoprotein and the antibody latex microsphere immunochromatography test paper have higher cost, and the method is generally used for clinical sample detection. And fourthly, other emerging microorganism detection technologies and methods, such as a microorganism matrix-assisted laser desorption ionization time-of-flight mass spectrometry, are used for identifying and typing microorganism strain levels by utilizing the stability and diversity of microorganism structural proteins. The method for identifying the microorganisms has the following main characteristics: and (1) the obtained result is rapid, the accuracy is high, the mass spectrum pretreatment only needs 1h, and the result can be obtained in the mass spectrum process for 3 min. (2) The database can be automatically established according to the separated microorganism source to obtain the characteristic key information of the microorganism, the identification rate accuracy is improved, and the microorganism information belonging to agricultural products is obtained according to the separated source of the microorganism, such as instant fruits and vegetables, and the specificity of the microorganism information is reserved.
In China and national standards, a specific sample processing method aiming at fruit and vegetable microorganism detection is not available, and the existing documents rarely carry out specific separation detection aiming at Klebsiella pneumoniae in fruits and vegetables.
Disclosure of Invention
The invention aims to provide a mass spectrometry method for detecting klebsiella pneumoniae in instant fresh fruits and vegetables, so as to solve the problem of detection of klebsiella pneumoniae in the instant fruits and vegetables.
A mass spectrum method for detecting Klebsiella pneumoniae in instant fresh fruits and vegetables comprises a Klebsiella pneumoniae separation culture medium and a method in the instant fruits and vegetables, and the pretreatment method and the mass spectrum condition for mass spectrum identification of the Klebsiella pneumoniae specifically comprise the following steps:
s1, specifically selecting and culturing Klebsiella pneumoniae in the instant fruits and vegetables;
s2, establishing a separation method for separating klebsiella pneumoniae from fruits and vegetables;
s3, extracting suspected Klebsiella pneumoniae protein components from fruits and vegetables;
and S4, collecting the protein map of the Klebsiella pneumoniae by matrix analysis ionization time-of-flight mass spectrometry, and identifying by adopting a characteristic peak fitting method.
Preferably, S1 is a specific culture medium for Klebsiella pneumoniae in the instant fresh fruits and vegetables, the culture medium is an improved citric acid liquid culture medium for pre-enrichment, and the formula of the culture medium is (/ L): 5.0g of sodium citrate, 1.0g of ammonium dihydrogen phosphate, 0.5g of dipotassium hydrogen phosphate, 1.5g of dipotassium hydrogen phosphate and 0.05g of bromothymol blue. The configuration method comprises the following steps: weighing the above materials, heating, stirring, dissolving in 1000ml distilled water, and autoclaving at pH 6.8 + -0.1,121 deg.C for 15 min.
As the specific separation method S2 for Klebsiella pneumoniae in the instant fresh fruits and vegetables, the separation procedure of the Klebsiella pneumoniae in the fruits and vegetables is as follows:
s21, weighing 25g of sample by aseptic technique, adding 225mL of improved citric acid liquid culture medium, homogenizing for 1 min-2 min, and culturing for 6-12 h at 36 +/-1 ℃.
S22, taking 1 mu L, streaking and inoculating the MAC plate, culturing at 36 +/-1 ℃ for 18-24 h, and observing the colony characteristics. On MAC agar plates, typical colonies are brick pink, round, raised, moist, shiny.
S23, selecting 10 suspicious colonies on the plate, streaking and inoculating the suspicious colonies on a rabbit blood agar plate, and culturing at 36 +/-1 ℃ for 18-24 h, wherein typical colonies are grey white, round, raised, flashing and grow vigorously to be cream. Purified suspect colonies were picked from rabbit blood agar plates for mass spectrometric identification.
As the mass spectrometric identification pretreatment S3 for Klebsiella pneumoniae in the instant fresh fruits and vegetables, the optimal selection process is as follows:
s31, adding appropriate amount of bacteria into 1.0ml ethanol water solution (V: 75: 25), performing 40KHz ultrasonic treatment for 10min, centrifuging at 8000r/min for 2min, and discarding the solution;
s32, adding 0.75ml of acetic acid aqueous solution (V: V is 50: 50), shaking for 20S, adding 0.75ml of acetonitrile, shaking uniformly, and centrifuging at 8000r/min for 1 min;
s33, taking 1 mu L of the supernatant, spotting the supernatant on a target plate, volatilizing, covering 1 mu L of CHCA, and volatilizing.
As mass spectrum identification mass spectrum condition S4 of Klebsiella pneumoniae in the instant fresh fruits and vegetables, the further scheme is as follows:
fixed focus at 338nm, linear mode of electron multiplier (multiplex dynode) detector, positive ion mode collection, laser beam energy frequency of 75-80 Hz, collection range of 2000-18000(m/z), 100 collected peak superpositions per sample (including 0.8% deviation ratio), and calibrator ATCC 8739 E.coli.
