Method for detecting early warning factor of IL-1beta gram-negative bacterium infection

Abstract

The invention discloses an IL-1beta gram-negative bacterium infection early warning factor detection method, and particularly relates to the technical field of IL-1beta gram-negative bacterium infection detection methods, the specific steps are as follows: step 1, collecting a fish sample; 2, extracting total RNA (Ribonucleic Acid); step 3, synthesizing cDNA (complementary deoxyribonucleic acid); step 4, cloning of IL-1beta (interleukin-1beta); step 5, detecting the tissue distribution of Il-1beta; and step 6, statistics and analysis. The method is helpful for knowing the immune state of the fish body, and lays a foundation for early prevention and treatment of IL-1beta in fish bacterial diseases.

Description

Technical Field

[0001] The invention relates to the technical field of IL-1β Gram-negative bacteria infection detection, and more specifically to a method for detecting an early warning factor of IL-1β Gram-negative bacteria infection. Background Art

[0002] Pelteobagrus vachellii belongs to the order Siluriformes, family Catfish, genus Pelteobagrus, and is the largest species in the genus Pelteobagrus, with a maximum length of 300 mm. Its meat is tender and delicious, rich in nutrition, and grows rapidly, which is deeply loved by consumers and breeders. It has now become an important freshwater famous and economically cultivated fish species. With the high-density artificial breeding of Pelteobagrus vachellii, the immunity of the farmed fish and the breeding environment have both declined, resulting in frequent diseases, causing serious losses to the breeding industry of Pelteobagrus vachellii.

[0003] In 1972, Grey et al. discovered that interleukin-1 (IL-1) is a pro-inflammatory cytokine that can activate a variety of immune cells and inflammatory cells. IL-1 is mainly produced by monocytes-macrophages and neutrophils. According to the structure and isoelectric point of its molecule, it can be divided into IL-1α and IL-1β. Among them, IL-1β has a wide range of biological activities and is one of the most powerful inflammatory mediators in the body. IL-1α has not yet been found in fish, but IL-1β has been found in rainbow trout (Oncorhynchus mykiss), and has been cloned in teleost fish such as salmon, cyprinids and perch. The concentration of IL-1β in normal blood is very low, but under the induction of physical, chemical and physiological factors, IL-1β can promote the expression of endothelial-leukocyte adhesion molecules, chemotactic neutrophils and other inflammatory cells into the body's lesions, and the expression of IL-1βmRNA, and its expression level is positively correlated with the degree of inflammation, so it is often used as an indicator for clinically judging the severity of the disease and the efficacy of treatment. Studies on the role of IL-1β in fish infection and immunity have found that the il-1βmRNA of yellow catfish was significantly upregulated in multiple organs after Edwardsiella ictaluri infection; rainbow trout rIL-1β can resist infection with Aeromonas salmonicida; channel catfish rIL-1β can be used as a vaccine adjuvant to improve fish innate immunity. At present, research on the il-1β gene of yellow catfish has not yet been carried out.

[0004] In the process of disease prevention and control, the improper use of antibiotics often causes fishery drug residues and bacterial resistance, which seriously affects the water environment and fish quality. In order to study the role of IL-1β in immunity and disease prevention and control of Pelteobagrus vachelli, the present invention proposes a method for detecting an early warning factor of IL-1β Gram-negative bacterial infection, which lays a foundation for the early prevention and control of bacterial diseases by IL-1β. Summary of the invention

[0005] In order to overcome the above-mentioned defects of the prior art, the present invention provides a method for detecting an early warning factor of IL-1β Gram-negative bacterial infection, which helps to understand the immune status of fish and lays a foundation for the early prevention and treatment of bacterial diseases in fish.

[0006] To achieve the above object, the present invention provides the following technical solution: a method for detecting an early warning factor of IL-1β Gram-negative bacterial infection, the specific steps of which are as follows:

[0007] Step 1, collecting fish samples;

[0008] Step 2, extracting total RNA;

[0009] Step 3, synthesizing cDNA;

[0010] Step 4: Cloning of IL-1β:

[0011] PCR amplification was performed using spleen cDNA as template and pf-il-1βF and pf-il-1βR as primers;

[0012] Step 5: Detection of tissue distribution of Il-1β:

[0013] Using cDNA of fish tissues and organs as templates, SYBR Green dye fluorescence quantitative PCR was performed using q-wa-il-1βF / R and internal reference primer β-actinF / R. Three parallel experiments were performed for each cDNA, and 2 -△△CT .

