CN115389669B - Chromatographic detection method for banana vascular wilt - Google Patents
Chromatographic detection method for banana vascular wilt Download PDFInfo
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- 235000018290 Musa x paradisiaca Nutrition 0.000 title claims abstract description 77
- 238000001514 detection method Methods 0.000 title claims abstract description 67
- 230000002792 vascular Effects 0.000 title claims abstract description 30
- 240000005561 Musa balbisiana Species 0.000 title 1
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 90
- UHPMCKVQTMMPCG-UHFFFAOYSA-N 5,8-dihydroxy-2-methoxy-6-methyl-7-(2-oxopropyl)naphthalene-1,4-dione Chemical compound CC1=C(CC(C)=O)C(O)=C2C(=O)C(OC)=CC(=O)C2=C1O UHPMCKVQTMMPCG-UHFFFAOYSA-N 0.000 claims abstract description 42
- 241000223218 Fusarium Species 0.000 claims abstract description 42
- 238000000605 extraction Methods 0.000 claims abstract description 34
- 238000000034 method Methods 0.000 claims abstract description 27
- DGMPVYSXXIOGJY-UHFFFAOYSA-N Fusaric acid Chemical compound CCCCC1=CC=C(C(O)=O)N=C1 DGMPVYSXXIOGJY-UHFFFAOYSA-N 0.000 claims abstract description 26
- 230000002378 acidificating effect Effects 0.000 claims abstract description 26
- 241000234295 Musa Species 0.000 claims abstract description 21
- 239000002689 soil Substances 0.000 claims abstract description 20
- 239000003053 toxin Substances 0.000 claims abstract description 19
- 231100000765 toxin Toxicity 0.000 claims abstract description 19
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- 238000000926 separation method Methods 0.000 claims abstract description 13
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- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 44
- 239000000243 solution Substances 0.000 claims description 35
- GYSCAQFHASJXRS-FFCOJMSVSA-N beauvericin Chemical compound C([C@H]1C(=O)O[C@@H](C(N(C)[C@@H](CC=2C=CC=CC=2)C(=O)O[C@@H](C(=O)N(C)[C@@H](CC=2C=CC=CC=2)C(=O)O[C@@H](C(=O)N1C)C(C)C)C(C)C)=O)C(C)C)C1=CC=CC=C1 GYSCAQFHASJXRS-FFCOJMSVSA-N 0.000 claims description 33
- GYSCAQFHASJXRS-UHFFFAOYSA-N beauvericin Natural products CN1C(=O)C(C(C)C)OC(=O)C(CC=2C=CC=CC=2)N(C)C(=O)C(C(C)C)OC(=O)C(CC=2C=CC=CC=2)N(C)C(=O)C(C(C)C)OC(=O)C1CC1=CC=CC=C1 GYSCAQFHASJXRS-UHFFFAOYSA-N 0.000 claims description 33
- 108010079684 beauvericin Proteins 0.000 claims description 33
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- 239000002253 acid Substances 0.000 claims description 29
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- 235000019253 formic acid Nutrition 0.000 claims description 22
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- 231100000614 poison Toxicity 0.000 claims description 2
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- 239000001509 sodium citrate Substances 0.000 claims description 2
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 2
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/30—Control of physical parameters of the fluid carrier of temperature
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/30—Control of physical parameters of the fluid carrier of temperature
- G01N2030/3007—Control of physical parameters of the fluid carrier of temperature same temperature for whole column
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- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
- G01N2030/324—Control of physical parameters of the fluid carrier of pressure or speed speed, flow rate
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Abstract
The invention discloses a chromatographic detection method for banana vascular wilt, which indirectly identifies fusarium wilt bacteria and occurrence degree thereof by simultaneously detecting metabolic toxins of banana vascular wilt, namely, white plasmin and fusaric acid, and specifically comprises the following steps: feeding the sample to be tested intoPreprocessing the rows; weighing a pretreated sample to be detected, proportionally adding an acidic acetonitrile extracting solution, adding a salt package after vortex extraction, continuing vortex, centrifuging to obtain a supernatant, and purifying the supernatant by a solid phase extraction column; the purification liquid is subjected to gradient elution and separation by adopting an ultrahigh pressure liquid chromatography tandem mass spectrometer, and electrospray ES is adopted + And (3) carrying out mass spectrum detection by using an ion source and multi-reaction monitoring. Compared with the traditional pathogenic bacteria identification, the method is simple and convenient to operate, high in sensitivity and good in repeatability, can be used for identification and quantitative analysis of fusarium wilt in bananas and other crops, and can provide detection data support for production and quality control of healthy seedlings of bananas and other crops and in the aspect of soil environmental condition risk assessment of crop planting fields.
