A method, reagent and kit for determining the reducing sugar content in pneumococcal activated sugar based on MBTH method

Abstract

The present application relates to the technical field of material detection, and specifically discloses a method, reagent and kit for determining the reducing sugar content in pneumococcal activated sugar based on the MBTH method. The method comprises the following steps: (1) establishing a linear regression equation; (2) determining the reducing sugar content: diluting the sample to be tested with water to obtain a sample solution to be tested; using water as a blank control, and determining the absorbance of the sample solution to be tested after pretreatment at a wavelength of 650nm; according to the linear regression equation obtained in step (1), calculating the concentration of the abscissa corresponding to the absorbance of the sample solution to be tested on the standard curve; the concentration of reducing sugar in the sample to be tested = the concentration of the sample solution to be tested × the dilution multiple of the sample to be tested. The reagents include hydrochloric acid solution, MBTH aqueous solution, and ferric chloride aqueous solution. The kit includes the above reagents. The present application improves the sensitivity of determining reducing sugars.

Description

Technical Field

[0001] The present application relates to the technical field of substance detection, and in particular to a method, reagent and kit for determining the reducing sugar content in pneumococcal activated sugar based on the MBTH method. Background Art

[0002] Streptococcus pneumoniae is an important human pathogen that can cause invasive diseases such as pneumonia, meningitis, bacteremia and otitis media. Infants and the elderly are the main susceptible populations. Every year, about 700,000 to 1 million children under the age of 5 die from diseases caused by Streptococcus pneumoniae. The most important virulence factor of Streptococcus pneumoniae is capsular polysaccharide. Polysaccharide is a T-cell-independent antigen, and the immune system of children under the age of 2 is not fully developed, lacking the ability to respond to T-cell-independent antigens. What is more serious is that the incidence of pathogenic pneumococcal infection is highest in children under the age of 2. However, the conjugate vaccine formed by covalently binding capsular polysaccharide to carrier protein can convert polysaccharide from T-cell-independent antigen to T-cell-dependent antigen, and can produce good immunogenicity and long-term protection in infants and young children.

[0003] The binding between polysaccharide antigen and protein antigen is achieved through covalent bonding using functional groups such as carboxyl, hemiacetal, amino / imino, sulfhydryl / disulfide bond, and hydroxyl groups in polysaccharide and protein molecules. This covalent bonding requires the polysaccharide to be activated first. The activators used at home and abroad include NaIO 4 , CNBr and CDAP, in which sodium periodate is used to oxidize the hydroxyl groups on the polysaccharide molecules and convert them into aldehyde groups or ketone groups to react with amino groups in proteins to form Schiff bases. The Schiff bases formed can be reduced to stable glycoprotein conjugates under the action of reducing agents (such as sodium cyanoborohydride). This binding method is called reductive amination. The other is cyanation, the principle of which is to use cyanogen bromide or 1-cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP) as activators, which use CNBr or CDAP to activate the hydroxyl groups on the polysaccharide molecules under alkaline conditions to form cyanate esters, and then react with adipic dihydrazide (ADH) to introduce ester hydrazide (AH) groups into polysaccharide molecules to form PS-AH derivatives, and finally form stable conjugates with protein carriers under the mediation of carbodiimide.

[0004] The amine reduction method is used in the conjugation process of pneumococcal polysaccharide conjugates. The most critical step is the oxidation of polysaccharides with sodium periodate. If the experimental conditions are not well controlled during the oxidation process, overoxidation will occur, causing the hydroxyl groups to be oxidized to carboxyl groups, and then the binding will decrease when binding to proteins. In addition, studies have shown that as the degree of oxidation increases, oxidation will cause more severe damage to the polysaccharide structure and greater damage to the antigenicity of the polysaccharide, so the degree of polysaccharide oxidation should be controlled within a certain range. Since sodium periodate oxidizes polysaccharides into aldehyde or ketone groups, the determination of the reducing sugar content can reflect the degree of oxidation of polysaccharides.

[0005] The main methods for determining reducing sugars are the 3,5-dinitrosalicylic acid (DNS) method, the potassium ferrocyanide method and the 3-methyl-2-benzothiazolinone hydrazone hydrochloride hydrate (MBTH) method. The precision and recovery of the above three methods are similar, but the MBTH method has the highest sensitivity for determining reducing sugars.

