CN111693687B - Preparation method of biological negative sample and biological standard substance and TMAO detection method - Google Patents
Preparation method of biological negative sample and biological standard substance and TMAO detection method Download PDFInfo
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Abstract
The invention discloses a preparation method of a biological negative sample and a biological standard substance and a TMAO detection method, and relates to the technical field of detection. The technical scheme of the invention has the advantages of ingenious design, simple and convenient operation and strong practicability, the whole process does not need expensive instruments or complex operation processes, the rapid and simple TMAO negative sample preparation can be realized, the microorganism can be integrated and uniformly treated after a plurality of biological samples are directly mixed, the TMAO is eliminated, and compared with the method for searching samples with low TMAO values one by one, the method is more convenient and economic and can realize batch preparation; and subsequently, excessive microorganisms can be removed through modes of inactivating or filtering microorganisms and the like, so that the method can be further applied to the preparation of the TMAO standard product.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a preparation method of a biological negative sample and a biological standard substance and a TMAO detection method.
Background
Trimethylamine oxide (TMAO) is a metabolite associated with the intestinal flora and is synthesized in the liver. In vivo alkaloids such as choline, L-carnitine and betaine are converted into Trimethylamine (TMA) under the action of intestinal flora, TMA is converted into trimethylamine oxide under the action of trimethylamine oxidase, and the trimethylamine oxide is metabolized by kidney and discharged with urine. Related studies indicate that TMAO is associated with cardiovascular diseases, chronic kidney disease, renal insufficiency and death risk, type II diabetes, insulin resistance, non-alcoholic fatty liver disease, and the development of certain cancers. Therefore, the detection of the content of the trimethylamine oxide in the biological sample has important significance.
In the design process of a detection experiment or a detection product, a negative control is usually needed, and a biological sample (such as blood, urine or interstitial fluid) is usually a complex system, wherein the biological sample contains a plurality of proteins and various chemical components, so that the conventional detection technology usually adopts blank biological matrixes such as water, Phosphate Buffer Solution (PBS) or human serum albumin and the like as the negative control, although the solutions can deduct background interference in the system to a certain extent, the conventional negative control still cannot eliminate the matrix effect due to the complex components of the biological sample, and the measurement result usually has poor methodological verification persuasiveness due to various errors, and especially cannot meet the explanation requirements for the matrix effect in the pharmacopoeia 'biological sample quantitative analysis method verification guidance principle'.
Some studies show that low TMAO value samples can be searched, however, the samples are limited in number and complicated in searching process, and therefore, the practicability is not strong. Chinese patent application CN109387410A discloses a TMAO negative sample and its preparation method and application, specifically discloses that adding raney salt into a biological sample to remove TMAO contained in the sample, although this method can achieve the purpose of eliminating TMAO background interference in blank matrix, there are obvious defects in using chemical complexing agent reaction, such as: 1. the reaction process is violent, a large amount of precipitates are generated after the reaction, and the precipitates can influence other components in the sample; 2. because the TMAO content in the biological sample is relatively low, the added reducing agent is easy to be excessive, and the excessive reducing agent is difficult to be completely removed or just completely reacted, the obtained negative sample cannot be added with TMAO in the later period to prepare the TMAO biological standard substance; 3. the Lee's salt is also highly toxic and is very easy to be inserted into eyes or adhered to skin to generate strong stimulation; the sample after reaction is extremely harmful to the water, even small-volume products can not contact underground water, water course or sewage system, therefore all there is very big potential safety hazard to experiment operating personnel and environment.
Therefore, it is of great significance to develop a preparation method of a biological sample for preparing a TMAO biological standard substance, which has mild reaction conditions, is convenient to operate, and is safe.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art. Therefore, the invention provides a preparation method of a biological negative sample, which can prepare a TMAO negative sample under mild conditions, and the negative sample can be further used for preparing a biological standard substance.
The invention also provides a method for preparing the TMAO biological standard substance by using the TMAO negative sample.
The invention also provides a TMAO detection method.
The method according to an embodiment of the first aspect of the invention comprises the following steps:
and (3) taking a biological sample, adding the microorganism with specificity removed of TMAO into the biological sample, and removing the microorganism after the reaction to obtain the biological negative sample.
According to some embodiments of the invention, the volume ratio of the microorganism to the biological sample is 1: 1000-1: 10000; preferably 1: 1000-1: 5000, wherein the concentration of the microorganisms is 1 x 108～1*1010CFU/mL. The TMAO content in the biological sample is lower, when adding the microorganism, the proportion of the microorganism that needs to be added is lower, the proportion of the metabolite produced by the added bacteria liquid culture medium or the added microorganism is extremely low relative to the negative sample, and the influence on the negative sample component change is smaller.
