Application of lgals2 gene reagent in preparation of product for diagnosing sepsis coagulopathy

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to application of a detection LGALS2 gene reagent in preparation of a reagent kit for detecting sepsis coagulopathy. The product diagnoses sepsis coagulopathy by detecting the transcription level of the LGALS2 gene in a sample, and when the transcription level of the LGALS2 gene is significantly reduced, the subject has sepsis coagulopathy. The application uses LGALS2 as a biomarker, and through a human experiment verification, the transcription level of peripheral blood LGALS2 of a sepsis coagulopathy patient is significantly lower than that of a patient without sepsis coagulopathy. The transcription level of LGALS2 in whole blood RNA has high specificity and sensitivity in diagnosis and prediction of the risk of sepsis coagulopathy. The LGALS2 gene and the transcription level thereof can be used for preparation of a reagent or a kit for early diagnosis of sepsis coagulopathy, provide reference information for giving individualized intervention treatment, and have important clinical application value and popularization.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of reagents for detecting the LGALS2 gene in the preparation of products for diagnosing septic coagulopathy. Background Technology

[0002] Sepsis is a life-threatening organ dysfunction caused by a dysregulated response to infection. Sepsis-induced coagulopathy (SIC) is a vascular endothelial cell damage and coagulation disorder resulting from sepsis. Delayed diagnosis and inadequate management of SIC can lead to disseminated intravascular coagulation (DIC), doubling the mortality rate. Therefore, the diagnosis and treatment of septic coagulopathy has become a major problem urgently needing to be solved in the medical field.

[0003] The pathophysiological mechanism of septic coagulopathy (SIC) is extremely complex. Currently, it is believed that SIC develops from moderate immune thrombosis into excessive thrombotic inflammation. Immune thrombosis is an immune-functional thrombus formed in the microvessels during the early stages of infection. Excessive formation of immune thrombosis can lead to dysregulation and spread of thrombotic inflammation, subsequently resulting in SIC. Monocytes play a crucial role in the formation of immune thrombosis. Monocytes are a potential source of tissue factor (TF) in circulating blood. Compared to subendothelial tissue factor expressed on fibroblasts and pericapillary cells, the expression of monocyte-associated tissue factor (TF) is usually very low, but increases significantly under pathogen stimulation. Furthermore, monocytes can promote massive TF activation through the pyroptosis pathway mediated by cysteine-containing aspartate protease 11 and the nucleotide oligomerization domain-like receptor thermoprotein domain-associated protein 3 inflammasome; this process is also known as TF "decryption." Once activated, monocyte-derived TF can activate FVII, thereby initiating the extrinsic coagulation pathway and forming an immune thrombus. However, the functional mechanisms of monocytes in septic coagulopathy are not yet fully understood, and the significant heterogeneity of monocytes poses challenges to their functional and mechanistic analysis. Therefore, elucidating the heterogeneity of monocytes in SIC has important clinical significance and will provide new molecular targets for the early diagnosis of SIC. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the purpose of this invention is to provide new biomarkers that can help in the early diagnosis of septic coagulopathy, specifically involving the application of the LGALS2 gene and its transcription level in the preparation of reagents or kits for the early diagnosis of septic coagulopathy.

[0005] The objective of this invention can be achieved through the following technical solutions:

[0006] Application of reagents for detecting the LGALS2 gene in the preparation of kits for detecting septic coagulopathy.

[0007] Furthermore, the reagents include reagent one, reagent two, and reagent three. Reagent one contains gDNAEraser, buffer one, and deionized water. Reagent two contains PrimeScript RT Enzyme Mix I, RTPrimer Mix, buffer two, and deionized water. Reagent three contains SYBR Premix Ex Taq and distilled water.

[0008] Reagent 1, Reagent 2, and Reagent 3 all contain two primers involved in the PCR reaction, namely LGALS2-F and LGALS2-R, wherein the sequence of LGALS2-F is SEQ ID NO: 1 and the sequence of LGALS2-R is SEQ ID NO: 2.

[0009] Furthermore, the preparation of the reagent includes:

[0010] Reagent 1 can be obtained by mixing gDNA Eraser, Buffer 1, deionized water, LGALS2-F and LGALS2-R;

[0011] Reagent 2 can be obtained by mixing PrimeScript RT Enzyme Mix I, RT Primer Mix, Buffer II, Deionized Water, LGALS2-F, and LGALS2-R.