[0006] However, in the relevant reports, the MBTH method uses xylose as the reference substance, and the concentration of the reference substance solution is 50 μg / ml xylose solution; the specific determination method is to accurately pipette 0, 50, 100, 150, 200, 250, 300, 350 μl of xylose reference substance solution, add distilled water to 600 μl, add 0.5 mol / L NaOH solution 600 μl and MBTH reagent (3 mg / ml MBTH solution and 1 mg / ml DTT solution are mixed in equal amounts) 600 μl, heat in 80°C water bath for 15 min, and then add ammonium ferric sulfate reagent (0.5 wt% [FeNH 4 (SO 4 ) 2 ]·12H 2 0, 0.5 wt% aminosulfonic acid, 0.5 mol / L hydrochloric acid) 1.2 ml, mix well, cool to room temperature and measure absorbance at 650 nm. The detection limit of xylose measured by this method is 0.3 μg / ml (the detection limit concentration converted to aldehyde group is 1.998 nmol / ml). Summary of the invention

[0007] In order to improve the sensitivity of reducing sugar determination, the present application provides a method, reagent and kit for determining the reducing sugar content in pneumococcal activated sugar based on the MBTH method.

[0008] In the first aspect, the present application provides a method for determining the reducing sugar content in pneumococcal activated sugar based on the MBTH method, which adopts the following technical scheme:

[0009] A method for determining the reducing sugar content in pneumococcal activated sugar, characterized in that the method comprises the following steps:

[0010] (1) Establishing the linear regression equation

[0011] Preparation of gradient standard solution: prepare glyceraldehyde as a standard substance into a gradient standard solution with a concentration of (40-320) nmol / ml;

[0012] Pretreatment of the gradient standard solution: first, add a hydrochloric acid solution with a concentration of (0.05-0.15) mol / L and an MBTH aqueous solution with a concentration of (0.2-0.3) wt% to the gradient standard solution, seal and mix, then react at a temperature of 35-40°C (for example: 37°C) for 20-40 minutes, cool to 20-30°C (for example: 20°C, 21°C, 21°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C); then add a hydrochloric acid solution with a concentration of (0.05-0.15) mol / L and an MBTH aqueous solution with a concentration of (0.2-0.3) wt% to the gradient standard solution, respectively. 7-1.0) wt% ferric chloride aqueous solution is sealed and mixed, then reacted at a temperature of 35-40° C. (for example, 37° C.) for 3-10 min, and cooled to 20-30° C. (for example, 20° C., 21° C., 21° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C., 30° C.); wherein the volume ratio of the gradient standard solution, the hydrochloric acid solution, the MBTH aqueous solution and the ferric chloride aqueous solution is 1:(2.925-3.575):(0.9-1.1):(0.9-1.1);

[0013] Obtaining the linear regression equation: using water as a blank control and measuring the absorbance of the pretreated gradient standard solution at a wavelength of 650 nm, plotting a standard curve with the concentration of the gradient standard solution as the abscissa and the absorbance of the gradient standard solution as the ordinate, and calculating the linear regression equation;

[0014] (2) Determination of reducing sugar content

[0015] Preparation of the sample solution to be tested: dilute the sample to be tested with water to obtain the sample solution to be tested;

[0016] Pretreatment of the sample solution to be tested: first, add a hydrochloric acid solution with a concentration of (0.05-0.15) mol / L and a MBTH aqueous solution with a concentration of (0.2-0.3) wt% to the sample solution to be tested, seal and mix, then react at a temperature of 35-40°C (for example: 37°C) for 20-40 minutes, cool to 20-30°C (for example: 20°C, 21°C, 21°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C); then ... 7-1.0) wt% ferric chloride aqueous solution is sealed and mixed, then reacted at a temperature of 35-40° C. (for example, 37° C.) for 3-10 min, and cooled to 20-30° C. (for example, 20° C., 21° C., 21° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C., 30° C.); wherein the volume ratio of the sample solution to be tested, the hydrochloric acid solution, the MBTH aqueous solution and the ferric chloride aqueous solution is 1:(2.925-3.575):(0.9-1.1):(0.9-1.1);

[0017] Obtaining the reducing sugar content: using water as a blank control and measuring the absorbance of the pretreated sample solution at a wavelength of 650 nm; calculating the concentration of the absorbance of the sample solution on the standard curve corresponding to the horizontal axis according to the linear regression equation obtained in step (1);

[0018] The concentration of reducing sugar in the sample to be tested = the concentration of the sample solution to be tested × the dilution factor of the sample to be tested.