According to some embodiments of the present invention, the temperature of the reaction system of the microorganism and the biological sample is 15-35 ℃, the reaction time is 0.5-4h, and the reaction pH is 5.0-9.0; preferably, the reaction system is shaken or rotated during the reaction process, and the rotating speed/shaking interval is 10-100 rmp.
According to some embodiments of the invention, the microorganisms include, but are not limited to: vibrio (Vibrio), thiophotosynthetic bacteria (also referred to as non-thiophotosynthetic bacteria), Escherichia coli (Enterobacteriaceae), Shewanella putrescentiae (Shewanella putreferans), Stenotrophomonas maltophilia (Stenotrophoromonas malthiophila), Providencia rettgeri (Providencia rettgeri), Salmonella typhimurium, Achromobacter (Achromobacter), Aeromonas aerogenes (Aeromonas), Alternaria (Alteromonas), Campylobacter (Campylobacter), Corynebacterium (Corynebacterium), Flavobacterium (Flavobacterium), Micrococcus (Micrococcus), Photobacterium (Photobacterium), Pseudomonas (Pseudomonas), Rhodopseudomonas (Rhodopseudomonas), Rhodospirillum (Rhodospirillum), or Shigella (Shigella); preferably, the microorganism is selected from at least one of vibrio, sulfur-free photosynthetic bacteria (also referred to as non-sulfur photosynthetic bacteria), escherichia coli, shewanella putrefaciens, maltophilia oligomonas, providencia rettgeri, salmonella typhimurium, achromobacter, aeromonas, alteromonas, campylobacter, micrococcus, photobacterium, pseudomonas, rhodopseudomonas, rhodospirillum, or shigella; more preferably, the microorganism is selected from at least one of Vibrio, sulfur-free photosynthetic bacteria, Escherichia coli, Shewanella putrefaciens, Salmonella typhimurium, Corynebacterium, or Flavobacterium; still more preferably, if included, the Vibrio is selected from Vibrio alginolyticus. The microorganisms of the present embodiment may be any microorganisms capable of utilizing TMAO, either as a single addition or as a synergistic combination of two or more.
According to some embodiments of the invention, the escherichia coli reaction pH is 5.0-8.0; the reaction pH of the salmonella typhimurium is 5.0-5.5; the pH value of the Shewanella putrefaciens reaction is 6.0-9.0; the pH value of the sulfur-free photosynthetic bacteria reaction is 7.5-8.5.
According to some embodiments of the invention, the microorganism is added to the biological sample in the form of a dry powder, a pellet, a colony, or a bacterial solution. Can be added in various forms, and has flexible and various operation modes. The microorganism dry powder does not need to be activated and can be directly added. The bacterial pellet, colony, and bacterial liquid are usually added after activation, and the medium for activation is LB medium.
According to some embodiments of the invention, the biological sample is typically a homologous sample to the sample to be tested; if the sample to be detected is a human body, the biological sample is taken from the human body; if the sample to be tested is derived from another organism, the biological sample is taken from another organism. TMAO negative samples which can be prepared from biological samples of the same source can ensure that the matrix components are relatively uniform, and can avoid the matrix effect caused by adopting other negative samples to the maximum extent in the application process of the negative samples which are used as samples to be detected of the type.
According to some embodiments of the invention, the biological sample is selected from at least one of plasma, serum, urine, lymph fluid, oral saliva, cerebrospinal fluid, ascites fluid, or dialysate. The biological sample may be selected from all samples that may contain TMAO, including but not limited to the above-mentioned examples, in the process of performing the microbial treatment on the biological sample, the microorganism does not necessarily need to be propagated in the biological sample, and the added microorganism itself has activity.
According to some embodiments of the invention, the biological negative sample satisfies at least the following conditions: a) the residual TMAO content value should be less than 20% of the lower limit of the desired assay standard curve; b) TMAO remains in the biological negative sample, but after exogenous TMAO is added to the biological negative sample, the difference between the actually detected TMAO concentration and the theoretical TMAO concentration after the addition is less than 10%, and the concentration difference is preferably less than 5%. According to the method for preparing the negative sample, the prepared biological negative sample is closer to the actual sample component to be detected after the microorganism is directly added into the biological sample for treatment.
According to some embodiments of the invention, the method of removing microorganisms is selected from at least one of membrane filtration, precipitant inactivation, or physical precipitation. In order to avoid excessive production of metabolites by the microorganisms in the biological sample, the reaction of the microorganisms with the biological sample is terminated after the microorganisms have reacted TMAO completely or to the limit requirements of the present invention. In order to avoid the damage of the original active substances in the biological samples, the method does not preferably inactivate the microorganisms by a high-temperature method, but when the microorganisms are filtered by a membrane-passing method and other methods, because the direct membrane-passing sterilization difficulty of partial biological samples such as serum and the like is higher, the operation is inconvenient and the membrane-passing sterilization is more difficult to complete. Therefore, when the sample is detected, the biological sample can be detected by adding a precipitator and an internal standard, and the addition of the precipitator can inactivate microorganisms, so that the microorganism is directly inactivated by adding the precipitator, and then the supernatant is obtained by high-speed centrifugation, thereby obtaining the effective TMAO negative sample.