[0014] Step 6: Statistics and analysis.

[0015] Preferably, in step 1, the fish samples include 12 samples of heart, liver, spleen, kidney, muscle, head kidney, foregut, midgut, hindgut, stomach, gill and brain, and the samples are placed in RNAstore and stored at 4°C overnight, and then transferred to -80°C low temperature storage after 24 hours.

[0016] Preferably, in step 2, the specific steps of extracting total RNA are as follows:

[0017] S1. After the sample is thawed, take 50-100 mg of tissue sample, add 1 mL of Trizol, and let it stand at room temperature for 5 minutes to allow it to fully lyse;

[0018] S2. Add 4 small steel beads to the sample EP tube, place it on a tissue homogenizer for 40 seconds, place it at 15-30℃ for 5 minutes (to completely separate the nucleoprotein complex), centrifuge it at 12000r / min4℃ for 5 minutes, take the supernatant, and discard the precipitate;

[0019] S3, add 200 μL chloroform to the supernatant, shake vigorously to mix, and leave at room temperature for 15 min;

[0020] S4, centrifuge at 4°C, 12000 rpm for 15 min, separate into three layers after centrifugation, aspirate the upper aqueous phase and transfer to another centrifuge tube;

[0021] S5. Add an equal volume of isopropanol, gently shake the centrifuge tube, place at room temperature for 5-10 minutes, centrifuge at 4°C, 12000r / min for 10 minutes, and discard the supernatant;

[0022] S6. Add 1 mL of 75% ethanol, gently shake the centrifuge tube, centrifuge for 5 min at 4°C, 12,000 r / min, discard the supernatant, and dry at room temperature for 5-10 min.

[0023] S7. Add 50 μL RNase-Free ddHO to the tube. 2 O dissolves the precipitate;

[0024] S8. Use 1% agarose gel electrophoresis to detect the quality of RNA, use a nucleic acid detector to determine the total RNA concentration, and store the extracted RNA at -80°C for future use.

[0025] Preferably, the specific steps of step 3 are: the total RNA extracted in step 2 is treated with RNase-Free ddH 2 O was diluted to a final concentration of 100 ng / μL, and cDNA reverse transcription was performed according to the cDNA reverse transcription kit.

[0026] Preferably, in step 4, the amplified product is subjected to agarose gel electrophoresis, gel recovery, T cloning, and sequencing of positive strains after ampicillin sodium screening. The sequenced sequence is uploaded to NCBI for Blast comparison, multiple homologous sequences of different similarities are downloaded, and DNAMAN is used to construct an evolutionary tree.

[0027] Preferably, in step 6, Prism 5.0 is used to perform significance analysis.

[0028] Technical effects and advantages of the present invention:

[0029] The method of the present invention clones the IL-1β gene of Pelteobagrus vachelli and analyzes its tissue distribution and mRNA level after lipopolysaccharide stimulation. The method of the present invention is helpful to understand the immune status of fish and lays a foundation for the early prevention and treatment of IL-1β in fish bacterial diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is the RNA concentration and dilution graph;

[0031] Figure 2 This is the PCR amplification product of il-1β;

[0032] Figure 3 Construct a diagram for the evolutionary tree of il-1β sequences;

[0033] Figure 4 The tissue distribution diagram of Il-1β detected by qRT-PCR in healthy Pelteobagrus vachelli;

[0034] Figure 5 This is the tissue distribution diagram of Il-1β detected by qRT-PCR after LPS stimulation in P. vachelli.

[0035] Figure 6 This is a diagram showing the difference in tissue expression of Il-1β before and after LPS stimulation. DETAILED DESCRIPTION

[0036] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] 1. Materials and Methods

[0038] 1 Healthy Yellow Catfish

[0039] Twenty fish weighing (8±2) g, healthy and disease-free, both male and female, were provided by the breeding base of the Sichuan Provincial Key Laboratory for the Conservation and Utilization of Fish Resources in the Upper Yangtze River. They were temporarily kept in the laboratory for 14 days (water temperature about 24℃, air pump inflation), and randomly divided into 2 groups (experimental group and control group), with 10 fish in each group.

