CN115389669A - Banana wilt chromatographic detection method - Google Patents
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- CN115389669A CN115389669A CN202211045408.1A CN202211045408A CN115389669A CN 115389669 A CN115389669 A CN 115389669A CN 202211045408 A CN202211045408 A CN 202211045408A CN 115389669 A CN115389669 A CN 115389669A
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Abstract
The invention discloses a method for detecting banana vascular wilt by chromatography, which indirectly identifies the bacterial wilt and the occurrence degree thereof by simultaneously detecting metabolic toxins of banana vascular wilt, namely Baijiaochun and fusaric acid, and specifically comprises the following steps: pretreating a sample to be detected; weighing a pretreated sample to be detected, adding an acidic acetonitrile extracting solution according to a proportion, adding a salt bag after vortex extraction, continuing vortex, centrifuging to obtain a supernatant, and purifying the supernatant through a solid-phase extraction column; using ultra-high pressure fluidPerforming gradient elution separation on the purified solution by a phase chromatography tandem mass spectrometer, and adopting electrospray ES + And (4) carrying out mass spectrum detection by using an ion source and multiple reaction monitoring. Compared with the traditional pathogen identification, the method has the advantages of simple and convenient operation, high sensitivity and good repeatability, can be used for identification and quantitative analysis of blight bacteria in bananas and other crops, and provides detection data support for production and quality control of healthy seedlings of bananas and other crops and evaluation of risks of soil environmental conditions in crop planting fields.
Description
Technical Field
The invention relates to the technical field of agriculture, in particular to a method for detecting banana vascular wilt by using a chromatogram.
Background
Bananas are widely planted in tropical and subtropical regions, are the fruits with the largest trade quantity and consumption quantity of fresh fruits in the world, and are also positioned as the fourth large grain crops after rice, wheat and corn in developing countries by the Food and Agriculture Organization (FAO) of the United nations. However, banana wilt disease caused by soil-borne fungus fusarium oxysporum cubeba transformation type (FOC) tropical No. 1 and No. 4 physiological races, especially No. 4 physiological races, rapidly erupts and prevails in 17 countries and regions including China in south east Asia in recent years, and a great threat is brought to the sustainable development of banana industry. Researches find that the fusarium oxysporum f.sp.cubense can infect almost all main cultivars of bananas, and particularly, secreted toxins of the fusarium oxysporum f.sp.cubense and fusaric acid play an important role in the pathogenic process of the fusarium oxysporum f.sp.cubense.
Fusarium Acid (FA) is also called wilting acid, 5-butylpyridine-2-carboxylic acid (C) 10 H 13 NO 2 ) The host non-specific toxin produced by fusarium oxysporum is secondarily metabolized, and researchers sequentially separate the compound from fusarium oxysporum or a culture medium thereof of crops such as sesame, bananas, cotton, balsam pears and the like.
Beauverine (BEA) is a cyclic hexapeptide of N-methyl amino acid biosynthesized by Beauveria bassiana, some fusarium and the like. The bacterial strain is firstly separated from insect pathogen beauveria bassiana, in recent years, researchers successively separate the pathogenic substance from culture solution for culturing No. 1 and No. 4 tropical physiological races of banana fusarium wilt, certain transformation exists, and the toxicity mechanism of pathogenic bananas is not completely clear.
At present, the banana vascular wilt is detected mainly through disease signs and laboratory molecular biology detection on the premise that the banana vascular wilt is damaged and shows obvious symptoms. The former has uncertainty and false positives, and the latter is time consuming and costly. Although liquid phase tandem mass spectrometry is adopted to detect fusarium wilt at present, a technology for simultaneously detecting fusaric acid and beauvericin aiming at banana wilt is not found, so that a banana wilt chromatographic detection method is researched to simultaneously and accurately extract, separate and detect the beauvericin and the fusaric acid in bananas and soil samples, and particularly, the indirect detection of the banana wilt is necessary.
Disclosure of Invention
The invention overcomes the defect that the banana vascular wilt can be detected through disease symptoms and laboratory molecular biology detection on the premise that the banana vascular wilt is damaged and shows obvious symptoms in the prior art. The chromatographic detection method for banana vascular wilt has the technical problems of uncertainty, false positive and time-consuming and high cost of the latter, and adopts an ultrahigh pressure liquid chromatography-mass spectrometry method to simultaneously and accurately extract, separate and detect beauvericin and fusaric acid in bananas and soil samples, thereby indirectly detecting banana vascular wilt.