According to some embodiments of the present invention, the specific operation of the precipitant deactivation method comprises adding precipitant, reacting, and removing precipitate by solid-liquid separation.
According to some embodiments of the invention, the method of solid-liquid separation is centrifugation.
According to some embodiments of the invention, the precipitating agent is selected from at least one of a phosphate, an oxalate, a sulfide, a strong hydroxide acid, a strong base, a heavy metal salt, urea, or an organic solvent; preferably, the organic solvent is selected from polar organic solvents; more preferably, the polar organic solvent is selected from one of methanol, acetonitrile or acetone.
According to some embodiments of the invention, if the precipitate is a solid, the mass-to-volume ratio of the added amount of the precipitating agent to the biological sample is 0.8-1.5%; if the biological sample contains the organic solvent, the adding amount of the organic solvent is 1/20-1/5 of the volume amount of the biological sample.
The preparation method according to the embodiment of the invention has at least the following beneficial effects: the method has the advantages of ingenious design, simple and convenient operation and strong practicability, the rapid and simple preparation of the TMAO negative sample can be realized without expensive instruments and equipment or complex operation processes in the whole process, multiple biological samples can be directly mixed and then are integrated with microorganisms for uniform treatment, the TMAO is eliminated, and compared with the method for searching samples with low TMAO values one by one, the method is more convenient and economic and can realize batch preparation; according to the scheme, the background interference of endogenous TMAO can be effectively eliminated, and the specificity of the microbial metabolic process is skillfully utilized to reduce, metabolize and decompose TMAO with oxidizability or add a group to change the structure for modification, so that the background interference caused by TMAO in a blank matrix is eliminated; and subsequently, excessive microorganisms can be removed through modes of inactivating or filtering microorganisms and the like, so that the method can be further applied to the preparation of the TMAO standard product.
The preparation method according to the second aspect embodiment of the present invention comprises the steps of:
s01, preparing a biological negative sample by adopting the method;
s02, TMAO is quantitatively added to the biological negative sample prepared in the step S01.
According to some embodiments of the present invention, the final concentration of the TMAO added in step S02 is between 1 ng/mL and 1000 ng/mL; preferably, the final concentration of the TMAO is between 2 and 500 ng/mL; more preferably, the final concentration of TMAO is selected from at least one of 2, 5, 10, 20, 50, 100, 200 and 500 ng/mL.
According to some embodiments of the present invention, the TMAO may be added in solid form or in solution form, and if in solution form, the solution may be prepared with a biological negative sample as a solvent or diluent.
The preparation method according to the embodiment of the invention has at least the following beneficial effects: the preparation of the negative sample by using the microorganism has the advantages that the reaction process is milder and controllable, and the prepared negative sample can be further made into a biological standard substance by inactivating the microorganism, so that the subsequent quantitative detection process is more accurate; the negative sample after TMAO is directly removed by the biological sample, a series of trimethylamine oxide standard solutions are added, and when the sample to be detected is homologous with the sample to be detected, the matrix effect brought by the standard solution prepared by adopting buffer solution or other solvents in the prior art can be greatly reduced, and meanwhile, the influence of the brought TMAO content when other samples are adopted as negative quality control substances or standard solution solvents can be avoided.
The detection method according to the third aspect embodiment of the present invention includes the steps of:
I. preparing a biological negative sample by adopting the method;
II. And (4) detecting the signal of the TMAO in the sample to be detected by taking the signal of the biological negative sample prepared in the step I as a background signal.
According to some embodiments of the present invention, the detection method is a quantitative detection method, a biological negative sample is added with a quantitative TMAO to prepare a biological standard in the detection process, the signal intensity of the TMAO in the biological standard is characterized, the TMAO signal intensity (which may be a TMAO detection method known in the art such as optical intensity, mass spectrum signal intensity or chromatographic signal intensity) at different concentrations is measured, and the TMAO in the sample to be detected is quantitatively analyzed.