Description
Technical Field
The invention relates to the technical field of agriculture, in particular to a chromatographic detection method for banana vascular wilt.
Background
Bananas are widely planted in tropical and subtropical areas, and become the fruit with the largest trade and consumption of fresh fruits worldwide. Researches show that banana fusarium wilt bacteria can infect almost all banana main cultivars, and especially the toxin secreted by the banana fusarium wilt bacteria, namely beauvericin and fusaric acid play an important role in the pathogenic process.
Fusarium Acid (FA) also known as wilting acid, 5-butylpyridine-2-carboxylic acid (C) 10 H 13 NO 2 ) Is a host non-specialization toxin secondary metabolism produced by fusarium oxysporum, and researchers separate the compound from crop fusarium wilt bacteria or culture mediums thereof, such as sesame, banana, cotton, balsam pear and the like.
Beauverine (BEA) is a cyclic hexapeptide of N-methyl amino acid which is biosynthesized by Beauverine, some fusarium and the like. Firstly, the bacterial strain is separated from beauveria bassiana which is an entomopathogenic fungus, and in recent years, researchers sequentially separate and separate the pathogenic substance from culture solution for culturing No. 1 and No. 4 physiological race of banana fusarium wilt, so that certain convertibility exists, and the pathogenic banana toxicity mechanism is not completely clear.
The detection of banana vascular wilt is currently realized mainly by the detection of signs and laboratory molecular biology on the premise that banana is harmed and obvious symptoms are displayed. The former suffers from uncertainty and false positives, and the latter is time consuming and costly. At present, although liquid phase tandem mass spectrometry is also adopted to detect fusarium toxin, a technology for simultaneously detecting fusarium acid and beauvericin aiming at banana wilt is not found, so that a banana wilt chromatographic detection method is researched, and accurate extraction, separation and detection are carried out on the beauvericin and the fusarium acid in banana and soil samples at the same time, and indirect detection of banana wilt is particularly necessary.
Disclosure of Invention
The invention overcomes the defect that the detection of banana vascular wilt in the prior art is realized by the detection of symptoms and laboratory molecular biology on the premise that the banana vascular wilt is harmed and obvious symptoms are displayed. The technical problems of uncertainty and false positive in the former and high time consumption and cost in the latter are solved, and the method for detecting the banana vascular wilt by using the ultra-high pressure liquid chromatography-mass spectrometry method is provided, and the beauvericin and the fusaric acid in banana and soil samples are simultaneously and accurately extracted, separated and detected, so that the banana vascular wilt is indirectly detected.
