A marker for diagnosing systemic inflammation and its application

Abstract

The present invention provides a marker for diagnosing systemic inflammation and its application, wherein the marker includes ADGRE3mRNA or ADGRE3 protein, or ADGRE3mRNA fragment, ADGRE3 protein fragment, and there is a significant difference in the content of the marker in samples of patients with systemic inflammation and healthy people. Clinical verification shows that the marker has high sensitivity and specificity in diagnosis, so systemic inflammation can be accurately diagnosed by detecting the ADGRE3mRNA transcription level or ADGRE3 protein expression level in individual samples. Compared with existing inflammatory markers, ADGRE3mRNA and ADGRE3 protein have higher sensitivity and specificity in diagnosing systemic inflammation.

Description

Technical Field

[0001] The present invention relates to a disease diagnosis reagent and method, and in particular to a marker for diagnosing systemic inflammation and its application. Background Art

[0002] The following background technology introduction is merely an introduction to background common sense and does not constitute any limitation to the present invention.

[0003] Systemic inflammation is an inflammatory response involving the entire body, usually a nonspecific response caused by infection, autoimmune disease, injury or other pathological conditions. This inflammatory response may lead to multi-organ dysfunction and even be life-threatening in severe cases. Therefore, timely and accurate diagnosis of systemic inflammation is crucial to guiding treatment and improving patient prognosis. In recent years, biomarkers have played an important role in the diagnosis of systemic inflammation, and they can provide important information about the inflammatory state, disease severity and treatment response.

[0004] Systemic inflammation is a protective response of the body to stimulation, involving immune cells, blood vessels and a variety of molecular mediators. In the process of systemic inflammation, after the body is stimulated by inflammation, immune cells such as monocytes / macrophages, neutrophils, and lymphocytes become activated, and release a large number of cytokines (such as IL-6, IL-1β, TNF-α and IFN-γ, etc.) to affect the synthesis of the body's acute phase reactants (APR), thereby changing their concentration in plasma. Changes in these cytokines and acute phase reactants reflect the body's response to inflammation and are currently used as important indicators for diagnosing and monitoring systemic inflammation.

[0005] C-reactive protein (CRP) is one of the acute phase reactive proteins derived from the liver and is currently one of the most widely used indicators for evaluating inflammation in clinical practice. When there is acute inflammation in the body, CRP begins to rise in 6 to 8 hours, and the peak value can be hundreds of times higher than the normal value, and it disappears quickly after the disease is cured. The increase in CRP is positively correlated with the degree of inflammation, which can timely make complication alarms and determine the effectiveness of treatment. Interleukin 6 (IL-6) is a key component of the cytokine network induced by IL-1 and TNF-α, and is also the main signaling molecule that mediates acute phase reactions. It can induce the body to produce APRs such as CRP, SAA and PCT. The increase in IL-6 is closely related to the degree of inflammation, especially in inflammation caused by systemic infections such as sepsis. Tumor necrosis factor-α (TNF-α) plays a key role in initiating the inflammatory cytokine storm reaction, and its rapid increase in levels reflects the acute stage of inflammation. Inhibitors targeting TNF-α have achieved varying degrees of success in the treatment of inflammatory diseases such as rheumatoid arthritis, ankylosing spondylitis, and inflammatory bowel disease. Erythrocyte sedimentation rate (ESR) is one of the traditional inflammatory markers, and its acceleration is usually associated with an increase in fibrinogen. Although the specificity of ESR is poor, it still has a certain value in the auxiliary reference of diagnosing inflammatory diseases. Procalcitonin (PCT) is another important inflammatory marker, especially in systemic inflammation caused by bacterial infection, the level of PCT is significantly increased. The determination of PCT has important clinical value for distinguishing bacterial and viral infections.

[0006] By measuring biomarkers such as CRP, IL-6, TNF-α, ESR and PCT, the degree of inflammation can be effectively assessed, disease progression and treatment response can be monitored. However, the above biomarkers only have a diagnostic and monitoring effect on inflammation, but do not distinguish between local inflammation and systemic inflammation, and have poor specificity. At the same time, the content of the above biomarkers in patients with systemic inflammation and healthy people is not significantly different, and the sensitivity is low. It is often necessary to combine multiple indicators and cooperate with imaging to make a diagnosis of systemic inflammation.

[0007] A variety of viral infections can lead to systemic inflammatory responses, such as influenza virus, novel coronavirus, dengue virus, respiratory syncytial virus, cytomegalovirus, Epstein-Barr virus, etc. In the systemic inflammatory response caused by viral infection, the immune system releases a variety of inflammatory mediators to activate immune cells, promote viral clearance and tissue repair. However, current conventional biomarkers such as PCT, HBP, and nCD64 infection index do not change significantly in viral infection, and CRP has low sensitivity. Therefore, there is still a lack of effective biomarkers in the clinic for the diagnosis of systemic inflammatory responses caused by viral infection and monitoring of the degree of inflammation to guide treatment adjustments and prognosis assessment.

[0008] Different from local inflammation, systemic inflammation is more serious and its diagnosis is more complicated. Timely and accurate diagnosis of systemic inflammation is crucial for guiding treatment and improving patient prognosis. Therefore, it is necessary to propose a highly sensitive and specific biomarker for systemic inflammation to address the current deficiencies in clinical medicine in the diagnosis of systemic inflammation. Summary of the invention

[0009] To solve the problems in the conventional technology, the present invention provides a marker for diagnosing systemic inflammation and its application, wherein the marker includes ADGRE3 mRNA or ADGRE3 mRNA fragment, or / and, may also include ADGRE3 protein or ADGRE3 protein fragment. By utilizing the changes of a single type of marker of the present invention, it is possible to diagnose whether an individual suffers from systemic inflammation, predict, monitor the severity, and guide specific treatment measures such as medication.

[0010] The present invention team found that there was a significant difference in the content of the marker in the samples of patients with systemic inflammation and healthy people, and the clinical validity of the marker diagnosis was verified. Therefore, by detecting ADGRE3 mRNA or ADGRE3 protein in individual samples, these markers can be used alone to diagnose whether an individual has systemic inflammation, and can also be used to diagnose the process of non-systemic inflammation developing into systemic inflammation, and can also be used to diagnose the effect of drug treatment for systemic inflammation. When used in combination with other markers, it can also distinguish whether the systemic inflammation is caused by viruses or bacteria.

[0011] The present invention team found that compared with existing inflammatory markers, ADGRE3 mRNA and / or ADGRE3 protein have higher sensitivity and specificity in diagnosing systemic inflammation.

[0012] High sensitivity has the following meanings: first, in the same group of patients with systemic inflammation, significance analysis shows that the ADGRE3 index (ADGRE3 mRNA or ADGRE3 protein) has decreased extremely significantly, while other inflammatory markers have a small change range, or, although other traditional inflammatory markers can diagnose systemic inflammation, the difference between healthy or local inflammation is not large, and some may not have significant differences, while the content difference of the markers of the present invention between samples of healthy or local inflammation patients and systemic inflammation patients is significant or extremely significant; second, in the same group of patients with systemic inflammation, the number of patients with an extremely significant decrease in the ADGRE3 index is greater, while the number of patients with changes in other inflammatory markers is small, so the use of the ADGRE3 index can more accurately diagnose systemic inflammation and reduce the misdiagnosis rate.

[0013] High specificity has the following meanings: first, in a case collection covering healthy people (no inflammation), non-systemic inflammation patients (patients with local inflammation or non-inflammatory diseases) and systemic inflammation patients, the ADGRE3 index can exclude non-systemic inflammation patients, including excluding local inflammation patients, which is impossible with existing inflammation indicators; second, within systemic inflammation patients, specific distinction can also be achieved for systemic inflammation of different infection types, for example, ADGRE3 indicators can be used to distinguish bacterial inflammation from non-bacterial inflammation, and assist clinical treatment. Although, when it is necessary to distinguish between bacterial infection or viral infection, the marker of the present invention needs to be combined with traditional bacterial markers, but the role and function of the marker of the present invention in the above diagnosis cannot be denied. Even among systemic inflammation patients, there are significant differences between different types of systemic inflammation, such as there are significant differences between sepsis patients and non-infectious systemic inflammation, so it can be understood that the marker can be used to distinguish sepsis from non-infectious systemic inflammation.

[0014] Therefore, one aspect of the present invention provides a use of a biomarker for preparing a reagent for diagnosing systemic inflammation, wherein the biomarker includes ADGRE3 mRNA or ADGRE3 mRNA fragment; or ADGRE3 protein, or protein fragment, or corresponding amino acid sequence or peptide fragment.

[0015] The detection of ADGRE3 mRNA or ADGRE3 mRNA fragments and the detection of ADGRE3 protein or ADGRE3 protein fragments in the present invention refers to the detection of the level or content of ADGRE3 mRNA; the level or content of ADGRE3 protein refers to the detection of the content of ADGRE3 mRNA or ADGRE3 mRNA fragments and ADGRE3 protein or ADGRE3 protein fragments in the sample. In some embodiments, the level of ADGRE3 mRNA is determined by the copy number of PCR amplification, and the expression level of ADGRE3 protein is determined by the mean fluorescence intensity (MFI) measured by flow cytometry. In some embodiments, thresholds are set for the copy number of ADGRE3 mRNA and the MFI value of ADGRE3 protein to classify the sample, so as to diagnose or distinguish systemic inflammation and related diseases. Of course, it can be understood that any other method that can characterize the content of ADGRE3 protein or mRNA in the sample is possible, such as concentration content, relative concentration content, and comparison with the normal level in healthy human samples can be used for testing.

[0016] In some embodiments, the present invention detects the content of ADGRE3 protein in neutrophils in blood samples of healthy non-inflammatory people by flow cytometry, calculates the mean fluorescence intensity (MFI) of ADGRE3 protein in each sample and obtains the average value of the mean fluorescence intensity (MFI) of ADGRE3 protein in healthy people, detects the mean fluorescence intensity (MFI) of neutrophil ADGRE3 protein in patient samples under the same conditions, and divides the mean fluorescence intensity (MFI) of neutrophil ADGRE3 protein in patients by the average value of the mean fluorescence intensity (MFI) of ADGRE3 protein in healthy people to obtain the patient neutrophil ADGRE3 protein expression index (nADGRE3), that is, nADGRE3 = patient neutrophil ADGRE3 protein MFI / healthy control neutrophil ADGRE3 protein MFI. Compare nADGRE3 and the set threshold to classify the samples to diagnose or distinguish systemic inflammation and related diseases.

[0017] Calculation of the neutrophil ADGRE3 protein expression index (nADGRE3) is only one of the threshold setting and sample diagnostic classification methods. ADGRE3 mRNA or ADGRE3 protein combined with any other threshold setting and sample classification known methods can obtain the same diagnostic results and are all within the scope of the present invention.

[0018] In some methods, white blood cells include five types: lymphocytes, neutrophils, monocytes, eosinophils, and basophils. Among them, neutrophils highly express ADGRE3 protein, and monocytes lowly express ADGRE3 protein. Therefore, neutrophils are mainly selected as samples for ADGRE3 protein content detection. By allowing any detection method to detect protein in neutrophils alone, monocytes also express ADGRE3 protein, and its expression level is correlated with the expression level of neutrophils, and therefore is also correlated with the occurrence of systemic inflammation. Therefore, monocytes can also be selected as samples; the distribution of mRNA is similar to that of protein, but the detection of mRNA cannot be subdivided into neutrophils or monocytes, so the detection object is usually all white blood cells, but this does not exclude the implementation method of detecting the ADGRE3 mRNA content in any type of white blood cells (lymphocytes, neutrophils, monocytes, eosinophils, basophils).

[0019] The sample in the present invention is not limited to neutrophils, monocytes or leukocytes, and can be any sample including or excluding neutrophils, monocytes or leukocytes. Detecting the leukocyte ADGRE3 mRNA transcription level or the neutrophil and monocyte ADGRE3 protein level in the sample only limits the ADGRE3 mRNA or ADGRE3 protein to come from neutrophils, monocytes or leukocytes, and ADGRE3 mRNA or ADGRE3 protein is not necessarily located in neutrophils, monocytes or leukocytes, but may also exist in the extracellular blood, tissue fluid and other physiological environments, and the content of ADGRE3 mRNA or ADGRE3 protein in the physiological environment can also be used to diagnose systemic inflammation; or, by rupturing neutrophils, monocytes or leukocytes or using other methods to extract substances from cells, the obtained sample also contains ADGRE3 mRNA and its fragments, ADGRE3 protein and its fragments, and the sample can also be used as a sample for diagnosing systemic inflammation.