[0040] 2 Primer design and synthesis

[0041] The full sequence primers pf-il-1βF and pf-il-1βR of the il-1β CDS region (Coding Sequence) of Pelteobagrus vachelli were designed with reference to the transcription data of Pelteobagrus vachelli. The primers (Table 1) were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0042] Table 1 Primer design for IL-1β of Pelteobagrus vachelli

[0043]

[0044]

[0045] 3 Main Materials

[0046] RNAstore, Trizol reagent, 2×PCRMix, SYBR Green qPCR mix, and cDNA reverse transcription kit were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., and the remaining reagents and consumables were commercially available.

[0047] 4 Experimental instruments

[0048] BHC-1300A2 biological safety cabinet; DQ ultrapure water instrument (Direct-Q3); FA2004N electronic analytical balance (Shanghai Jinghai Instrument Co., Ltd.); agarose gel electrophoresis (Bio-rad), CFX100 fluorescence quantitative PCR instrument (Bio-rad), and nucleic acid protein instrument.

[0049] Second, this embodiment provides a method for detecting an early warning factor of IL-1β Gram-negative bacterial infection, and the specific steps are as follows:

[0050] Step 1: Fish sample collection

[0051] After the experimental fish were anesthetized with para-acetylphenyl ether, 200 μL / tail of lipopolysaccharide (LPS 10 μg / mL) was intraperitoneally injected into the experimental group (LPS injection group), and 200 μL / tail of normal saline was intraperitoneally injected into the control group (healthy group). After recovery, the fish were returned to the culture barrel for continued culture under the same culture conditions and the fish body condition was observed.

[0052] After 4 hours, the experimental fish were anesthetized with paraacetylphenyl ether and killed. The fish were dissected and 12 samples including heart, liver, spleen, kidney, muscle, head kidney, anterior, middle and posterior intestine, stomach, gill and brain were collected and stored in RNAstore at 4°C overnight. After 24 hours, they were transferred to -80°C low-temperature storage until RNA extraction.

[0053] Step 2: Extraction of total RNA

[0054] Four fish tissues and organs were randomly selected from the experimental group and the control group for RNA extraction. The extraction method was based on the Trizol method, and the specific steps were as follows:

[0055] (1) After thawing, take 50-100 mg of tissue sample, add 1 mL of Trizol, and let stand at room temperature for 5 min to allow for complete lysis;

[0056] (2) Add 4 small steel beads to the sample EP tube, place it on a tissue homogenizer for 40 seconds, place it at 15-30°C for 5 minutes (to completely separate the nucleoprotein complex), centrifuge it at 12000r / min at 4°C for 5 minutes, take the supernatant, and discard the precipitate;

[0057] (3) Add 200 μL of chloroform to the supernatant, shake vigorously to mix, and leave at room temperature for 15 min;

[0058] (4) Centrifuge at 4°C, 12,000 rpm for 15 min. The mixture is separated into three layers. The upper aqueous phase is aspirated and transferred to another centrifuge tube.

[0059] (5) Add an equal volume of isopropanol, gently shake the centrifuge tube, place at room temperature for 5-10 min, centrifuge at 4°C, 12000 r / min for 10 min, and discard the supernatant;

[0060] (6) Add 1 mL of 75% ethanol, gently shake the centrifuge tube, centrifuge at 4°C, 12,000 rpm, centrifuge for 5 min, discard the supernatant, and air-dry at room temperature for 5-10 min;

[0061] (7) Add 50 μL RNase-Free ddHO to the tube. 2 O dissolves the precipitate;

[0062] (8) The quality of RNA was tested by 1% agarose gel electrophoresis, and the total RNA concentration was determined by a nucleic acid detector. The extracted RNA was stored at -80°C for future use;

[0063] Step 3: Synthesis of Pelteobagrus vachelli cDNA

[0064] The total RNA extracted in step 2 was purified by RNase-Free ddH 2 O was diluted to a final concentration of 100 ng / μL, and cDNA reverse transcription was performed according to the cDNA reverse transcription kit. The reaction system and procedure are shown in Table 2.

[0065] Table 2 cDNA synthesis reaction system and procedure

[0066]

[0067]

[0068] Step 4. Cloning of IL-1β

[0069] PCR amplification was performed using the spleen cDNA of Pelteobagrus vachelli as a template and pf-il-1βF and pf-il-1βR as primers. The reaction procedure and system are shown in Table 3. The amplified products were obtained by agarose gel electrophoresis, gel recovery, and T cloning. The positive strains screened with ampicillin sodium (1 μg / mL) were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequence was uploaded to NCBI for Blast comparison, and multiple homologous sequences of different similarities were downloaded, and the evolutionary tree was constructed using DNAMAN.