The detection method provided by the embodiment of the invention has at least the following beneficial effects: the detection method of the invention does not aim at disease diagnosis and treatment, and the detection process directly prepares a biological negative sample from a homologous sample, thereby avoiding unreliable detection results caused by matrix effect when other simulation samples are used as negative quality control products or standard solution solvents in the traditional technology; the detection method can realize quantitative detection and qualitative detection; the biological sample can be reduced, decomposed or modified by adding microorganisms into the biological sample, so that TMAO in the biological sample is removed, and the biological sample without TMAO or the biological sample with residual TMAO which is negligible when used as negative sample contrast is obtained, thereby meeting the requirements of the invention. The invention has the advantages of mild reaction conditions of the microorganism and the biological sample, high reaction efficiency and small damage degree to the biological sample matrix, so that the matrix effect background of the obtained biological negative sample can be deducted to the maximum extent. Meanwhile, the product of the reaction of the microorganisms and the TMAO or other metabolites generated in the reaction process of the microorganisms can be effectively separated from the detection peak time of the TMAO by optimizing the detection conditions, so that the quantitative detection of the TMAO is not influenced. In addition, the action mechanism of the reaction of the microorganisms and the TMAO is not limited to reduction or decomposition of the TMAO, and a new substance can be generated by modifying the TMAO, so that the microorganisms and the TMAO can be separated in the detection process to achieve the effect that the TMAO cannot be detected in a negative sample, and when the mechanism of the microorganisms for processing the TMAO is different, a plurality of microorganisms can be added simultaneously to perform synergistic reaction to enhance the reaction effect.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
FIG. 1 is a graph showing the change of TMAO concentration after different reaction times in example 1 of the present invention;
FIG. 2 is a chromatogram of TMAO in a sample after a reaction of a microorganism detected in example 2 of the present invention;
FIG. 3 is a chromatogram of an internal standard detected in example 2 of the present invention;
FIG. 4 is a standard curve measured in example 2 of the present invention.
Detailed Description
In order to explain technical contents, achieved objects, and effects of the present invention in detail, the following description is made with reference to the embodiments and the accompanying drawings. The test methods used in the examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available reagents and materials unless otherwise specified.
The most key concept of the invention is as follows: the microbial reaction process is mild, the microorganisms are easy to remove, TMAO contained in the biological sample is removed by a microbiological method, the batch production of biological negative samples can be realized, and meanwhile, the residual microorganisms after the reaction are easy to remove, so the negative quality control product prepared by the scheme can also be used for the subsequent preparation and detection of biological standard products.
The first embodiment of the invention is as follows: a biological negative sample for TMAO detection and a preparation method thereof are provided, the biological negative sample for TMAO detection is prepared by the following method, and the method comprises the following steps:
1.1 biological sample collection: the method comprises the following steps of clinically collecting whole blood samples (subjected to relevant pathogenic microorganism detection and free of relevant infection) of 18 Chinese healthy people, taking 5mL of blood from each sample, separating blood plasma to obtain about 2mL of blood plasma, and uniformly mixing the blood plasma of each sample to obtain 35mL of biological sample mixture.
1.2 standard curve drawing: the methanol water solution is prepared into gradient verification standard sample solutions with TMAO concentrations of 2, 5, 10, 20, 50, 100, 200 and 500ng/mL respectively. A200. mu.L standard sample was taken and 600. mu.L TMAO-D9 (600. mu.L 2 ng. mL) was added-1) Internal standard working solution is shaken for 30s, 100 mu L of supernatant is taken, 900 mu L of diluent (ultrapure water) is added, vortex mixing is carried out, and 2 mu L of sample injection is used for determination. The content was measured by a Liquid chromatograph-Mass spectrometer (HPLC-MS) and a TMAO standard curve was plotted. Wherein, the volume percentage of the methanol in the methanol water solution is 40 percent (30-60 percent).
1.3 biological sample TMAO assay: and (3) quantitatively detecting the TMAO content in the biological sample mixture obtained in the step 1.1, and repeating the step 10 times to obtain an average value (the sample amount is 200 mu L each time).
1.4 microbial culture:
group one (vibrio alginolyticus culture):
preparing LBS (high-salt) culture medium: 25g of agar, 10g of peptone, 10g of meat extract, 5g of yeast extract, 20g of glucose, 80mL of tween and K2HPO42g, 5g sodium acetate, 2g triammonium citrate and MgSO4·7H2O 200mg，MnSO4·4H2O50 mg, acetic acid 3.7mL, sodium chloride 10g, refined water 1000 mL. Heating to dissolve and correct pH to 6.0-6.5, sterilizing at 121.3 deg.C for 20 min. Cooling to about 50 deg.C, and adding acetic acid. After mixing well, the mixture was dispensed to a plate.
Inoculation: selecting a Vibrio alginolyticus single colony to be placed in 10mL of liquid culture medium, and carrying out activated culture for 24 hours under the conditions of 30 ℃ and 200 rpm;
obtaining thalli: inoculating 100 μ L of the bacterial liquid into new 100mL liquid culture medium for secondary activation, culturing at 30 deg.C and 150rpm for 12h, and centrifuging to obtain thallus precipitate. Washed with 1.5mL of 1mol/L pre-cooled sorbitol and resuspended for use.