[0019] Optionally, in step (1), the number of the gradient standard solutions is at least 3, for example: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc. The concentrations of the gradient standard solutions can be 40nmol / ml, 80nmol / ml, 120nmol / ml, 160nmol / ml, 200nmol / ml, 240nmol / ml, 280nmol / ml, 320nmol / ml, respectively.

[0020] Optionally, in step (1), the concentration of the hydrochloric acid solution is (0.08-0.12) mol / L, for example: 0.09 mol / L, 0.10 mol / L, 0.11 mol / L.

[0021] Optionally, in step (1), the concentration of the MBTH aqueous solution is (0.23-0.27) wt%, for example: 0.24 wt%, 0.25 wt%, 0.26 wt%.

[0022] Optionally, in step (1), the concentration of the ferric chloride aqueous solution is (0.8-0.9) wt%, for example: 0.81 wt%, 0.82 wt%, 0.83 wt%, 0.84 wt%, 0.85 wt%, 0.86 wt%, 0.87 wt%, 0.88 wt%, 0.89 wt%.

[0023] Optionally, in step (1), the volume ratio of the gradient standard solution, the hydrochloric acid solution, the MBTH aqueous solution and the ferric chloride aqueous solution is 1:(3.0875-3.4125):(0.95-1.05):(0.95-1.05), for example: the volume ratio of the gradient standard solution, the hydrochloric acid solution, the MBTH aqueous solution and the ferric chloride aqueous solution is 1:3.25:1:1.

[0024] Optionally, the linear regression equation obtained in step (1) is y=0.00532x+0.02236.

[0025] Optionally, the goodness of fit R of the linear regression equation y=0.00532x+0.02236 2 =0.99908.

[0026] Optionally, in step (2), the concentration of the hydrochloric acid solution is (0.08-0.12) mol / L, for example: 0.09 mol / L, 0.10 mol / L, 0.11 mol / L.

[0027] Optionally, in step (2), the concentration of the MBTH aqueous solution is (0.23-0.27) wt%, for example: 0.24 wt%, 0.25 wt%, 0.26 wt%.

[0028] Optionally, in step (2), the concentration of the ferric chloride aqueous solution is (0.8-0.9) wt%, for example: 0.81 wt%, 0.82 wt%, 0.83 wt%, 0.84 wt%, 0.85 wt%, 0.86 wt%, 0.87 wt%, 0.88 wt%, 0.89 wt%.

[0029] Optionally, in step (2), the volume ratio of the sample solution to be tested, the hydrochloric acid solution, the MBTH aqueous solution and the ferric chloride aqueous solution is 1:(3.0875-3.4125):(0.95-1.05):(0.95-1.05), for example: the volume ratio of the sample solution to be tested, the hydrochloric acid solution, the MBTH aqueous solution and the ferric chloride aqueous solution is 1:3.25:1:1.

[0030] In a second aspect, the present application provides a reagent for determining the reducing sugar content in pneumococcal activated sugar based on the MBTH method, which adopts the following technical scheme:

[0031] A reagent for determining the reducing sugar content in pneumococcal activated sugar, the reagent is based on the above method, and the reagent comprises a hydrochloric acid solution with a concentration of (0.05-0.15) mol / L, an MBTH aqueous solution with a concentration of (0.2-0.3) wt%, and a ferric chloride aqueous solution with a concentration of (0.7-1.0) wt%.

[0032] In a third aspect, the present application provides a kit for determining the reducing sugar content in pneumococcal activated sugar based on the MBTH method, which adopts the following technical scheme:

[0033] A kit for determining the reducing sugar content in pneumococcal activated sugar comprises the above reagent.

