Analysis method for detecting lipopolysaccharide content in bacteria

Abstract

The invention belongs to the technical field of bacteria detection, and discloses an analysis method for detecting lipopolysaccharide (LPS) in bacteria. The method comprises the following steps: adding an acid solution into a bacterial culture solution, carrying out acidolysis in a water bath of 50-100 DEG C, cooling to room temperature, adding a purifying agent, carrying out vortex, standing for layering, discarding a supernatant, taking a lower-layer bacterial hydrolysate, adding ammonia water, and carrying out vortex mixing uniformly to obtain a standby solution; adding a 4, 5-methylenedioxy-1, 2-phenylenediamine hydrochloride derivating agent solution into the standby liquid, uniformly mixing in a vortex manner, carrying out a derivative reaction at 40-100 DEG C to obtain a 4, 5-methylenedioxy-1, 2-phenylenediamine hydrochloride labeled fluorescent derivative of 3-deoxy-D-mann-2-octanone acid ammonium, and carrying out filter membrane treatment to obtain a filtrate, namely the 4, 5-methylenedioxy-1, 2-phenylenediamine hydrochloride-labeled fluorescent derivative of 3-deoxy-D-mann-2-octanone acid ammonium salt and 4, 5-methylenedioxy-1, 2-phenylenediamine hydrochloride-labeled fluorescent derivative; separating under an isocratic elution condition through a liquid chromatographic column, and detecting the content of lipopolysaccharide in the bacteria through fluorescence. According to the method, the LPS content in the bacteria is detected through a liquid chromatography-fluorescence method, and a new way is provided for bacteria classification and identification.

Description

Technical Field

[0001] The invention belongs to the field of bacteria detection, and in particular relates to an analytical method for detecting lipopolysaccharide content in bacteria. Background Art

[0002] Bacterial lipopolysaccharide (LPS), also known as endotoxin, is a key virulence factor responsible for clinically recognized sepsis, septicemia, and multiple organ failure. Even trace amounts of LPS can cause fever, inflammation, tissue damage, and even death. In intensive, large-scale livestock and poultry farming, the accumulation of endotoxins in livestock housing and the surrounding environment is a major cause of sub-health outcomes and vaccine failure. LPS is composed of three structural domains: lipid A, core polysaccharide, and O-antigen. 3-Deoxy-D-mannose-octulose (Kdo) is a key biomarker within the lipid A molecule. Measuring the free Kdo following LPS hydrolysis reflects the bacterial outer membrane LPS content. Therefore, to minimize the harmful effects of endotoxins on humans and other animals, developing sensitive, accurate, and reliable methods for detecting and analyzing bacterial LPS is crucial.

[0003] Currently, LPS analysis and detection methods are broadly divided into two categories based on their principles. The first category encompasses common methods based on the biological activity characteristics of LPS, such as the rabbit pyrogenicity test, monocyte viability test, Limulus amebocyte lysate test, and biosensor assays. The second category, based on the structural characteristics of LPS itself, utilizes characteristic hydroxy fatty acids in LPS as chemical substances for quantitative analysis, such as chromatography, chromatography-mass spectrometry, and capillary electrophoresis. These methods primarily focus on biological analysis, but they have numerous limitations, including expensive and difficult-to-obtain reagent preparation materials, a narrow linear range for sample analysis, and poor reproducibility of analytical results. Summary of the Invention

[0004] This invention aims to overcome the shortcomings of existing detection technologies and provides an analytical method for detecting lipopolysaccharide in bacteria. This method, based on the principle of acid hydrolysis combined with a derivatization strategy, establishes a high-performance liquid chromatography-fluorescence method with high sensitivity, good stability, and strong operability for the determination of lipopolysaccharide in bacteria.

[0005] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0006] An analytical method for detecting the lipopolysaccharide content in bacteria comprises the following steps:

[0007] S1. Culture the bacteria at a constant temperature of 35-40°C, pick a single colony and inoculate it into LB broth. Cultivate the culture medium at 35-40°C in a shaking incubator until the bacteria reach the logarithmic growth phase.