Second group (non-sulfur photosynthetic bacteria culture):
preparing a culture medium: 3g of sodium acetate, 3g of ammonium chloride, 0.1g of yeast extract and KH2PO4 0.2g，MgSO4·7H2O5 mg, sodium carbonate 0.2g, 1mL of inorganic salt solution, and 1000mL of refined water. The composite inorganic salt solution comprises the following components: EDTA 0.5g, FeSO4·7H2O 0.2g，ZnSO4·7H20.01g of O, 0.03g of boric acid, MnCl2·4H2O 0.003g，CaCl2·2H2O 0.02g，NiCl2·6H2O 0.002g，CuCl2·2H2O 0.001g，Na2MoO4·2H20.003g of O, 100mL of purified water, and pH adjusted to 3.0 with HCl. Sterilizing at 121 deg.C for 20 min. After cooling and full mixing, the mixture is injected into a flat plate;
inoculation: selecting a single bacterial colony of non-sulfur photosynthetic bacteria in 10mL of liquid culture medium, performing activation culture for 120h under the conditions of 30 ℃ and 150rpm under the illumination of 800-10000 lux in an illumination incubator;
obtaining thalli: inoculating 100 μ L of the bacterial liquid into new 100mL liquid culture medium for secondary activation, culturing at 30 deg.C and 150rpm for 24h, and centrifuging to obtain thallus precipitate. Washed with 1.5mL of 1mol/L pre-cooled sorbitol and resuspended for use.
Third group (E.coli culture):
preparing LBS (low-salt) culture medium: 25g of agar, 10g of peptone, 10g of meat extract, 5g of yeast extract, 20g of glucose, 80mL of tween and K2HPO42g, 5g sodium acetate, 2g triammonium citrate and MgSO4·7H2O 200mg，MnSO4·4H2O50 mg, acetic acid 3.7mL, sodium chloride 0.5g, refined water 1000 mL. Heating to dissolve and correct pH to 6.0Sterilizing at 121 deg.C for 20min at 6.5 deg.C. Cooling to about 50 deg.C, and adding acetic acid. After fully mixing, the mixture is injected into a flat plate;
inoculation: selecting single escherichia coli colonies, placing the single escherichia coli colonies in 10mL of liquid culture medium, and performing activated culture for 18h at 37 ℃ under the condition of 200 rpm;
sixthly, obtaining thalli: inoculating 100 μ L of the bacterial liquid into new 100mL liquid culture medium for secondary activation, culturing at 37 deg.C under 200rpm for 12h, and centrifuging to obtain thallus precipitate. Washed with 1.5mL of 1mol/L pre-cooled sorbitol and resuspended for use.
Fourth group (Shewanella putrefaciens culture):
preparing a CM0847 culture medium: tryptone 15g, soybean peptone 5g, sodium chloride 5g, agar 13g, refined water 1000 mL. Heating to dissolve and correct to pH7.3 + -0.2, 121 deg.C, sterilizing for 20 min. Cooling and then distributing on a flat plate;
inoculation: selecting a Shewanella putrefaciens single colony to be placed in 10mL of liquid culture medium, and performing activated culture for 24h under the conditions of 25 ℃ and 150 rpm;
obtaining thalli: inoculating 100 μ L of the bacterial liquid into new 100mL liquid culture medium for secondary activation, culturing at 25 deg.C and 150rpm for 12h, and centrifuging to obtain thallus precipitate. Washed with 1.5mL of 1mol/L pre-cooled sorbitol and resuspended for use.
1.5 reaction
Carrying out microbial reaction: respectively taking 6mL and 4 parts of 1.3 mixed samples, respectively adding about 3 microliter of each thallus precipitate prepared in 1.4, and then placing the mixture under appropriate conditions (namely the optimum temperature and pH value of each bacteria, the pH value of the vibrio alginolyticus reaction is 6.0, the pH value of the non-sulfur photosynthetic bacteria reaction is 3.0, the pH value of the escherichia coli reaction is 6.0, the pH value of the Shewanella putrefaciens reaction is 7.5, and the temperature of each group is 30 ℃) for reaction. The reaction solution was shaken up and down once every 30s, and 200. mu.L of the solution was sampled every 2min for TMAO content determination.
② control group (Leeb's salt precipitation): taking 6mL of 1.3 mixed sample, adding 0.6mL of Rayleigh salt solution (the concentration is determined according to the analysis result of 1.3, the molar ratio of Rayleigh salt to TMAO is not lower than 1: 1), adjusting the pH value to 1 by using concentrated hydrochloric acid, and reacting at 4 ℃. The reaction solution was shaken up and down once every 30s, and 200. mu.L of the solution was sampled every 2min for TMAO content determination.
1.6 detection of TMAO content in plasma samples: collecting 200 μ L reacted plasma sample, centrifuging at 4 deg.C and 4000rpm for 20min, collecting supernatant, adding acetonitrile (20 μ L) for precipitation, centrifuging at 4 deg.C and 12000rpm for 10min, collecting supernatant, adding 600 μ L TMAO-D9(600μL 2ng·mL-1) And oscillating the internal standard working solution for 30s, taking 100 mu L of supernatant, adding 900 mu L of diluent (ultrapure water), uniformly mixing by vortex, introducing 2 mu L of sample for HPLC-MS detection, and calculating the content of the TMAO in the sample according to a verification standard curve.