[0020] The fragment of ADGRE3 mRNA or ADGRE3 protein refers to any fragment of ADGRE3 mRNA or ADGRE3 protein naturally present in an individual.

[0021] Taking ADGRE3 protein as an example, on the one hand, ADGRE3 protein is a membrane protein, which is distributed in the cell membrane, across the membrane, and outside the membrane. Intramembrane means that part of the protein is located inside the cell membrane, and extramembrane means that part of the protein is located outside the cell membrane. Transmembrane means that part of the protein is located inside the cell membrane and the other part is located outside the cell membrane; therefore, the transmembrane protein outside the membrane, inside the membrane, and transmembrane parts (i.e., fragments of ADGRE3 protein) can be detected to determine the content of ADGRE3 protein. Of course, in some methods, cells can be ruptured to extract membrane proteins and then test the protein content. In other methods, under the action of various enzymes in vivo or in vitro, ADGRE3 protein will be enzymatically hydrolyzed into various fragments, and detecting these fragments or the content of any one of the fragments can also determine the content of ADGRE3 protein, thereby diagnosing systemic inflammation.

[0022] The content of ADGRE3 mRNA or ADGRE3 protein in the sample has the function of diagnosing systemic inflammation, and the mRNA fragments and protein fragments have homology with the overall nucleotide sequence of mRNA and the overall amino acid sequence of protein. Therefore, in theory, the content determination results of ADGRE3 mRNA fragments or ADGRE3 protein fragments can also diagnose systemic inflammation.

[0023] In some embodiments, the reagent for diagnosing systemic inflammation is used to detect ADGRE3 mRNA or ADGRE3 mRNA fragments in leukocytes of an individual.

[0024] In some embodiments, the reagent for diagnosing systemic inflammation is used to detect ADGRE3 mRNA or ADGRE3 mRNA fragments in an individual sample; the sample includes any one or more of peripheral blood, urine, secretions, pus, body fluids, spinal cord, and fresh tissue.

[0025] Leukocytes are present in various tissues and body fluids of the human body. In healthy humans, they are mainly distributed in peripheral blood. Urine, secretions, pus, body fluids, spinal cord, and fresh tissues also contain leukocytes. In patients with systemic inflammation, leukocytes infiltrate the inflammatory tissues. Therefore, leukocytes can be detected in various types of clinical samples. Any sample containing leukocytes or intraleukocyte substances (intraleukocyte substances refer to substances that originally come from leukocytes) can be used as a sample for detecting ADGRE3 mRNA or ADGRE3 mRNA fragments to diagnose systemic inflammation.

[0026] In some embodiments, the systemic inflammation includes infectious systemic inflammation or non-infectious systemic inflammation.

[0027] In some embodiments, the infectious systemic inflammation includes any one or more of bacterial infection, viral infection, fungal infection, and parasitic infection. Systemic inflammation may be caused by the above infections.

[0028] In some embodiments, the viral infection includes any one of influenza virus infection, dengue virus infection, novel coronavirus infection, respiratory syncytial virus, cytomegalovirus, Epstein virus, and new Bunyavirus infection.

[0029] The above-mentioned various viral infections are typical viral infections that cause systemic inflammation. In addition to the systemic inflammation caused by the above-mentioned influenza virus infection, dengue virus infection, new coronavirus infection, respiratory syncytial virus, cytomegalovirus, Epstein-Barr virus, and new Bunyavirus infection, systemic inflammation caused by other viral infections can also be diagnosed by detecting the ADGRE3 mRNA or ADGRE3 mRNA fragment, ADGRE3 protein or ADGRE3 protein fragment discovered in the present invention.

[0030] In some embodiments, the non-infectious inflammation includes any one or more of inflammation caused by autoimmune diseases, transplant organ rejection, inflammatory bowel disease, allergic reactions, metabolic diseases, and tissue damage.

[0031] In some embodiments, the autoimmune disease includes any one or more of systemic lupus erythematosus, rheumatoid arthritis, vasculitis, and ankylosing spondylitis.

[0032] In some embodiments, the systemic inflammation caused by transplant organ rejection includes systemic inflammation caused by any one or more of bone marrow transplantation, kidney transplantation and liver transplantation.

[0033] In addition to the inflammation (systemic inflammation) caused by bone marrow transplantation, kidney transplantation and liver transplantation mentioned above, inflammation caused by rejection of other transplanted organs can also be diagnosed by detecting the ADGRE3 mRNA or ADGRE3 mRNA fragment, ADGRE3 protein or ADGRE3 protein fragment discovered by the present invention.

[0034] In some embodiments, the systemic inflammation comprises sepsis.

[0035] In some embodiments, the sepsis is caused by any one or more of lung infection, biliary tract infection, urinary tract infection, digestive tract infection, bloodstream infection, reproductive tract infection, abdominal infection, central nervous system infection, skin and soft tissue infection, and suppurative osteomyelitis.

[0036] In addition to the sepsis caused by the above-mentioned lesions, sepsis caused by any other lesions can be diagnosed by detecting the ADGRE3 mRNA or ADGRE3 mRNA fragment, ADGRE3 protein or ADGRE3 protein fragment discovered in the present invention.

[0037] The second aspect of the present invention provides a use of a biomarker for preparing a reagent for diagnosing whether an individual suffers from systemic inflammation caused by bacterial infection or systemic inflammation caused by viral infection, wherein the biomarker comprises ADGRE3 mRNA or ADGRE3 mRNA fragment; or, ADGRE3 protein or ADGRE3 protein fragment.

[0038] In some embodiments, the present invention provides a use of a biomarker for preparing a reagent for distinguishing and diagnosing an individual suffering from systemic inflammation caused by Gram-positive bacterial infection or systemic inflammation caused by H1N1 infection, wherein the biomarker comprises ADGRE3 mRNA or ADGRE3 mRNA fragment; or, ADGRE3 protein or ADGRE3 protein fragment.

[0039] The clinical symptoms of systemic inflammation caused by Gram-positive bacterial infection or H1N1 infection are similar. The treatment of systemic inflammation requires clear location and type of lesions. Therefore, ADGRE3 mRNA or ADGRE3 protein can be used to distinguish Gram-positive bacterial infection or H1N1 infection, thereby providing targeted treatment for systemic inflammation.

[0040] In some embodiments, the present invention provides a use of a biomarker for preparing a reagent for diagnosing whether an individual has dengue fever systemic inflammation, wherein the biomarker comprises ADGRE3 mRNA or ADGRE3 mRNA fragment; or ADGRE3 protein or ADGRE3 protein fragment.

[0041] In some embodiments, the present invention provides a use of a biomarker for preparing a reagent for diagnosing whether an individual has systemic inflammation caused by COVID-19 infection, wherein the biomarker includes ADGRE3 mRNA or ADGRE3 mRNA fragment; or, ADGRE3 protein or ADGRE3 protein fragment.

[0042] Furthermore, the diagnosis of whether an individual suffers from systemic inflammation caused by COVID-19 infection includes diagnosing patients with asymptomatic systemic inflammation caused by COVID-19 infection, or diagnosing patients with symptomatic systemic inflammation caused by COVID-19 infection.

[0043] In some ways, some COVID-19 infected people are asymptomatic, that is, they will not develop clinical symptoms of COVID-19 infection, so they are difficult to detect clinically; patients with systemic inflammation are likely to be accompanied by asymptomatic COVID-19 infection. According to public data from the World Health Organization, COVID-19 can also cause or aggravate systemic inflammation, so asymptomatic COVID-19 infection needs to be diagnosed, but there is currently a lack of such detection indicators in clinical practice; and the ADGRE3 mRNA or ADGRE3 protein proposed in the present invention can diagnose asymptomatic COVID-19 infected people.

[0044] In some embodiments, the present invention provides a use of a biomarker for preparing a reagent for diagnosing whether an individual has systemic inflammation caused by respiratory syncytial virus infection, wherein the biomarker includes ADGRE3 mRNA or ADGRE3 mRNA fragment; or, ADGRE3 protein or ADGRE3 protein fragment.

[0045] Furthermore, the diagnosis of whether an individual suffers from systemic inflammation caused by syncytial virus infection includes: diagnosing whether the individual has acute syncytial virus infection (a type of systemic inflammation), or diagnosing whether the individual has recovered from systemic inflammation caused by respiratory syncytial virus infection.

[0046] There are extremely significant differences in the ADGRE3 mRNA transcription levels in samples from patients with acute respiratory syncytial virus infection and those who have recovered from systemic inflammation caused by respiratory syncytial virus infection. Therefore, the detection of ADGRE3 mRNA can monitor the changes in the condition of individual syncytial virus infection and provide data support for clinical treatment.

[0047] In some embodiments, the present invention provides a use of a biomarker for preparing a reagent for diagnosing whether an individual has systemic inflammation caused by cytomegalovirus infection, wherein the biomarker includes ADGRE3 mRNA or ADGRE3 mRNA fragment; or ADGRE3 protein or ADGRE3 protein fragment.

[0048] In some embodiments, the present invention provides a use of a biomarker for preparing a reagent for diagnosing whether an individual suffers from systemic inflammation caused by Epstein-Barr virus infection, wherein the biomarker comprises ADGRE3 mRNA or ADGRE3 mRNA fragment; or ADGRE3 protein or ADGRE3 protein fragment.

[0049] In some embodiments, the present invention provides a use of a biomarker for preparing a reagent for distinguishing and diagnosing an individual as being healthy, a non-infected systemic inflammatory response syndrome patient, or a sepsis patient, wherein the biomarker comprises ADGRE3 mRNA or an ADGRE3 mRNA fragment; ADGRE3 protein or an ADGRE3 protein fragment.

[0050] There are extremely significant differences between the leukocyte ADGRE3 mRNA transcription levels of healthy people, non-infectious systemic inflammatory response syndrome patients, and sepsis patients, specifically, they are in a decreasing relationship. The present invention has clinically verified the diagnosis of these three, and the diagnostic AUC value for distinguishing healthy people from sepsis patients is 0.920, the diagnostic AUC value for distinguishing sepsis from non-infectious systemic inflammatory response syndrome is 0.864, and the diagnostic AUC value for diagnosing sepsis from the entire sample set is 0.862, indicating that ADGRE3 mRNA can accurately diagnose non-infectious systemic inflammatory response syndrome and sepsis. Similarly, the test of ADGRE3 protein or ADGRE3 protein fragment also has similar results.

[0051] In some embodiments, the present invention provides a use of a biomarker for preparing a reagent for distinguishing and diagnosing whether an individual suffers from bacterial systemic inflammation, wherein the biomarker comprises ADGRE3 mRNA or ADGRE3 mRNA fragment.

[0052] The third aspect of the present invention provides a method for diagnosing whether an individual has systemic inflammation, the method comprising: providing a test sample, detecting the content of ADGRE3 mRNA or ADGRE3 mRNA fragments; or ADGRE3 protein or ADGRE3 protein fragments in the sample; comparing the tested content with a preset threshold value, and diagnosing that the individual does not suffer from systemic inflammation when the content is greater than or equal to the threshold value, and diagnosing that the individual suffers from systemic inflammation when the content is less than the threshold value.

[0053] In some embodiments, the detection of the content of ADGRE3 mRNA or ADGRE3 mRNA fragments in the sample is to detect the content of ADGRE3 mRNA or ADGRE3 mRNA fragments, or the content of ADGRE3 protein or ADGRE3 protein fragments in leukocytes in the individual sample.

[0054] In some embodiments, the sample includes any one or more of peripheral blood, urine, secretions, pus, body fluids, spinal cord, and fresh tissue.

[0055] In some embodiments, the systemic inflammation includes infectious systemic inflammation or non-infectious systemic inflammation.

[0056] In some embodiments, the infectious systemic inflammation includes systemic inflammation caused by any one or more of bacterial infection, viral infection, fungal infection, and parasitic infection.