[0008] S2. Add the bacterial culture to an acid solution and acid-hydrolyze in a water bath at 50-100°C. After cooling to room temperature, add a purifier and vortex. Let stand to separate and discard the supernatant. Take the lower bacterial hydrolyzate and add ammonia water. Vortex and mix to obtain a standby solution.

[0009] S3. Add the reserved solution to the 4,5-methylenedioxy-1,2-phenylenediamine hydrochloride derivatizing agent solution and vortex mix thoroughly. Derivatize at 40-100°C to obtain a fluorescent derivative of 3-deoxy-D-manno-2-octulose acid ammonium (Kdo) labeled with 4,5-methylenedioxy-1,2-phenylenediamine hydrochloride (DMB), abbreviated as Kdo-DMB derivative. Filter through a syringe filter to obtain a filtrate.

[0010] S4. The filtrate is separated by a liquid chromatography column and isocratically eluted with acetonitrile, methanol and pure water as mobile phases, and the content of lipopolysaccharide in the bacteria is detected by fluorescence.

[0011] Preferably, the bacteria in step S1 are Gram-negative bacteria.

[0012] More preferably, the Gram-negative bacteria are Escherichia coli, Salmonella, Pseudomonas aeruginosa, and Klebsiella pneumoniae.

[0013] Preferably, the constant temperature culture time in step S1 is 6 to 24 hours, and the shaking culture time is 2 to 10 hours.

[0014] Preferably, the acid solution in step S2 is hydrochloric acid, sulfuric acid, phosphoric acid or trifluoroacetic acid, and the purifier is n-pentane, n-hexane, heptane, octane or ethyl acetate.

[0015] Preferably, the concentration of the acid solution in step S2 is 0.01-1 mol / L, the concentration of the ammonia water is 0.2-5 mol / L; the volume ratio of the bacterial culture solution to the acid solution is 5:(1-10), the acid hydrolysis time is 30-100 min, and the volume ratio of the purifier to the ammonia water is (1-6) mL:(50-500) μL.

[0016] Preferably, the 4,5-methylenedioxy-1,2-phenylenediamine hydrochloride derivatizing agent solution in step S3 is prepared by adding 4,5-methylenedioxy-1,2-phenylenediamine hydrochloride to β-mercaptoethanol and using a hydrochloric acid solution; the concentration of the 4,5-methylenedioxy-1,2-phenylenediamine hydrochloride derivatizing agent solution is 0.4-1.5 mmol / L; the volume ratio of the 4,5-methylenedioxy-1,2-phenylenediamine hydrochloride derivatizing agent solution to the reserve solution is (1-10):4, the derivatization reaction time is 0.5-2 h, and the diameter of the organic filter membrane is 0.22 μm.

[0017] Preferably, the volumes of acetonitrile, methanol and pure water in step S4 are (0-1):(2-4):(5-8).

[0018] Preferably, the column temperature of the liquid chromatography in step S4 is 15-35° C., the flow rate is 0.4-1.2 mL / min, the injection volume is 2-10 μL, the excitation wavelength of the fluorescence detection is 300-400 nm, and the emission wavelength is 420-465 nm.

[0019] The analytical method is used to detect the lipopolysaccharide content in bacteria.

[0020] The lipopolysaccharide (LPS) described in this study is composed of three structural domains: lipid A, core polysaccharide, and O-antigen. 3-Deoxy-D-mannose-octulose (KdO) is an important biomarker within the lipid A molecule. Measuring the free KdO after LPS hydrolysis can reflect the LPS content in the bacterial outer membrane. 4,5-Methylenedioxy-1,2-phenylenediamine (DMB) is a carbonyl derivatization reagent that reacts with α-keto acids such as KdO to form fluorescent derivatives. Based on this, a sensitive, reliable, and stable high-performance liquid chromatography-fluorimetry method for the detection of LPS in bacteria was established by optimizing bacterial culture conditions and hydrolysis and derivatization reaction conditions. The derivatives were separated on an Agilent HB-C8 column (250×4.6 mm ID, 5 μm) and measured using a fluorescence detector. KdO content was quantitatively analyzed using the standard addition method, thereby indirectly detecting the LPS content in the bacteria.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. The present invention hydrolyzes lipopolysaccharide in an acid solution to release Kdo, which then reacts with the derivatizing agent DMB at 40-100°C to generate a Kdo-DMB derivative with stable fluorescence.