If the TMAO content in the plasma sample is higher than 1.6ng/mL, the TMAO in the sample is not completely reacted, and at the moment, the sample reaction time needs to be prolonged and then the sampling detection is continued until the TMAO content is equal to or lower than 1.6 ng/mL.
1.7 negative sample preparation: and (3) centrifuging the reaction sample reaching the requirement of the step 1.6 for 20min at 4 ℃ and 4000rpm, taking the supernatant, adding acetonitrile (about one tenth of the volume of the biological sample) to precipitate, centrifuging the reaction sample at 4 ℃ and 12000rpm for 10min, and taking the supernatant to obtain the TMAO negative sample.
1.8TMAO quantitative standard solution preparation: and (3) adding TMAO negative samples prepared by the two methods in the step 1.7 with different concentrations to prepare TMAO quantitative standard solution with the concentration range of 2-500ng/mL, or adding high-concentration TMAO to prepare high-concentration TMAO solution, and diluting the TMAO negative samples prepared in the step 1.7 into TMAO quantitative standard solution with the concentration range of 2-500 ng/mL.
The equipment and parameter information adopted in the embodiment is as follows:
1) apparatus and device
Waters TQ-D Mass Spectrometry System (Watts, USA); waters UPLC I-Class liquid phase system (Watts, U.S.); waters UNIFI software; (Watts, USA) Thermo Fisher Thermo ST16R model high speed refrigerated centrifuge (Sammerfei, USA); XW-80A vortex mixer (Ningbo Xinzhi Biotech Co., Ltd., China); pipettes (Gilson, usa).
2) Reagent and material
Trimethylamine oxide standard: tokyo chemical industryManufactured by tradename, purity 99.5%, lot number: 3 EVJG-MC; deuterated trimethylamine oxide (TMAO-D)9): toronto Research Chemicals, purity 98%, batch number: 3-LXM-155-1; ammonium acetate: analytically pure, national medicine, batch number: 20160310, respectively; acetonitrile: gradient for liquid chromatography, Merck; formic acid: LC grade, ACS.
3) Conditions for liquid chromatography and mass spectrometry
Chromatographic conditions
A chromatographic column: ACQUITYBEH C18(2.1X 50mm, 1.7 μm); mobile phase: 10mM ammonium acetate aqueous solution: 0.1% formic acid in acetonitrile by volume 80:20 (v/v); the flow rate is 0.2 mL/min; column temperature: 30 ℃; sample chamber temperature: 10 ℃; sample introduction amount: 3 mu L of the solution; needle washing liquid: 80% methanol water.
Conditions of Mass Spectrometry
Detecting in positive ion MRM mode by using electrospray ion source (ESI); trimethylamine oxide: monitor ion m/z 75.99 → m/z 58.6; taper hole voltage: 18V, collision energy: 12 eV; Oxetamine-D9: monitor ion m/z 85.1 → m/z 68, cone voltage: 18V, collision energy: 13 eV; capillary voltage: 0.5 kV; taper hole airflow: 60L/h; desolventizing the gas stream: 1000L/h; ion source temperature: 150 ℃; the temperature of the desolvation: at 500 ℃.
The results of this example were analyzed as follows:
the average TMAO concentration of the mixture measured in the step 1.3 of this example is 120.76ng/mL, and after the above steps, the TMAO content finally measured in each group 1.6 is shown in Table 1 and FIG. 1:
TABLE 1
In the above table, 1-4 groups represent the correspondingly numbered microorganisms of step 1.4, and group 5 is a control group.
According to the experimental results, the consumption rates of 1-4 groups of TMAO are obviously higher than those of the 5 th group, which indicates that the efficiency of the microorganism catalysis of TMAO is higher than that of the Rayleigh salt precipitation reaction. Wherein group 2 reached the lower limit of detection by the sixth minute of reaction progress; the groups 1 and 4 reached the lower limit of detection by the time the reaction proceeded to the 8 th minute. Reaction to the tenth minute, the concentration of TMAO in all groups was below the lower limit of detection, at which point the reaction was terminated.
After the experiment is finished, 4.6mL of TMAO negative samples are obtained in groups 1-4 respectively; in group 5, a large amount of precipitate is generated due to the direct reaction of TMAO and Rayleigh salt, and only 3.8mL of TMAO negative sample is obtained.
In addition, the raney salts are extremely toxic compared to microorganisms, and are easily inserted into the eye or adhered to the skin to cause strong irritation; the sample after the reaction of the raney salt is extremely harmful to the water body, and even a small amount of products cannot contact underground water, a water channel or a sewage system. The preparation of TMAO invisible samples by using microorganisms is also superior to the Lee's salt precipitation method from the viewpoint of protecting operators and environment.