In order to solve the problems, the invention adopts the following technical scheme:
a chromatographic detection method for banana vascular wilt, which separates and simultaneously detects the metabolic toxins of banana vascular wilt, namely beauvericin and fusaric acid, specifically comprises the following steps:
s1, pretreatment of a sample: adding distilled water into a sample to be measured, homogenizing or directly homogenizing/uniformly mixing to obtain a pretreatment sample to be measured;
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 extraction solution, adding a salt package into the extraction solution, continuously performing vortex for 1min, centrifuging for 2-10 min at 4000r/min, and loading the supernatant fluid into a container C 18 Purifying the PSA by a solid phase extraction column, collecting the purified liquid, and then passing through a 0.22 mu m organic microporous filter membrane; the ratio of the pretreatment sample to be detected to the extraction solvent is 1 g:1-4 ml; the extraction solvent is acidic acetonitrile extraction solution; the acidic acetonitrile extracting solution contains 0.05 to 2 mass percent of acidic substances;
s3, separation and identification:
and (2) carrying out gradient elution separation and mass spectrum detection on the purifying liquid passing through the organic microporous filter membrane in the step (S2) by adopting an ultrahigh-pressure liquid chromatography-mass spectrometer, wherein the liquid phase elution separation conditions are as follows:
chromatographic column: ACQUITYUPLC@HSS T 3 ；
Mobile phase a: formic acid aqueous solution, formic acid volume percentage is 0.05-0.5%;
mobile phase B: acetonitrile formate solution with formic acid volume percentage of 0.05-0.5%;
flow rate: 0.25mL/min, column temperature of 35 ℃ and sample injection amount of 2 mu L;
gradient elution procedure:
beauvericin retention time is 4.68min, and Fusarium acid retention time is 2.71min;
the mass spectrum detection conditions are as follows:
mass spectrometry conditions: using electrospray ion sources ES + Multiple Reaction Monitoring (MRM) detection;
capillary voltage: 3.0KV;
ion source temperature: 350 ℃;
desolventizing gas is N 2 ；
Desolventizing gas flow rate: n (N) 2 ，400～1000L/h；
Taper hole air flow rate: n (N) 2 ，150L/h；
Collision gas flow rate: ar,0.10mL/Min;
mass spectral parameters of toxins were tuned as follows:
beauvericin: quantitative ion pair 784.27>134.09, cone aperture voltage 54V, collision energy 30eV; qualitative ion pair 784.27>244.05, cone aperture voltage 54V, collision energy 24eV;
fusarium acid: the quantitative ion pair is 180.08>65.06, the taper hole voltage is 74V, and the collision energy is 50eV; qualitative ion pair 180.08>92.03, cone aperture voltage 74V, collision energy 24eV.
In step S2, the acidic acetonitrile extracting solution contains an acidic substance, wherein the acidic substance is one of formic acid, acetic acid or hydrochloric acid; the volume percentage of the acidic substance is 0.05-2%.
In step S2, the acidic acetonitrile extracting solution contains 0.5% by volume of acetic acid.
Further, the salt comprises one or more of anhydrous sodium chloride, anhydrous sodium sulfate, anhydrous magnesium sulfate, sodium citrate and disodium citrate.
Further, the solid phase extraction column is filled with 50mg C 18 50mg PSA, 150mg anhydrous magnesium sulfate.
Further, in step S3, the volume percentage of formic acid in the formic acid aqueous 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, and the molecular mass deviation of the ion pairs is +/-1 respectively, so that the method is an indirect detection method for confirming banana vascular wilt.
Further, in the step S1, the samples are banana samples and soil samples containing fusarium wilt metabolic toxicant, wherein the banana samples comprise stems, leaves and fruits of bananas.
Further, in the step S1, distilled water is added into the stem, leaf and fruit samples of the bananas according to the weight ratio of 1:1 for full homogenization; grinding the soil sample in the shade, sieving with a 40-mesh sieve, mixing, and adding distilled water for wetting before extraction.
Compared with the prior art, the invention has the following beneficial effects:
(1) The invention adopts the ultrahigh-pressure liquid chromatography-mass spectrometry to detect the beauvericin and the fusaric acid in the sample simultaneously, can finish the extraction, purification, separation and detection of the sample within 40 minutes, and has the advantages of high detection speed, high sensitivity, good accuracy, simple operation and low detection cost compared with the traditional pathogen separation and molecular identification method, and has advantages in identification efficiency, cost and accuracy. The invention directly detects the content of pathogenic bacteria metabolite of banana vascular wilt in the sample, and the detection result can be directly used for evaluating the safety of the soil environment condition of the crop planting field; the method can also be used for quality control of banana healthy seedling production, and is used for judging whether the produced banana healthy seedlings carry fusarium wilt pathogenic bacteria or not, and the method is used for providing detection data support for banana and other crop healthy seedlings in terms of condition risk assessment.