[0057] In some embodiments, the viral infection includes any one or more of influenza virus infection, dengue virus infection, novel coronavirus infection, respiratory syncytial virus, cytomegalovirus, Epstein-Barr virus, and new Bunyavirus infection.

[0058] In some embodiments, the non-infectious systemic inflammation includes systemic inflammation caused by any one or more of autoimmune diseases, transplant organ rejection, inflammatory bowel disease, allergic reactions, metabolic diseases, and inflammation caused by tissue damage.

[0059] In some embodiments, the autoimmune disease includes any one or more of systemic lupus erythematosus, rheumatoid arthritis, vasculitis, and ankylosing spondylitis.

[0060] In some embodiments, the systemic inflammation caused by transplant organ rejection includes systemic inflammation caused by any one or more of bone marrow transplantation, kidney transplantation and liver transplantation.

[0061] In some embodiments, the systemic inflammation comprises sepsis.

[0062] In some embodiments, the present invention provides a method for diagnosing whether an individual suffers from systemic inflammation caused by bacterial infection or viral infection, the method comprising: providing a test sample, detecting the content of ADGRE3 mRNA or ADGRE3 mRNA fragment, or ADGRE3 protein or ADGRE3 protein fragment in the sample; comparing the tested content with a preset threshold value, and diagnosing that the individual does not suffer from systemic inflammation caused by bacterial infection or viral infection when the content is greater than or equal to the threshold value, and diagnosing that the individual suffers from systemic inflammation caused by bacterial infection or viral infection when the content is less than the threshold value.

[0063] In some embodiments, the present invention provides a method for diagnosing whether an individual suffers from systemic inflammation caused by Gram-positive bacteria infection or systemic inflammation caused by H1N1 infection, the method comprising: providing a test sample, detecting the content of ADGRE3 mRNA or ADGRE3 mRNA fragments in the sample; or the content of ADGRE3 protein or ADGRE3 protein fragments; comparing the tested content with a preset threshold value, and diagnosing that the individual does not suffer from systemic inflammation caused by Gram-positive bacteria infection or systemic inflammation caused by H1N1 infection when the content is greater than or equal to the threshold value, and diagnosing that the individual suffers from systemic inflammation caused by Gram-positive bacteria infection or systemic inflammation caused by H1N1 infection when the content is less than the threshold value.

[0064] On the other hand, the present invention provides a method for distinguishing and diagnosing whether a patient with systemic inflammation has a Gram-positive bacterial infection or an H1N1 infection, the method comprising: providing a test sample, detecting the content of ADGRE3 mRNA or ADGRE3 mRNA fragments, or the content of ADGRE3 protein or ADGRE3 protein fragments in the sample; comparing the tested content with a preset threshold value, and diagnosing the patient with a Gram-positive bacterial infection when the content is greater than the threshold value, and diagnosing the patient with an H1N1 infection when the content is less than the threshold value.

[0065] On the other hand, the present invention provides a method for diagnosing whether an individual suffers from dengue fever systemic inflammation, the method comprising: providing a test sample, detecting the content of ADGRE3 mRNA or ADGRE3 mRNA fragment or ADGRE3 protein or ADGRE3 protein fragment in the sample; comparing the tested content with a preset threshold value, and diagnosing that the individual does not suffer from dengue fever systemic inflammation when the content is greater than or equal to the threshold value, and diagnosing that the individual suffers from dengue fever systemic inflammation when the content is less than the threshold value.

[0066] On the other hand, the present invention provides a method for diagnosing whether an individual has systemic inflammation caused by COVID-19 infection with a reagent, the method comprising: providing a test sample, detecting the content of ADGRE3 mRNA or ADGRE3 mRNA fragments, or the content of ADGRE3 protein or ADGRE3 protein fragments in the sample; comparing the tested content with a preset threshold value, and diagnosing that the individual does not have systemic inflammation caused by COVID-19 infection when the content is greater than or equal to the threshold value, and diagnosing that the individual has systemic inflammation caused by COVID-19 infection when the content is less than the threshold value.

[0067] Furthermore, the diagnosis of whether an individual suffers from systemic inflammation caused by COVID-19 infection includes diagnosing patients with asymptomatic systemic inflammation caused by COVID-19 infection or patients with systemic inflammation caused by symptomatic COVID-19 infection.

[0068] On the other hand, the present invention provides a method for diagnosing whether an individual suffers from systemic inflammation caused by respiratory syncytial virus infection, the method comprising: providing a test sample, detecting the content of ADGRE3 mRNA or ADGRE3 mRNA fragments, or the content of ADGRE3 protein or ADGRE3 protein fragments in the sample; comparing the tested content with a preset threshold value, and diagnosing that the individual does not suffer from systemic inflammation caused by respiratory syncytial virus infection when the content is greater than or equal to the threshold value, and diagnosing that the individual suffers from systemic inflammation caused by respiratory syncytial virus infection when the content is less than the threshold value.

[0069] Furthermore, the diagnosing whether an individual suffers from systemic inflammation caused by respiratory syncytial virus infection also includes: diagnosing whether an individual has acute systemic inflammation caused by syncytial virus infection, or diagnosing whether an individual has recovered from systemic inflammation caused by syncytial virus infection.

[0070] On the other hand, the present invention provides a method for diagnosing whether an individual suffers from systemic inflammation caused by cytomegalovirus infection, the method comprising: providing a test sample, detecting the content of ADGRE3 mRNA or ADGRE3 mRNA fragments in the sample; or the content of ADGRE3 protein or ADGRE3 protein fragments; comparing the tested content with a preset threshold value, and diagnosing that the individual does not suffer from systemic inflammation caused by cytomegalovirus infection when the content is greater than or equal to the threshold value, and diagnosing that the individual suffers from systemic inflammation caused by cytomegalovirus infection when the content is less than the threshold value.

[0071] On the other hand, the present invention provides a method for diagnosing whether an individual suffers from systemic inflammation caused by Epstein-Barr virus infection, the method comprising: providing a test sample, detecting the content of ADGRE3 mRNA or ADGRE3 mRNA fragments in the sample; or the content of ADGRE3 protein or ADGRE3 protein fragments; comparing the tested content with a preset threshold value, and diagnosing that the individual does not suffer from systemic inflammation caused by Epstein-Barr virus infection when the content is greater than or equal to the threshold value, and diagnosing that the individual suffers from systemic inflammation caused by Epstein-Barr virus infection when the content is less than the threshold value.

[0072] On the other hand, the present invention provides a method for distinguishing an individual as being healthy, a non-infectious systemic inflammatory response syndrome patient, or a sepsis patient, the method comprising: providing a test sample, detecting the content of ADGRE3 mRNA or ADGRE3 mRNA fragment in the sample; or the content of ADGRE3 protein or ADGRE3 protein fragment; comparing the tested content with a preset threshold, the threshold comprising a threshold for distinguishing healthy people from non-infectious systemic inflammatory response syndrome patients, and a threshold for distinguishing non-infectious systemic inflammatory response syndrome patients from sepsis patients.

[0073] In some embodiments, the individual is healthy when the tested amount is greater than or equal to a threshold value that distinguishes healthy people from non-infectious systemic inflammatory response syndrome patients.

[0074] In some embodiments, when the amount detected is less than a threshold value that distinguishes between non-infectious systemic inflammatory response syndrome patients and sepsis patients, the individual is a sepsis patient.

[0075] In some embodiments, when the tested amount is less than a threshold value for distinguishing healthy people from non-infectious SIRS patients and greater than a threshold value for distinguishing non-infectious SIRS patients from sepsis patients, the individual is a non-infectious SIRS patient.

[0076] On the other hand, the present invention provides a method for diagnosing whether an individual suffers from systemic inflammation caused by transplant organ rejection, the method comprising: providing a test sample, detecting the content of ADGRE3 mRNA or ADGRE3 mRNA fragments in the sample; or the content of ADGRE3 protein or ADGRE3 protein fragments; comparing the tested content with a preset threshold value, and diagnosing that the individual does not suffer from systemic inflammation caused by transplant organ rejection when the content is greater than or equal to the threshold value, and diagnosing that the individual suffers from systemic inflammation caused by transplant organ rejection when the content is less than the threshold value.

[0077] In the above study, we found and verified that ADGRE3 mRNA; or ADGRE3 protein or ADGRE3 protein fragment; can diagnose systemic inflammation, as well as the above-mentioned other related diseases; ADGRE3 mRNA is transcribed from the ADGRE3 gene, and the production of ADGRE3 protein is translated from the above ADGRE3 mRNA, but the protein content is not completely determined by the mRNA content, and the content of ADGRE3 protein is also affected by protein binding, degradation, etc. Although there are such reasons, we have also proved that ADGRE3 protein or ADGRE3 protein fragments also have the function of diagnosing systemic inflammation by verifying the function.

[0078] On the other hand, the present invention provides a use of a biomarker for preparing a reagent for diagnosing systemic inflammation, wherein the biomarker includes an ADGRE3 protein or an ADGRE3 protein fragment, or an amino acid sequence or a peptide fragment corresponding to ADGRE3.

[0079] The diagnosis of systemic inflammation includes: diagnosing whether an individual suffers from systemic inflammation, or predicting the risk of an individual suffering from systemic inflammation, or monitoring the disease progression of patients with systemic inflammation, or determining the prognosis of patients with systemic inflammation, or providing medication guidance for patients with systemic inflammation, one or more of the above.

[0080] The reagent for diagnosing systemic inflammation is used to detect ADGRE3 protein or ADGRE3 protein fragments in individual samples. Preferably, the ADGRE3 protein or ADGRE3 protein fragments are present on or in neutrophils or monocytes in the sample.

[0081] ADGRE3 protein is mainly expressed in neutrophils and also in monocytes, but the detection of ADGRE3 protein or ADGRE3 protein fragments in individual neutrophils or monocytes is not a limitation on the test samples. Any sample containing neutrophils or monocytes, or substances in neutrophils or monocytes (substances in neutrophils or monocytes refer to substances originally derived from neutrophils or monocytes) can be used as a sample for detecting ADGRE3 protein or ADGRE3 protein fragments in individual neutrophils or monocytes to diagnose systemic inflammation.

[0082] Detecting the ADGRE3 protein or ADGRE3 protein fragment of individual neutrophils or monocytes means: detecting the ADGRE3 protein or ADGRE3 protein fragment derived from neutrophils or monocytes. The ADGRE3 protein or ADGRE3 protein fragment may exist inside the cell membrane, outside the cell membrane or across the membrane, and the above may be measured by flow cytometry or other well-known techniques.

[0083] In some embodiments, the sample includes any one or more of peripheral blood, urine, secretions, pus, body fluids, spinal cord, and fresh tissue.

[0084] By detecting the level or content of neutrophil ADGRE3 protein in healthy people and clinical patients, it was found that there were extremely significant differences in the expression levels of neutrophil ADGRE3 protein between patients with various types of systemic inflammation and healthy people. Therefore, systemic inflammation can be diagnosed by measuring the content of neutrophil ADGRE3 protein; the expression levels of ADGRE3 protein in neutrophils and monocytes are correlated, so systemic inflammation can also be diagnosed by measuring the expression level of monocyte ADGRE3 protein.

[0085] Combining multiple existing inflammation indicators, the present invention found that ADGRE3 protein has higher diagnostic sensitivity; by adding interference cases of patients with local inflammation, the diagnosis of healthy people, patients with local inflammation, and patients with systemic inflammation was verified, and it was found that ADGRE3 protein can correctly classify two types of inflammation, indicating that ADGRE3 protein has high specificity in diagnosing systemic inflammation.

[0086] In some embodiments, the systemic inflammation includes infectious systemic inflammation or non-infectious systemic inflammation.

[0087] In some embodiments, the infectious systemic inflammation includes any one or more of bacterial infection, viral infection, fungal infection, and parasitic infection.

[0088] In some embodiments, the viral infection includes any one or more of influenza virus infection, new coronavirus infection, dengue virus infection, new bunyavirus infection, respiratory syncytial virus infection, cytomegalovirus, and Epstein-Barr virus.