[0023] 2. This method uses LB broth to culture Gram-negative bacteria such as Escherichia coli, Salmonella, Pseudomonas aeruginosa, and Klebsiella pneumoniae. By optimizing the reaction conditions for the hydrolysis of lipopolysaccharide (LPS) and the derivatization of the characteristic product KdO, a KdO-DMB derivative containing a fluorescent probe was developed for indirect determination of LPS in bacteria using high-performance liquid chromatography-fluorescence detection. This method is highly sensitive and operational, providing a new approach for the classification and identification of bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the effect of hydrochloric acid and trifluoroacetic acid on the hydrolysis of lipopolysaccharide in Example 1.

[0025] Figure 2 The following are the effects of different hydrolysis conditions on the reaction in Example 1.

[0026] Figure 3 Schematic diagram of the derivatization reaction of Kdo and DMB.

[0027] Figure 4 The figure shows the effects of different derivatization conditions on the derivatization reaction in Example 1.

[0028] Figure 5 This is the mass spectrometry structure identification diagram of the Kdo-DMB derivative in Example 1.

[0029] Figure 6 This is a typical HPLC chromatogram of the selectivity study of Example 2. DETAILED DESCRIPTION

[0030] The present invention will be further described below in conjunction with specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0031] The instruments used in the examples are as follows: Agilent 1260 series high performance liquid chromatograph with fluorescence detector (Agilent Technologies, USA); triple quadrupole mass spectrometer, API 5500 (Applied Biosystems, USA); AUW120D electronic analytical balance (sensitivity 0.00001 g, Shimadzu Corporation, Japan); YP10002B electronic balance (sensitivity 0.01 g, Shanghai Lichen Bangxi Instrument Technology Co., Ltd.); Thermos D-37520 high-speed refrigerated centrifuge (Thermo Scientific, USA); constant temperature incubator (Thermo Scientific, USA) Scientific Company); MTX-2500 mini mixer (Shanghai Qiqian Electronic Technology Co., Ltd.); SHA-B constant temperature water bath oscillator (Changzhou Aohua Instrument Co., Ltd.); DHG-9055A electric blast drying oven (Shanghai Yiheng Scientific Instrument Co., Ltd.); KH7200DB CNC ultrasonic cleaner (Jiangsu Kunshan Hechuang Ultrasonic Instrument Co., Ltd.); Milli-Q Advantage A10 ultrapure water system (Millipore Corporation, USA); WD-12 nitrogen blow dryer (Hangzhou Aosheng Instrument Co., Ltd.); HHVE-50 high pressure sterilizer (HIRAYAMA, Japan); THZ-C constant temperature shaker (Guangzhou Shenhua Biotechnology Co., Ltd.); PE multifunctional enzyme label analyzer (PerkinElmer Co., Ltd.); Haier -20℃ upright freezer (Haier Group, China); nylon 66 filter membrane (pore size 0.22 μm, Tianjin Jinteng Company).