[0034] In summary, this application has the following beneficial effects:

[0035] First, the reaction principle of the method established in this application is: first, MBTH reacts with reducing sugar to form oxazine, and then the excess MBTH is reacted with Fe 3+ Oxidized to cations, the cations react with oxazine to generate cyan blue compounds, the cyan blue compounds have maximum absorption at 650nm, and within a certain range, the amount of reducing sugar and the color intensity of the reaction solution are in direct proportion. The method established in the present application uses glyceraldehyde as a reference substance, and the concentration of the detection limit calculated by the aldehyde group is 0.877nmol / ml, which improves the sensitivity of the determination of reducing sugars.

[0036] Second, the method established in the present application only requires three reagents (MBTH, hydrochloric acid, ferric chloride), and the reaction conditions are mild (35-40° C., for example: 37° C.), which not only saves experimental costs but also facilitates operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 is the standard curve of the gradient standard solution. DETAILED DESCRIPTION

[0038] The present application is further described in detail below with reference to the accompanying drawings and embodiments.

[0039] Instruments and materials

[0040] Glyceraldehyde and MBTH were purchased from Sigma; hydrochloric acid (superior grade) and ferric chloride hexahydrate were purchased from Sinopharm Chemical Reagent Co., Ltd.; UV-visible spectrophotometer was purchased from Agilent; and electric constant temperature water bath was purchased from Shanghai Yiheng Scientific Instrument Co., Ltd.

[0041] Preparation of hydrochloric acid solution

[0042] Take 5 ml of concentrated hydrochloric acid and slowly add it into 595 ml of water, mix well, and obtain a hydrochloric acid solution with a concentration of 0.1 mol / L.

[0043] Preparation of MBTH aqueous solution

[0044] Weigh 0.25 g of MBTH, dissolve it in purified water and make it up to 100 ml, mix well, and obtain an MBTH aqueous solution with a concentration of 0.25 wt%.

[0045] Preparation of ferric chloride aqueous solution

[0046] Weigh 0.83 g of ferric chloride, dissolve it in purified water and make up to 100 ml, mix well, and obtain an aqueous solution of ferric chloride with a concentration of 0.83 wt%.

[0047] Building a linear regression equation

[0048] Preparation of gradient standard solution: Accurately weigh 90 mg of glyceraldehyde, dissolve it in water and make up to 100 ml to obtain a glyceraldehyde reference stock solution with a concentration of 10 mmol / L.

[0049] The 10 mmol / L glyceraldehyde reference substance stock solution was diluted 25 times to obtain a 400 nmol / ml glyceraldehyde reference substance solution.

[0050] 0.1ml, 0.2ml, 0.3ml, 0.4ml, 0.5ml, ml, 0.6ml, 0.7ml, 0.8ml of 400nmol / ml glyceraldehyde reference solution were measured and placed in stoppered glass test tubes and fixed to 1.0ml to obtain eight gradient standard solutions. The concentrations of the eight gradient standard solutions were 40nmol / ml, 80nmol / ml, 120nmol / ml, 160nmol / ml, 200nmol / ml, 240nmol / ml, 280nmol / ml, 320nmol / ml.

[0051] Pretreatment of the gradient standard solution: first, add 3.25 ml of a 0.1 mol / L hydrochloric acid solution and 1.0 ml of a 0.25 wt% MBTH aqueous solution to the eight stoppered glass test tubes, seal with stoppers and mix thoroughly, then place the eight stoppered glass test tubes in a 37°C water bath to react for 30 min, and cool to 25°C; then, add 1.0 ml of a 0.83 wt% ferric chloride aqueous solution to the eight stoppered glass test tubes, seal with stoppers and mix thoroughly, then place the eight stoppered glass test tubes in a 37°C water bath to react for 5 min, and cool to 25°C.

[0052] Obtaining the linear regression equation: Using water as the blank control, and measuring the absorbance of the pretreated gradient standard solution at a wavelength of 650nm, using the concentration of the gradient standard solution as the abscissa and the absorbance of the gradient standard solution as the ordinate, plotting the following: Figure 1 The standard curve is shown. Figure 1 As is known, the gradient standard solution shows a good linear relationship in the range of (0-320) nmol / ml.

[0053] According to the standard curve, the linear regression equation y=0.00532x+0.02236 was obtained, and the goodness of fit of the linear regression equation R 2 =0.99908.