In order to solve the problems, the invention adopts the following technical scheme:
a method for detecting banana vascular wilt by chromatography, which separates and simultaneously detects metabolic toxins of banana vascular wilt, namely beauvericin and fusaric acid, comprises the following steps:
s1, sample pretreatment: adding distilled water into a sample to be detected, and then homogenizing or directly homogenizing/uniformly mixing to obtain a pretreatment sample to be detected;
s2, extracting and purifying a sample:
accurately weighing the pretreatment sample to be detected obtained in the step S1, adding an extraction solvent into the pretreatment sample to be detected, performing vortex extraction for 0.5-2.0 min to obtain an extracting solution, adding a salt bag into the extracting solution, continuing to perform vortex extraction for 1min, centrifuging at 4000r/min for 2-10 min, and passing the supernatant through a container C 18 Purifying by a PSA solid phase extraction column, collecting the purified solution, and filtering by a 0.22 mu m organic microporous filter membrane; the proportion of the pretreatment sample to be detected to the extraction solvent is 1g; the extraction solvent is an acidic acetonitrile extracting solution; the acidic acetonitrile extracting solution contains 0.05 to 2 mass percent of acidic substances;
s3, separation and identification:
and (3) performing gradient elution separation and mass spectrometry detection on the purified liquid passing through the organic microporous filter membrane in the step (S2) by using an ultrahigh pressure LC-MS, wherein the conditions of liquid phase elution separation are as follows:
a chromatographic column: ACQUITYUPLC @ HSS T 3 ；
Mobile phase A: formic acid aqueous solution, wherein the volume percentage of formic acid is 0.05-0.5%;
mobile phase B: formic acid acetonitrile solution, the volume percentage of formic acid is 0.05% -0.5%;
flow rate: 0.25mL/min, the column temperature is 35 ℃, and the sample injection amount is 2 mu L;
gradient elution procedure:
the retention time of beauvericin is 4.68min, and the retention time of fusaric acid is 2.71min;
the mass spectrum detection conditions are as follows:
mass spectrum conditions: using electrospray ion source ES + Multiple Reaction Monitoring (MRM) detection;
capillary voltage: 3.0KV;
ion source temperature: 350 ℃;
desolvation gas is N 2 ；
Desolventizing air flow rate: n is a radical of hydrogen 2 ，400～1000L/h；
Taper hole air flow velocity: n is a radical of 2 ，150L/h；
Collision airflow rate: ar,0.10mL/Min;
the mass spectral parameters of the toxin were tuned as follows:
beauvericin: quantifying an ion pair 784.27 >; the qualitative ion pair 784.27 >;
fusaric acid: the quantitative ion pair is 180.08 >; qualitative ion pair 180.08>, 92.03, cone voltage 74V, collision energy 24eV.
Further, in step S2, the acidic acetonitrile extracting solution contains an acidic substance, wherein the acidic substance is one of formic acid, acetic acid and hydrochloric acid; the volume percentage of the acidic substance is 0.05-2%.
Further, in step S2, the acidic acetonitrile extract contains 0.5% by volume of acetic acid.
Further, the salt package contains one or more of anhydrous sodium chloride, anhydrous sodium sulfate, anhydrous magnesium sulfate, sodium citrate and disodium citrate.
Further, 50mg of C is filled in the solid phase extraction column 18 50mg PSA, 150mg anhydrous magnesium sulphate.
Further, in step S3, the volume percentage of formic acid in the aqueous formic acid solution is 0.1%; in the formic acid acetonitrile solution, the volume percentage of formic acid is 0.1%.
Further, in the step S3, qualitative and quantitative ion pairs of beauvericin and fusaric acid are detected simultaneously, the molecular masses of the ion pairs deviate from +/-1 respectively, and the method is a method for indirectly detecting and confirming the banana vascular wilt.
Further, in the step S1, the samples are banana samples and soil samples containing metabolic toxicants of fusarium oxysporum, and the banana samples include stems, leaves and fruits of bananas.
Further, in the step S1, adding distilled water into the stem, the leaf and the fruit sample of the banana according to the weight ratio of 1; and (3) drying the soil sample in the dark indoors, grinding and sieving the dried soil sample with a 40-mesh sieve, uniformly mixing, and adding distilled water for fully wetting before extraction.
