CN116087490A

CN116087490A - Application of polysaccharide substance detection reagent in preparation of intracranial infection detection reagent

Info

Publication number: CN116087490A
Application number: CN202211593560.3A
Authority: CN
Inventors: 叶雷; 杜卫东; 程宏伟; 季学飞; 李平平; 管辽; 赵亮
Original assignee: First Affiliated Hospital of Anhui Medical University
Current assignee: First Affiliated Hospital of Anhui Medical University
Priority date: 2022-12-13
Filing date: 2022-12-13
Publication date: 2023-05-09

Abstract

The invention belongs to the technical field of medicine, and particularly discloses application of a polysaccharide substance detection reagent in preparation of an intracranial infection detection reagent. The invention provides a detection reagent for polysaccharide substances, wherein the polysaccharide substances are one or more of Gal beta 3GalNAc, gal beta 3GalNAc-Ser/Thr and alpha-fucose, the detection reagent is applied to preparation of detection of intracranial cerebrospinal fluid infection caused by bacteriality, and the detection reagent comprises a lectin chip. The invention uses lectin chip method to detect polysaccharide substance in cerebrospinal fluid, has specific diagnosis post-operation bacterial intracranial infection marker, can increase diagnosis sensitivity and specificity, and can diagnose neurosurgery post-operation bacterial intracranial infection.

Description

Application of polysaccharide substance detection reagent in preparation of intracranial infection detection reagent

Technical Field

The invention belongs to the technical field of medicine, and particularly discloses application of a polysaccharide substance detection reagent in preparation of an intracranial infection detection reagent.

Background

Central nervous system infection is a serious nervous system infectious disease, although surgery-related intracranial infection conditions are more complex than idiopathic meningitis. First, primary diseases of the nervous system can produce neuroinflammation, which can also occur in surgery, and post-operative patients often exhibit aseptic inflammation, often similar to the symptoms of intracranial infections, such as fever, headache, and the like. Current diagnostic methods for intracranial infections rely mainly on two approaches: bacterial culture, which is highly specific but very low in positive rate, shows that in all laboratory data, the positive rate of cerebrospinal bacterial culture is typically less than 30%; biochemical indicators of cerebrospinal fluid, such as cerebrospinal fluid leukocytes, sugars, chlorine, total proteins, polymorphonuclear cells, cerebrospinal fluid sugar to blood sugar ratio, etc. But this method is less specific. Some novel biomarkers, such as cerebrospinal fluid/haemolactic acid, procalcitonin, etc., can be affected by cerebrospinal fluid properties, such as hemolysis, etc., affecting the final result.

Glycosylation is a very common post-translational modification of biological proteins, which is widely occurring in mammals and bacteria, and is a major polysaccharide substance involved in glycosylation, which plays an extremely important role in various biological processes, such as cell adhesion, anti-inflammatory clearance, cell signaling, etc. The bacterial surface is rich in polysaccharide or polysaccharide-binding substances, which can be used as antigens to generate innate immune response to the organism. Furthermore, the polysaccharide structure of the outer surface of these bacteria is involved in the movement, adhesion and virulence of the bacteria and mediates interactions between the bacteria and the surrounding environment and bacteria. In addition, polysaccharide substances can also be used as potential targets for vaccine development.

Diagnosis of bacterial intracranial infections after neurosurgery is difficult, on the one hand, both primary diseases of the nervous system and surgical procedures can cause fever, headache and other clinical symptoms similar to intracranial infections. On the other hand, the primary diseases and the operation process of the nervous system can possibly cause the phenomena of increased white blood cells of the cerebrospinal fluid, increased total proteins and the like, and the diagnostic method has more identity with the diagnosis of the cerebrospinal fluid of the bacterial intracranial infection. The existing method for detecting polysaccharide substances in a sample mainly comprises mass spectrometry, chromatography and other methods, and the method needs large-scale equipment, needs professional operators to operate, has complicated procedures and cannot meet the clinical rapid diagnosis requirement of diseases. Lectin is a substance capable of specifically combining with polysaccharide, and can be used for detecting polysaccharide substances, and a lectin chip is a novel biochip platform, so that a result can be obtained after 2 hours under the condition that a chip probe is prepared, and the clinical requirement is met. In addition, in general mass spectrometry detection, more samples need to be detected, and for cerebrospinal fluid, the samples are very precious, and the cerebrospinal fluid needs to be obtained through lumbar puncture or lumbar cistern drainage, and in general, if the drainage amount is large, intracranial pressure can be influenced, and cranium low pressure is caused, and even life and health are endangered. There is no report on polysaccharide substances in cerebrospinal fluid for bacterial intracranial infection after neurosurgery.