[0054] Determination of reducing sugar content

[0055] Preparation of the sample solution to be tested: dilute the sample to be tested with water to obtain the sample solution to be tested;

[0056] Pretreatment of the sample solution to be tested: 1.0 ml of the sample solution to be tested is measured and placed in a stoppered glass test tube, and first 3.25 ml of a hydrochloric acid solution with a concentration of 0.1 mol / L and 1.0 ml of a MBTH aqueous solution with a concentration of 0.25 wt% are added to the stoppered glass test tube respectively, the stopper is sealed and fully mixed, and then the stoppered glass test tube is placed in a 37° C. water bath to react for 30 min, and cooled to 25° C.; then 1.0 ml of a ferric chloride aqueous solution with a concentration of 0.83 wt% is added to the sample solution to be tested, the stopper is sealed and fully mixed, and then the stoppered glass test tube is placed in a 37° C. water bath to react for 5 min, and cooled to 25° C.

[0057] Obtaining the reducing sugar content: using water as a blank control and measuring the absorbance of the pretreated sample solution at a wavelength of 650 nm; calculating according to the linear regression equation y=0.00532x+0.02236, obtaining the concentration of the absorbance of the sample solution on the standard curve corresponding to the horizontal coordinate; wherein, the concentration of reducing sugar in the sample to be tested=the concentration of the sample solution to be tested×the dilution multiple of the sample to be tested.

[0058] Method validation test

[0059] 1. Detection limit and quantification limit test

[0060] According to the Chinese Pharmacopoeia guidelines "based on the standard deviation of the response value and the slope of the standard curve" to determine the limit of detection (LOD) and limit of quantification (LOQ):

[0061]

[0062]

[0063] Where δ is the standard deviation of the response value; S is the slope of the standard curve.

[0064] Take 20 blank samples and measure them according to the method for measuring reducing sugar. Calculate the detection limit and quantification limit based on the measurement results. The detection limit and quantification limit test results are shown in Table 1.

[0065] Table 1: Results of detection limit and quantification limit tests

[0066]

[0067] 2. Specificity test

[0068] Take the reagents used in the process of activating sugars for pneumococcal types 3, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 15B, 19A, 19F, 22F, 23F, and 33F (0.1 mmol / L acetic acid aqueous solution, 0.1 mmol / L NaOH aqueous solution, 0.1 mg / ml NaIO 4 Aqueous solution) was used to carry out a specificity test according to the above method for determining the reducing sugar content. The reagents (0.1mmol / L acetic acid aqueous solution, 0.1mmol / L NaOH aqueous solution, 0.1mg / ml NaIO 4 The specificity test results are shown in Table 2.

[0069] Table 2: Specificity test results

[0070]

[0071] It can be seen from Table 2 that 0.1mmol / L acetic acid aqueous solution, 0.1mmol / L NaOH aqueous solution, 0.1mg / ml NaIO 4 The reducing sugar contents of the aqueous solutions were 0.648 nmol / ml, -0.663 nmol / ml, and -0.016 nmol / ml, respectively, all lower than the quantification limit LOQ = 2.657 nmol / ml of the method. This indicates that the reagents used in the process of activating sugars for pneumococci of types 3, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 15B, 19A, 19F, 22F, 23F, and 33F do not interfere with the determination of reducing sugar content.

[0072] 3. Accuracy test

[0073] Take 3, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 15B, 19A, 19F, 22F, 23F, 33F pneumococcal activated sugar samples respectively, and dilute the reducing sugar content of the above pneumococcal activated sugar samples to the linear range to obtain pneumococcal activated sugar sample solutions; add 200nmol / ml glyceraldehyde reference solution to the pneumococcal activated sugar sample solution in equal volume, and the ratio of the added reference to the amount of the component to be determined in the sample is controlled at about 1.2:1, 1:1, and 0.8:1, and 3 portions are prepared for each ratio, and the reducing sugar content of the sample and its spiked sample is determined, and the recovery rate and relative standard deviation (RSD) of the determination results are calculated. The recovery rate should be (80-120)%, and the relative standard deviation RSD<10%.

[0074]

[0075] In the formula, A is the amount of the measured component contained in the test sample, B is the amount of reference substance added, and C is the measured value.