(2) In the step S2, acetonitrile is adopted as an extraction solvent, and the acetonitrile is mainly used for being mutually dissolved with an aqueous solution in the early extraction stage (salt is not added), so that the acetonitrile can be fully contacted with an extracted substance, after salt is added, the solubility of acetonitrile in a water phase is rapidly reduced, after centrifugal treatment, the acetonitrile and the salt are layered, and supernatant fluid is subjected to through-type solid-phase dispersion extraction and purification, and then is subjected to machine detection after a filtering membrane. The sample extraction and purification detection technology adopted by the invention is the same as the classical sample pretreatment scheme: compared with the method of purifying, eluting, concentrating and constant volume by adopting liquid-liquid extraction and concentrating extract liquid and solid-phase extraction small column and filtering membrane on-machine detection, the method reduces the pretreatment step of on-machine detection, reduces the loss of the to-be-detected object in the purifying process and improves the extraction rate of the to-be-detected object; the pretreatment time of the detection of the machine is controlled within 30min, and the conventional pretreatment generally needs 3-6 h, so that the working efficiency is greatly improved.
(3) In the step S2, acidic acetonitrile is used as an extraction solvent, mainly the beauvericin and the Fusarium acid maintain the physicochemical properties of the organic acid in the extraction solution, and a higher extraction rate is obtained, particularly the beauvericin has good water solubility and has not been found for a long time, and the main reason is that the beauvericin is difficult to extract from the aqueous solution only by the organic solvent. The invention adopts acidic acetonitrile extracting solution, wherein the acidic acetonitrile extracting solution contains 0.05 to 2 mass percent of acidic substances; preferably, the acid acetonitrile extracting solution contains 0.5% of formic acid, acetic acid, hydrochloric acid and other volatile acids, so that the acid stability of two toxin metabolites in a sample extracting solution system can be improved.
(4) The applicant found when establishing the instrument mass spectrometry method that in step S3 of the present invention, the mobile phase a adopts formic acid aqueous solution (0.05-0.5% by volume of formic acid), the mobile phase B adopts formic acid acetonitrile solution (0.05-0.5% by volume of formic acid), and compared with the case that only the mobile phase a adopts formic acid aqueous solution (0.05-0.5% by volume of formic acid), the mobile phase B also adopts formic acid acetonitrile solution, the signal of two toxin metabolites is stronger, the peak shape is narrow and symmetrical, and the mass spectrometry detection effect is better.
Drawings
FIG. 1 is a graph showing the MRM multi-reaction monitoring of Fusarium acid and Beauverine in example 2 of the present invention.
Detailed Description
The invention is further illustrated by the following examples and experiments.
Example 1:
the embodiment provides a method for detecting banana vascular wilt in a soil sample, which comprises the following steps:
s1, pretreatment of a sample: collecting a sample by a four-division method, drying the sample in the shade at room temperature after collecting the soil sample, grinding the sample, sieving the ground sample by a 40-mesh sieve, uniformly mixing the ground sample to obtain a soil pretreatment sample to be detected, and writing a label for later use;
s2, extracting and purifying a sample:
accurately weighing 10.00g of the soil pretreatment sample to be detected obtained in the step S1, adding 2-3 ml of distilled water into the soil according to the dryness of the soil, and fully wetting the soil; the ratio of the pretreatment sample to be tested of the soil to the acidic acetonitrile extracting solution is 1g: adding 20.0ml of acidic acetonitrile extract into a pretreated sample to be tested for soil in 2ml, performing vortex extraction for 2.0min to obtain an extract, adding 3.0g of sodium chloride powder (anhydrous sodium chloride powder) dried at 350 ℃ into the extract, performing vortex for 1min, centrifuging at 4000r/min for 2min to obtain 2.0ml of supernatant, and loading 50mgC to the supernatant 18 Purifying 50mgPSA and 150mg anhydrous magnesium sulfate by a solid phase extraction column, collecting the purified liquid, and then passing through a 0.22 μm organic microporous filter membrane;