[0032] The following drugs were used in the examples: MacConkey agar powder medium (containing glucose), LB agar powder medium (containing glucose), and LB broth (containing glucose) were purchased from Guangdong Huankai Microbiological Technology Co., Ltd.; 3-deoxy-D-manno-2-octulose acid ammonium reference substance, 4,5-methylenedioxy-1,2-phenylenediamine dihydrochloride, and bacterial lipopolysaccharide standard were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; methanol (chromatographic grade), acetonitrile (chromatographic grade), β-mercaptoethanol (chromatographic grade), sodium dithionite (analytical grade), and trifluoroacetic acid (analytical grade) were purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; sodium hydroxide (analytical grade), n-pentane (analytical grade), n-hexane (analytical grade), ethyl acetate (analytical grade), and concentrated hydrochloric acid (superior grade) were purchased from Guangzhou Chemical Reagent Factory; and water for bacterial endotoxin testing was purchased from Zhanjiang Bokang Marine Biological Co., Ltd. Water for bacterial endotoxin testing is water specifically used for bacterial endotoxin testing and is typically purified water that has been rigorously treated and filtered to ensure that no substances that may interfere with or contaminate the results are introduced during the test process.

[0033] Some of the Gram-negative bacteria, including Escherichia coli, Salmonella, Pseudomonas aeruginosa, and Klebsiella pneumoniae, were preserved in our laboratory, and some were purified and isolated from a poultry farm in Guangdong Province, totaling 200 strains.

[0034] Example 1 Selection and optimization of sample pretreatment conditions

[0035] 1. Determination of bacterial culture time: In order to explore the specific culture time when bacteria grow best and ensure that lipopolysaccharide is not easily released during the bacterial culture process, the 24-hour growth curve of the bacterial standard strain corresponding to the wild strain in the application part was measured and analyzed. Finally, the optimal bacterial culture time of 6 hours was selected as the sampling time for the subsequent hydrolysis test.

[0036] 2. Optimization of hydrolysis conditions: From the hydrolysis tests of various acids and enzyme preparations, hydrochloric acid and trifluoroacetic acid were screened out as relatively better. Therefore, further comparative tests were conducted on the two acids. Figure 1 The effects of hydrochloric acid and trifluoroacetic acid on hydrolysis in Example 1 are shown in Figure 1. a and b are chromatograms of derivatives obtained by hydrochloric acid hydrolysis and trifluoroacetic acid hydrolysis, respectively. Figure 1 As can be seen from the results, the peak area of the target compound obtained by trifluoroacetic acid hydrolysis is higher than that obtained by hydrochloric acid hydrolysis, but there are more interfering peaks and the chromatographic resolution is also lower than that obtained by hydrochloric acid hydrolysis. Therefore, hydrochloric acid was ultimately selected as the hydrolysis solution. Subsequently, based on the hydrochloric acid hydrolysis, the time, temperature, and hydrochloric acid concentration that affect the hydrolysis were optimized. Figure 2 The effects of different hydrolysis conditions on the reaction in Example 1 are shown below. Wherein, a, b, and c correspond to the hydrolysis time, hydrolysis temperature, and hydrochloric acid concentration conditions, respectively. Figure 2It can be seen that 0.1 mol / L hydrochloric acid, hydrolysis temperature of 80 °C and hydrolysis time of 80 min are selected as the optimal hydrolysis reaction conditions.

[0037] 3. Optimization of Purification Conditions: Since the hydrolysis of samples using 0.1 mol / L hydrochloric acid solution easily produces a large amount of fat-soluble impurities, which interfere with subsequent HPLC detection, preliminary purification of the hydrolyzate is an effective measure to reduce or avoid impurity interference. Purifiers immiscible with the hydrolyzate, including n-pentane, n-hexane, heptane, octane, and ethyl acetate, were compared to determine their purification effects and their impact on recovery. The results showed that alkanes performed better than ethyl acetate in purification, but heptane provided the highest recovery. Therefore, heptane was ultimately selected as the purification agent.