[0070] Table 3 cDNA synthesis reaction system and procedure

[0071]

[0072] Step 5: Tissue distribution of Il-1β

[0073] Using the cDNA of various tissues and organs of the control group (healthy) Pelteobagrus vachelli as templates, SYBR Green dye fluorescence quantitative PCR was performed using q-va-il-1βF / R and internal reference primers β-actinF / R. The reaction procedures and systems are shown in Table 4. Three parallel experiments were performed for each cDNA. The experimental results were calculated using β-actin as the first reference and brain as the second reference. -△△CT .

[0074] Table 4 qRT-PCR reaction system and procedure for tissue distribution of IL-1β

[0075]

[0076] Step 6. Expression profile of IL-1β after LPS stimulation

[0077] Using cDNA of various tissues and organs of P. vachelli in the experimental group (LPS injection group) as templates, SYBR Green dye fluorescence quantitative PCR was performed using q-va-il-1βF / R and internal reference primer β-actinF / R, respectively. The reaction procedures and systems were the same as those in Table 4. Three parallel experiments were performed for each cDNA. The experimental results were calculated using β-actin as the first control reference and the control group (healthy group) as the second control reference. -△△CT .

[0078] Step 7: Statistics and analysis

[0079] The qRT-PCR experimental results were analyzed and plotted using Prism5.0.

[0080] 3. Results

[0081] 1. Sample collection, RNA extraction and cDNA synthesis

[0082] After intraperitoneal injection of LPS and saline, the fish in the experimental and control groups showed no clinical lesions within 4 hours. After dissection and collection of various tissues, RNA was extracted with Trizol, and the RNA concentration was measured using a micro-nucleic acid protein analyzer. The minimum RNA concentration was 228.6 ng / μL (muscle tissue of the control group) and the maximum was 5171.1 ng / μL (liver tissue of the experimental group). All RNA concentrations were greater than 200 ng / μL, which was suitable for cDNA reverse transcription. The obtained RNA was diluted with RNase-Freewater and successfully synthesized into cDNA ( Figure 1 ).

[0083] 2. Il-1β CDS region

[0084] Using the spleen cDNA of P. vachelli as template and pf-il-1βF / R as primers, a specific band of about 750 bp was obtained by PCR amplification. Figure 2 The product was obtained by agarose gel electrophoresis, gel recovery, and T cloning. The positive strains screened with ampicillin sodium (1 μg / mL) were sent to Shanghai Biotech Co., Ltd. for sequencing. The sequence was uploaded to NCBI for Blast comparison, and multiple homologous sequences of different similarities were downloaded. The evolutionary tree was constructed using DNAMAN. The results showed that the IL-1β of P. vachelli was clustered with that of P. fulvidraco, and the similarity with the IL-1β of mammals, amphibians, reptiles, and birds was not high ( Figure 3 ).

[0085] 3. Tissue distribution of Il-1β in healthy P. vachelli

[0086] QRT-PCR was used to detect the expression of IL-1β in the heart, liver, spleen, kidney, muscle, head kidney, anterior, middle and posterior intestine, stomach, gill and brain of healthy P. vachelli. The internal reference gene β-actin was used as the first control reference, and the brain was used as the second control reference. The 2 -△△CT , the results show (Table 5, Figure 4 ): In a healthy state, the highest expression level of il-1β was in the liver (7.09±5.21), followed by the spleen (8.48±3.46), and the lowest expression level was in the midgut (0.61±0.25). The results of differential analysis using Prism5.0 software showed that the expression level of il-1β in the liver and spleen of healthy P. vachelli was significantly higher than that in other organs.

[0087] Table 5 qRT-PCR detection of Il-1β in healthy P. vachelli tissues

[0088]

[0089]

[0090] 4. Tissue distribution of Il-1β after LPS stimulation

[0091] QRT-PCR was used to detect the expression of IL-1β in the heart, liver, spleen, kidney, muscle, head kidney, anterior, middle and posterior intestine, stomach, gill and brain of P. vachelli stimulated with LPS. The internal reference gene β-actin was used as the first control reference, and the brain was used as the second control reference. The 2 -△△CT The results show (Table 6, Figure 5 ): After LPS stimulation, the highest expression of il-1β was in the liver (151.62±37.17), followed by the spleen (83.57±31.94), and the lowest expression was in the brain (0.61±0.25). The results of differential analysis using Prism5.0 software showed that the expression of il-1β in the liver, spleen and heart was significantly higher than that in other organs of P. vachelli after LPS stimulation.