Drawings
FIG. 1 is a procedure for detection of Klebsiella pneumoniae;
FIG. 2 is a mass spectrum of Klebsiella pneumoniae;
FIG. 3 the morphology of Klebsiella pneumoniae in MAC colonies;
FIG. 4 colony morphology of Klebsiella pneumoniae in blood plates.
Detailed Description
The principles and features of this invention are described below in conjunction with examples, which are set forth to illustrate, but are not to be construed to limit the scope of the invention.
The principles and features of the present invention are described below in conjunction with examples to make the technical aspects, the creative features, the achievement purposes and the efficacies of the present invention easy to understand, and are not used for limiting the scope of the present invention.
1 separation and mass spectrum identification of klebsiella pneumoniae in strawberries
1 abbreviations the following abbreviations apply to this document.
MAC MacConey Agar culture medium
CHCA alpha-Cyano-4-hydroxycinnamic Acid alpha-Cyano-4-hydroxyycinnamic Acid
2 apparatus and materials
Except for the conventional sterilization and culture equipment in the microbiological laboratory, other equipment and materials are as follows:
2.1 constant temperature incubator at 36 ℃. + -. 1 ℃.
2.2 refrigerator at 2-5 deg.C.
2.3 electronic balance, the sensitive quantity is 0.1g and 0.01 g.
2.4 homogenizer.
2.5 oscillator.
2.6 sterile pipette 1mL (with 0.01mL scale), 10mL (with 0.1mL scale) or micropipette and tip.
2.7 sterile homogenizing cup or sterile homogenizing bag with 500mL capacity.
2.8 sterile petri dishes with a diameter of 90 mm.
2.9 pH meter or precision pH paper.
2.10 microcentrifuge tube, 1.5mL or 2.0 mL.
2.11 looper 1. mu.L.
2.12 the low-temperature high-speed centrifuge, the rotating speed is more than or equal to 13000r/min, and the temperature is controlled between 4 and 8 ℃.
2.13 matrix analysis ionization time-of-flight mass spectrometry
2.14 micropipette and suction head 0.5-2. mu.L, 2-20. mu.L, 20-200. mu.L, 200-1000. mu.L.
3 culture media and reagents
3.1 improved citric acid liquid Medium
The components: 5.0g of sodium citrate, 1.0g of ammonium dihydrogen phosphate, 0.5g of dipotassium hydrogen phosphate, 1.5g of dipotassium hydrogen phosphate and 0.05g of bromothymol blue. 1000mL of distilled water.
The configuration method comprises the following steps: mixing the above materials, heating to dissolve, adjusting pH to 6.8 + -0.1,121 deg.C, and autoclaving for 15 min.
3.2 MacConkey Agar (MAC):
the components: 20.0g of peptone, 10.0g of lactose, 1.5g of No. 3 bile salt, 5.0g of sodium chloride, 0.03g of neutral red, 0.001g of crystal violet and 15.0g of agar. 1000mL of distilled water.
The preparation method comprises the following steps: autoclaving at 121 deg.C for 15 min. Cooling to 45-50 deg.c and pouring into plate.
3.3 Rabbit blood agar
The components: 100mL of bean flour agar (pH7.5 +/-0.2), 5-10 mL of defibered sheep blood (or rabbit blood) and 1000mL of distilled water.
The preparation method comprises the following steps: heating to melt agar, cooling to 50 deg.C, adding defibrinated rabbit blood under aseptic condition, shaking, and pouring into plate.
3.4 nutrient agar
The components: 10.0g of peptone, 3.0g of beef extract, 5.0g of sodium chloride and 15.0-20.0 g of agar.
1000mL of distilled water
The preparation method comprises the following steps: the components except agar were dissolved in distilled water, and about 2mL of 15% sodium hydroxide solution was added to adjust the pH to 7.3. + -. 0.2.
3.50.85% sterilized normal saline
The components: 10.0g of peptone, 3.0g of beef extract, 5.0g of sodium chloride and 15.0-20.0 g of agar. 1000mL of distilled water.
The preparation method comprises the following steps: the components except agar were dissolved in distilled water, and about 2mL of 15% sodium hydroxide solution was added to adjust the pH to 7.3. + -. 0.2. Autoclaving at 121 deg.C for 15 min. Cooling to 45-50 deg.c and pouring into plate.
4 operating procedure (see FIG. 1)
Sampling and enriching bacteria: weighing 25g of strawberry sample by aseptic operation, adding into a homogenizing bag filled with 225mL of improved citric acid liquid culture medium, homogenizing for 1-2 min, and culturing at 36 +/-1 ℃ for 6-12 h.