[0076] Table 3-1: Results of the accuracy test of reducing sugar content in activated sugar for type 3 pneumococcus

[0077]

[0078] Table 3-2: Results of the accuracy test of reducing sugar content in activated sugar for type 5 pneumococcus

[0079]

[0080] Table 3-3: Results of the accuracy test of reducing sugar content in activated sugars of type 6A pneumococcal bacteria

[0081]

[0082] Table 3-4: Results of the accuracy test of reducing sugar content in activated sugars of type 6B pneumococcus

[0083]

[0084] Table 3-5: Results of the accuracy test of reducing sugar content in activated sugar of 7F pneumococcal bacteria

[0085]

[0086] Table 3-6: Results of the accuracy test of reducing sugar content in activated sugar for type 8 pneumococcus

[0087]

[0088] Table 3-7: Results of the accuracy test of reducing sugar content in activated sugar of 9V type pneumococcus

[0089]

[0090] Table 3-8: Results of the accuracy test of reducing sugar content in activated sugars of type 10A pneumococcal bacteria

[0091]

[0092] Table 3-9: Results of the accuracy test of reducing sugar content in activated sugars of type 10A pneumococcal bacteria

[0093]

[0094] Table 3-10: Results of the accuracy test of reducing sugar content in activated sugar for 15B pneumococcal infection

[0095]

[0096] Table 3-11: Results of the accuracy test of reducing sugar content in activated sugars of 19A pneumococcal bacteria

[0097]

[0098] Table 3-12: Results of the accuracy test of reducing sugar content in activated sugar of 19F pneumococcal bacteria

[0099]

[0100] Table 3-13: Results of the accuracy test of reducing sugar content in activated sugar of 22F pneumococcal bacteria

[0101]

[0102] Table 3-14: Results of the accuracy test of reducing sugar content in activated sugar of 23F pneumococcal bacteria

[0103]

[0104] Table 3-15: Results of the accuracy test of reducing sugar content in activated sugar of 33F pneumococcal bacteria

[0105]

[0106] It can be seen from Table 3-1 to Table 3-15 that the spiked recoveries of low, medium and high concentration spiked experiments of activated sugar samples of pneumococcal types 3, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 15B, 19A, 19F, 22F, 23F and 33F were all within the range of 90-115%, and the average spiked recoveries of each type were 100.0%, 102.0%, 101.0%, 103.4%, 102.9% and 105.1%, respectively. , 108.9%, 94.1%, 104.6%, 105.8%, 105.0%, 101.7%, 106.3%, 109.6%, 104.2%; the RSDs of the spiked recoveries of each type were 1.46%, 2.19%, 1.21%, 1.66%, 0.67%, 1.37%, 1.40%, 2.79%, 1.65%, 1.73%, 1.98%, 2.68%, 2.55%, 1.77%, 1.42%, respectively. This shows that the method has good accuracy in determining the content of activated sugar and reducing sugar in pneumococcal types 3, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 15B, 19A, 19F, 22F, 23F, and 33F.

[0107] 4. Precision test

[0108] Six test solutions were prepared from each of the activated sugar samples of pneumococci types 3, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 15B, 19A, 19F, 22F, 23F, and 33F, and the reducing sugar content was determined. The relative standard deviation (RSD < 10%) of the six determination results was calculated to examine the repeatability of the method.

[0109] On different dates, different analysts took 3, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 15B, 19A, 19F, 22F, 23F, and 33F pneumococcal activated sugar samples to prepare 6 test solutions, determined the reducing sugar content, and calculated the relative standard deviation of the 6 measurement results (RSD < 10%). At the same time, the reducing sugar content of 12 samples of 3, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 15B, 19A, 19F, 22F, 23F, and 33F pneumococcal activated sugars determined by different analysts was calculated, and the relative standard deviation of the measurement results (RSD < 10%) was calculated to investigate the intermediate precision of the method.