[0092] Table 6 qRT-PCR detection of Il-1β tissue distribution in P. vachelli after LPS stimulation

[0093]

[0094]

[0095] 5. Analysis of the difference in tissue expression of Il-1β before and after LPS stimulation

[0096] Prism5.0 software was used to compare and analyze the expression differences of Il-1β in various tissues before and after LPS stimulation. β-actin was used as the first reference and the control group (healthy group) was used as the second reference. -△△CT The results showed that after LPS stimulation, the expression of Il-1β in liver, spleen, kidney, head kidney, muscle, heart, foregut and hindgut increased significantly, and the expression in brain, gill, midgut and stomach did not change significantly (Table 7, Figure 6 ).

[0097] Table 7 Analysis of differences in tissue expression of Il-1β before and after LPS stimulation

[0098]

[0099]

[0100] Discussion

[0101] 1. Sequence analysis of fish IL-1β gene

[0102] This embodiment cloned and identified the CDS sequence of the il-1β gene of Pelteobagrus vachelli. The CDS sequence is a coding sequence region that can be translated into protein in mature mRNA. Compared with ORF (Open reading Frame), it can more accurately reflect the primary structure of the protein, and there are no other sequences unrelated to the protein. The CDS sequence length of the il-1β gene of Pelteobagrus vachelli obtained is 729bp, which is 15bp longer than the reported il-1β gene of Pelteobagrus vachelli (Genbank No.JQ730738.1) and 114bp shorter than the il-1β gene of Pelteobagrus vachelli (Genbank No.XM_027134319.2). Further through multiple sequence alignment, it was found that the sequence had a high similarity with catfish such as Pelteobagrus vachelli, Channel Catfish, and Macrofin Catfish, suggesting that the il-1β gene of Pelteobagrus vachelli may be missing a section compared to Pelteobagrus vachelli. Liao Chenlei et al. (Liao Chenlei, Cloning and Expression Analysis of IL-1β and IL-1RI Genes in Pelteobagrus fulvidraco [D]. Huazhong Agricultural University, 2018.) found that the amino acid sequence of il-1β in yellow catfish lacks aspartic acid in the ICE site in mammals, which is consistent with the results of this embodiment. Evolutionary tree analysis found that the il-1β gene of Pelteobagrus fulvidraco is quite different from the il-1β gene of humans, cattle, birds (sparrows) and reptiles (snakes).

[0103] 2. Analysis of IL-1β mRNA expression profile in fish

[0104] The expression profile of il-1β mRNA in fish varies greatly among different species: grass carp, crocodile icefish and semi-smooth tongue sole have high expression of il-1β in head kidney and spleen, while yellowfin sea bream has very low expression in kidney and spleen, and even cannot be detected in liver, muscle and heart. The results of this experiment are similar to the expression profile of il-1β in yellow catfish, which is expressed in all the tissues tested, with high expression in body kidney, head kidney, heart, muscle, liver and spleen; the difference is that the expression level of il-1β in yellow catfish is highest in liver, while that in yellow catfish is highest in head kidney. This may be related to the age of the fish. The yellow catfish used in this experiment is a juvenile, while the yellow catfish used by Liao Chenlei et al. is an adult fish.

