Application of reagent for detecting LGALS2 gene in preparation of product for diagnosing sepsis coagulopathy

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of a reagent for detecting an LGALS2 gene in preparation of a kit for detecting sepsis coagulopathy. According to the product, the sepsis coagulopathy is diagnosed by detecting the transcriptional level of the LGALS2 gene in a sample, and when the transcriptional level of the LGALS2 gene is remarkably reduced, a subject suffers from the sepsis coagulopathy. According to the application disclosed by the invention, LGALS2 is adopted as a biomarker, and the transcriptional level of LGALS2 in peripheral blood of a patient suffering from sepsis coagulopathy is obviously lower than that of a patient not suffering from sepsis coagulopathy through crowd experiments. The transcriptional level of the LGALS2 in the whole blood RNA has high specificity and sensitivity on diagnosis and prediction of the generation risk of sepsis coagulopathy. The LGALS2 gene and the transcriptional level thereof can be used for preparing a reagent or a kit for early diagnosis of sepsis coagulopathy, reference information is provided for individualized intervention treatment, and the LGALS2 gene has important clinical application value and generalization performance.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of a reagent for detecting the LGALS2 gene in the preparation of a product for diagnosing septic coagulopathy. Background Art

[0002] Sepsis is a life-threatening organ dysfunction caused by an imbalanced response of the body to infection. Sepsis-induced coagulopathy (SIC) is endothelial cell damage and coagulation disorder caused by sepsis. According to statistics, 24.0%-60.0% of sepsis patients worldwide develop SIC, while in my country the rate is as high as 67.9%. Delayed diagnosis and improper treatment of SIC can develop into disseminated intravascular coagulation (DIC), which doubles the mortality rate of patients. Therefore, the diagnosis and treatment of sepsis-induced coagulopathy has become a major problem that needs to be solved urgently in the medical community.

[0003] The pathophysiological mechanism of septic coagulopathy is extremely complex. It is currently believed that SIC is a process that develops from moderate immune thrombosis to excessive thrombotic inflammation. Immune thrombosis is a thrombus with immune function formed in the microvessels by the body in the early stage of infection. Excessive formation of immune thrombosis can lead to the disorder and spread of thrombotic inflammation, and then SIC occurs. Monocytes play an important role in the formation of immune thrombosis. Monocytes are a potential source of "tissue factor" in circulating blood. Compared with the subendothelial tissue factor expressed on fibroblasts and capillary pericytes, the expression of monocyte-related tissue factor is usually very low, but it will increase significantly under the stimulation of pathogens. In addition, monocytes can promote the massive activation of TF through the cysteine-containing aspartate protease 11-mediated cell pyroptosis pathway and the nucleotide oligomerization domain-like receptor pyrin domain-associated protein 3 inflammasome, a process also known as TF "decryption". Once the monocyte-derived tissue factor is activated, it can activate FVII to initiate the extrinsic coagulation pathway and form immune thrombosis. However, the functional mechanism of monocytes in septic coagulopathy has not been fully elucidated, and monocytes have great heterogeneity, which makes it difficult to analyze their functions and mechanisms. Therefore, analyzing 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 deficiencies of the prior art, the object of the present invention is to provide a new biomarker that is helpful for the early diagnosis of septic coagulopathy, specifically to use the LGALS2 gene and its transcription level in the preparation of an early diagnostic reagent or kit for septic coagulopathy.

[0005] The object of the present invention can be achieved by the following technical solutions:

[0006] Use of a reagent for detecting the LGALS2 gene in the preparation of a kit for detecting septic coagulopathy.

[0007] Furthermore, the reagent comprises Reagent 1, Reagent 2 and Reagent 3. Reagent 1 contains gDNAEraser, Buffer 1 and deionized water. Reagent 2 contains PrimeScript RT Enzyme Mix I, RT Primer Mix, Buffer 2 and deionized water. Reagent 3 contains SYBR Premix Ex Taq and distilled water;

[0008] Both Reagent 1, Reagent 2 and Reagent 3 contain two primers participating in the PCR reaction, namely 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.

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

[0010] Mix gDNA Eraser, Buffer 1, deionized water, LGALS2-F and LGALS2-R to obtain Reagent 1;

[0011] Mix PrimeScript RT Enzyme Mix I, RT Primer Mix, Buffer 2, deionized water, LGALS2-F and LGALS2-R to obtain Reagent 2;

[0012] Mix SYBR Premix Ex Taq, distilled water, LGALS2-F and LGALS2-R to obtain Reagent 3;

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

[0014] Furthermore, Buffer 1 in Reagent 1 is 5×gDNAEraserBuffer.

[0015] Furthermore, Buffer 2 in Reagent 2 is 5×PrimeScriptBuffer 2.

[0016] Furthermore, the deionized water in Reagent 1 and Reagent 2 is RNase Free dH 2 O.

[0017] Furthermore, the distilled water in Reagent 3 is ddH2 O。

[0018] Furthermore, the reaction conditions for Reagent 1 to participate in the reaction are as follows: The reaction is divided into two stages. The first stage: 42 °C, 2 min; the second stage: 4 °C, and this temperature is maintained until the next reaction.