Compared with the prior art, the invention has the following beneficial effects:
(1) The method adopts an ultrahigh pressure liquid chromatography-mass spectrometry method to simultaneously detect the beauvericin and the fusaric acid in the sample, can finish the extraction, purification, separation and detection of the sample within 40min, has the advantages of high detection speed, high sensitivity, good accuracy, simple operation and low detection cost compared with the traditional pathogenic bacteria separation and molecular identification method, and has advantages in identification efficiency, cost and accuracy. The content of metabolites of pathogenic bacteria of banana vascular wilt in a sample is directly detected, and the detection result can be directly used for evaluating the safety of soil environment conditions of a crop planting field; the method can also be used for quality control of banana healthy seedling production to judge whether the produced banana healthy seedlings carry blight pathogenic bacteria or not, and provides detection data support for banana and other crop healthy seedlings and condition risk assessment.
(2) In the step S2, acetonitrile is used as an extraction solvent, and the acetonitrile and an aqueous solution can be mutually dissolved in the early stage of extraction (without adding salt), so that the acetonitrile can be fully contacted with an extracted substance, the acetonitrile solubility in the aqueous phase is sharply reduced after the salt is added, the acetonitrile and the aqueous solution are layered after the centrifugal treatment, and the supernatant is purified by passing-type solid-phase dispersion extraction, filtered and then subjected to machine detection. The invention adopts the sample extraction and purification detection technology, and compared with the classic sample pretreatment scheme: compared with the method that liquid-liquid extraction and extraction liquid concentration are adopted, a solid phase extraction column is adopted for purification, elution, concentration and volume fixing, and a filter membrane is used for detection on a computer, the pretreatment step of detection on the computer is reduced, the loss of the object to be detected in the purification process is reduced, and the extraction rate of the object to be detected is improved; the pretreatment time of the on-machine detection is controlled within 30min, while the conventional pretreatment generally needs 3-6 h, and the working efficiency is greatly improved.
(3) In the step S2 of the invention, acidic acetonitrile is used as an extraction solvent, so that physical and chemical properties of organic acids in the beauvericin and fusaric acid in the extraction solution are mainly maintained, and a higher extraction rate is obtained, particularly beauvericin which is not found for a long time due to good water solubility is mainly difficult to extract from an aqueous solution only through the organic solvent. The invention adopts an acidic acetonitrile extracting solution which contains 0.05 to 2 mass percent of acidic substances; preferably, the acidic acetonitrile extract containing 0.5% of volatile acids such as formic acid, acetic acid, and hydrochloric acid can improve the acid stability of two toxin metabolites in the sample extract system.
(4) The applicant finds that when the instrument mass spectrometry method is established, in the step S3 of the invention, the mobile phase a adopts a formic acid aqueous solution (the volume percentage of formic acid is 0.05-0.5%), the mobile phase B adopts a formic acid acetonitrile solution (the volume percentage of formic acid is 0.05-0.5%), and compared with the case that the mobile phase a only adopts a formic acid aqueous solution (the volume percentage of formic acid is 0.05-0.5%), the mobile phase B of the invention also adopts a formic acid acetonitrile solution, so that the signals of two toxin metabolites are stronger, the peak shapes are narrow and symmetrical, and the mass spectrometry detection effect is better.
Drawings
FIG. 1 is the MRM multiple reaction monitoring ion diagram of fusaric acid and beauvericin in example 2 of the present invention.
Detailed Description
The present invention will be further described with reference to examples and tests.