[0038] 4. Optimization of derivatization reaction conditions: Figure 3 Schematic diagram of the derivatization reaction between Kdo and DMB, where Kdo represents 3-deoxy-D-manno-2-octulose acid ammonium and DMB represents 4,5-methylenedioxy-1,2-phenylenediamine dihydrochloride. Under acidic conditions, DMB maximizes its reaction with α-ketoacid structures, resulting in a more stable product. The effects of the derivatization agent dosage, derivatization time, and derivatization temperature on the derivatization reaction were investigated under hydrochloric acid conditions at pH 2. Figure 4 The effect of a single factor on the derivatization reaction in Example 1. Among them, a, b, and c are the amount of derivatizing agent, derivatization temperature, and derivatization time, respectively. Figure 4 As can be seen from the data, when the derivatization agent dosage of DMB was 100 μL, the derivative peak area reached its highest level and then declined. With increasing derivatization temperature, the peak area of the target analyte gradually decreased, and the optimal temperature for this derivatization reaction was 80°C. With increasing derivatization time, the peak area of the target analyte also gradually decreased, with a negative correlation between the two. The optimal derivatization reaction time was 60 minutes. Taking all these factors into consideration, the optimal derivatization reaction conditions for fluorescent labeling of Kdo with DMB were pH = 2, 100 μL of derivatization agent, 80°C, and 60 minutes. Figure 5 Figure 2 is the mass spectrometry structure identification diagram of the Kdo-DMB derivative in Example 1. Figure 5 It can be seen that the molecular ion peak [MH + ] - The fragment ion m / z 203.1 is the result of σ bond cleavage at the methylene group of m / z 353.0 (red mark at ①) and CC bond closure (hydroxycyclopropyl) with the carbon in C=O. The fragment ion m / z 245.0 is the result of dehydration of the hydroxyl group of m / z 353.0, σ bond cleavage (green mark at ②) and proton transfer.

[0039] Example 2

[0040] 1. Preparation of standard solution

[0041] (a) Dissolve 3-deoxy-D-manno-2-octanolone (Kdo) standard in bacterial endotoxin test water. Transfer to a 10-mL brown glass volumetric flask and dilute to volume to prepare a 1 mg / mL Kdo standard solution. Store in a -20°C refrigerator protected from light.

[0042] (b) Dissolve the LPS standard in a small amount of bacterial endotoxin test water, transfer to a 10 mL brown glass volumetric flask, and dilute to volume to prepare a 1 mg / mL LPS standard solution. Store in a -20°C refrigerator protected from light.

[0043] 2. Solution Preparation

[0044] (a) Weigh 7.9 mg of 4,5-methylenedioxy-1,2-phenylenediamine hydrochloride (DMB) derivatizing agent and add 3.5 mL of β-mercaptoethanol. Dose 0.01 mol / L hydrochloric acid to make 50 mL of DMB derivatizing agent solution with a concentration of 0.7 mmol / L.

[0045] (b) Prepare 100 mL of 0.01 mol / L hydrochloric acid by adding 83 μL of concentrated hydrochloric acid (12 mol / L) to 100 mL of ultrapure water.

[0046] (c) Add 2.7 mL of concentrated aqueous ammonia to 37.3 mL of ultrapure water and vortex to mix thoroughly to prepare 40 mL of aqueous ammonia with a concentration of 1 mol / L.

[0047] (d) Weigh 18.0 g of LB agar powder and add it to 500 mL of deionized water, stirring to dissolve. Sterilize the mixture at 121°C for 20 min. When cooled to approximately 60°C, pour the mixture into a 90 mm sterilized Petri dish and allow it to solidify into LB agar medium for later use.

[0048] (e) Weigh 10.5 g of LB broth powder and add it to 500 mL of deionized water. Stir to dissolve the mixture, then autoclave at 121°C for 20 min to prepare LB broth.