[0012] Reagent 3 can be obtained by mixing SYBR Premix Ex Taq, distilled water, LGALS2-F, and LGALS2-R.

[0013] The sequence of LGALS2-F is SEQ ID NO: 1, and the sequence of LGALS2-R is SEQ ID NO: 2.

[0014] Furthermore, the buffer solution in reagent one is 5×gDNA Eraser Buffer.

[0015] Furthermore, the buffer solution in reagent two is 5×PrimeScript Buffer 2.

[0016] Furthermore, the deionized water in reagent 1 and reagent 2 is RNase-free dH2O.

[0017] Furthermore, the distilled water in reagent three is ddH2O.

[0018] Furthermore, the reaction conditions for reagent one are as follows: the reaction is divided into two stages: the first stage: 42℃, 2min; the second stage: 4℃, maintaining this temperature until the next reaction.

[0019] Furthermore, the reaction conditions for reagent 2 are as follows: the reaction conditions are divided into three stages: the first stage: 37℃, 15min; the second stage: 85℃, 5sec; and the third stage: 4℃.

[0020] A reagent for diagnosing septic coagulopathy, the reagent comprising a reagent for detecting the LGALS2 gene.

[0021] The beneficial effects of this invention are as follows: Using LGALS2 as a biomarker, this invention has verified through population-based experiments that the peripheral blood LGALS2 transcription level in patients with septic coagulopathy is significantly lower than that in patients without septic coagulopathy. The LGALS2 transcription level in whole blood RNA has high specificity and sensitivity for predicting the risk of septic coagulopathy. The LGALS2 gene and its transcription level can be used to prepare reagents or kits for the early diagnosis of septic coagulopathy, providing reference information for personalized intervention and treatment, and has significant clinical application value and potential for widespread application. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of this disclosure or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, those skilled in the art can obtain other drawings based on these drawings without creative effort.

[0023] Figure 1 Figure A shows the expression patterns of LGALS2 in SIC and NSIC in this invention; Figure A shows that the expression of LGALS2 in SIC is significantly lower than that in Nsic; Figure B and D show the ROC curve analysis of LGALS2, APTT, and D-dimer in SIC and NSIC.

[0024] Figure 2 The proportion of the monocyte subset (cluster 2) that highly expresses LGALS2+ in sepsis-associated coagulopathy is reduced in this invention; where A is the UMAP diagram of monocyte clusters; B is the bubble diagram of marker genes of each monocyte subset; C is the proportion of each monocyte subset in the peripheral blood of healthy individuals (HC), patients with sepsis (NSIC), and patients with sepsis-associated coagulopathy (NSIC). Detailed Implementation

[0025] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] Example 1: Method for obtaining diagnostic cell subsets and related genes from peripheral blood mononuclear cells in patients with septic coagulopathy

[0027] Single-cell sequencing was performed on peripheral blood mononuclear cells from healthy individuals, sepsis patients, and patients with septic coagulopathy. Cell populations were annotated using the Seurat package to identify different cell types. Monocyte populations were then extracted and further subpopulation clustered, identifying eight monocyte subpopulations, named Clusters 1-8 (e.g., ...). Figure 2 A). The FindALLMarker function was used to identify Cluster 1-8 specific genes. For example, the LGALS2 gene was mainly enriched in the Cluster 2 subpopulation, and its expression was low in other subpopulations (e.g., Figure 2 B). The `wilcox_test` function was used to calculate the significant differences in the proportion of each subpopulation among healthy individuals (HC), patients with septicemia (NSIC), and patients with septic coagulopathy (SIC). It can be seen that the proportion of the Cluster 2 subpopulation is significantly reduced in SIC patients (e.g., ...). Figure 2 C).