Example 1:
the embodiment provides a method for detecting banana vascular wilt bacteria in a soil sample, which comprises the following steps:
s1, sample pretreatment: collecting a sample by a quartering method, fully drying in the shade at room temperature after collecting a soil sample, grinding and sieving by a 40-mesh sieve, uniformly mixing to obtain a pre-treatment sample to be detected of the soil, and writing a label for later use;
s2, extracting and purifying a sample:
accurately weighing 10.00g of the soil to be detected pretreated sample obtained in the step S1, and adding 2-3 ml of distilled water into the soil according to the dryness of the soil to fully wet the soil; according to the proportion of the soil to be detected pretreatment sample to the acidic acetonitrile extracting solution of 1g:2ml of acidic acetonitrile extracting solution is added into a pre-treated sample to be detected of soil, 20.0ml of acidic acetonitrile extracting solution is added into the pre-treated sample to be detected of soil, vortex extraction is carried out for 2.0min to obtain the extracting solution, 3.0g of sodium chloride powder (namely anhydrous sodium chloride powder) dried at 350 ℃ is added into the extracting solution, then vortex is carried out for 1min, centrifugation is carried out for 2min at 4000r/min to obtain 2.0ml of supernatant, and the supernatant is filled with 50mgC 18 Purifying with 50mg PSA and 150mg anhydrous magnesium sulfate by passing solid phase extraction column, collecting the purified solution, and filtering with 0.22 μm organic microporous membrane;
wherein the acidic acetonitrile extracting solution is an extracting solvent and contains 0.5 percent of acetic acid by volume percentage;
s3, separation and determination:
and (3) performing gradient elution separation and mass spectrum detection on the purified liquid passing through the organic microporous membrane in the step (S2) by using an ultrahigh pressure liquid mass spectrometer, wherein the liquid phase elution separation conditions are as follows:
a chromatographic column: ACQUITYUPLC @ HSST 3 ；
A mobile phase A: formic acid aqueous solution, wherein the volume percentage of formic acid is 0.1%;
mobile phase B: formic acid acetonitrile solution, wherein the volume percentage of formic acid is 0.1%;
flow rate: 0.25mL/min, the column temperature is 35 ℃, and the sample injection amount is 2 mu L;
gradient elution procedure:
the retention time of beauvericin is 4.68min, and the retention time of fusaric acid is 2.71min;
the mass spectrum detection conditions are as follows:
mass spectrum conditions: using electrospray ion source ES + Multiple Reaction Monitoring (MRM) detection;
capillary voltage: 3.0KV;
ion source temperature: 350 ℃;
desolventizing gas is N 2 ；
Desolventizing air flow rate: n is a radical of 2 ，700L/h；
Taper hole air flow velocity: n is a radical of 2 ，150L/h；
Collision airflow rate: ar,0.10mL/Min;
the mass spectral parameters of the toxin were tuned as follows:
beauvericin: quantifying the ion pair 784.27 >; 784.27 >;
fusaric acid: the quantitative ion pair is 180.08 >; the qualitative ion pair 180.08 >.
Example 2:
the embodiment provides a method for detecting banana fusarium oxysporum in bulbs and leaves of bananas, which comprises the following steps:
s1, sample pretreatment: collecting samples by a quartering method, and according to the banana samples: adding distilled water into a banana sample to be detected according to the mass ratio of 1;
s2, extracting and purifying a sample:
accurately weighing 20.00g of the banana to-be-detected pretreatment sample obtained by fully homogenizing in the step S1, wherein the weight ratio of the banana to-be-detected pretreatment sample to the acidic acetonitrile extracting solution is 1g:2ml of acidic acetonitrile extracting solution which is 40.0ml is added into a pretreatment sample to be detected of the banana; vortex extracting for 2.0min to obtain extractive solution, adding 6g sodium chloride powder (anhydrous sodium chloride powder) dried at 350 deg.C, and continuously vortex for 1minCentrifuging at 4000r/min for 10min to obtain supernatant 2ml, and passing the supernatant through a container containing 50mgC 18 Purifying with 50mg PSA and 150mg anhydrous magnesium sulfate by passing solid phase extraction column, collecting the purified solution, and filtering with 0.22 μm organic microporous membrane;
wherein the acidic acetonitrile extracting solution is an extracting solvent which contains 0.5 percent by mass of acetic acid;
in this embodiment, S3 is the same as step S3 in embodiment 1;
the ultrahigh pressure LC-MS is adopted to analyze and detect fusaric acid and beauvericin which are metabolic poisons of banana vascular wilt, the MRM multi-reaction monitoring ion map of fusaric acid and beauvericin is shown in figure 1, the retention time of beauvericin is 4.68min, the retention time of fusaric acid is 2.71min, and the peak shapes are narrow and symmetrical in figure 1.
Test No.)