Disclosure of Invention

The invention aims to provide application of a polysaccharide substance detection reagent in preparation of an intracranial infection detection reagent.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

the application of a detection reagent of polysaccharide substances in preparing an intracranial infection detection reagent is characterized in that the polysaccharide substances are one or more of Gal beta 3GalNAc, gal beta 3GalNAc-Ser/Thr and alpha-fuse.

Preferably, the infection is a bacterially induced intracranial cerebrospinal fluid infection in a brain tumor patient.

Preferably, the intracranial cerebrospinal fluid infection causes an increase in the level of the polysaccharide.

Preferably, the detection reagent is a detection reagent for qualitative detection or quantitative detection of the polysaccharide substance.

Preferably, the detection reagent comprises a lectin chip, a gene chip or a protein chip.

Preferably, the surface of the lectin chip is coated with one or more of peanut lectins, jackfruit and/or European hundred vein root lectins, wherein the peanut lectins correspond to polysaccharide substances Gal beta 3GalNAc, the jackfruit corresponds to polysaccharide substances Gal beta 3GalNAc-Ser/Thr, and the European hundred vein root lectins correspond to polysaccharide substances alpha-fucose.

Preferably, the preparation method of the lectin chip comprises the following steps:

s1, taking a glass sheet as a substrate, covering a gold foil chip on the substrate, and forming a protein chip by using a TEFLON film with array holes above the gold foil chip in a regional manner;

s2, immersing the protein chip obtained in the S1 into the modification liquid for the first time, cleaning after the first immersing is finished, immersing the protein chip into the mixed modification liquid again for the second immersing, and cleaning after the second immersing is finished, so as to obtain the modified protein chip.

Preferably, in S1, the gold foil chip has a thickness of 10nm, the array is 8 rows by 12 columns, the array aperture is 1.25mm, and the TEFLON film has a thickness of 50 μm.

Preferably, in S2, the modification solution is an ethanol solution of 16-amino-1-hexadecanethiol, the concentration is 0.8-1.2mM, and the time for the first soaking is 8-12h.

Preferably, in S2, the mixed modification solution is formed by dissolving 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester and 4- (dimethylamino) pyridine in N, N-dimethylformamide, and the mass-volume ratio of 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester, 4- (dimethylamino) pyridine and N, N-dimethylformamide is 100mg:50mg:10mL, and the time for the second soaking is 6-8h.

Compared with the prior art, the invention has the following beneficial effects:

the invention discovers that polysaccharide substances in cerebrospinal fluid can be used as independent risk factors for diagnosing bacterial intracranial infection after neurosurgery, and the lectin chip method is applied to detecting the polysaccharide substances in the cerebrospinal fluid, so that the marker for specifically diagnosing the bacterial intracranial infection after neurosurgery can increase diagnosis sensitivity and specificity.

The invention provides a general and specific polysaccharide type related to bacterial infection, and the polysaccharide substance in cerebrospinal fluid has diagnostic value in bacterial intracranial infection after brain tumor neurosurgery, participates in various vital activities, has stronger relevance with bacterial virulence and organism immune response in the bacterial infection process, is one of the bases of vaccine research and development, can diagnose bacterial intracranial infection after neurosurgery, and can also provide potential value for vaccine development for preventing intracranial infection.

Drawings

FIG. 1 is a graph showing polysaccharide level analysis in cerebrospinal fluid after neurosurgical treatment;

FIG. 2 is a graph of fluorescence value analysis of polysaccharide in tumor patients after neurosurgical treatment.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

It should be noted that the technical terms used in the present invention are only for describing specific embodiments, and are not intended to limit the scope of the present invention, and various raw materials, reagents, instruments and equipment used in the following embodiments of the present invention may be purchased commercially or prepared by existing methods unless otherwise specifically described.

Example 1

The infection is a bacterial infection of cerebral tumor patient's intracranial cerebrospinal fluid.

Intracranial cerebrospinal fluid infection causes an increase in the level of the polysaccharide.

The detection reagent is used for carrying out qualitative detection or quantitative detection on the polysaccharide substances.

The detection reagent is a lectin chip.

The surface of the lectin chip is coated with one or more of peanut lectin, jackfruit lectin and/or European Baical root lectin, wherein the peanut lectin corresponds to a polysaccharide substance Galbeta 3GalNAc, the jackfruit lectin corresponds to a polysaccharide substance Galbeta 3GalNAc-Ser/Thr, and the European Baical root lectin corresponds to a polysaccharide substance alpha-fucose.