[0089] In some embodiments, the non-infectious systemic inflammation includes any one or more of inflammation caused by autoimmune diseases, transplant organ rejection, inflammatory bowel disease, allergic reactions, metabolic diseases, and tissue damage.

[0090] In some embodiments, the autoimmune disease includes any one or more of systemic lupus erythematosus, rheumatoid arthritis, vasculitis, ankylosing spondylitis, and autoimmune hepatitis.

[0091] In addition to the inflammation (systemic inflammation) caused by the above-mentioned systemic lupus erythematosus, rheumatoid arthritis, vasculitis, ankylosing spondylitis, and autoimmune hepatitis, systemic inflammation caused by other autoimmune diseases can also be diagnosed by detecting the ADGRE3 mRNA or ADGRE3 mRNA fragments, ADGRE3 protein or ADGRE3 protein fragments discovered by the present invention.

[0092] In some embodiments, the inflammation caused by rejection of the transplanted organ includes systemic inflammation caused by any one or more rejection reactions of liver transplantation, kidney transplantation, and bone marrow transplantation.

[0093] In some embodiments, the systemic inflammation comprises sepsis.

[0094] In some embodiments, the sepsis is caused by any one or more of lung infection, biliary tract infection, urinary tract infection, digestive tract infection, bloodstream infection, reproductive tract infection, abdominal infection, central nervous system infection, skin and soft tissue infection, and suppurative osteomyelitis.

[0095] In addition to the sepsis caused by the above-mentioned lesions, sepsis caused by any other lesions can be diagnosed by detecting the ADGRE3 mRNA or ADGRE3 mRNA fragment, ADGRE3 protein or ADGRE3 protein fragment discovered by the present invention.

[0096] In some embodiments, the sepsis includes any one or more of gram-positive bacterial infection sepsis, gram-negative bacterial infection sepsis, and fungal infection sepsis.

[0097] In some embodiments, the sepsis comprises septic shock.

[0098] In some embodiments, the present invention provides a use of a biomarker for preparing a reagent for distinguishing and diagnosing whether an individual is a healthy person or a patient with systemic inflammation caused by a viral infection, wherein the biomarker includes an ADGRE3 protein or an ADGRE3 protein fragment, or an amino acid sequence or peptide fragment corresponding to ADGRE3.

[0099] In some embodiments, the reagent can also distinguish and diagnose patients with systemic inflammation caused by viral infection as one of mild patients, severe patients and critical patients.

[0100] In some embodiments, the present invention provides a use of a biomarker for preparing a reagent for diagnosing whether an individual has systemic lupus erythematosus systemic inflammation, wherein the biomarker includes ADGRE3 protein or ADGRE3 protein fragment, or an amino acid sequence or peptide fragment corresponding to ADGRE3.

[0101] In some embodiments, the present invention provides a use of a biomarker for preparing a reagent for diagnosing whether a patient with systemic lupus erythematosus systemic inflammation is a stable patient or an active patient, wherein the biomarker includes an ADGRE3 protein or an ADGRE3 protein fragment, or an amino acid sequence or peptide fragment corresponding to ADGRE3.

[0102] In some embodiments, the present invention provides a use of a biomarker for preparing a reagent for diagnosing whether an individual has systemic inflammation of rheumatoid joints, wherein the biomarker includes ADGRE3 protein or ADGRE3 protein fragment, or an amino acid sequence or peptide fragment corresponding to ADGRE3.

[0103] In some embodiments, the present invention provides a use of a biomarker for preparing a reagent for diagnosing whether a patient with systemic inflammation of rheumatoid joints is a stable patient or an active patient, wherein the biomarker includes an ADGRE3 protein or an ADGRE3 protein fragment, or an amino acid sequence or peptide fragment corresponding to ADGRE3.

[0104] Systemic lupus erythematosus and rheumatoid arthritis can be divided into a stable period and an active period according to the course of the disease. The stable period refers to the period during which the condition no longer improves or worsens significantly, and the active period refers to the period other than the stable period. In the stable period and the active period, the treatment plans for patients are different. For example, patients in the stable period of systemic lupus erythematosus need to reduce the dosage of medication during the stable period, while maintaining more frequent disease monitoring. Therefore, it is clinically necessary to diagnose the stable period and active period of systemic lupus erythematosus and rheumatoid arthritis, but there is currently no indicator that can make a clear diagnosis of them. The present invention finds that ADGRE3 protein can be used as a marker to diagnose the stable period and active period of systemic lupus erythematosus and rheumatoid arthritis.

[0105] In some embodiments, the present invention provides a use of a biomarker for preparing a reagent for distinguishing and diagnosing whether an individual is a healthy person or a sepsis patient or a septic shock patient, wherein the biomarker includes an ADGRE3 protein or an ADGRE3 protein fragment, or an amino acid sequence or peptide fragment corresponding to ADGRE3.

[0106] Sepsis can be divided into sepsis, severe sepsis and septic shock according to the severity of the disease. The condition of patients with septic shock is extremely serious and requires more medical resources for emergency treatment. Therefore, when sepsis occurs, patients need to be classified according to the severity of their condition. The present invention finds that ADGRE3 protein can distinguish and diagnose septic shock, thereby assisting in clinical patient classification.

[0107] In some embodiments, the present invention provides a use of a biomarker for preparing a reagent for predicting the risk of death in sepsis patients, wherein the biomarker includes an ADGRE3 protein or an ADGRE3 protein fragment, or an amino acid sequence or a peptide fragment corresponding to ADGRE3.

[0108] The present invention also found that the risk of death in sepsis patients is highly correlated with ADGRE3 protein, and the course of sepsis disease is also highly correlated with changes in the content of ADGRE3 protein.

[0109] In some embodiments, the use further includes: if the content of ADGRE3 protein or ADGRE3 protein fragment, or the amino acid sequence or peptide fragment corresponding to ADGRE3 in the sample on the 5th day after the individual is diagnosed with the disease is higher than the content in the sample on the 3rd day, it is predicted that the individual's sepsis will be improved; otherwise, it is predicted that the individual's sepsis will not be improved or the individual will die.

[0110] On the other hand, the present invention provides a use of a biomarker combination for preparing a reagent for diagnosing whether a patient with systemic inflammation is a patient with systemic inflammation caused by bacterial infection, wherein the biomarker combination includes an ADGRE3 protein or an ADGRE3 protein fragment, or an amino acid sequence or peptide fragment corresponding to ADGRE3, and the biomarker combination also includes any one or more markers for specific diagnosis of bacterial inflammation. Markers for specific diagnosis of bacterial inflammation are: when an individual has bacterial inflammation, the content of the marker changes significantly, and when the individual does not have inflammation or has non-bacterial inflammation, the content of the marker does not change significantly, that is, it is specific to bacterial inflammation, and there are currently many such markers, such as CD64, PCT, HBP, etc.

[0111] In some embodiments, the markers for specific diagnosis of bacterial inflammation include any one or more of CD64, PCT and HBP.

[0112] In some methods, when the content of ADGRE3 protein or ADGRE3 protein fragment, or the amino acid sequence or peptide fragment corresponding to ADGRE3 in the sample is lower than a preset threshold, and the content of markers for specific diagnosis of bacterial inflammation in the sample does not belong to the content range of healthy people, it can be determined that the patient has systemic inflammation caused by bacterial infection.

[0113] In some methods, when the content of ADGRE3 protein or ADGRE3 protein fragment, or the amino acid sequence or peptide fragment corresponding to ADGRE3 in the sample is lower than a preset threshold, and the content of markers for specific diagnosis of bacterial inflammation in the sample belongs to the content range of healthy people, it can be determined that the patient has systemic inflammation caused by viral infection or non-infectious disease.

[0114] In some embodiments, the threshold value is the level of ADGRE3 protein or ADGRE3 protein fragment in healthy people, or the amino acid sequence or peptide fragment corresponding to ADGRE3 in the sample.

[0115] The present invention simultaneously measures the content of neutrophil ADGRE3 protein and the content of markers for specific diagnosis of bacterial inflammation in the sample, and finds that when the content of ADGRE3 protein decreases, if the content of markers for specific diagnosis of bacterial inflammation changes or also significantly increases, the patient is classified as having bacterial systemic inflammation; if the content of markers for specific diagnosis of bacterial inflammation is normal, the patient is classified as having non-bacterial systemic inflammation; therefore, the content of ADGRE3 protein or ADGRE3 protein fragments can be combined with markers for specific diagnosis of bacterial inflammation to distinguish between systemic inflammation caused by bacterial infection and systemic inflammation caused by viral infection or non-infectious diseases.

[0116] Regarding the content of markers for specific diagnosis of bacterial inflammation, it does not belong to the content range of healthy people. For different indicators, it can include two situations: high (higher than the healthy range) and low (lower than the healthy range). Any of the two situations of markers for specific diagnosis of bacterial inflammation indicates the occurrence of bacterial inflammation. For example, an increase in the detection value of the nCD64 indicator represents the occurrence of bacterial inflammation.

[0117] On the other hand, the present invention provides a method for diagnosing whether an individual suffers from systemic inflammation, the method comprising: providing a test sample, detecting the content of ADGRE3 protein or ADGRE3 protein fragment, or the content of the amino acid sequence or peptide fragment corresponding to ADGRE3 in the sample; comparing the tested content with a preset threshold value, and diagnosing that the individual does not suffer from systemic inflammation when the content is greater than or equal to the threshold value, and diagnosing that the individual suffers from systemic inflammation when the content is less than the threshold value.

[0118] Regarding the detection of the content of ADGRE3 protein or ADGRE3 protein fragments in a sample, the present invention adopts a flow cytometer to set a gate on the cells in the sample, and can specifically detect the content of ADGRE3 protein or ADGRE3 protein fragments in neutrophils or monocytes in the sample. The protein or protein fragment can exist outside the cell, or across the membrane, or inside the cell, and its content can be analyzed by flow cytometry. The detection of the content of ADGRE3 protein or ADGRE3 protein fragments in the sample in combination with any known detection method to diagnose systemic inflammation falls within the protection scope of the present invention.

[0119] Regarding setting a threshold for the content of ADGRE3 protein or ADGRE3 protein fragments, since there will be differences in the mean fluorescence intensity (MFI) directly measured in different batches of experiments, different flow cytometers and different detection methods, the present invention adopts the expression index of neutrophil ADGRE3 protein (nADGRE3), that is, nADGRE3 = patient neutrophil ADGRE3 protein MFI / healthy control neutrophil ADGRE3 protein MFI. The present invention found that when the threshold value of nADGRE3 is 0.8361, the sensitivity, specificity and accuracy of diagnosing systemic inflammation are the highest; combined with any other known method for setting a threshold, the use of ADGRE3 protein or ADGRE3 protein fragment content to diagnose systemic inflammation falls within the scope of protection of the present invention.

[0120] Preferably, the ADGRE3 protein or ADGRE3 protein fragment is present on or in neutrophils or monocytes in the sample.

[0121] In some embodiments, the sample includes any one or more of peripheral blood, urine, secretions, pus, body fluids, spinal cord, and fresh tissue.

[0122] In some embodiments, the systemic inflammation includes infectious systemic inflammation or non-infectious systemic inflammation.

[0123] In some embodiments, the infectious systemic inflammation includes systemic inflammation caused by any one or more of bacterial infection, viral infection, fungal infection, and parasitic infection.

[0124] In some embodiments, the viral infection includes any one or more of influenza virus infection, novel coronavirus infection, dengue virus infection, and new bunyavirus infection.

[0125] In some embodiments, the non-infectious systemic inflammation includes systemic inflammation caused by any one or more of autoimmune diseases, transplant organ rejection, inflammatory bowel disease, allergic reactions, metabolic diseases, and inflammation caused by tissue damage.

[0126] In some embodiments, the autoimmune disease includes any one or more of systemic lupus erythematosus, rheumatoid arthritis, vasculitis, ankylosing spondylitis, and autoimmune hepatitis.

[0127] In some embodiments, the systemic inflammation caused by transplant organ rejection includes systemic inflammation caused by any one or more of bone marrow transplantation, kidney transplantation and liver transplantation.

[0128] In some embodiments, the systemic inflammation comprises sepsis.