The sample pretreatment methods of examples 1 and 2 and the instrument conditions of step S3 were used to perform the additive recovery and precision evaluation experiments on banana soil, bulbs, and whole fruit samples, and the results are shown in table 1. As can be seen from Table 1, at the sample addition levels of 0.01, 0.04 and 0.2mg/kg, the average recovery rates of beauvericin and fusaric acid in soil are 93.29-97.09%, the relative standard deviation is 1.64-8.23%, the average recovery rate in bulbs is 95.45-106.62%, the relative standard deviation is 0.84-5.00%, the average recovery rate in whole fruits is 87.26-106.98%, and the relative standard deviation is 0.74-4.61%.
The signal-to-noise ratio of the matrix standard sample detected by the instrument is calculated to obtain: the minimum detection concentrations of beauvericin and fusaric acid in the soil are 0.01ug/kg and 4.38ug/kg respectively. The minimum detection concentrations in the corms are 0.04ug/kg and 7.79ug/kg respectively, and the minimum detection concentrations in the whole fruits of the bananas are 0.03ug/kg and 7.63ug/kg respectively, which completely meet the detection requirements, so that the detection method established by the invention has high sensitivity and good repeatability, and can accurately detect the metabolic toxicants of the banana vascular wilt.
TABLE 1 recovery of Fusarium acid and Beauverine addition in different banana samples (n = 5)
Test No. two
According to the detection method of example 2, the addition recovery and precision evaluation experiments were performed on acidic acetonitrile extract solutions of different concentrations. As can be seen from Table 2, the acetonitrile formate and the acetonitrile acetate with different concentrations have certain influence on the extraction of the beauvericin and the fusaric acid, when the concentration of the acid in the acetonitrile is more than 0.05%, the addition recovery rates of the two toxins reach more than 80%, and when the concentration of the acetic acid in the acetonitrile is 0.5%, the addition recovery rates of the two toxins exceed 93.35%, while the acetonitrile formate with the same concentration has no difference on the extraction of the beauvericin, and the extraction rate of the acetonitrile formate with the same concentration does not exceed 88%. After taking into account the acetonitrile containing 0.5% acetic acid was taken as the best extraction solvent.
Table 2 recovery of different concentrations of acid acetonitrile on fusaric acid and beauvericin addition (n = 3)
Experiment three
Extraction and detection of toxin in culture bacterial liquid of banana wilt
1. Activation and culture of banana wilt bacteria
Preparation of potato solid Medium (PDA): weighing 200g of potato fragments, adding the potato fragments into 1000mL of distilled water, heating and boiling for 30min, filtering the boiled soup through four layers of gauze, taking the lower layer of filtrate, adding 20g of glucose and 15-20 g of agar powder into the filtrate, continuously stirring and dissolving, adding water to a constant volume of 2000mL, subpackaging, performing high-pressure sterilization in a high-pressure steam sterilization pot at 121 ℃ for 20min, and cooling for later use.
Preparing a Chao's culture medium: weighing 3g of sodium nitrate, 1g of dipotassium phosphate, 0.5g of magnesium sulfate heptahydrate, 0.5g of potassium chloride, 0.01g of ferrous sulfate and 30g of sucrose, heating in 1000mL of deionized water, dissolving, subpackaging, sterilizing at 121 ℃ for 20min, and cooling for later use.
2. The banana wilt disease pathogenic bacterium fusarium oxysporum cuba specialization type (FOC 4) is provided by the fungus laboratory of the department of Phytopathology of southern China university of agriculture. A small amount of Foc4 is picked from the test tube and streaked on PDA, and the streaked product is cultured in a biochemical incubator under dark conditions at 28 ℃ for three generations to complete the strain activation. The 6mm dish was removed by a punch and placed in the Czochralski medium, and the plate was shake-cultured in a shaking incubator at 28 ℃ and 180rpm in the dark. Periodically take out FOC4 and put into superclean bench, observe and count with the blood count board under the optical microscope of 40 times, to the fungus liquid of collecting in the same period, accurately weigh 5.00g, according to the fungus liquid: adding 10mL of acetonitrile acetate solution containing 0.5 mass percent of acetonitrile acetate in a weight ratio of 1 18 50mg PSA and 150mg anhydrous magnesium sulfate, purifying the purified solution by a passing solid phase extraction cartridge, passing the purified solution through a 0.22 μm organic microporous filter membrane, and performing machine detection according to the instrument conditions of the step S3 in the example 2. The accumulated content of toxin metabolites of the activated fusarium oxysporum cubeba specialization type of the banana vascular wilt pathogenic bacteria in the chai's culture solution at different times is shown in table 3 below.