In conclusion, the scheme of the invention can rapidly prepare a large amount of TMAO negative samples, and the reaction speed, the preparation efficiency and the environmental protection are all obviously superior to those of the Reye salt precipitation method.
The second embodiment of the invention is a TMAO detection method, which comprises the following steps:
2.1 drawing of quantitative standard curve: taking a proper amount of biological negative samples in example 1, adding TMAO to prepare quantitative standard solutions (except different TMAO contents, the contents of other components are consistent) containing TMAO with different concentrations (2, 5, 10, 20, 50, 100, 500ng/mL), and detecting the TMAO and the internal standard substance in each standard solution by HPLC-MS. And (3) adding an internal standard substance into a sample obtained after 1.5 parts of the group 4 microorganisms in example 1 react for 10min for HPLC-MS test, wherein the detection results of the TMAO and the internal standard substance are shown in figures 2 and 3, the figure 2 is a map of the TMAO in the sample, and the result of the internal standard substance is shown in figure 3. And (3) drawing a standard curve by taking the ratio of the chromatographic peak area of the TMAO in each standard solution of the standard substance prepared after the 4 th group of microorganisms react to the chromatographic peak area of the internal standard substance as a vertical coordinate and the ratio of the concentration of the TMAO in the corresponding standard solution to the concentration of the internal standard substance as a horizontal coordinate, as shown in FIG. 4.
2.2 method accuracy verification: biological negative samples were prepared by repeating the procedure of example 1, and TMAO series samples were prepared at concentrations of 2, 5, 10, 20, 50, 100, 200 and 500ng/mL (sequentially labeled as cure 01 to cure 08), and low, medium and high quality control samples (sequentially labeled as Q1, Q2, Q3) at concentrations of 6, 75 and 400 ng/mL. Quantitative analysis of TMAO content of the system sample was performed according to quantitative standard curves obtained by plotting samples of example 1 in section 2.1, respectively, to verify the accuracy of the method.
2.3 analysis of results:
quantitative standard curve linear relationship:
in fig. 4, the equation obtained by the quantitative standard curve fitting of the negative sample of example 1 is Y ═ 0.37x +0.00685, r2Is 0.9999. The biological negative sample prepared by the scheme of the invention has good linear correlation when used for quantitative detection of TMAO concentration at 2-500ng/mL, thereby showing that the negative sample prepared by the scheme of the invention can obtain wider linear range and lower sensitivity when used for quantitative detection.
And (3) testing accuracy:
TMAO with known concentration is added into the negative sample prepared by the 2.2, and quantitative analysis is carried out by combining with the quantitative standard curve of the 2.1 sample, so as to verify the stability and reliability of the scheme of the invention, and the data is shown in the following table 2:
table 2 accuracy testing experiment data table
| Sample (I) | Theoretical concentration (ng/mL) | Determination of concentration (ng/mL) | Accuracy (%) |
| curve-01 | 2 | 2.008 | 100.40 |
| curve-02 | 5 | 4.899 | 97.98 |
| curve-03 | 10 | 10.175 | 101.75 |
| curve-04 | 20 | 20.028 | 100.14 |
| curve-05 | 50 | 50.829 | 101.66 |
| curve-06 | 100 | 98.534 | 98.53 |
| curve-07 | 200 | 197.729 | 98.86 |
| curve-08 | 500 | 503.289 | 100.66 |
| |
6 | 6.165 | 102.75 |
| |
6 | 5.964 | 99.40 |
| |
6 | 6.202 | 103.37 |
| |
75 | 73.618 | 98.16 |
| |
75 | 74.709 | 99.61 |
| |
75 | 75.143 | 100.19 |
| |
400 | 407.001 | 101.75 |
| |
400 | 394.435 | 98.61 |
| |
400 | 401.261 | 100.32 |
As can be seen from Table 2, the accuracy of the detection method of the embodiment of the invention is between 95% and 105%, and the detection method belongs to a reasonable error range, so that the result of the TMAO quantitative analysis of the biological negative sample detected by the TMAO prepared by the scheme of the invention is accurate and reliable.
In summary, the biological negative sample for detecting TMAO, the preparation method and the application thereof provided by the invention can realize accurate detection of TMAO in the biological sample, and the preparation process is simple and convenient.
In the above embodiments, blood is used as a sample source, and other body fluids may be used as a sample source, which also has similar effects to the above samples.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all equivalent changes made by using the contents of the present specification and the attached drawings, or directly or indirectly applied to the related technical fields, are included in the scope of the present invention.