wherein the acidic acetonitrile extracting solution is an extracting solvent and contains 0.5 percent of acetic acid by volume;
s3, separation and determination:
and (2) carrying out gradient elution separation and mass spectrum detection on the purifying liquid passing through the organic microporous filter membrane in the step (S2) by adopting an ultrahigh-pressure liquid chromatography-mass spectrometer, wherein the liquid phase elution separation conditions are as follows:
chromatographic column: ACQUITYUPLC@HSS T 3 ；
Mobile phase a: formic acid aqueous solution, formic acid volume percentage is 0.1%;
mobile phase B: acetonitrile formate solution, formic acid volume percent is 0.1%;
flow rate: 0.25mL/min, column temperature of 35 ℃ and sample injection amount of 2 mu L;
gradient elution procedure:
beauvericin retention time is 4.68min, and Fusarium acid retention time is 2.71min;
the mass spectrum detection conditions are as follows:
mass spectrometry conditions: using electrospray ion sources ES + Multiple Reaction Monitoring (MRM) detection;
capillary voltage: 3.0KV;
ion source temperature: 350 ℃;
desolventizing gas is N 2 ；
Desolventizing gas flow rate: n (N) 2 ，700L/h；
Taper hole air flow rate: n (N) 2 ，150L/h；
Collision gas flow rate: ar,0.10mL/Min;
mass spectral parameters of toxins were tuned as follows:
beauvericin: quantitative ion pair 784.27>134.09, cone aperture voltage 54V, collision energy 30eV; qualitative ion pair 784.27>244.05, cone aperture voltage 54V, collision energy 24eV;
fusarium acid: the quantitative ion pair is 180.08>65.06, the taper hole voltage is 74V, and the collision energy is 50eV; qualitative ion pair 180.08>92.03, cone aperture voltage 74V, collision energy 24eV.
Example 2:
the embodiment provides a method for detecting banana fusarium wilt in banana corms and leaves, which comprises the following steps:
s1, pretreatment of a sample: the sample is collected by a quartering method according to the banana sample: distilled water is added into the banana sample to be detected according to the mass ratio of distilled water of 1:1, and then the banana sample to be detected is placed into a broken wall food processor for homogenization to obtain a banana pretreatment sample to be detected;
s2, extracting and purifying a sample:
accurately weighing 20.00g of banana pretreatment sample to be detected, which is obtained by fully homogenizing in the step S1, wherein the ratio of the banana pretreatment sample to be detected to the acidic acetonitrile extracting solution is 1g: adding 40.0ml of acidic acetonitrile extracting solution into 2ml of the banana pretreatment sample to be detected; vortex extracting for 2.0min to obtain extractive solutionAdding 6g sodium chloride powder (anhydrous sodium chloride powder) dried at 350deg.C into the extractive solution, further swirling for 1min, centrifuging at 4000r/min for 10min to obtain 2ml supernatant, and loading 50mgC 18 Purifying 50mgPSA and 150mg anhydrous magnesium sulfate by a solid phase extraction column, collecting the purified liquid, and then passing through a 0.22 μm organic microporous filter membrane;
wherein the acidic acetonitrile extracting solution is an extracting solvent and contains 0.5% of acetic acid by mass percent;
in this embodiment, S3 is separated and detected in the same manner as in step S3 in embodiment 1;
the super high pressure liquid chromatography-mass spectrometry instrument is adopted to analyze and detect the metabolism toxicants of the banana vascular wilt, namely, the fusaric acid and the beauvericin, wherein a multi-reaction monitoring ion chart of the fusaric acid and the beauvericin MRM is shown in a figure 1, the beauvericin retention time is 4.68min, the fusaric acid retention time is 2.71min, and the peak shape is narrow and symmetrical.