[0129] In some embodiments, the sepsis includes any one or more of gram-positive bacterial infection sepsis, gram-negative bacterial infection sepsis, and fungal infection sepsis.

[0130] In some embodiments, the sepsis comprises septic shock.

[0131] In some embodiments, the present invention provides a method for diagnosing whether an individual suffers from systemic inflammation caused by a viral infection, the method comprising: providing a test sample, detecting the content of ADGRE3 protein or ADGRE3 protein fragment, or the content of the amino acid sequence or peptide fragment corresponding to ADGRE3 in the sample; comparing the tested content with a preset threshold value, and diagnosing that the individual does not suffer from systemic inflammation caused by a viral infection when the content is greater than or equal to the threshold value, and diagnosing that the individual suffers from systemic inflammation caused by a viral infection when the content is less than the threshold value.

[0132] In some embodiments, the present invention provides a method for distinguishing patients with systemic inflammation caused by viral infection as having mild, severe, or critical illness, the method comprising: providing a test sample, detecting the content of ADGRE3 protein or ADGRE3 protein fragment in the sample, or the content of the amino acid sequence or peptide fragment corresponding to ADGRE3; comparing the tested content with a preset threshold, the threshold comprising a threshold for distinguishing mild from severe illness, and a threshold for distinguishing severe from critical illness.

[0133] In some embodiments, when the tested amount is greater than a threshold value that distinguishes mild from severe disease, the patient is classified as having a mild disease.

[0134] In some embodiments, when the tested amount is less than a threshold value that distinguishes severe from critical illness, the patient is classified as critically ill.

[0135] In some embodiments, when the tested amount is less than a threshold value for distinguishing mild from severe disease and greater than a threshold value for distinguishing severe from critical disease, the patient is classified as severe.

[0136] On the other hand, the present invention provides a method for distinguishing patients with systemic inflammation of systemic lupus erythematosus as being in a stable stage or an active stage, the method comprising: providing a test sample, detecting the content of ADGRE3 protein or ADGRE3 protein fragment, or the content of the amino acid sequence or peptide fragment corresponding to ADGRE3 in the sample; comparing the tested content with a preset threshold, and classifying the patient as a stable stage patient when the content is greater than or equal to the threshold, and classifying the patient as an active stage patient when the content is less than the threshold.

[0137] On the other hand, the present invention provides a method for distinguishing patients with systemic inflammation of rheumatoid arthritis as being in a stable stage or an active stage, the method comprising: providing a test sample, detecting the content of ADGRE3 protein or ADGRE3 protein fragment in the sample, or the content of the amino acid sequence or peptide fragment corresponding to ADGRE3; comparing the tested content with a preset threshold, and classifying the patient as a stable stage patient when the content is greater than or equal to the threshold, and classifying the patient as an active stage patient when the content is less than the threshold.

[0138] On the other hand, the present invention provides a method for diagnosing whether an individual suffers from sepsis, the method comprising: providing a test sample, detecting the content of ADGRE3 protein or ADGRE3 protein fragment, or the content of amino acid sequence or peptide fragment corresponding to ADGRE3 in the sample; comparing the tested content with a preset threshold value, and diagnosing that the individual does not suffer from sepsis when the content is greater than or equal to the threshold value, and diagnosing that the individual suffers from sepsis when the content is less than the threshold value.

[0139] On the other hand, the present invention provides a method for predicting the risk of death of a patient with sepsis, the method comprising: providing a test sample, detecting the content of ADGRE3 protein or ADGRE3 protein fragment, or the content of the amino acid sequence or peptide fragment corresponding to ADGRE3 in the sample; comparing the tested content with a preset threshold value, when the content is greater than or equal to the threshold value, the risk of death of the patient with sepsis is low, and when the content is less than the threshold value, the risk of death of the patient with sepsis is high.

[0140] Furthermore, the method also includes: if the content of ADGRE3 protein or ADGRE3 protein fragment, or the content of the amino acid sequence or peptide fragment corresponding to ADGRE3 in the sample on the 5th day after the patient is diagnosed with the disease is higher than that in the sample on the 3rd day, it is predicted that the patient's sepsis will be improved; otherwise, it is predicted that the patient's sepsis will not be improved or will die.

[0141] On the other hand, the present invention provides a method for diagnosing whether an individual suffers from systemic inflammation caused by transplant organ rejection, the method comprising: providing a test sample, detecting the content of ADGRE3 protein or ADGRE3 protein fragment, or the content of the amino acid sequence or peptide fragment corresponding to ADGRE3 in the sample; comparing the tested content with a preset threshold value, and diagnosing that the individual does not suffer from systemic inflammation caused by transplant organ rejection when the content is greater than or equal to the threshold value, and diagnosing that the individual suffers from systemic inflammation caused by transplant organ rejection when the content is less than the threshold value.

[0142] In another aspect, the present invention provides a method for diagnosing whether an individual suffers from bacterial systemic inflammation, the method comprising: providing a test sample, detecting the content of ADGRE3 protein or ADGRE3 protein fragment, or the content of the amino acid sequence or peptide fragment corresponding to ADGRE3 in the sample; comparing the tested content with a preset threshold value, and diagnosing that the individual does not suffer from bacterial systemic inflammation when the content is greater than or equal to the threshold value;

[0143] When the level is less than the threshold value, any one or more markers for specific diagnosis of bacterial inflammation in the sample are detected. If the content of the markers for specific diagnosis of bacterial inflammation does not fall within the content range of healthy people, the individual is diagnosed with bacterial systemic inflammation. If the content of the markers for specific diagnosis of bacterial inflammation falls within the content range of healthy people, the individual is diagnosed as not suffering from bacterial systemic inflammation.

[0144] In some embodiments, the markers for specific diagnosis of bacterial inflammation include any one or more of CD64, PCT, and HBP.

[0145] The ADGRE3 gene corresponds to a variety of mRNA transcripts and transcript variants, and any ADGRE3 mRNA and its fragments can be used as a marker of the present invention; the ADGRE3 proteins translated from the various mRNAs can have different amino acid sequences, and any ADGRE3 protein translated from the ADGRE3 mRNA and its fragments can be used as a marker of the present invention.

[0146] In some embodiments, the ADGRE3 mRNA has a nucleotide sequence as shown in any one or more of XM_054322398.1, XM_047439546.1, XM_011528374.3, NM_001289158.2, NM_001289159.2, and NM_032571.5 (RefSeq database).

[0147] In some embodiments, the ADGRE3 protein has the amino acid sequence shown in SEQ ID NO: 1; or, the ADGRE3 protein is translated from any one of the mRNAs XM_054322398.1, XM_047439546.1, XM_011528374.3, NM_001289158.2, NM_001289159.2, and NM_032571.5.

[0148] Beneficial Effects

[0149] For the first time, a marker for diagnosing systemic inflammation was proposed. The transcription level or expression level of ADGRE3 mRNA or ADGRE3 protein in leukocytes, neutrophils, and monocytes can highly specifically distinguish between common inflammation and systemic inflammation. It has a high sensitivity for indicating systemic inflammation, far exceeding the existing series of diagnostic indicators, and its specificity for the diagnosis of systemic inflammation has been clinically verified. BRIEF DESCRIPTION OF THE DRAWINGS

[0150] Figure 1 : Volcano plot of differentially expressed genes in peripheral blood of patients with sepsis and patients with non-infectious systemic inflammatory response;

[0151] Figure 2 :Volcano plot of differentially expressed genes in peripheral blood between sepsis patients and healthy people;

[0152] Figure 3 : Comparison of ADGRE3 mRNA transcription levels in leukocytes between healthy controls, patients with systemic inflammation caused by bacterial infection, and patients with systemic inflammation caused by viral infection;

[0153] Figure 4 : Comparison of ADGRE3 mRNA transcription levels in leukocytes between healthy controls, patients with systemic inflammation caused by Gram-negative bacteria infection, and patients with systemic inflammation caused by H1N1 infection;

[0154] Figure 5 : Comparison of ADGRE3 mRNA transcription levels in leukocytes between healthy controls and patients with dengue systemic inflammation;

[0155] Figure 6 : Comparison of leukocyte ADGRE3 mRNA transcription levels in healthy controls, patients with asymptomatic COVID-19 infection-induced systemic inflammation, and patients with symptomatic COVID-19 infection-induced systemic inflammation;

[0156] Figure 7 : Comparison of ADGRE3 mRNA transcription levels in leukocytes of healthy controls, patients with acute RSV-infected systemic inflammation, and people who recovered from RSV-infected systemic inflammation;

[0157] Figure 8 : Comparison of ADGRE3 mRNA transcription levels in leukocytes between healthy controls and patients with systemic inflammation caused by cytomegalovirus infection;

[0158] Fig. 9 : Comparison of ADGRE3 mRNA transcription levels in leukocytes between healthy controls and patients with systemic inflammation caused by EBV infection;

[0159] Fig.10 : Comparison of leukocyte ADGRE3 mRNA transcription levels between healthy controls and patients with systemic inflammation caused by liver transplant rejection;

[0160] Fig.11 : Comparison of leukocyte ADGRE3 mRNA transcription levels in healthy controls, non-infectious systemic inflammatory response syndrome patients, and sepsis patients;

[0161] Fig.12 : ROC curve of leukocyte ADGRE3 mRNA transcription level between healthy controls and sepsis patients;

[0162] Fig.13 :ROC curve of leukocyte ADGRE3 mRNA transcription level for distinguishing and diagnosing patients with non-infectious systemic inflammatory response syndrome and sepsis;

[0163] Fig.14 : Comparison of leukocyte ADGRE3 mRNA transcription levels in healthy controls, patients with systemic inflammation caused by influenza A and B viruses, and patients with systemic inflammation caused by novel coronavirus infection;

[0164] Fig.15 : Comparison of leukocyte ADGRE3 mRNA transcription levels in healthy controls, patients with systemic lupus erythematosus, patients with systemic inflammation due to rheumatoid arthritis, patients with systemic inflammation due to vasculitis, and patients with systemic inflammation due to ankylosing spondylitis;

[0165] Fig.16 : Comparison of ADGRE3 mRNA transcription levels in leukocytes between healthy controls and sepsis patients;

[0166] Fig.17 : Comparison of ADGRE3 protein expression levels in peripheral blood neutrophils among healthy controls, patients with systemic inflammation caused by influenza A and B viruses, patients with systemic inflammation caused by SARS-CoV-2, patients with systemic inflammation caused by dengue fever, and patients with systemic inflammation caused by new bunyavirus;

[0167] Fig.18 :ROC curve of neutrophil ADGRE3 protein expression for distinguishing and diagnosing healthy people and patients with systemic inflammation caused by viral infection;

[0168] Fig.19 : Comparison of neutrophil ADGRE3 protein expression levels between healthy controls and patients with systemic inflammation caused by viral infection (mild patients, severe patients, and critical patients);

[0169] Fig. 20 : Comparison of neutrophil ADGRE3 protein expression levels in patients with systemic inflammation of autoimmune diseases (systemic lupus erythematosus, rheumatoid arthritis, vasculitis, ankylosing spondylitis, autoimmune hepatitis);

[0170] Fig.21 : Comparison of neutrophil ADGRE3 protein expression levels in healthy controls, patients with systemic inflammation induced by kidney transplantation, and patients with systemic inflammation induced by bone marrow transplantation;

[0171] Fig. 22 : ROC curve of neutrophil ADGRE3 protein expression level for distinguishing healthy people and non-infectious systemic inflammation;

[0172] Fig.23 : Comparison of ADGRE3 protein expression levels in neutrophils between healthy controls and patients with sepsis;

[0173] Fig.24 : ROC curve of neutrophil ADGRE3 protein expression for distinguishing healthy people and sepsis patients;

[0174] Fig.25 : Comparison of ADGRE3 protein expression levels in neutrophils of healthy controls, Gram-positive bacteria sepsis, Gram-negative bacteria sepsis, and fungal sepsis;

[0175] Fig.26 : Comparison of ADGRE3 protein expression levels in neutrophils between healthy controls and sepsis patients (sepsis patients, septic shock patients);

[0176] Fig. 27 : Comparison of neutrophil ADGRE3 protein expression in sepsis survivors and patients who died of sepsis;

[0177] Fig.28 : ROC curve of neutrophil ADGRE3 protein for predicting the risk of death in patients with sepsis;

[0178] Fig.29 : Comparison of neutrophil ADGRE3 protein expression between patients with improved sepsis and those without improved or dead patients;

[0179] Fig.30 : Comparison of neutrophil ADGRE3 protein expression between healthy controls and patients with local inflammation (patients with hepatitis B, hepatitis C, and hepatitis E);

[0180] Fig.31 : Flowchart of classification of patients with systemic inflammatory response caused by bacterial infection according to nADGRE3 and other inflammatory indicators;

[0181] Fig.32 : Flowchart of classification of patients with systemic inflammatory response caused by viral infection according to nADGRE3 and other inflammatory indicators;

[0182] Fig.33 : Flowchart of classification of patients with systemic inflammatory response caused by autoimmune diseases and transplant rejection according to nADGRE3 and other inflammatory markers;

[0183] Fig.34 : Flowchart for the diagnostic classification of patients suspected of systemic inflammatory response according to nADGRE3 and other bacterial inflammation indicators.