Claims (9)
1. A method for preparing a biological negative sample, comprising: the method comprises the following steps:
taking a biological sample, adding a microorganism with specificity for removing TMAO into the biological sample, and removing the microorganism after the reaction to obtain the biological negative sample; the volume ratio of the microorganisms to the biological sample is 1: 1000-1: 10000, and the concentration of the microorganisms is 1 x 108~1*1010CFU/mL; the method for removing microorganisms is at least one selected from the group consisting of a precipitant inactivation method and a physical precipitation methodOne kind of the material is selected;
the microorganism is selected from one of Vibrio alginolyticus, non-sulfur photosynthetic bacteria, Escherichia coli and Shewanella putrefaciens;
the biological negative sample meets the following conditions: a) the residual TMAO content value is less than 20% of the lower limit of the required determination standard curve; b) TMAO remains in the biological negative sample, but after exogenous TMAO is added to the biological negative sample, the difference between the actual detected TMAO concentration and the theoretical TMAO concentration after the addition is less than 10%.
2. The method for preparing a biological negative sample according to claim 1, wherein: the microorganism is added into the biological sample in the form of dry powder, sediment, colony or bacteria liquid.
3. The method for preparing a biological negative sample according to claim 1, wherein: the specific operation of the precipitant inactivation method comprises the steps of adding the precipitant for reaction, and then carrying out solid-liquid separation to remove the precipitate.
4. The method for preparing a biological negative sample according to claim 3, wherein: the precipitant is at least one selected from phosphate, oxalate, sulfide, hydroxide, urea or organic solvent.
5. The method for preparing a biological negative sample according to claim 4, wherein: the organic solvent is selected from one of methanol, acetonitrile or acetone.
6. The method for preparing a biological negative sample according to claim 4, wherein: the adding amount of the organic solvent is 1/20-1/5 of the volume amount of the biological sample.
7. A preparation method of a biological standard substance is characterized by comprising the following steps: the method comprises the following steps:
s01, preparing a biological negative sample by the method of any one of claims 1 to 6;
s02, TMAO is quantitatively added to the biological negative sample prepared in the step S01.
8. A TMAO detection method is characterized in that: the method comprises the following steps:
I. preparing a biological negative sample using the method of any one of claims 1 to 6;
II. And (4) detecting the signal of the TMAO in the sample to be detected by taking the signal of the biological negative sample prepared in the step I as a background signal.
9. The TMAO detection method of claim 8, wherein: the detection method is a quantitative detection method, a biological standard substance is prepared by adding quantitative TMAO into a biological negative sample in the detection process, the signal intensity of the TMAO in the biological standard substance is characterized, the TMAO signal intensity under different concentrations is measured, and the TMAO in the sample to be detected is quantitatively analyzed.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107340341A (en) * | 2017-06-27 | 2017-11-10 | 长沙都正生物科技有限责任公司 | A kind of TMAO immue quantitative detection reagent box and method |
| CN108507984A (en) * | 2018-02-28 | 2018-09-07 | 山东大学第二医院 | A kind of method and its application of enzyme process detection trimethylamine oxide TMAO |
| CN109163951A (en) * | 2018-11-05 | 2019-01-08 | 长沙都正医学检验有限责任公司 | A kind of preparation facilities of TMAO negative sample |
| CN109387410A (en) * | 2018-09-27 | 2019-02-26 | 长沙都正医学检验有限责任公司 | A kind of TMAO negative sample and the preparation method and application thereof |
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| CN102533582A (en) * | 2011-09-20 | 2012-07-04 | 北京师范大学 | Efficient degradation bacteria for PBDEs (polybrominated diphenyl ethers) and application thereof |
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| FR2998799B1 (en) * | 2012-11-30 | 2015-09-04 | Univ Dauvergne Clermont I | USE OF MICROORGANISMS TO DECREASE TRIMETHYLAMINE RATE IN INTESTINES, TREATMENT OF TRIMETHYLAMINUREMIA AND PREVENTION OF ATHEROMIC PLATE FORMATION |
| CN105110486B (en) * | 2015-09-01 | 2017-11-03 | 哈尔滨工业大学 | A kind of method that tetracycline in water removal is removed with microbial flocculant |
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-
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- 2020-05-28 CN CN202010465577.5A patent/CN111693687B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107340341A (en) * | 2017-06-27 | 2017-11-10 | 长沙都正生物科技有限责任公司 | A kind of TMAO immue quantitative detection reagent box and method |
| CN108507984A (en) * | 2018-02-28 | 2018-09-07 | 山东大学第二医院 | A kind of method and its application of enzyme process detection trimethylamine oxide TMAO |
| CN109387410A (en) * | 2018-09-27 | 2019-02-26 | 长沙都正医学检验有限责任公司 | A kind of TMAO negative sample and the preparation method and application thereof |
| CN109163951A (en) * | 2018-11-05 | 2019-01-08 | 长沙都正医学检验有限责任公司 | A kind of preparation facilities of TMAO negative sample |
Non-Patent Citations (1)
| Title |
|---|
| 氧化三甲胺代谢相关菌的研究;郭修娟;《中国优秀硕士学位论文全文数据库》;20120415(第2012/04期);摘要，第9页最后一段-第10页第1段，第65页第1段，第68页 * |
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