The preparation method of the lectin chip comprises the following steps:

s1, soaking a gold foil chip in an acetone solution for 5min, then cleaning the gold foil chip with deionized water for 3 times, each time for 2 min, and drying the gold foil chip for later use by using nitrogen;

a glass sheet is taken as a substrate, a layer of gold foil chip with the thickness of 10nm is covered on the substrate, an array TEFLON film is arranged above the gold foil chip in a regional mode, the array mode is 8 rows by 12 columns, the aperture of the array is 1.25mm, round holes (96 holes by 2) are formed in the TEFLON film, and the diameter of each round hole is 50 mu m, so that a lectin chip is formed;

s2, immersing the lectin chip obtained in the S1 into 0.8mM of ethanol solution modification solution of 16-amino-1-hexadecanethiol for 12 hours, washing with absolute ethanol after the first immersing is finished, drying, immersing 100mg of 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester and 50mg of 4- (dimethylamino) pyridine into mixed modification solution formed by 10mM of LN, N-dimethylformamide for the second immersing for 8 hours, and washing with N, N-dimethylformamide and deionized water in sequence after the second immersing is finished, drying to obtain the modified lectin chip.

Example 2

A method of using a modified lectin chip comprising the steps of:

after the modified lectin chip was washed with absolute ethanol and dried with nitrogen, wheat germ lectin (Wheat GermAgglutinin, WGA), lablab album (Lens CulinarisAgglutinin, LCA), peanut lectin (PeanutAgglutinin, PNA), ricin 1 (Ricinus Communis Agglutinin I, RCA-I), jackfruit lectin (Jacalin), faba lectin (ViciaVillosa Lectin, VVL), sambucus nigra lectin (Sambucus Nigra Lectin, SNA), sophora japonica lectin 1 (Maackia Amurensis Lectin I, MAL-I), european vein root lectin (Lotus Tetragonolobus Lectin, LTL), colchicine lectin (Narcissus Pseudonarcissus Lectin, NPL) and bean lectin (Phaseolus Vulgaris Leucoagglutinin, PVL) were diluted to 1mg/mL with 10mM HEPES buffer (ph 8.5) (containing 0.001% BSA), respectively, and then dropped onto round holes in different areas of the chip surface, and further coated on the lectin chip surface obtained in example 1, and incubated for 2 hours under a normal temperature and humidity room environment; meanwhile, using an IgG and albumin removal kit to remove high kurtosis protein in cerebrospinal fluid, incubating the treated cerebrospinal fluid sample with an equal volume of cy3 marked by N-hydroxysuccinimide (NHS) for 1h in a room temperature and humidity environment, coating the eluent on a chip with lectin probes through a G25 desalting column, incubating for 0.5h, and then washing for 3 times by using phosphate buffer solution-tween 20, drying and waiting for detection.

Example 3

General clinical data of patients

A total of 136 patients receiving neurosurgical treatment were collected in this study.

136 patients are all patients with cerebral arterial thrombosis, the age range is 13-88 years old, the average age is 56.9 years + -16.0 years old, 73 men and 63 women; wherein 53 people in the infected group are aged in the range of 13-88 years, with average age of 55.7+ -17.1 years, 28 men, 25 women; 83 persons in the non-infected group, ages in the range of 14-83 years, average age 57.7+ -15.4 years, 45 men, 38 women.

Sample acquisition

The ridge liquid is collected in the process of invasive lumbar puncture when the patient is suspected of intracranial infection after neurosurgery operation, and a sterile tube is adopted for collection.

According to the diagnostic criteria, the diagnostic criteria are "diagnosis and treatment of central nervous system infections in neurosurgery" national expert consensus (2021 edition) published in journal of neurosurgery, volume 37, 202, pages 2-15. Mainly based on the result of bacterial culture of cerebrospinal fluid or the conventional +cerebrospinal fluid biochemical index, 53 people are diagnosed with bacterial intracranial infection after neurosurgery and 83 people have no infection.

Samples of cerebrospinal fluid from all patients were extracted, after two steps of removing kurtosis protein and cy3 labeling, spotted on the chip loaded with 11 lectin probes of example 2, all samples were tested, and two sets of fluorescence values were compared, and polysaccharide substances corresponding to WGA probe (p=0.002), LCA probe (p=0.002), PNA probe (p=0.002), RCA-I probe (p=0.027), jacalin probe (P < 0.001), SNA probe (p=0.009), MAL-I probe (p=0.048) and LTL probe (P < 0.001) were shown to be statistically different between the two sets. The levels of the infected group were higher than the non-infected group, while the polysaccharide corresponding to NPL probe (p=0.073) and PHA-L probe (p=0.086) were statistically different between the two groups, as shown in fig. 1.

To confirm which of these can be used for acute independent diagnosis of disease, we found that in single factor analysis (single factor analysis of variance) there was a significant difference in polysaccharide corresponding to WGA probe (p=0.002), LCA probe (p=0.002), PNA probe (p=0.002), RCA-I probe (p=0.027), jacalin probe (P < 0.001), SNA probe (p=0.009), MAL-I probe (p=0.048) and LTL probe (P < 0.001) between the infected and non-infected groups. The data were recalculated using a multifactor analysis method (logistic regression) and the main objective was to see if the data would affect each other, the results of the multifactor analysis (multifactor anova) were considered significant, as independent risk factors between the two groups (i.e. intracranial infection), and as shown in table 1, only the polysaccharide corresponding to the PNA probe (p=0.034), the Jacalin probe (P < 0.034) and the LTL probe (p=0.001) showed statistical differences between the two groups (table below). It is suggested that the polysaccharide corresponding to the three lectin probes can be used as independent risk factors in diagnosis of intracranial infection after neurosurgery.