[0049] 3. Sample Pretreatment

[0050] Use a 1 μL inoculating loop to scrape a bacterial sample and streak it on an LB agar plate. Incubate it in a constant temperature incubator at 37°C for 16-18 hours. Pick a single colony and inoculate it into 5 mL of LB broth medium. Incubate it in a shaker at 37°C for 6 hours until the bacteria reach the logarithmic growth phase to obtain a bacterial culture solution. Take 500 μL of the bacterial culture solution in the logarithmic growth phase and LB broth medium (as a blank control) into a 15 mL sterile, enzyme-free centrifuge tube, add 500 μL of 0.1 mol / L hydrochloric acid, and acidify it in an 80°C water bath for 80 minutes. After cooling to room temperature, add 3 mL of heptane and vortex for 2 minutes. Let it stand until stratification occurs and discard the supernatant. Take 200 μL of the lower layer of hydrolyzate into a 2 mL centrifuge tube, add 200 μL of 1 mol / L ammonia water, vortex mix and set aside; take 100 μL of the reserve solution into a brown glass graduated test tube, add 100 μL of 0.7 mmol / L DMB derivatization agent solution, vortex mix and react at 80 ° C for 1 h, cool, and pass the derivatized product through a 0.22 μm organic filter membrane, transfer to a brown injection bottle, and wait for liquid chromatography analysis.

[0051] 4. Chromatographic conditions: An Agilent HB-C8 column (250 × 4.6 mm id, 5 μm) was used. The column temperature was 25°C, the flow rate was 0.8 mL / min, the mobile phase A was acetonitrile, the mobile phase B was methanol, and the mobile phase C was water. The elution procedure was isocratic, and the volume ratio of phases A, B, and C was 9:21:71. The fluorescence detection was performed at an excitation wavelength of 371 nm and an emission wavelength of 451 nm.

[0052] 5. Selectivity investigation test: Using blank LB broth culture medium after high-pressure sterilization as blank sample, 20 blank LB broths from different sources were collected, and the sample pretreatment steps were followed. The blank samples were analyzed by high-performance liquid chromatography to investigate the selectivity of the analytical method.

[0053] Figure 6 The following is a typical HPLC chromatogram of the selectivity study of Example 2, wherein a is a blank of the Kdo standard reagent; b is a blank of the Kdo standard reaction process; c is a blank of the DMB derivatization reagent; d is a blank of the DMB derivatization reaction process; and e is a sample of blank LB broth medium supplemented with Kdo (the Kdo concentration is 0.1 μg / mL, and the chromatographic peak of the Kdo-DMB derivative is indicated by the arrow). Figure 6 The chromatographic analysis results showed that blank samples a, b, c and d did not show any interfering peaks at ±2.5% of the retention time of the Kdo-DMB derivative (e), indicating that the method had good selectivity.

[0054] Example 3 Standard Curve, Detection Limit and Quantification Limit

[0055] According to Example 2, LB broth was hydrolyzed and purified to prepare a blank LB broth extract. Seven portions of 100 μL were taken, and 100 μL of 0.2, 0.4, 1.0, 2.0, 4.0, 10.0, and 20.0 μg / mL Kdo standard solutions were added in sequence. After vortex mixing, the target analyte was treated according to the derivatization method of Example 2 to prepare 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, and 10.0 μg / mL Kdo derivatized standard solutions for HPLC analysis. The peak area of the chromatogram of the target analyte was used as the ordinate and the corresponding standard solution content was used as the abscissa. Linear regression analysis was performed to obtain the regression equation and correlation coefficient (r 2 The results showed that when the concentration of Kdo standard solution was in the range of 0.1 to 10 μg / mL, there was a good linear relationship between the target analyte content (x) and its corresponding peak area (y). The fitting linear equation was: y = 3.23x + 0.645, and the correlation coefficient r 2 =0.9983.

[0056] To 100 μL of blank LB broth culture medium, 20-100 μL of a 0.1-10 μg / mL Kdo standard solution was added. The solution was hydrolyzed, purified, and derivatized according to the method described in Example 2, and then analyzed by high-performance liquid chromatography. The detection and quantification limits of the method were determined based on the concentration of Kdo that produced a signal-to-noise ratio (S / N) greater than or equal to 3 and 10, respectively. The results showed that the detection and quantification limits of Kdo in LB broth were 0.04 μg / mL and 0.1 μg / mL, respectively, demonstrating the high sensitivity of this method, which meets the requirements for detecting lipopolysaccharide in bacteria.