TABLE 3 Beauverine and Fusarium acid contents in the inoculum solution at different incubation times (n = 3)
As can be seen from Table 3, the contents of beauvericin and fusaric acid increased continuously with the increase of the culture time, fusarium reached a maximum at day 3 of the culture and then decreased slightly with the increase of the culture time, while the content of beauvericin increased gradually during the culture.
Experiment four
And (3) detecting and comparing banana wilt samples:
the unit has carried out related experimental research on the detection and identification of banana fusarium wilt pathogen molecules in the day ahead, and related contents are disclosed in the literature, "molecular detection and identification of No. 4 physiological races of banana fusarium wilt pathogens in Guangxi", moyou base workers and the like, southern agricultural science, 2012, no. 43. This test utilizes the same germ strain infected banana samples in this document for comparative experiments. In this document, the banana wilt disease samples are: 6 parts of banana body samples suspected of blight in Guangxi banana main producing area are collected, the number of the banana body samples is 1-6 correspondingly, and banana varieties mainly comprise Williams banana and West tribute banana. In the literature, diseased plant bulbs and pathogens are separated and purified to obtain pathogen strains, the experiment adopts banana wilt samples infected by the same pathogenic strain in the literature to detect, wherein the corresponding serial numbers are the same, negative controls are also non-susceptible potted Williams banana seedlings, positive controls are also potted Williams banana seedlings which are inoculated with No. 1 and No. 4 physiological races of banana wilt pathogens and show obvious symptoms, the bulb samples are taken to be homogenized and prepared during the experiment, and qualitative and quantitative detection of toxin metabolites beauvericin and fusaric acid is carried out.
Among them, the detection method and the detection result for detecting and identifying the molecule of the banana vascular wilt pathogen are also described in the above documents. According to the detection method of the embodiment 2 of the present invention, the different bulb samples are homogenized and sampled, qualitative and quantitative detection of the toxin metabolites beauvericin and fusaric acid is performed, and the extraction detection results of beauvericin and fusaric acid in the different samples are shown in the following table 4:
TABLE 4 Beauverine and Fusarium acid content in different samples (n = 2)
"-" indicates no detection or below the instrument detection limit.
As can be seen from Table 4, except for the negative control M, other samples detected both the metabolic toxins of banana vascular wilt, beauvericin and fusaric acid, the detected concentration of beauvericin of the fusarium oxysporum f.sp.cubense 1 special type physiological race was slightly higher than that of fusaric acid, while other samples included the detected concentration of fusaric acid of the fusarium oxysporum f.sp.cubense 4 special type physiological race, which was higher than that of beauvericin. The method is basically consistent with the PCR detection results recorded in the above documents, but the physiological race cannot be distinguished according to the detection concentration, but two pathogenic physiological races are the main pathogenic bacteria on bananas in China at present.
According to the comparison between the chromatographic detection method and the traditional pathogen detection result, the method for detecting banana vascular wilt provided by the invention is feasible by adopting the chromatographic detection method to identify toxin, and compared with the traditional pathogen detection, the method has the advantages of rapidness, convenience and accuracy, and has advantages in identification efficiency, cost and accuracy. Because the invention relates to the simultaneous detection of two toxins, the beauvericin is rarely found before, and no false positive phenomenon exists at present, the detection result of the invention can be directly used for evaluating the safety of soil environmental conditions of a crop planting field, and can also be used for quality control of banana healthy seedling production to judge whether the produced banana healthy seedling carries blight pathogenic bacteria. The method can provide detection data support for healthy seedlings of bananas and other crops and condition risk assessment.
The above description is for the purpose of illustrating the preferred embodiments of the present invention, but the present invention is not limited thereto, and all changes and modifications that can be made within the spirit of the present invention should be included in the scope of the present invention.