TABLE 1 multifactor analysis results

Multi-factor analysis	Pvalue
		WGA	0.830
LCA	0.423
		PNA	0.034
RCAI	0.676
		Jacalin	0.034
EBL	0.431
		MAL	0.220
LTL	0.001

We used the subject work curve (ROC curve) to perform a diagnostic efficacy analysis on these three indicators. The working curve of the subject is a statistical method, a certain item of data of two groups (an infected group and a non-infected group) is input into SPSS software, one group is defined as a normal group, the data of the disease group is compared with the data of the normal group, so that a about sign index (which can be understood as the total inflection point of the two curves) can be calculated, and the corresponding cut-off value (cut-off value), sensitivity and specificity can be found according to the about sign index. As shown in fig. 2, the fluorescence values of the infected group and the fluorescence values of the non-infected group are input into statistical software to generate results, and fig. 2 is an image of a subject work curve (ROC), the area from the broken line to the bottom side is the area under the curve (AUC), the sensitivity is the ordinate, (1-specificity) is the abscissa, and the larger the area under the curve is, the higher the diagnosis accuracy is. On the ROC curve, the point closest to the upper left of the graph of fig. 2 is a critical value with higher sensitivity and specificity.

The diagnostic efficacy of each index was also analyzed as shown in table 2.

TABLE 2 diagnostic efficacy of levels of polysaccharide substances in cerebrospinal fluid

Wherein the AUC value suggests diagnostic accuracy, the closer to 1 the better, the cut-off value represents "over this fluorescence value, and can be judged as an infection".

Table 2 is primarily a diagnostic efficacy analysis from which it can be inferred that the effect of different markers in diagnosing intracranial infections, sensitivity, specificity and cutoff were all obtained by a Johnson index in the subject's working curve (ROC). The highest value for specificity and sensitivity is 100%, with approximately near 100% being better. As shown in Table 2, the sensitivity of PNA probe diagnosis was highest, and the specificity of both the Jacalin probe and LTL probe diagnosis was high. Besides polysaccharide substances corresponding to the three probes, the effect of judging intracranial infection by combining the three probes is analyzed, and the overall effect is better.

It should be noted that, when numerical ranges are referred to in the present invention, it should be understood that two endpoints of each numerical range and any numerical value between the two endpoints are optional, and because the adopted step method is the same as the embodiment, in order to prevent redundancy, the present invention describes a preferred embodiment. While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.

Claims

1. The application of the detection reagent of the polysaccharide substance in preparing the detection reagent of intracranial infection is characterized in that the polysaccharide substance is one or more of Gal beta 3GalNAc, gal beta 3GalNAc-Ser/Thr and alpha-fucose.

2. The use according to claim 1, wherein the infection is a bacterial infection of the intracranial cerebrospinal fluid in a brain tumor patient.

3. The use according to claim 2, wherein the intracranial cerebrospinal fluid infection causes an increase in the level of the polysaccharide.

4. The use according to claim 1, wherein the detection reagent is a detection reagent for qualitative or quantitative detection of the polysaccharide substance.

5. The use according to claim 1, wherein the detection reagent comprises a lectin chip, a gene chip or a protein chip.

6. The use according to claim 5, wherein the surface of the lectin chip is coated with one or more of peanut lectin, jackfruit lectin, and european hundred vein root lectin, the peanut lectin corresponds to polysaccharide galβ3galnac, the jackfruit lectin corresponds to polysaccharide galβ3galnac-Ser/Thr, and the european hundred vein root lectin corresponds to polysaccharide α -fucose.

7. The use according to claim 5, characterized in that the method for preparing a lectin chip comprises the following steps:

8. The use according to claim 7, wherein in S1, the gold foil chip has a thickness of 10nm, the array is 8 rows by 12 columns, the array aperture is 1.25mm, and the TEFLON film has a thickness of 50 μm.

9. The use according to claim 7, wherein in S2 the modification solution is an ethanol solution of 16-amino-1-hexadecanethiol at a concentration of 0.8-1.2mM, and the first soaking time is 8-12h.

10. The use according to claim 7, wherein in S2, the mixed modification solution is formed by dissolving 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester and 4- (dimethylamino) pyridine in N, N-dimethylformamide, and the mass/volume ratio of 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester, 4- (dimethylamino) pyridine and N, N-dimethylformamide is 100mg:50mg:10mL, and the time for the second soaking is 6-8h.