[0028] Therefore, it can be seen that the cluster subset with high expression of LGALS2 has reduced specificity in septic coagulopathy. The specific method is as follows:

[0029] 1. Single-cell sequencing was performed on peripheral blood mononuclear cells from healthy individuals, sepsis patients, and patients with septic coagulopathy. Cell populations were annotated using the Seurat package to identify different cell types. Mononuclear cell populations were then extracted and subpopulations were clustered separately, identifying eight mononuclear cell subpopulations, named Clusters 1-8. The code is as follows: `DimPlot( object, dims = c(1, 2), cells = NULL, cols = NULL, pt.size = NULL, reduction = NULL, group.by = NULL, split.by = NULL, shape.by = NULL, order = NULL, shuffle = FALSE, seed = 1, label = FALSE, label.size = 4, label.color = "black", label.box = FALSE, repel = FALSE, alpha = 1, cells.highlight = NULL, cols.highlight = "#DE2D26", sizes.highlight = 1,na.value = "grey50", ncol = NULL, combine = TRUE, raster = NULL,raster.dpi = c(512, 512))

[0030] 2. The FindALLMarker function was used to identify specific genes in Clusters 1-8. For example, the LGALS2 gene was mainly enriched in the Cluster 2 subpopulation, and its expression was low in other subpopulations. The code is: DotPlot( object, features,assay = NULL, cols = c("lightgrey", "blue"), col.min = -2.5, col.max =2.5, dot.min = 0, dot.scale = 6, idents = NULL, group.by = NULL,split.by = NULL, cluster.idents = FALSE, scale = TRUE, scale.by = "radius", scale.min = NA, scale.max = NA)

[0031] Example 2: Analysis of early diagnosis of septic coagulopathy using LGALS2 transcription levels

[0032] Single-cell sequencing analysis revealed that Cluster 2, characterized by high LGALS2 expression, showed decreased specificity in SIC patients. Therefore, LGALS2 was selected as a candidate gene to analyze its sensitivity and specificity in distinguishing between sepsis and septic coagulopathy. Peripheral blood samples from 30 sepsis patients and 39 septic coagulopathy patients were collected, and RNA was extracted.

[0033] Therefore, the transcriptional level of LGALS2 can be used as a biomarker for the early diagnosis of septic coagulopathy. The specific method is as follows:

[0034] 1.1.1 Collect 250 μl of human peripheral blood using an EDTA anticoagulant tube.

[0035] 1.1.2 Extraction of peripheral blood RNA

[0036] (1) Add 750 μl of Trizol, mix well by pipetting, and let stand at room temperature for 5 min to allow the sample to be fully lysed.

[0037] (2) Add 0.2 mL of chloroform to each 1 mL of Trizol used, shake vigorously for 15 seconds, and let stand at room temperature for 5 min.

[0038] (3) Centrifuge at 4℃ and 13000 rpm for 15 min. After centrifugation, the sample is divided into three layers: the bottom layer is a red organic phase, the top layer is a colorless aqueous phase, and an intermediate layer. DNA is in the intermediate layer and the organic layer.

[0039] (4) Transfer the aqueous phase to a new tube, add 0.5 mL of isopropanol, invert and mix well, and let stand at room temperature for 10 min or more.

[0040] (5) Centrifuge at 4℃ and 13000 rpm for 10 min. No RNA precipitate can be seen before centrifugation. After centrifugation, if there is a large amount of RNA, a white precipitate may appear at the bottom of the tube. Carefully discard the supernatant.

[0041] (6) Wash the RNA precipitate with 75% ethanol (prepared with DEPC water). Add 1 mL of 75% ethanol (prepared with DEPC water), gently vortex the centrifuge tube to suspend the precipitate. Centrifuge at 4°C for no more than 7500×g for 5 min, and carefully discard the supernatant.

[0042] (7) Repeat step 6 once.

[0043] (8) Wash the RNA precipitate once more with anhydrous ethanol, add 1 mL of anhydrous ethanol, gently shake the centrifuge tube to suspend the precipitate. Centrifuge at 4°C for no more than 7500×g for 5 min, and carefully discard the supernatant.

[0044] (9) Allow the RNA precipitate to dry at room temperature or vacuum dry for about 5-10 minutes. Add an appropriate amount of DEPC H2O to dissolve (20-50 μL / tube), detect the RNA concentration, and store at -80℃.

[0045] 1.1.3 RNA reverse transcription into cDNA

[0046] RNA reverse transcription to cDNA requires two primers, LGALS2-F and LGALS2-R. The sequence of LGALS2-F is SEQ ID NO: 1, and the sequence of LGALS2-R is SEQ ID NO: 2.