Claims (7)
1. A method for detecting banana vascular wilt by chromatography is characterized in that the method separates and simultaneously detects metabolic toxins of banana vascular wilt, namely beauvericin and fusaric acid, and specifically comprises the following steps:
s1, sample pretreatment: adding distilled water into a sample to be detected, and then homogenizing or directly homogenizing/uniformly mixing the sample to be detected to obtain a pretreatment sample to be detected;
s2, extracting and purifying a sample:
accurately weighing the pretreatment sample to be detected obtained in the step S1, adding an extraction solvent into the pretreatment sample to be detected, performing vortex extraction for 0.5-2.0 min to obtain an extracting solution, adding a salt bag into the extracting solution, continuing to perform vortex extraction for 1min, centrifuging at 4000r/min for 2-10 min, and performing solid-phase extraction on the supernatantPurifying with column, collecting the purified solution, and filtering with 0.22 μm organic microporous membrane; the proportion of the pretreatment sample to be detected to the extraction solvent is 1g; the extraction solvent is an acidic acetonitrile extraction solution; the solid phase extraction column is provided with C 18 PSA, anhydrous magnesium sulfate solid phase extraction column;
s3, separation and determination:
and (3) performing gradient elution separation and mass spectrum detection on the purified liquid passing through the organic microporous membrane in the step (S2) by using an ultrahigh pressure liquid mass spectrometer, wherein the liquid phase elution separation conditions are as follows:
a chromatographic column: ACQUITY UPLC @ HSS T 3 ；
A mobile phase A: formic acid aqueous solution, wherein the volume percentage of formic acid is 0.05-0.5%;
and (3) mobile phase B: formic acid acetonitrile solution, the volume percentage of formic acid is 0.05% -0.5%;
flow rate: 0.25mL/min, column temperature 35 ℃, sample injection amount of 2 muL;
gradient elution procedure:
the retention time of beauvericin is 4.68min, and the retention time of fusaric acid is 2.71min;
the mass spectrum detection conditions are as follows:
mass spectrum conditions: using electrospray ion source ES + Multiple Reaction Monitoring (MRM) detection;
capillary voltage: 3.0KV;
ion source temperature: 350 ℃;
desolvation gas is N 2 ；
Desolventizing air flow rate: n is a radical of 2 ，400～1000L/h；
Taper hole air flow rate: n is a radical of hydrogen 2 ，150L/h；
Collision airflow rate: ar,0.10mL/Min;
the mass spectral parameters of the toxin were tuned as follows:
beauvericin: quantifying the ion pair 784.27 >; 784.27 >;
fusaric acid: the quantitative ion pair is 180.08 >; the qualitative ion pair 180.08 >.
2. The method for detecting banana vascular wilt chromatography according to claim 1, wherein in step S2, said acidic acetonitrile extracting solution contains an acidic substance, said acidic substance is one of formic acid, acetic acid or hydrochloric acid; the volume percentage of the acidic substance is 0.05-2%.
3. The method for detecting banana wilt chromatography according to claim 2, wherein in step S2, said acidic acetonitrile extracting solution contains 0.5% by volume of acetic acid.
4. The method for detecting the banana wilt chromatogram of claim 1, wherein the salt comprises one or more of anhydrous sodium chloride, anhydrous sodium sulfate, anhydrous magnesium sulfate, sodium citrate and disodium citrate.
5. The method for detecting banana vascular wilt spectrum according to claim 1, wherein 50mg of C is filled in said solid phase extraction column 18 50mg PSA, 150mg anhydrous magnesium sulphate.
6. The method for detecting banana vascular wilt spectrum according to claim 1, wherein in step S3, the formic acid in said aqueous solution is 0.1% by volume formic acid; in the acetonitrile formic acid solution, the volume percentage of formic acid is 0.1 percent.
7. The method for detecting banana wilt spectra according to claim 1, wherein in step S1, said samples are banana samples containing metabolic poison of fusarium oxysporum and soil samples, and said banana samples include stems, leaves and fruits of bananas.