[0019] Furthermore, the reaction conditions for Reagent 2 to participate in the reaction are as follows: The reaction conditions are divided into three stages. The first stage: 37 °C, 15 min; the second stage: 85 °C, 5 sec; the third stage: 4 °C.

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

[0021] Advantages of the present invention: The present invention uses LGALS2 as a biomarker. Through population experiments, it is verified that the transcriptional level of LGALS2 in the peripheral blood of patients with septic coagulopathy is significantly lower than that of patients without septic coagulopathy. The transcriptional level of LGALS2 in whole blood RNA has high specificity and sensitivity for the diagnosis and prediction of the risk of septic coagulopathy. The LGALS2 gene and its transcriptional level can be used to prepare reagents or kits for the early diagnosis of septic coagulopathy, providing reference information for individualized intervention and treatment, and having important clinical application value and promotion potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present disclosure or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, for those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0023] Figure 1 This is the expression pattern of LGALS2 in SIC and NSIC in the present invention; wherein Figure A shows that the expression of LGALS2 in SIC is significantly lower than that in Nsic; Figures B - D are the ROC curve analyses of LGALS2, APTT, and D - dimer in SIC and NSIC.

[0024] Figure 2 This shows that the proportion of monocytes subsets (cluster 2) with high expression of LGALS2+ is decreased in septic coagulopathy in the present invention; wherein A is the UMAP map of monocyte clustering; B is the bubble map of marker genes of each monocyte subset; C is the proportion of each monocyte subset in the peripheral blood of healthy people (HC), sepsis (NSIC), and septic coagulopathy (SIC) patients. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0025] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0026] Example 1: Method for obtaining cell subsets and related genes related to the diagnosis of peripheral blood monocytes in patients with septic coagulopathy

[0027] Single-cell sequencing was performed on peripheral blood mononuclear cells of healthy people, sepsis patients, and patients with septic coagulopathy. The cell populations were annotated through the Seurat package to distinguish different types of cells. Then, the monocyte population was extracted and subclustered separately, and 8 monocyte subsets were identified and named Cluster 1-8 (as Figure 2 A). The specific genes of Cluster 1-8 were identified using the FindALLMarker function. For example, the LGALS2 gene was mainly enriched in the Cluster 2 subset, and its expression in other subsets was relatively low (as Figure 2 B). The wilcox_test function was used to calculate the significant difference in the cell proportions of each subset among healthy people (HC), sepsis (NSIC) patients, and septic coagulopathy (SIC) patients. It can be seen that the proportion of the Cluster 2 subset in SIC patients was significantly decreased (as Figure 2 C).

[0028] Thus, it can be seen that the Cluster subset with high expression of LGALS2 is specifically decreased in septic coagulopathy. The specific method is as follows:

[0029] 1. Single-cell sequencing was performed on peripheral blood mononuclear cells of healthy individuals, sepsis patients, and patients with septic coagulopathy. The cell populations were annotated using the Seurat package to distinguish different types of cells. Then, the monocyte population was extracted and subclustered separately, and 8 monocyte subpopulations were identified and named Cluster 1-8. The code is: 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 specific genes of Cluster 1-8 were identified using the FindALLMarker function. For example, the LGALS2 gene was mainly enriched in the Cluster 2 subpopulation and was expressed at a low level 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: Early diagnosis analysis of septic coagulopathy using the transcriptional level of LGALS2

[0032] From single-cell sequencing analysis, it can be seen that Cluster 2, characterized by high expression of LGALS2, is specifically reduced in SIC patients. Therefore, LGALS2 was selected as a candidate gene to analyze its sensitivity and specificity in differentiating sepsis and septic coagulopathy patients. Peripheral blood from 30 sepsis patients and 39 septic coagulopathy patients was collected, and RNA was extracted.

[0033] Thus, it can be seen that the early diagnosis of septic coagulopathy can be carried out by using the transcriptional level of LGALS2 as a biomarker. 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 Extract RNA from peripheral blood

[0036] (1) Add 750 μl of Trizol, pipette and mix well, and let it stand at room temperature for 5 min to fully lyse the sample.

[0037] (2) Add 0.2 ml of chloroform for every 1 ml of Trizol used, shake vigorously for 15 seconds, and place at room temperature for 5 min.

[0038] (3) Centrifuge at 13,000 rpm for 15 min at 4°C. After centrifugation, the sample is divided into three layers: the bottom layer is the red organic phase, the upper layer is the 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 it stand at room temperature for 10 min or more.

[0040] (5) Centrifuge at 13,000 rpm for 10 min at 4°C. Before centrifugation, no RNA precipitate can be seen. When the amount of RNA is large after centrifugation, a white precipitate may appear at the bottom of the tube. Carefully pour off 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 shake the centrifuge tube to suspend the precipitate. Centrifuge at 4°C at no more than 7,500 × g for 5 min, and carefully discard the supernatant.

[0042] (7) Repeat step 6 once.