[0184] Description of symbols and contents in the drawings:

[0185] Figure 1 and Figure 2 In the figure, each point corresponds to a gene. A point on the left side of the origin of the horizontal axis represents a decrease in the expression of the corresponding gene, and a point on the right side of the origin of the horizontal axis represents an increase in the expression of the corresponding gene. The color of the point represents that the change in the expression of the corresponding gene is more than 2 times and has a statistically significant difference (p<0.05). Blue represents a decrease in expression of more than 2 times, and red represents a significant increase in expression of more than 2 times. The farther the point is from the origin of the horizontal axis, the greater the change in the expression of the corresponding gene. When the absolute value of the horizontal axis of the point is greater than or equal to 1, it means that there is a difference of more than 2 times, and when the absolute value is greater than or equal to 2, it means that there is a change of more than 4 times.

[0186] like Figure 3 In the figure of mRNA expression level of the present invention, the ordinate is the mRNA expression level, specifically the copy number of mRNA, and "GSE+number" is the corresponding data set number in the GEO database; Fig.15 In the graph of protein expression level of the present invention, the ordinate is the relative expression amount of protein (nADGRE3), and the calculation of nADGRE3 has been introduced in the content of the invention.

[0187] The significance of mRNA or protein expression levels between groups is indicated by ns or several *s; ns means no significant difference (p>0.05), “*” means significant difference (p<0.05), “**” means extremely significant difference (p<0.01), “***” means higher significance (p<0.001), and “****” means extremely high significance (p<0.0001).

[0188] Detailed description

[0189] The structures involved in the present invention or the technical terms used are further explained below. If not otherwise specified, the terms are interpreted according to the general understanding of the art.

[0190] diagnosis

[0191] The "diagnosis" in the present invention is an evaluation concept of health status, including prediction of whether an individual is healthy or unhealthy, and also includes the evaluation of the therapeutic effect of the disease during the treatment process. Therefore, the diagnosis of the present invention includes at least one or more of the following aspects, including:

[0192] First, diagnose whether an individual has systemic inflammation or a specific type of systemic inflammation, including: distinguishing between healthy people and patients with systemic inflammation, distinguishing between patients with local inflammation or non-inflammatory diseases and patients with systemic inflammation, and also distinguishing between systemic inflammation caused by different etiologies (such as distinguishing between bacterial systemic inflammation and non-bacterial systemic inflammation, between viruses and bacteria), etc.; or,

[0193] Second, predicting the risk of an individual suffering from systemic inflammation or a specific type of systemic inflammation, including: predicting the risk of patients with non-systemic inflammation suffering from systemic inflammation, predicting the risk of local inflammation developing into systemic inflammation, predicting the risk of patients with chronic diseases suffering from systemic inflammation, etc.; or,

[0194] Third, monitoring the disease progression of patients with systemic inflammation, judging whether the condition of patients with systemic inflammation is worsening or improving, and judging whether patients with systemic inflammation are at risk of worsening to sepsis; or judging the prognosis of patients with systemic inflammation, judging the development trend of the condition of patients with systemic inflammation, and thus predicting the risk of death of patients with systemic inflammation, etc.; or,

[0195] Fourth, drug guidance for patients includes: when patients with non-systemic inflammation are found to have a tendency to develop systemic inflammation, drug intervention is used for prevention; when patients with systemic inflammation are found to have their condition worsen, the drug dosage is increased to improve the treatment effect; when patients with systemic inflammation are found to have their condition improved, the drug dosage is reduced to reduce the probability of adverse drug reactions or drug resistance in patients; the above-mentioned drugs can be any drugs for treating systemic inflammation, including but not limited to antibiotics, antiviral drugs, and hormone drugs.

[0196] ADGRE3 mRNA or encoded protein

[0197] The ADGRE3 gene (gene ID number for ADGRE3 is ENSG00000131355) is located on chromosome 19 and has been identified as a protein-coding gene. The gene structure consists of multiple exons, and alternative splicing produces multiple mRNA transcript variants encoding different isoforms. The expression profile of ADGRE3 suggests that it plays a role in phagocyte function, cell-cell interactions, and / or signaling during inflammation. The gene is mainly expressed by cells of the immune system, and its expression in synovial granulocytes is higher than that in peripheral blood granulocytes, which may be due to the inflammatory microenvironment in the synovial fluid.

[0198] The ADGRE3 protein is a G protein-coupled receptor that may play a role in myeloid-myeloid interactions during immune and inflammatory responses. The ligand for the soluble form of this receptor is present on the surface of monocyte-derived macrophages and activated neutrophils. Diseases associated with the gene encoding ADGRE3 include inflammatory bowel disease. The involvement of this gene in these diseases may be related to its role in immune and inflammatory responses. ADGRE3 participates in multiple pathways, including the innate immune system and GPCR downstream signaling. The protein product of this gene interacts with other proteins in the immune system. The ADGRE3 protein is expressed in multiple tissues, including bone marrow and lymphoid tissues, with a tissue-enhanced expression pattern. The protein is expected to be secreted, with some isoforms localized on the membrane.

[0199] In summary, ADGRE3 is a protein-coding gene that plays a vital role in the immune system, particularly in phagocyte function, cell-cell interactions, and signaling during inflammation. The gene is expressed primarily in cells of the immune system and is involved in multiple pathways related to immune and inflammatory responses.

[0200] ADGRE3 gene has multiple mRNA transcript variants, any mRNA and its fragments can be used as a marker of the present invention; one of the main mRNA transcript sequences is NM_032571.5, and the mRNA targets in the present invention include the following six: XM_054322398.1, XM_047439546.1, XM_011528374.3, NM_001289158.2, NM_001289159.2, NM_032571.5. The above mRNA transcript sequences are provided by the RefSeq database. It can be understood that any method that can specifically detect the content of the mRNA and the number of copies can be used in the diagnostic method of the present invention, such as commonly used variable temperature amplification, PCR, isothermal amplification, and gene editing technology. Any method or supporting reagents for nucleic acid testing can be used in the present invention for testing or detection. When amplification is required, it is easy to design suitable primers.

[0201] Since the ADGRE3 gene has multiple mRNA transcript variants, the ADGRE3 proteins translated from different mRNAs may have different amino acid sequences, so any ADGRE3 protein translated from any mRNA and its fragments can be used as a marker of the present invention. It can be understood that any method that can specifically detect the amount of ADGRE3 protein can be used in the diagnostic method of the present invention, such as flow cytometry, Coomassie Brilliant Blue method, etc. Any method or supporting reagent for protein testing can be used in the present invention for testing or detection.

[0202] An amino acid sequence of human ADGRE3 protein (SEQ ID NO: 1):

[0203] MQGPLLLPGLCFLLSLFGAVTQKTKTSCAKCPPNASCVNNTHCTCNHGYTSGSGQKLFTF

[0204] PLETCNDINECTPPYSVYCGFNAVCYNVEGSFYCQCVPGYRLHSGNEQFSNSNENTCQDT

[0205] TSSKTTEGRKELQKIVDKFESLLTNQTLWRTEGRQEISSTATTILRDVESKVLETALKDP

[0206] EQKVLKIQNDSVAIETQAITDNCSEERKTFNLNVQMNSMDIRCSDIIQGDTQGPSAIAFI

[0207] SYSSLGNIINATFFEEMDKKDQVYLNSQVVSAAIGPKRNVSLSKSVTLTFQHVKMTPSTK

[0208] KVFCVYWKSTGQGSQWSRDGCFLIHVNKSHTMNCSHLSSFAVLMALTSQEEDPVLTVIT

[0209] YVGLSVSLLCLLLAALTFLLCKAIRNTSTSLHLQLSLCLFLAHLLFLVGIDRTEPKVLCS

[0210] IIAGALHYLYLAAFTWMLLEGVHLFLTARNLTVVNYSSINRLMKWIMFPVGYGVPAVTVA

[0211] ISAASWPHLYGTADRCWLHLDQGFMWSFLGPVCAIFSANLVLFILVFWILKRKLSSLNSE

[0212] VSTIQNTRMLAFKATAQLFILGCTTWCLGLLQVGPAAQVMAYLFTIINSLQGFFIFLVYCL

[0213] LSQQVQKQYQKWFREIVKSKSESETYTLSSKMGPDSKPSEGDVFPGQVKRKY.

[0214] According to the characteristics of different fragments or regions, the human ADGRE3 protein having the amino acid sequence shown in SEQ ID NO: 1 can be divided into multiple fragments or regions as shown below:

[0215]

[0216]

[0217]

[0218] Features are used to describe different regions and domains in protein sequences. Signal peptide sequence (FTSIGNAL): a short amino acid sequence that can guide newly synthesized proteins into the endoplasmic reticulum-Golgi pathway for protein secretion or localization. Mature protein sequence (FTCHAIN): after the protein enters the secretory pathway, the signal peptide will be recognized and removed by a special signal peptidase. After the signal peptide is removed, what remains is the mature protein sequence. The mature protein sequence usually contains the functional domain and other important regions of the protein, which determine the final conformation and biological function of the protein. Transmembrane region (FTTRANSMEM) refers to the hydrophobic α-helix or β-helix fold region in the protein sequence that is embedded in the cell membrane. α-helix or β-helix fold is a common secondary structure of proteins and can be embedded in the lipid bilayer of the cell membrane. Topological domain (FTTOPO_DOM) describes the topological arrangement of proteins on or in the membrane, such as intracellular, extracellular or transmembrane regions. Functional domain (FTTRANSMEM) DOMAIN refers to an independent foldable structural unit with specific biological functions; region (FTREGION) describes a fragment in the sequence that has certain common characteristics but unclear functions, such as low-complexity regions and repetitive sequences.

[0219] The fragment position represents the position of the first and last amino acids of the fragment or region in the full-length amino acid sequence of the protein.

[0220] Extracellular refers to the region of the protein sequence that is located outside the cell membrane; helical refers to the region of the protein sequence that forms the α-helical structure. The α-helix is ​​one of the common secondary structures of proteins and has a spiral spatial arrangement. Many transmembrane proteins contain multiple α-helical regions, which can be embedded in the lipid bilayer of the cell membrane to form a transmembrane domain; cytoplasmic refers to the region of the protein sequence that is located in the cytoplasm, which contains domains and regulatory regions related to intracellular signal transduction and may interact with effector molecules in the cell to transmit signals. Epidermal growth factor-like domain (EGF-like) is an independent folding structural module widely present in many proteins, which is usually involved in protein-protein interactions. EGF-like domains usually contain 6 conserved cysteine ​​residues, forming 3 pairs of disulfide bonds (1-3, 2-4, 5-6). Some EGF-like domains also have calcium-binding ability; GPS domain is a conserved sequence of about 40 amino acid residues, rich in canonical cysteine ​​and tryptophan residues, and is the most highly conserved part of G protein-coupled receptors (GPCRs); disordered or irregular structural regions refer to disordered or irregular structural regions, which lack fixed three-dimensional structures and have high conformational flexibility and dynamics; disordered regions are usually rich in polar and charged residues, and play an important role in protein folding, interaction and regulation.