Test one
According to the sample pretreatment methods of example 1 and example 2 and the instrument conditions of step S3, the banana soil, bulb and whole fruit samples were subjected to additive recovery and precision evaluation experiments, and the results are shown in table 1. As is clear from Table 1, at the sample addition levels of 0.01, 0.04, and 0.2mg/kg, the average recovery rate of beauvericin and Fusarium acid in the soil was 93.29% to 97.09%, the relative standard deviation was 1.64% to 8.23%, the average recovery rate in the bulb was 95.45% to 106.62%, the relative standard deviation was 0.84% to 5.00%, and the average recovery rate in the whole fruit was 87.26% to 106.98%, and the relative standard deviation was 0.74% to 4.61%.
The signal to noise ratio of the substrate standard sample is calculated by an instrument to obtain: the minimum detection concentration of the beauvericin and the fusaric acid in the soil is 0.01ug/kg and 4.38ug/kg respectively. The minimum detection concentration in the corm is 0.04ug/kg and 7.79ug/kg respectively, and the minimum detection concentration in the whole banana fruit is 0.03ug/kg and 7.63ug/kg respectively, so that the detection requirements are completely met, and the detection method established by the invention has high sensitivity and good repeatability, and can accurately detect the metabolic poison of the banana wilt.
Table 1 results of recovery of Fusarium acid and beauvericin addition in different banana samples (n=5)
Test II
According to the detection method of example 2, the addition recovery and precision evaluation experiments were performed on the acidic acetonitrile extracts of different concentrations. As can be seen from Table 2, acetonitrile formate and acetonitrile acetate at different concentrations have a certain influence on the extraction of beauvericin and Fusarium acid, when the concentration of the acetonitrile in the solution is more than 0.05%, the recovery rate of the two toxins added is more than 80%, and when the concentration of the acetic acid in the acetonitrile is 0.5%, the recovery rate of the two toxins added is more than 93.35%, and the acetonitrile formate at the same concentration has no difference in the extraction of beauvericin, and the extraction of Fusarium acid is not more than 88%. Acetonitrile containing 0.5% acetic acid was taken after comprehensive consideration as the optimal extraction solvent.
TABLE 2 recovery results of acid acetonitrile at different concentrations for Fusarium acid and Beauverine (n=3)
Test three
Extraction and detection of toxins in culture bacteria liquid of banana vascular wilt
1. Activation and culture of banana fusarium wilt
Potato solid medium (PDA) was prepared: 200g of potato fragments are weighed and added into 1000mL of distilled water, heated and boiled for 30min, the boiled soup is filtered through four layers of gauze, the lower filtrate is taken, 20g of glucose and 15-20 g of agar powder are added into the filtrate, stirring and dissolving are continued, water is added for constant volume to 2000mL, split charging is carried out, and high-pressure sterilization is carried out for 20min in a high-pressure steam sterilization pot at 121 ℃ after split charging, and cooling is carried out for standby.
Preparation of a culture medium: 3g of sodium nitrate, 1g of dipotassium hydrogen phosphate, 0.5g of magnesium sulfate heptahydrate, 0.5g of potassium chloride and 0.01g of ferrous sulfate are weighed, 30g of sucrose is heated in 1000mL of deionized water, dissolved and packaged, sterilized for 20min at 121 ℃, and cooled for later use.
2. Fusarium oxysporum dedicated (FOC 4), a pathogenic bacterium for banana vascular wilt, is provided by the plant pathogenicity system fungi laboratory at agricultural university of North China. A small amount of Foc4 was picked from the tube and streaked on PDA, and cultured in a biochemical incubator under dark conditions at 28℃for three generations to complete strain activation. Taking out 6mm fungus dish with puncher, placing into a culture medium of Nahnia, shake culturing at 28deg.C, 180rpm in dark condition in shaking incubator. FOC4 is taken out periodically and put into an ultra-clean workbench, observed under a 40-time optical microscope and counted by a blood cell counting plate, and 5.00g of bacterial liquid collected synchronously is accurately weighed according to the bacterial liquid: adding 10mL acetonitrile solution containing 0.5% of acetic acid by mass percentage into acetonitrile solution with the weight ratio of 1:2, performing vortex extraction for 2min, adding 3.0g sodium chloride powder dried at 350 ℃ for further vortex for 1min, centrifuging for 5min 4000r/min, collecting supernatant, and loading 50mgC 18 Purifying with 50mg PSA and 150mg anhydrous magnesium sulfate by a solid phase extraction column, and passing the purified solution through a 0.22 μm organic microporous filter membrane, and performing mechanical detection under the instrument condition of the step S3 in example 2. The accumulation levels of toxin metabolites of activated banana vascular wilt pathogenic bacteria Fusarium oxysporum dedicated to different times in the culture solution of Chlamydia are shown in Table 3 below.