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106243024A (en) * | 2016-07-29 | 2016-12-21 | 河南省农业科学院芝麻研究中心 | Fusaric acid and the method for dehydrogenation fusaric acid is separated from wilt toxin |
| CN106596785A (en) * | 2016-12-23 | 2017-04-26 | 南京市食品药品监督检验院 | Method for rapidly measuring fusarium toxins in cereal grains |
| CN108226328A (en) * | 2017-12-22 | 2018-06-29 | 国家食品安全风险评估中心 | The collaborative detection method of beauvericin and enniatin in grain and its product |
| CN110286178A (en) * | 2019-08-05 | 2019-09-27 | 弘正道（中国）中药研究有限公司 | The high throughput analysis method of 22 kinds of mycotoxins in a kind of detection health-care food oral liquid |
| CN112839514A (en) * | 2018-07-24 | 2021-05-25 | 斯托克顿(以色列)有限公司 | Treatment of banana fusarium sp.4 small seeds |
| CN113234188A (en) * | 2021-04-02 | 2021-08-10 | 华南理工大学 | Molecularly imprinted photonic crystal and preparation method and application thereof |
| CN113881571A (en) * | 2021-03-05 | 2022-01-04 | 中国热带农业科学院热带生物技术研究所 | Method for quickly separating and quantitatively detecting banana fusarium wilt and application |
| US20220074900A1 (en) * | 2020-03-17 | 2022-03-10 | Innov Ad N.V. | Monitoring mycotoxins and its metabolites in the blood of pigs or broiler chickens |
-
2022
- 2022-08-30 CN CN202211045408.1A patent/CN115389669B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106243024A (en) * | 2016-07-29 | 2016-12-21 | 河南省农业科学院芝麻研究中心 | Fusaric acid and the method for dehydrogenation fusaric acid is separated from wilt toxin |
| CN106596785A (en) * | 2016-12-23 | 2017-04-26 | 南京市食品药品监督检验院 | Method for rapidly measuring fusarium toxins in cereal grains |
| CN108226328A (en) * | 2017-12-22 | 2018-06-29 | 国家食品安全风险评估中心 | The collaborative detection method of beauvericin and enniatin in grain and its product |
| CN112839514A (en) * | 2018-07-24 | 2021-05-25 | 斯托克顿(以色列)有限公司 | Treatment of banana fusarium sp.4 small seeds |
| CN110286178A (en) * | 2019-08-05 | 2019-09-27 | 弘正道（中国）中药研究有限公司 | The high throughput analysis method of 22 kinds of mycotoxins in a kind of detection health-care food oral liquid |
| US20220074900A1 (en) * | 2020-03-17 | 2022-03-10 | Innov Ad N.V. | Monitoring mycotoxins and its metabolites in the blood of pigs or broiler chickens |
| CN113881571A (en) * | 2021-03-05 | 2022-01-04 | 中国热带农业科学院热带生物技术研究所 | Method for quickly separating and quantitatively detecting banana fusarium wilt and application |
| CN113234188A (en) * | 2021-04-02 | 2021-08-10 | 华南理工大学 | Molecularly imprinted photonic crystal and preparation method and application thereof |
Non-Patent Citations (8)
| Title |
|---|
| THUKKARAM DAMODARAN等: "Biological Management of Banana Fusarium Wilt Caused by Fusarium oxysporum f. sp. cubense Tropical Race 4 Using Antagonistic Fungal Isolate CSR-T-3 (Trichoderma reesei)", FRONTIERS IN MICROBIOLOGY, vol. 11, pages 1 - 19 * |
| 夏倩等: "UHPLC-Q-TOF-MS 法测定僵蚕及其制剂中白僵菌素", 中成药, vol. 42, no. 10, pages 2652 - 2655 * |
| 曹永军;程萍;喻国辉;黎永坚;杨紫红;: "香蕉枯萎病菌菌株致病力分化及其原因研究", 热带作物学报, vol. 32, no. 08, pages 143 - 147 * |
| 李春雨;陈石;左存武;邝瑞彬;易干军;: "香蕉枯萎病菌新毒素――白僵菌素的鉴定", 园艺学报, vol. 38, no. 11, pages 2092 - 2098 * |
| 杨军;孙小杰;胡文彦;王玉梅;: "基于QuEChERS提取的超高效液相色谱-串联质谱法测定小麦粉中5种镰刀菌毒素", 分析测试学报, vol. 36, no. 07, pages 882 - 886 * |
| 杨可心等: "棉花枯萎病菌新生理型菌株毒素鉴定及其活性测定", 棉花学报, vol. 33, no. 3, pages 258 - 268 * |
| 杨迪等: "香蕉枯萎病菌分子检测研究进展", 热带作物学报, vol. 41, no. 12, pages 2582 - 2590 * |
| 王玉娇;聂继云;闫震;李志霞;程杨;张晓男;: "超高效液相色谱-串联质谱法同时检测干果中16种真菌毒素", 分析化学, vol. 45, no. 10, pages 145 - 152 * |
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