[0105] 3. Application of fish IL-1β

[0106] IL-1β is mainly produced by macrophages and neutrophils during inflammation. It is an important cytokine involved in biological functions such as inflammatory response, immune regulation and metabolic regulation. Appropriate amounts of IL-1β can stimulate immunity and improve immune capacity: intraperitoneal injection of rIL-1β in eels significantly upregulated liver NF-kB, and inhibited AKR and Nrf2 genes. Wu Jie et al. (Wu Jie. Study on cloning and expression of IL-1β gene in spotted catfish and its immune effect as a subunit vaccine adjuvant [D].) and Yan Chunmei (Yan Chunmei, Zheng Wei, Du Xiaoyan, et al. Construction and combined application of recombinant plasmids of fish interleukin (IL-1β) and interferon (IFN) [J]. Heilongjiang Fisheries, 2015, 6: 36-42.) found that rIL-1β can significantly improve the autoimmunity of spotted catfish and rainbow trout. After the use of LPS intraperitoneal injection of Pelteobagrus vachelli in this embodiment, except for the brain, stomach, gills and midgut, the remaining organs showed a significant upregulation of il-1β, indicating that the fish's immune system is in an activated state. Although IL-1β can enhance immunity, excessive IL-1β may promote the occurrence of infection: studies have found that inhibiting the activity of IL-1β, such as blocking IL-1β receptors or using inhibitors, can promote cell autophagy and accelerate the clearance of intracellular bacteria. Therefore, studying the threshold of IL-1β in Pelteobagrus vachelli will help understand the immune status of the fish and provide a reference for the diagnosis of diseases in Pelteobagrus vachelli.

[0107] Finally: The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting an early warning factor of IL-1β Gram-negative bacterial infection, characterized in that: The specific steps are as follows: Step 1, collecting fish samples; Step 2, extracting total RNA; Step 3, synthesizing cDNA; Step 4: Cloning of IL-1β: PCR amplification was performed using spleen cDNA as template and pf-il-1βF and pf-il-1βR as primers; Step 5: Detection of tissue distribution of Il-1β: Using cDNA of fish tissues and organs as templates, SYBR Green dye fluorescence quantitative PCR was performed using q-wa-il-1βF / R and internal reference primer β-actinF / R. Three parallel experiments were performed for each cDNA, and 2 -△△CT ; Step 6: Statistics and analysis.

2. The method for detecting an early warning factor of IL-1β Gram-negative bacterial infection according to claim 1, characterized in that: In step 1, the fish samples included 12 samples of heart, liver, spleen, kidney, muscle, head kidney, foregut, midgut, hindgut, stomach, gill and brain. The samples were stored in RNAstore at 4°C overnight and transferred to -80°C low temperature storage after 24 hours.

3. The method for detecting an early warning factor of IL-1β Gram-negative bacterial infection according to claim 2, characterized in that: In step 2, the specific steps of total RNA extraction are as follows: S1. After the sample is thawed, take the tissue sample and add Trizol, then let it stand at room temperature for 5 minutes to allow it to fully lyse; S2. Add 4 small steel beads to the sample EP tube, place it on a tissue homogenizer for 40 seconds, place it at 15-30℃ for 5 minutes to completely separate the nucleoprotein complex, centrifuge it at 12000r / min, 4℃ for 5 minutes, take the supernatant, and discard the precipitate; S3. Add chloroform to the supernatant, shake vigorously to mix, and leave at room temperature for 15 min. S4, centrifuge at 4°C, 12000 rpm for 15 min, separate into three layers after centrifugation, aspirate the upper aqueous phase and transfer to another centrifuge tube; S5. Add an equal volume of isopropanol, gently shake the centrifuge tube, place at room temperature for 5-10 minutes, centrifuge at 4°C, 12000r / min for 10 minutes, and discard the supernatant; S6. Add 75% ethanol, gently shake the centrifuge tube, centrifuge for 5 min at 4°C, 12,000 r / min, discard the supernatant, and dry at room temperature for 5-10 min; S7, add RNase-Free ddH2O to the tube to dissolve the precipitate; S8. After testing the quality and concentration of RNA, store the RNA at -80°C for future use.

4. The method for detecting an early warning factor of IL-1β Gram-negative bacterial infection according to claim 1, characterized in that: The specific steps of step 3 are: dilute the total RNA extracted in step 2 to a final concentration of 100 ng / μL using RNase-Free ddH2O, and perform cDNA reverse transcription according to the cDNA reverse transcription kit.

5. The method for detecting an early warning factor of IL-1β Gram-negative bacterial infection according to claim 1, characterized in that: In step 4, the amplified product was subjected to agarose gel electrophoresis, gel recovery, and T cloning, and the positive strains were sequenced after ampicillin sodium screening. The sequenced sequence was uploaded to NCBI for Blast comparison, and multiple homologous sequences of different similarities were downloaded, and the evolutionary tree was constructed using DNAMAN.

6. The method for detecting an early warning factor of IL-1β Gram-negative bacterial infection according to claim 1, characterized in that: In step 6, Prism 5.0 was used for significance analysis.

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