[0221] Of course, it is understood that the corresponding ADGRE3 protein sequence in mammals may have a small number of amino acid differences from that in humans, and the ADGRE3 protein, protein fragment, amino acid or amino acid fragment (polypeptide fragment) in mammals can be used with the marker of the present invention to be tested or detected.

[0222] In the subsequent description of the present invention, if not otherwise specified, the term "gene" refers to mRNA or mRNA fragments, and "protein" refers to full-length protein or protein fragments, or full-length peptides or peptide fragments, or full-length amino acids or amino acid fragments. The so-called "fragment" is some shorter sequence relative to the full length, such as a gene sequence or an amino acid sequence, such as an extramembrane protein fragment or a peptide fragment.

[0223] White blood cells and neutrophils

[0224] White blood cells are a type of colorless, spherical, nucleated blood cells. White blood cells are not a uniform cell group. They can be divided into three categories based on their morphology, function, and source: granulocytes, monocytes, and lymphocytes. Granulocytes can be divided into three types: neutrophils, eosinophils, and basophils based on the different staining properties of the granules in the cytoplasm.

[0225] White blood cells generally have active mobility. They can migrate from inside blood vessels to outside blood vessels, or from extravascular tissues to inside blood vessels. Therefore, in addition to being present in blood and lymph, white blood cells are also widely present in tissues outside blood vessels and lymphatic vessels. Most white blood cells only stay in the blood for a short time before entering tissues to play a role.

[0226] Neutrophils are a type of white blood cell. Their main function is to resist the invasion of microorganisms. They have the ability to deform and phagocytose, and are very important in resisting diseases and protecting the body. In healthy people, they are usually present in the blood, but in patients with diseases, especially those with systemic inflammation, white blood cells will quickly infiltrate the inflammatory site. Therefore, white blood cells are often found in various tissues of patients. Therefore, white blood cells and neutrophils, monocytes, etc. in the white blood cell population can be detected in different types of clinical samples.

[0227] When the present invention uses ADGRE3 mRNA as a marker, it generally detects the level of transcription in white blood cells. For example, if the transcription level is lower than the normal level, it indicates the possibility of systemic inflammation. It is understandable that the protein translated by its mRNA can also be used as a marker, and we have found that the use of protein as a marker has higher sensitivity and specificity. Therefore, in theory, it is possible to use proteins in any cell in the white blood cell group, such as any one of granulocytes, monocytes and lymphocytes, or any one of the three neutrophils, eosinophils and basophils in granulocytes. Although the protein content translated by different cells is different, it is still of diagnostic value to use the same cell for testing and comparison, such as testing the protein expression of monocytes in healthy and local inflammation or systemic inflammation populations, and there are still significant differences. Of course, in some ways, it is also possible to use proteins in neutrophils for testing.

[0228] ADGRE3 mRNA or ADGRE3 protein as marker

[0229] The present invention finds that ADGRE3 Gene transcription mRNA The content in peripheral blood leukocytes shows a downward trend as inflammation progresses, especially when local inflammation develops into systemic inflammation. At the same time, there are significant differences even between different types of systemic inflammation caused by different reasons.

[0230] In order to find a gene that plays an important role in the pathogenesis of sepsis, the present invention team initially analyzed and compared the changes in peripheral blood gene expression profiles of sepsis patients and patients with non-infectious systemic inflammation in the public database, and found that the expression of this gene in the peripheral blood of sepsis patients changed significantly ( Figure 1 );from Figure 1 It can be seen that among the two genes significantly downregulated, the gene of the present invention was found to be significantly downregulated, while the other gene (Membrane Metalloendopeptidase, MME) was downregulated but not suitable for use as a systemic inflammatory marker because the analysis of data cohorts of other infections and sepsis found that there was no significant difference in the expression level of the MME gene between sepsis and healthy controls. Figure 1 It can be clearly seen that the downregulation of the ADGRE3 gene of the present invention can be used as a marker to distinguish patients with sepsis from patients with non-infectious systemic inflammation. Of course, the corresponding protein can also be used as a marker to distinguish patients with sepsis from patients with non-infectious systemic inflammation. Then, we analyzed and compared the differences in gene regulation between sepsis patients and healthy people, and also found that the gene was significantly downregulated ( Figure 2 ). This suggests that this gene can be used to distinguish sepsis patients from healthy patients. Figure 2 Many down-regulated genes were found, but some could not be used as markers, and some were well-known down-regulated genes.

[0231] In subsequent experiments, we singled out this gene or protein and analyzed the changes in gene expression in different infection type data in public data. We also collected samples from patients in our hospital to verify mRNA and protein expression, and compared them with existing clinical inflammatory indicators. We found that this indicator has a very high sensitivity for indicating systemic inflammation, exceeding the existing clinical indicators, which cannot improve the sensitivity and specificity of diagnosing inflammatory diseases. We also found that the transcription level of ADGRE3 mRNA in the peripheral blood of patients with systemic inflammation caused by bacterial infection and viral infection was significantly lower than that in healthy controls. The same was true for Gram-positive bacteria infection, H1N1 infection, dengue fever, COVID-19, RSV infection, cytomegalovirus infection, Epstein-Barr virus infection and influenza virus infection, thus verifying the effectiveness of ADGRE3 mRNA in diagnosing these patients with systemic inflammation caused by infectious inflammation. In addition, it was found that the transcription level of ADGRE3 mRNA can distinguish patients with Gram-positive bacteria infection from patients with H1N1 infection, asymptomatic COVID-19 patients from symptomatic COVID-19 patients, and patients with acute RSV infection from those who recovered from RSV infection. Therefore, ADGRE3 mRNA or ADGRE3 protein can not only diagnose patients with systemic inflammation caused by infectious inflammation, but also diagnose the lesions of systemic inflammation caused by infectious inflammation, so as to implement targeted treatment plans and monitor the progression of the disease.

[0232] In addition, clinical validation of healthy people, non-infectious systemic inflammatory response syndrome (SIRS) and sepsis patients demonstrated that the leukocyte transcription levels or protein contents of ADGRE3 mRNA or protein in these three groups of people were significantly different from each other, so ADGRE3 mRNA or protein can be used as a marker for distinguishing non-infectious systemic inflammatory response syndrome and sepsis. The diagnostic results of the validation set showed that the diagnostic AUC value for distinguishing healthy people from sepsis patients was 0.920, the diagnostic AUC value for distinguishing sepsis from SIRS was 0.864, and the diagnostic AUC value for diagnosing sepsis from the above-mentioned entire sample set was 0.862, indicating that the leukocyte ADGRE3 mRNA transcription level can accurately diagnose sepsis.

[0233] Inflammation

[0234] Inflammation is a physiological response of the body to injury, infection or irritation, aimed at eliminating pathogens, repairing tissues, and restoring tissue function. Inflammation usually manifests as local symptoms such as redness, swelling, heat, pain and functional impairment.

[0235] The occurrence of inflammation usually includes the following steps: Inflammation triggers: These can be external or internal factors such as infection, trauma, chemical stimulation, allergic reaction, etc. Release of inflammatory mediators: Damaged cells release inflammatory mediators, such as histamine, prostaglandins, cytokines, etc., which cause vasodilation, increased vascular permeability and leukocyte infiltration. Leukocyte infiltration: Leukocytes, especially neutrophils, enter tissues through the blood vessel wall to remove pathogens and cell debris. Tissue repair: During the inflammation process, the body will initiate tissue repair mechanisms, including fibrin deposition, cell proliferation and repair processes.

[0236] Treatments for inflammation depend on the cause and severity of the inflammation. Common treatments include: Nonsteroidal anti-inflammatory drugs (NSAIDs): such as ibuprofen and paracetamol, used to relieve pain and inflammation. Steroid hormones: such as prednisone, used to control inflammatory responses. Immunosuppressants: used to control overactive immune systems. Antibiotics: used to treat inflammatory infections. Physical therapy: such as ice and heat can help reduce inflammation and pain.

[0237] Systemic inflammation

[0238] Systemic inflammation is a systemic inflammatory response that is not limited to specific tissues or organs, but involves the inflammatory process of the entire body. This inflammatory response is usually caused by the body's non-specific response to infection or inflammation-inducing factors, such as microorganisms such as bacteria, fungi and viruses, trauma, burns, surgery and other stimuli. Systemic inflammation is relative to local specific tissues or organs (specific). Of course, it is understandable that local inflammation can develop into systemic severe inflammation, which is a more severe inflammation in the development of inflammation.

[0239] Generally, 100% of the body's tissues or organs have an inflammatory response, which is one mode of the present invention. Of course, it is also possible that more than 50% to 100% of the tissues or organs have an inflammatory response, which is also covered by the present invention. From another perspective, the process from specific inflammation to nonspecific inflammatory response is also one of the embodiments of the present invention. The above ranges all belong to the term " Systemic inflammation "For example, when local inflammation occurs, the indicator of the present invention is used to monitor the development of the disease. At this time, the marker of the present invention may not be significantly reduced. Once the measured level is significantly lower than that of the previous test, it can be determined that systemic inflammation has occurred. Or if the current test level is always on a downward trend, it can be predicted that local inflammation may lead to systemic inflammation, and intervention treatment can be performed early to prevent or prevent the occurrence of systemic inflammation.

[0240] Characteristics of systemic inflammatory response include: Release of inflammatory mediators: In systemic inflammatory response, the body releases a variety of inflammatory mediators, such as cytokines, chemokines, interleukins, etc., which can cause local and systemic inflammatory response. Systemic symptoms: Patients may show systemic symptoms, such as fever, chills, general fatigue, increased heart rate, shortness of breath, increased or decreased peripheral white blood cells, etc. Impaired organ function: In severe systemic inflammatory response, excessive release of inflammatory mediators may lead to multiple organ dysfunction syndrome (MODS), which seriously threatens the patient's life. Immune system disorders: During systemic inflammatory response, the immune system may be in a hyperactive state, leading to immune dysfunction and even autoimmune response. Life-threatening in severe cases: In some cases, such as systemic inflammatory response caused by severe infection or bacterial toxins, it may evolve into severe sepsis or septic shock, which endangers the patient's life.

[0241] The key to managing systemic inflammatory response lies in early identification and active intervention. The goals of treatment include controlling inflammatory response, maintaining organ function, symptomatic supportive treatment, and treating the cause. Commonly used clinical treatment methods include antibiotic treatment, hemodynamic support, immunomodulatory therapy, etc. Early intervention can effectively reduce the severity of systemic inflammatory response and improve the patient's survival rate and prognosis.

[0242] Distinguishing between systemic and nonsystemic inflammation, or systemic and nonsystemic inflammation

[0243] On the one hand, from a medical perspective: when distinguishing between systemic and non-systemic inflammation, or between systemic and non-systemic inflammation, the following aspects need to be considered:

[0244] 1. Medical history and clinical manifestations: Systemic inflammation: Patients may present with systemic symptoms, such as fever, fatigue, general malaise, etc. Inflammation may involve multiple organ systems. Systemic inflammation is often accompanied by systemic symptoms and increased inflammatory markers. Non-systemic inflammation: Inflammation may be confined to specific parts or organs, manifesting as local symptoms, such as local redness, swelling, pain, limited function, etc.

[0245] 2. Laboratory examination: Systemic inflammation: often accompanied by abnormal complete blood count, elevated inflammatory markers (such as C-reactive protein, procalcitonin), increased release of systemic inflammatory mediators, etc. Non-systemic inflammation: Laboratory test results may show that the inflammatory response is limited to the affected area, and the complete blood count and inflammatory markers may not be as significantly elevated as systemic inflammation.

[0246] 3. Imaging examination: Systemic inflammation: Imaging examination may show inflammation manifestations involving multiple organ systems, such as multi-organ infection foci in systemic inflammation. Non-systemic inflammation: Imaging examination results may only show inflammatory changes in the affected area, such as local infection or inflammatory foci.

[0247] 4. Pathogen detection: Systemic inflammation: Pathogens may be detected in multiple parts of the body through blood culture or other detection methods. Non-systemic inflammation: Pathogen detection results may be positive only in the affected area or specific area.

[0248] 5. Treatment response: Systemic inflammation: Systemic inflammation may require systemic antibiotics or immunomodulatory therapy. Non-systemic inflammation: Non-systemic inflammation can usually be effectively controlled by local treatment (such as local antibiotics, local anti-inflammatory drugs).