TABLE 3 Beauverine and Fusarium acid content (n=3) in bacterial solutions at different culture times
As is clear from Table 3, the content of beauvericin and Fusarium acid increased continuously with the increase of the culture time, and the content of beauvericin increased gradually during the culture period, with the maximum value of Fusarium at the 3 rd day of culture and then decreased slightly with the increase of the time.
Test four
Detection and comparison of banana vascular wilt samples:
related experimental researches on detection and identification of banana fusarium wilt pathogen molecules are carried out in the unit before the day, and related contents are disclosed, and the related contents are specifically disclosed in the literature of molecular detection and identification of Guangxi banana fusarium wilt pathogen No. 4 physiological race, mo Jianyou et al, southern agricultural journal, 2012, 43. The test uses banana samples infected by the same bacterial strains in the document for comparison experiments. In this document, banana vascular wilt samples are: 6 parts of banana disease body samples with the corresponding numbers 1-6 and the banana varieties mainly comprising Williams and tribute bananas are collected in the main production area of Guangxi bananas. The corms of the disease strains are separated from pathogen purification to obtain pathogen strains, the test is carried out by adopting banana wilt samples infected by the same pathogen strains in the document, wherein the corresponding numbers are the same, negative control is also noninductive potted wilt banana seedlings, positive control is also potted wilt banana seedlings which are inoculated with physiological micro-species No. 1 and No. 4 of banana wilt bacteria and show obvious symptoms, and the corm samples are taken for homogenate sample preparation during the test to carry out qualitative and quantitative detection of toxin metabolites beauvericin and fusaric acid.
Among them, detection methods and detection results for detecting and identifying banana vascular wilt pathogen molecules are described in the above-mentioned documents. According to the detection method of the embodiment 2 of the present invention, samples of different corms are subjected to homogenization and sample preparation, qualitative and quantitative detection of toxin metabolites beauvericin and Fusarium acid are performed, and the extraction and detection results of beauvericin and Fusarium acid in different samples are shown in the following table 4:
table 4 beauvericin and Fusarium acid content (n=2) in different samples
"-" indicates no detection or below the instrument detection limit.
As can be seen from Table 4, except for the negative control M, other samples simultaneously detected the banana fusarium wilt metabolic toxins, namely beauvericin and Fusarium acid, the detection concentration of the beauvericin in the banana fusarium wilt Fusarium oxysporum dedicated type 1 physiological race was slightly higher than that of the Fusarium acid, and the detection concentration of the other samples including the banana fusarium wilt Fusarium oxysporum dedicated type 4 physiological race was higher than that of the Beauverine acid. The PCR detection results are basically consistent with those described in the above documents, but the physiological species cannot be distinguished according to the detection concentration, but two pathogenic physiological species are the main pathogenic bacteria on bananas in China at present.
According to the comparison of the results of the chromatographic detection method and the traditional pathogenic bacteria detection, the toxin is identified by adopting the chromatographic detection method, the detection of banana vascular wilt is feasible, and compared with the traditional pathogenic bacteria detection, the method has the advantages of rapidness, simplicity, convenience and accuracy, and has the advantages of identification efficiency, cost and accuracy. The invention relates to simultaneous detection of two toxins, and the beauveria bassiana extract is rarely found before, and no false positive phenomenon exists at present, so that the detection result of the invention can be directly used for evaluating the safety of the soil environment condition 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 fusarium wilt pathogenic bacteria. The method can provide detection data support for healthy seedlings of bananas and other crops and in terms of condition risk assessment.