Separation: taking 1 mu L, streaking and inoculating the MAC plate, culturing for 18-24 h at 36 +/-1 ℃, and selecting a red, round, bulged, wet and flash colony on the MAC agar plate for continuous culture. Selecting suspicious colonies on the MAC plate, streaking and inoculating the suspicious colonies on a rabbit blood agar plate, culturing at 36 +/-1 ℃ for 18-24 h, selecting gray white, round, raised, flashing and growing cream (shown in figure 4), and performing mass spectrum identification on the suspicious colonies.
Mass spectrum confirmation: pretreatment: adding appropriate amount of bacteria into 1.0ml ethanol water solution (V: 75: 25), performing 40KHz ultrasonic treatment for 10min, centrifuging at 8000r/min for 2min, and discarding solution; adding 0.75ml of acetic acid aqueous solution (V: V is 50: 50), shaking for 20s, adding 0.75ml of acetonitrile, shaking uniformly, and centrifuging at 8000r/min for 1 min; apply 1. mu.L of supernatant to the target plate, evaporate to dryness, cover with 1. mu.L of CHCA, and evaporate to dryness.
Mass spectrum conditions: fixed focus at 338nm, linear mode of detector electron multiplier (multiple dynode), positive ion mode collection, laser beam energy frequency of 75-80 Hz, collection range of 2000-18000(m/z), 100 collected peak superpositions per sample (including 0.8% deviation ratio), and calibrator ATCC 8739 E.coli. Isolation of Klebsiella pneumoniae Mass Spectroscopy Peak plot (FIG. 2)
5 results report: the Klebsiella pneumoniae 5 strain is detected from 20 strawberry samples, the detection rate and the addition rate are the same, and the recovery requirement is met.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (5)
1. A mass spectrometry method for detecting Klebsiella pneumoniae in instant fresh fruits and vegetables comprises a Klebsiella pneumoniae separation culture medium and a method in the instant fruits and vegetables, a pretreatment method for mass spectrometry identification of the Klebsiella pneumoniae and mass spectrometry conditions. The method is characterized by comprising the following steps:
s1, specifically selecting and culturing Klebsiella pneumoniae in the instant fruits and vegetables;
s2, establishing a separation method for separating klebsiella pneumoniae from fruits and vegetables;
s3, extracting suspected Klebsiella pneumoniae protein components from fruits and vegetables;
and S4, collecting the protein map of the Klebsiella pneumoniae by matrix analysis ionization time-of-flight mass spectrometry, and identifying by adopting a characteristic peak fitting method.
2. The screening culture medium for specificity of klebsiella pneumoniae in instant fresh fruits and vegetables according to claim 1, wherein the preferred S1 is a modified citric acid liquid culture medium, and S1 is characterized by formula and efficacy (/ L): 5.0g of sodium citrate as a carbon source, 1.0g of ammonium dihydrogen phosphate as a nitrogen source, 0.5g of dipotassium hydrogen phosphate as a buffering agent, 1.5g of dipotassium hydrogen phosphate as a buffering agent and 0.05g of bromothymol blue as a color developing agent.
3. The screening method S2, S2 for detecting Klebsiella pneumoniae in the instant fresh fruits and vegetables according to claim 1 is characterized by comprising the following steps:
s21, culturing the pre-enrichment liquid in an improved citric acid liquid culture medium at 36 +/-1 ℃ for 6-12 h;
s22, separating the MAC selective culture medium, and culturing for 18-24 h at 36 +/-1 ℃;
s23, carrying out passage on the rabbit blood agar culture medium, and culturing for 18-24 h at 36 +/-1 ℃.
4. The mass spectrometry identification method S3 for detecting Klebsiella pneumoniae in instant fresh fruits and vegetables according to claim 1, is characterized by comprising the following steps:
s31 precipitating saccharide substance of Klebsiella pneumoniae by 75% alcohol, and centrifuging for 2min at 8000r/min under 40KHz ultrasonic for 10 min;
s32, 70 percent formic acid and 100 percent acetonitrile (1:1) are centrifuged at 8000r/min for 1min, and the protein of the Klebsiella pneumoniae is extracted;
s33, CHCA is used as an auxiliary analysis matrix of the Klebsiella pneumoniae protein.
5. The mass spectrum condition S4 for the Klebsiella pneumoniae separated from fruits and vegetables according to claim 1, is characterized by fixed focus at 338nm, linear mode of electron multiplier (multiplydonode) of detector, positive ion mode collection, laser beam energy frequency of 75-80 Hz, and 100 collected peak superpositions per sample (including 0.8% deviation rate).
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| CN107904189A (en) * | 2017-11-13 | 2018-04-13 | 浙江工业大学 | acid-producing Klebsiella and application thereof |
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| CN101935619A (en) * | 2009-10-20 | 2011-01-05 | 中南林业科技大学 | Klebsiella sp.A-08 for fermentation and application thereof |
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