[0057] Example 4 Accuracy and Precision

[0058] Multiple 100 μL tubes of blank LB broth were spiked with 10 to 50 μL of a 0.2 to 2.0 μg / mL Kdo standard solution. Kdo-spiked sample solutions at concentrations of 0.1, 0.2, and 1.0 μg / mL were prepared. Six replicates were run at each concentration level, resulting in three batches. The samples were processed according to the method described in Example 2 and analyzed by HPLC. The accuracy of the method was evaluated by calculating the average sample recoveries, as shown in Table 1. As shown in Table 1, the average intra-batch recoveries of Kdo in LB broth at the three spike levels of 0.1 μg / mL, 0.2 μg / mL, and 1.0 μg / mL were 86.2 to 99.6% within the batches, and 90.5 to 94.6% between the batches. The intra-batch and inter-batch relative standard deviations (RSDs) were less than 11.2% and 10.8%, respectively.

[0059] Table 1 Recovery and precision of Kdo in bacteria

[0060]

[0061] Application Example 1

[0062] 200 selected wild-type Gram-negative bacterial strains were cultured in LB broth with shaking for 6 hours. Chromatographic analysis was performed according to the pretreatment method steps in Example 2 to obtain the Kdo concentration in the actual sample. Assuming that each lipopolysaccharide molecule contains two Kdo groups, and assuming that the relative molecular mass of lipopolysaccharide is 10,000, the amount of the introduced substance and the relative molecular mass ratio of the two were used to calculate the lipopolysaccharide content in the bacterial sample.

[0063]

[0064] Where: m LPS is the mass of lipopolysaccharide, in μg

[0065] C Kdo The mass concentration of Kdo was measured by HPLC, in μg / mL;

[0066] M LPS is the relative molecular mass of lipopolysaccharide (10000);

[0067] M Kdo is the relative molecular mass of Kdo (238.07);

[0068] V sample is the sample collection volume for pretreatment, in μL;

[0069] K is the dilution factor during the hydrolysis process.

[0070] The wet weight method for bacteria was used to determine the lipopolysaccharide content per unit mass ( / g) of bacteria (wet weight). The strain was inoculated into 5 mL of LB broth and cultured at 37°C with shaking for 6 hours. The resulting bacterial suspension was centrifuged at 5000 rpm for 10 minutes, and the supernatant was discarded. The wet weight of the bacteria in the 5 mL of culture at 6 hours was 3 mg. The actual sample volume was then used for calculation to determine the lipopolysaccharide content per gram of bacteria (wet weight).

[0071] According to the calculation, the lipopolysaccharide content per gram of bacteria (wet weight) in the 200 wild-type Gram-negative bacteria was 3.67×10 1 ~3.08×10 2 The lipopolysaccharide content of the most widely distributed 177 strains of Escherichia coli in the environment was consistent with the content range of the 200 negative bacteria. The lipopolysaccharide content of the remaining 23 strains of Klebsiella pneumoniae was 9.80×10 1 ~2.18×102 μg / g.

[0072] In summary, the present invention establishes a sensitive, accurate, and reliable liquid chromatography-fluorescence analysis method for the indirect detection of lipopolysaccharide in bacteria based on the high-temperature hydrolysis of bacterial liquid in a hydrochloric acid solution and the derivatization with 4,5-methylenedioxy-1,2-phenylenediamine dihydrochloride (DMB). Based on this method, the lipopolysaccharide content level in Gram-negative bacteria such as Escherichia coli and Klebsiella pneumoniae can be calculated, providing a new possible approach for the identification and classification of bacteria.