The foregoing description is directed to the preferred embodiments of the present invention, but the embodiments are not intended to limit the scope of the invention, and all equivalent changes or modifications made under the technical spirit of the present invention should be construed to fall within the scope of the present invention.
Claims (7)
1. A chromatographic detection method for banana vascular wilt is characterized by separating and simultaneously detecting the metabolic toxins of banana vascular wilt, namely beauvericin and fusaric acid, and specifically comprises the following steps:
s1, pretreatment of a sample: adding distilled water into a sample to be measured and homogenizing or directly homogenizing/uniformly mixing the sample to be measured to obtain a pretreatment sample to be measured;
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 extraction solution, adding a salt package into the extraction solution, performing vortex for 1min, performing centrifugation for 2-10 min at 4000r/min, purifying the supernatant by a solid phase extraction column, and collecting the purified solution and then passing through a 0.22 mu m organic microporous filter membrane; the ratio of the pretreatment sample to be detected to the extraction solvent is 1 g:1-4 ml; the extraction solvent is acidic acetonitrile extraction solution; the solid phase extraction column is provided with C 18 Solid phase extraction column of PSA and anhydrous magnesium sulfate;
s3, separation and determination:
and (2) carrying out gradient elution separation and mass spectrum detection on the purifying liquid passing through the organic microporous filter membrane in the step (S2) by adopting an ultrahigh-pressure liquid chromatography-mass spectrometer, wherein the liquid phase elution separation conditions are as follows:
chromatographic column: ACQUITY UPLC@HSS T 3 ；
Mobile phase a: formic acid aqueous solution, formic acid volume percentage is 0.05-0.5%;
mobile phase B: acetonitrile formate solution with formic acid volume percentage of 0.05-0.5%;
flow rate: 0.25mL/min, column temperature 35 ℃, and sample injection amount 2 mu L;
gradient elution procedure:
Beauvericin retention time is 4.68min, and Fusarium acid retention time is 2.71min;
the mass spectrum detection conditions are as follows:
mass spectrometry conditions: using electrospray ion sources ES + Multiple Reaction Monitoring (MRM) detection;
capillary voltage: 3.0KV;
ion source temperature: 350 ℃;
desolventizing gas is N 2 ；
Desolventizing gas flow rate: n (N) 2 ，400～1000 L/h；
Taper hole air flow rate: n (N) 2 ，150 L/h；
Collision gas flow rate: ar,0.10mL/Min;
mass spectral parameters of toxins were tuned as follows:
beauvericin: quantitative ion pair 784.27>134.09, cone aperture voltage 54V, collision energy 30eV; qualitative ion pair 784.27>244.05, cone aperture voltage 54V, collision energy 24eV;
fusarium acid: the quantitative ion pair is 180.08>65.06, the taper hole voltage is 74V, and the collision energy is 50eV; qualitative ion pair 180.08>92.03, cone aperture voltage 74V, collision energy 24eV.
2. The method for detecting banana vascular wilt by chromatography according to claim 1, wherein in step S2, the acidic acetonitrile extract contains an acidic substance, and the acidic substance is one of formic acid, acetic acid and hydrochloric acid; the volume percentage of the acidic substance is 0.05-2%.
3. The method for detecting banana vascular wilt by chromatography according to claim 2, wherein in step S2, the acidic acetonitrile extract contains 0.5% by volume of acetic acid.
4. The method for detecting banana vascular wilt by chromatography according to claim 1, wherein the salt contains 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 by chromatography according to claim 1, wherein the solid phase extraction column is filled with 50mg C 18 50mg PSA, 150mg anhydrous magnesium sulfate.
6. The method for detecting banana vascular wilt by chromatography according to claim 1, wherein in step S3, the formic acid aqueous solution has a volume percentage of 0.1%; in the formic acid acetonitrile solution, the volume percentage of formic acid is 0.1%.
7. The method according to claim 1, wherein in step S1, the sample is a banana sample or a soil sample containing a metabolic poison of fusarium wilt, and the banana sample includes stems, leaves, and fruits of bananas.
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