[0043] (8) Wash the RNA precipitate with absolute ethanol once more. Add 1 ml of absolute ethanol, gently shake the centrifuge tube to suspend the precipitate. Centrifuge at 4°C at no more than 7,500 × g for 5 min, and carefully discard the supernatant.

[0044] Air-dry the RNA pellet at room temperature or dry it under vacuum for about 5 - 10 minutes. Add an appropriate amount of DEPC H₂O to dissolve it (20 - 50 μL / tube), measure the RNA concentration, and store it at -80 °C.

[0045] 1.1.3 Reverse transcription of RNA into cDNA

[0046] Two primers are required for the reverse transcription of RNA into cDNA, namely 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 the following reaction system:

[0048] Component Volume (μl) 5× gDNA Eraser Buffer 2.0 gDNA Eraser 1.0 RNA 2.0 RNase Free dH2O 5.0

[0049] Perform the reaction on a Bio-Rad PCR instrument with the prepared PCR system. The reaction conditions are divided into two stages. The first stage: 42 °C for 2 minutes; the second stage: 4 °C, maintain this temperature until the next reaction.

[0050] Take out the reaction product from the previous step and prepare the following reaction system:

[0051]

[0052] Perform the reaction on a Bio-Rad PCR instrument with the prepared PCR system. The reaction conditions are divided into three stages. The first stage: 37 °C for 15 minutes; the second stage: 85 °C for 5 seconds; the third stage: 4 °C, can be stored in a 4 °C refrigerator.

[0053] 1.1.4 Quantitative qPCR

[0054] Prepare the following reaction system:

[0055]

[0056]

[0057] Perform the reaction on a Roche qPCR instrument with the prepared PCR system. The conditions are as follows:

[0058] Stage 1: Pre-denaturation

[0059] 95 °C for 30 seconds at 20 °C / second

[0060] 1 Cycle

[0061] Stage 2: PCR reaction

[0062] 95°C for 5 seconds at 20°C / second (denaturation)

[0063] 60°C for 20 seconds at 20°C / second (annealing and extension)

[0064] 40 Cycles

[0065] Stage 3: Melting curve analysis

[0066] 95°C for 0 seconds at 20°C / second

[0067] 65°C for 15 seconds at 20°C / second

[0068] 95°C for 0 seconds at 0.1°C / second

[0069] The transcriptional level of LGALS2 was detected by qPCR, and it was found that its level in SIC was significantly lower than that in NSIC( Figure 2 A). Through ROC curve analysis, it was found that the AUC of LGALS2 was 0.730, which was significantly higher than the commonly used thrombus-related indicators APTT (AUC = 0.565) and D-dimer (AUC = 0.467).

[0070] In the description of this specification, the descriptions referring to terms such as "one embodiment", "example", "specific example", etc. mean that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in a suitable manner in any one or more embodiments or examples.

[0071] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art of this industry should understand that the present invention is not limited by the above embodiments. The above embodiments and the descriptions in the specification only illustrate the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed.

Claims

1. Application of a reagent for detecting the LGALS2 gene in the preparation of a kit for detecting septic coagulopathy.

2. The use according to claim 1, characterized in that: The reagents include reagent 1, reagent 2 and reagent 3, wherein reagent 1 contains gDNA Eraser, buffer 1 and deionized water, reagent 2 contains PrimeScript RTEnzyme Mix I, RT Primer Mix, buffer 2 and deionized water, and reagent 3 contains SYBR Premix ExTaq and distilled water; The reagents one, two and three 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.

3. The use according to claim 1, characterized in that: The reagent configuration includes: Mix gDNA Eraser, buffer 1, deionized water, LGALS2-F and LGALS2-R to obtain reagent 1; Mix PrimeScript RT Enzyme Mix I, RT Primer Mix, Buffer II, deionized water, LGALS2-F, and LGALS2-R to obtain Reagent II; Mix SYBR Premix Ex Taq, distilled water, LGALS2-F and LGALS2-R to obtain reagent three; The sequence of LGALS2-F is SEQ ID NO: 1, and the sequence of LGALS2-R is SEQ ID NO:

2.

4. The use according to claim 2 or 3, characterized in that: The buffer solution in the reagent one is 5×gDNAEraser Buffer.

5. The use according to claim 2 or 3, characterized in that: The buffer 2 in the reagent 2 is 5×PrimeScript Buffer 2.

6. The use according to claim 2 or 3, characterized in that: The deionized water in reagents 1 and 2 is RNaseFree dH2O.

7. The use according to claim 2 or 3, characterized in that: The distilled water in the reagent three is ddH2O.

8. The use according to claim 2 or 3, characterized in that: The reaction conditions when reagent 1 participates in the reaction are: the reaction is divided into two stages, the first stage: 42°C, 2 minutes; the second stage: 4°C, maintaining this temperature until the next reaction.

9. The use according to claim 2 or 3, characterized in that: The reaction conditions when reagent 2 participates in the reaction are: the reaction conditions are divided into three stages, the first stage: 37°C, 15min; the second stage: 85°C, 5sec; the third stage 4°C.

10. A reagent for diagnosing septic coagulopathy, characterized in that: The reagents include reagents for detecting the LGALS2 gene.

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