[0047] Dilute the RNA to 500 ng / μL and prepare it according to the following system:

[0048]

[0049] The prepared PCR system was used to perform the reaction using a Bio-Rad PCR instrument. The reaction conditions were divided into two stages: the first stage was 42℃ for 2 min; the second stage was 4℃, and this temperature was maintained until the next reaction.

[0050] Remove the reaction product from the previous step from the PCR instrument and prepare it according to the following system:

[0051]

[0052] The prepared PCR system was reacted using a Bio-Rad PCR instrument. The reaction conditions were divided into three stages: the first stage was 37°C for 15 min; the second stage was 85°C for 5 sec; and the third stage was 4°C. The system can be stored in a 4°C refrigerator.

[0053] 1.1.4 Quantitative qPCR

[0054] Prepare according to the following system:

[0055]

[0056] The prepared PCR system was reacted using a Roche qPCR instrument under the following conditions:

[0057] Stage 1: Pre-variation

[0058] 95℃ 30 seconds 20℃ / second

[0059] 1 Cycle

[0060] Stage 2: PCR reaction

[0061] 95℃ for 5 seconds, 20℃ / second (denaturation)

[0062] 60℃ for 20 seconds (annealing and extension)

[0063] 40 Cycles

[0064] Stage 3: Melting Curve Analysis

[0065] 95℃ 0 seconds 20℃ / second

[0066] 65℃ 15 seconds 20℃ / second

[0067] 95℃ 0 seconds 0.1℃ / second

[0068] Using qPCR to detect LGALS2 transcription levels, it was found that its level in SIC was significantly lower than that in NSIC. Figure 1 A). ROC curve analysis revealed that the AUC of LGALS2 was 0.730, which was significantly higher than that of commonly used thrombosis-related indicators APTT (AUC=0.565) and D-dimer (AUC=0.467).

[0069] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0070] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. Application of reagents for detecting LGALS2 gene expression levels in the preparation of kits for detecting septic coagulopathy; The reagents include reagent one, reagent two, and reagent three. Reagent one contains gDNA Eraser, buffer one, and deionized water. Reagent two contains PrimeScript RT Enzyme Mix I, RT Primer Mix, buffer two, and deionized water. Reagent three contains SYBR Premix Ex Taq and distilled water. Reagent 1, Reagent 2, and Reagent 3 all contain two primers involved in the PCR reaction, namely LGALS2-F and LGALS2-R, wherein the sequence of LGALS2-F is SEQ ID NO: 1 and the sequence of LGALS2-R is SEQ ID NO:

2.

2. The application according to claim 1, characterized in that, The preparation of the reagent includes: Reagent 1 can be obtained by mixing gDNA Eraser, Buffer 1, deionized water, LGALS2-F and LGALS2-R; Reagent 2 can be obtained by mixing PrimeScript RT Enzyme Mix I, RT Primer Mix, Buffer II, Deionized Water, LGALS2-F, and LGALS2-R. Reagent 3 can be obtained by mixing SYBR Premix Ex Taq, distilled water, LGALS2-F, and LGALS2-R. The sequence of LGALS2-F is SEQ ID NO: 1, and the sequence of LGALS2-R is SEQ ID NO:

2.

3. The application according to claim 1 or 2, characterized in that, The buffer solution in reagent one is 5×gDNAEraser Buffer.

4. The application according to claim 1 or 2, characterized in that, The buffer solution in reagent two is 5×PrimeScript Buffer 2.

5. The application according to claim 1 or 2, characterized in that, The deionized water in Reagent 1 and Reagent 2 is RNase-free dH2O.

6. The application according to claim 1 or 2, characterized in that, The distilled water in reagent three is ddH2O.

7. The application according to claim 1 or 2, characterized in that, The reaction conditions for reagent 1 are as follows: the reaction is divided into two stages. The first stage is 42℃ for 2 min. The second stage is 4℃, and this temperature is maintained until the next reaction.

8. The application according to claim 1 or 2, characterized in that, The reaction conditions for reagent 2 are as follows: The reaction conditions are divided into three stages: the first stage: 37℃, 15min; the second stage: 85℃, 5sec; and the third stage: 4℃.

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