[0073] The above embodiments are preferred implementations of the present invention, but the implementations of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. An analytical method for detecting the content of lipopolysaccharide in bacteria, characterized in that: The following steps are involved: S1. Culture the bacteria at a constant temperature of 35-40°C, pick a single colony and inoculate it into LB broth. Cultivate the culture medium at 35-40°C in a shaking incubator until the bacteria reach the logarithmic growth phase. S2. Add the bacterial culture to an acid solution and acid-hydrolyze in a water bath at 50-100°C. After cooling to room temperature, add a purifier and vortex. Let stand to separate and discard the supernatant. Take the lower bacterial hydrolyzate and add ammonia water. Vortex and mix to obtain a standby solution. S3. Add the reserved solution to the 4,5-methylenedioxy-1,2-phenylenediamine hydrochloride derivatizing agent solution and vortex mix thoroughly. Derivatize at 40-100°C to obtain a fluorescent derivative of 3-deoxy-D-manno-2-octulose acid ammonium labeled with 4,5-methylenedioxy-1,2-phenylenediamine hydrochloride, abbreviated as Kdo-DMB derivative. Filter through a syringe filter to obtain a filtrate. S4. The filtrate is separated by a liquid chromatography column and isocratically eluted with acetonitrile, methanol and pure water as mobile phases, and the content of lipopolysaccharide in the bacteria is detected by fluorescence.

2. The analytical method for detecting lipopolysaccharide content in bacteria according to claim 1, characterized in that: The bacteria described in step S1 are Gram-negative bacteria.

3. The analytical method for detecting lipopolysaccharide content in bacteria according to claim 2, characterized in that: The Gram-negative bacteria are Escherichia coli, Salmonella, Pseudomonas aeruginosa or Klebsiella pneumoniae.

4. The analytical method for detecting lipopolysaccharide content in bacteria according to claim 1, characterized in that: The constant temperature culture time in step S1 is 6 to 24 hours, and the shaking culture time is 2 to 10 hours.

5. The analytical method for detecting lipopolysaccharide content in bacteria according to claim 1, characterized in that: In step S2, the acid solution is hydrochloric acid, sulfuric acid, phosphoric acid or trifluoroacetic acid, and the purifier is n-pentane, n-hexane, heptane, octane or ethyl acetate.

6. The analytical method for detecting lipopolysaccharide content in bacteria according to claim 1, characterized in that: In step S2, the concentration of the acid solution is 0.01 to 1 mol / L, and the concentration of the ammonia water is 0.2 to 5 mol / L; the volume ratio of the bacterial culture solution to the acid solution is 5:(1 to 10), the acid hydrolysis time is 30 to 100 minutes, and the volume ratio of the purifier to the ammonia water is (1 to 6) mL:(50 to 500) μL.

7. The analytical method for detecting lipopolysaccharide content in bacteria according to claim 1, characterized in that: The 4,5-methylenedioxy-1,2-phenylenediamine hydrochloride derivatizing agent solution in step S3 is prepared by adding 4,5-methylenedioxy-1,2-phenylenediamine hydrochloride to β-mercaptoethanol and using a hydrochloric acid solution; the concentration of the 4,5-methylenedioxy-1,2-phenylenediamine hydrochloride derivatizing agent solution is 0.4 to 1.5 mmol / L; the volume ratio of the 4,5-methylenedioxy-1,2-phenylenediamine hydrochloride derivatizing agent solution to the reserve solution is (1 to 10):4; the derivatization reaction time is 0.5 to 2 hours; and the diameter of the organic filter membrane is 0.22 μm.

8. The analytical method for detecting lipopolysaccharide content in bacteria according to claim 1, characterized in that: The volumes of acetonitrile, methanol and pure water in step S4 are (0-1):(2-4):(5-8).

9. The analytical method for detecting lipopolysaccharide content in bacteria according to claim 1, characterized in that: The temperature of the liquid chromatography column in step S4 is 15-35° C., the flow rate is 0.4-1.2 mL / min, the injection volume is 2-10 μL, the excitation wavelength of the fluorescence detection is 300-400 nm, and the emission wavelength is 420-465 nm.

10. Use of the analytical method according to any one of claims 1 to 9 in detecting the lipopolysaccharide content in bacteria.

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