[0110] Table 4-1: Results of precision test on reducing sugar content in activated sugar for type 3 pneumococcus

[0111]

[0112] Table 4-2: Results of precision test on reducing sugar content in activated sugar for type 5 pneumococcus

[0113]

[0114] Table 4-3: Results of precision test on reducing sugar content in activated sugar for type 6A pneumococcus

[0115]

[0116] Table 4-4: Results of precision test on reducing sugar content in activated sugar for type 6B pneumococcus

[0117]

[0118] Table 4-5: Results of precision test on reducing sugar content in activated sugar of 7F pneumococcal strain

[0119]

[0120] Table 4-6: Results of precision test on reducing sugar content in activated sugar for type 8 pneumococcus

[0121]

[0122] Table 4-7: Results of precision test on reducing sugar content in activated sugar for 9V pneumococcal infection

[0123]

[0124] Table 4-8: Results of precision test on reducing sugar content in activated sugar for type 10A pneumococcal infection

[0125]

[0126] Table 4-9: Results of precision test on reducing sugar content in activated sugar of 12F pneumococcal bacteria

[0127]

[0128] Table 4-10: Results of precision test on reducing sugar content in activated sugar for 15B pneumococcal infection

[0129]

[0130] Table 4-11: Results of precision test on reducing sugar content in activated sugar for 19A pneumococcal infection

[0131]

[0132] Table 4-12: Results of precision test on reducing sugar content in activated sugar for 19F pneumococcal infection

[0133]

[0134] Table 4-13: Results of precision test on reducing sugar content in activated sugar of 22F pneumococcal bacteria

[0135]

[0136] Table 4-14: Results of precision test on reducing sugar content in activated sugar of 23F pneumococcal bacteria

[0137]

[0138] Table 4-14: Results of precision test on reducing sugar content in activated sugar of 33F pneumococcal strain

[0139]

[0140] It can be seen from Tables 4-1 to 4-15 that the reducing sugar content in activated sugar of types 3, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 15B, 19A, 19F, 22F, 23F, and 33F pneumococcal strains was tested for repeatability, and the relative standard deviations (RSDs) of the test results were 0.93%, 1.52%, 2.00%, 0.30%, 1.48%, 0.66%, 0.96%, 0.55%, 1.04%, 1.95%, 1.17%, 1.30%, 0.56%, 1.49%, and 0.85%, respectively, indicating that the method has good repeatability; different testers tested the reducing sugar content in activated sugar of types 3, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 15B, 19A, 19F, 22F, 23F, and 33F pneumococcal strains at different times. An intermediate precision test was carried out on the reducing sugar content in the activated saccharide of pneumococcal cocci of types 23F, 23F and 33F. The relative standard deviations (RSDs) of the determination results were 0.81%, 0.81%, 0.86%, 0.53%, 1.40%, 0.97%, 0.94%, 0.53%, 0.64%, 1.39%, 0.81%, 0.98%, 1.34%, 0.62% and 0.86%, respectively; the relative standard deviations (RSDs) of 12 determination results of the same test sample by different analysts were 2.77%, 1.31%, 1.53%, 0.50%, 2.86%, 1.73%, 2.24%, 4.68%, 1.99%, 2.25%, 3.07%, 2.07%, 3.96%, 1.63% and 0.81%, respectively. The results showed that this method could determine the reducing sugar content in activated sugar of types 3, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 15B, 19A, 19F, 22F, 23F, and 33F pneumococci with good precision.

[0141] It is to be understood that the above embodiments are merely exemplary embodiments used to illustrate the principles of the present invention, but the present invention is not limited thereto. For those of ordinary skill in the art, various modifications and improvements can be made without departing from the spirit and essence of the present invention, and these modifications and improvements are also considered to be within the scope of protection of the present invention.

Claims

1. A method for determining the reducing sugar content in pneumococcal activated sugar based on the MBTH method, characterized in that: The method comprises the following steps: (1) Establishing the linear regression equation Preparation of gradient standard solution: prepare glyceraldehyde as a standard substance into a gradient standard solution with a concentration of (40-320) nmol / ml; Pretreatment of the gradient standard solution: first, add a hydrochloric acid solution with a concentration of (0.05-0.15) mol / L and an MBTH aqueous solution with a concentration of (0.2-0.3) wt% to the gradient standard solution, seal and mix, then react at a temperature of 35-40° C. for 20-40 min, and cool to 20-30° C.; then, add a ferric chloride aqueous solution with a concentration of (0.7-1.0) wt% to the gradient standard solution, seal and mix, then react at a temperature of 35-40° C. for 3-10 min, and cool to 20-30° C.; wherein the volume ratio of the gradient standard solution, the hydrochloric acid solution, the MBTH aqueous solution and the ferric chloride aqueous solution is 1:(2.925-3.575):(0.9-1.1):(0.9-1.1); Obtaining the linear regression equation: using water as a blank control and measuring the absorbance of the pretreated gradient standard solution at a wavelength of 650 nm, plotting a standard curve with the concentration of the gradient standard solution as the abscissa and the absorbance of the gradient standard solution as the ordinate, and calculating the linear regression equation; (2) Determination of reducing sugar content Preparation of the sample solution to be tested: dilute the sample to be tested with water to obtain the sample solution to be tested; Pretreatment of the sample solution to be tested: first, add a hydrochloric acid solution with a concentration of (0.05-0.15) mol / L and an MBTH aqueous solution with a concentration of (0.2-0.3) wt% to the sample solution to be tested, seal and mix, then react at a temperature of 35-40° C. for 20-40 min, and cool to 20-30° C.; then, add a ferric chloride aqueous solution with a concentration of (0.7-1.0) wt% to the sample solution to be tested, seal and mix, then react at a temperature of 35-40° C. for 3-10 min, and cool to 20-30° C.; wherein the volume ratio of the sample solution to be tested, the hydrochloric acid solution, the MBTH aqueous solution and the ferric chloride aqueous solution is 1:(2.925-3.575):(0.9-1.1):(0.9-1.1); Obtaining the reducing sugar content: using water as a blank control and measuring the absorbance of the pretreated sample solution at a wavelength of 650 nm; calculating the concentration of the absorbance of the sample solution on the standard curve corresponding to the horizontal axis according to the linear regression equation obtained in step (1); The concentration of reducing sugar in the sample to be tested = the concentration of the sample solution to be tested × the dilution multiple of the sample to be tested.

2. The method according to claim 1, characterized in that In step (1), the number of the gradient standard solutions is at least 3.

3. The method according to claim 1, characterized in that In step (1), the volume ratio of the gradient standard solution, the hydrochloric acid solution, the MBTH aqueous solution and the ferric chloride aqueous solution is 1:(3.0875-3.4125):(0.95-1.05):(0.95-1.05).

4. The method according to claim 3, characterized in that In step (1), the volume ratio of the gradient standard solution, the hydrochloric acid solution, the MBTH aqueous solution and the ferric chloride aqueous solution is 1:3.25:1:

1.

5. The method according to claim 1, characterized in that The linear regression equation obtained in step (1) is y=0.00532x+0.02236.

6. The method according to claim 5, characterized in that The goodness of fit R of the linear regression equation y=0.00532x+0.02236 2 =0.99908.

7. The method according to claim 1, characterized in that In step (2), the volume ratio of the sample solution to be tested, the hydrochloric acid solution, the MBTH aqueous solution and the ferric chloride aqueous solution is 1:(3.0875-3.4125):(0.95-1.05):(0.95-1.05).

8. The method according to claim 7, characterized in that In step (2), the volume ratio of the sample solution to be tested, the hydrochloric acid solution, the MBTH aqueous solution and the ferric chloride aqueous solution is 1:3.25:1:

1.

9. The method according to claim 1, characterized in that: In step (1) and / or step (2), the concentration of the hydrochloric acid solution is (0.08-0.12) mol / L.

10. The method according to claim 9, characterized in that In step (1) and / or step (2), the concentration of the hydrochloric acid solution is 0.09 mol / L, 0.10 mol / L, or 0.11 mol / L.

11. The method according to claim 1, characterized in that: In step (1) and / or step (2), the concentration of the MBTH aqueous solution is (0.23-0.27) wt%.

12. The method according to claim 11, characterized in that In step (1) and / or step (2), the concentration of the MBTH aqueous solution is 0.24wt%, 0.25wt%, or 0.26wt%.

13. The method according to claim 1, characterized in that In step (1) and / or step (2), the concentration of the ferric chloride aqueous solution is (0.8-0.9) wt%.

14. The method according to claim 13, characterized in that In step (1) and / or step (2), the concentration of the ferric chloride aqueous solution is 0.81wt%, 0.82wt%, 0.83wt%, 0.84wt%, 0.85wt%, 0.86wt%, 0.87wt%, 0.88wt%, or 0.89wt%.

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