[0249] By comprehensively analyzing information such as medical history, clinical manifestations, laboratory tests, imaging examinations, pathogen detection, and treatment response, infectious medicine experts can more accurately distinguish between systemic and non-systemic inflammation, or systemic and non-systemic inflammation, and develop appropriate treatment plans.

[0250] Secondly, from an immunological perspective: When distinguishing between systemic and non-systemic inflammation, or between systemic and non-systemic inflammation, the following aspects need to be considered: 1. Scope of immune response: Systemic / systemic inflammation: Involves abnormal activity or dysregulation of the immune system throughout the body, resulting in a generalized immune response to multiple organs and tissues. Immune cells and inflammatory mediators may be active in multiple parts of the body. Non-systemic / non-systemic inflammation: The immune response is confined to specific parts or organs, and the inflammatory response is mainly confined to the affected local area.

[0251] 2. Involvement of immune cells: Systemic inflammation: Involves abnormal activity of multiple immune cell types, which may include T lymphocytes, B lymphocytes, neutrophils, macrophages, etc., leading to systemic immune response and inflammation. Non-systemic inflammation: Immune cell activity is mainly confined to the affected area, such as a specific tissue or organ.

[0252] 3. Release of inflammatory mediators: Systemic inflammation: Increased release of inflammatory mediators throughout the body may lead to systemic symptoms and elevated inflammatory markers. Non-systemic inflammation: Release of inflammatory mediators is primarily limited to the affected local area, and inflammatory markers may not be as elevated at the systemic level as in systemic inflammation.

[0253] 4. Immunomodulatory mechanisms: Systemic inflammation: may involve comprehensive regulation of the immune system, requiring comprehensive regulation of immune cell activity and inflammatory mediator release. Non-systemic inflammation: treatment may be more localized, with the focus on controlling local inflammatory responses without interfering with systemic immune balance.

[0254] By comprehensively considering factors such as the scope of immune response, immune cell involvement, release of inflammatory mediators and immune regulatory mechanisms, immunologists can more accurately distinguish between systemic and non-systemic inflammation, or systemic and non-systemic inflammation, and formulate corresponding immune regulatory treatment plans.

[0255] Local inflammation and systemic inflammation considered from any of the above perspectives belong to the scope of the present invention. The markers of the present invention can not only distinguish local inflammation from systemic inflammation, but also distinguish patients with systemic inflammation from healthy people.

[0256] Infectious inflammation

[0257] Infectious inflammation can develop from local infection to systemic inflammation or systemic inflammation. Infectious inflammation is an inflammatory response caused by infection. Infection refers to the process by which pathogens (such as bacteria, viruses, fungi or parasites) invade the host body and multiply. When the body is infected, the immune system will initiate an inflammatory response to eliminate pathogens and repair damaged tissues.

[0258] Characteristics of infectious inflammation include: Pathogen invasion: Infectious inflammation is caused by pathogens invading the body, which can be bacteria, viruses, fungi or parasites. Inflammatory response: The inflammatory response caused by infection is a natural defense mechanism of the immune system, including the release of inflammatory mediators, activation and migration of white blood cells. Local symptoms: Infectious inflammation often manifests as local symptoms, such as redness, swelling, pain, heat and dysfunction. These symptoms are part of the body's response to fight infection. Systemic inflammation / systemic inflammation: Severe infection may cause systemic symptoms, such as fever, chills, general fatigue, headache, nausea, vomiting, etc.

[0259] Complications in severe cases: Severe infections may lead to severe complications such as sepsis and septic shock, leading to multiple organ dysfunction and even life-threatening. The above also belong to systemic inflammation, and are classified according to the severity.

[0260] The key to managing infectious inflammation is early identification and timely treatment of infection. Treatment methods include antibiotics (for bacterial infections), antiviral drugs (for viral infections), antifungal drugs (for fungal infections), etc., and supportive treatment is required to maintain the patient's vital signs. The key to preventing infectious inflammation includes maintaining good personal hygiene habits, vaccination, avoiding contact with known sources of infection, and timely treatment of infections. For susceptible groups (such as those with weakened immune function, patients with chronic diseases, etc.), it is necessary to pay close attention to measures to prevent infection.

[0261] Non-infectious inflammation

[0262] Non-infectious inflammation can develop from local infection to systemic inflammation or systemic inflammation. Non-infectious inflammation refers to an inflammatory response that is not caused by external pathogens (such as bacteria, viruses, fungi or parasites). This inflammation may be caused by the body's own abnormal immune response, transplant organ rejection, tissue damage, chemical stimulation or other non-infectious factors. Some common non-infectious inflammatory diseases include: Autoimmune diseases: such as rheumatoid arthritis, systemic lupus erythematosus, vasculitis, autoimmune hepatitis, autoimmune thyroiditis (Hashimoto's thyroiditis) and Graves' disease, multiple sclerosis (Multiple Sclerosis), Sjögren's syndrome and transplant organ rejection, etc. These diseases are caused by the immune system mistakenly attacking body tissues. Inflammatory bowel disease: such as Crohn's disease and ulcerative colitis, these diseases are caused by chronic inflammation of the intestines.

[0263] Allergic reactions: Allergic diseases such as asthma and allergic rhinitis are caused by abnormal immune system reactions caused by allergens. Metabolic diseases: For example, diabetes, obesity, etc., these diseases are also closely related to inflammatory reactions. Inflammation caused by tissue damage: For example, trauma, chemical stimulation, radiation damage, etc. may all cause non-infectious inflammation. Non-infectious inflammation and infectious inflammation are different in etiology and pathogenesis, but there may be some similarities in clinical manifestations, such as fever, pain, redness and swelling and other inflammatory symptoms.

[0264] Methods for managing non-infectious inflammation usually include controlling inflammatory responses, relieving symptoms, and reducing tissue damage. The specific treatment method depends on the type of disease and the severity of the condition. For autoimmune diseases, conventional treatments include immunosuppressants, anti-inflammatory drugs, etc. For other types of non-infectious inflammation, treatments may include medication, physical therapy, surgical treatment, etc. Timely diagnosis and treatment of non-infectious inflammation can help alleviate symptoms, control disease progression, and improve the patient's quality of life.

[0265] Sepsis

[0266] Sepsis is a systemic inflammatory response syndrome, a more severe type of systemic inflammation, commonly seen in patients with severe trauma or infectious diseases. Causes of the disease include infections caused by bacteria, fungi, viruses, and parasites, which lead to inflammatory responses and imbalances in immune regulation. Sepsis can develop into severe sepsis and septic shock, which can lead to organ dysfunction and circulatory disorders, threatening life.

[0267] Sepsis refers to a systemic inflammatory response syndrome caused by a clear or suspected infection. Severe sepsis refers to sepsis with resulting organ dysfunction and tissue hypoperfusion. Septic shock refers to sepsis with resulting hypotension that cannot be reversed by fluid therapy.

[0268] Diagnostic criteria for sepsis: the presence of clear or suspected infection and one or more of the following clinical features:

[0269] Signs and clinical symptoms: Body temperature: body temperature>38.3℃ or body temperature<36℃; heart rate>90 times / min, or greater than the two standard vertebral differences of normal values ​​for different ages; shortness of breath: breathing>20 times / min or hyperventilation, PaCO2<32mmHg; changes in mental status;

[0270] Significant edema or fluid leveling: more than 20 ml / kg in 24 hours; hyperglycemia without a history of diabetes. Hemodynamics: Hypotension: systolic blood pressure <90 mmHg, mean arterial pressure (MAP) <70 mmHg or systolic blood pressure in adults drops by more than 40 mmHg or is two standard deviations below the normal value for age.

[0271] Laboratory examination: Inflammatory response indicators: Leukocytosis: white blood cells (WBC) > 12000 / μl; Leukocytopenia: WBC < 4000 / μl; WBC normal but the total number of immature white blood cells exceeds 10%; plasma C-reactive protein > normal two standard deviations; plasma procalcitonin > normal two standard deviations.

[0272] Indicators of organ dysfunction: hypoxemia: arterial oxygen partial pressure (PaO2) / oxygen concentration (FiO2) <300 mmHg; acute oliguria: even with adequate fluid resuscitation, urine output is still low; blood creatinine >44.2 μmol / L (0.5 mg / dl); abnormal coagulation function; intestinal obstruction: disappearance of bowel sounds; thrombocytopenia: platelets (PLT) <10000 / μl; hyperbilirubinemia.

[0273] Tissue perfusion indicators: hyperlactatemia (lactate>1mmoL / L); decreased capillary reperfusion capacity or ecchymosis formation. SOFA score: For patients with infection or suspected infection, sepsis can be diagnosed when the sequential (Sepsis-related) Organ Failure Assessment, SDFA] score increases by ≥2 points compared with the baseline. Because the SOFA score is relatively complicated to operate, the bedside quick SOFA (qSOFA) standard is often used clinically to identify critically ill patients. If at least two of the qSOFA criteria are met, combined with the results of the organ dysfunction assessment, a preliminary judgment of sepsis is made. The qSOFA criteria are as follows: respiratory rate ≥22 times / min; changes in consciousness; systolic blood pressure ≤100mmHg;

[0274] Diagnostic criteria for severe sepsis and septic shock: sepsis with organ dysfunction and / or tissue hypoperfusion caused by sepsis, any of the following; signs and clinical symptoms: hypotension caused by sepsis; urine output <0.5 ml / kg / h for at least 2 hours even with adequate fluid resuscitation; acute lung injury not caused by pneumonia with PaO2 / FiO2 <250 mmHg; acute lung injury caused by pneumonia with PaO2 / FiO2 <200 mmHg; laboratory tests: lactate greater than normal; serum creatinine >176.8 μmol / L (2.0 mg / dl); bilirubin >34.2 μmol / L (2 mg / dl); PLT <100000μl; coagulation disorder (international normalized ratio> 1.5).

[0275] At present, the treatment of this disease can be roughly divided into three parts: etiological treatment, supportive treatment and immunomodulatory treatment. Etiological treatment includes early removal of infected lesions and the use of effective antibiotics. Supportive treatment includes early circulatory resuscitation, mechanical ventilation, renal replacement, metabolic support, anticoagulation and other measures for damage to different organs and systems. Immunomodulatory treatment mainly refers to treatment methods that can affect the body's immune inflammatory response.

[0276] The content of ADGRE3 mRNA and / or ADGRE3 protein discovered in the present invention can reflect the etiology type of patients with systemic inflammation to a certain extent. On the one hand, it refers to distinguishing infectious inflammation and non-infectious inflammation from a large classification perspective, and distinguishing the severity of systemic inflammation, such as distinguishing sepsis and septic shock, thereby avoiding the use of the above-mentioned complex diagnostic indicators and improving the sensitivity, specificity and accuracy of diagnosis; on the other hand, it refers to finding the cause of the disease, such as distinguishing infectious inflammation caused by different pathogens, thereby assisting in etiological treatment and supportive treatment.

[0277] Sepsis has an extremely high mortality rate, but there is currently no technology for predicting the risk of death in patients with sepsis. The content of the ADGRE3 protein discovered in the present invention can predict the risk of death in patients; at the same time, by monitoring the expression level of the patient's neutrophil ADGRE3 protein every day, it is possible to accurately judge whether the patient's sepsis has improved, thereby assisting clinical treatment.

[0278] Threshold (cut-off)

[0279] Threshold definition: The threshold is the value that converts a continuous variable into a discrete variable and is used to determine whether the result is positive or negative. It determines the classification of the result.

[0280] Setting the threshold (cut-off): Based on the reference range: The reference normal range is an important basis for setting the threshold. The normal reference range is determined by statistical analysis of samples from the healthy population and used as an important reference for the threshold. Based on disease characteristics: Understanding the characteristics of the disease being tested is crucial for setting the threshold. Some diseases may require higher or lower thresholds for accurate diagnosis. Based on the receiver operating characteristic curve (ROC curve): The ROC curve can help evaluate the sensitivity and specificity under different thresholds and find the best threshold to achieve the best diagnostic effect. Based on clinical experience: Clinical experience plays a key role in setting the threshold. Laboratory experts should consider the needs and actual conditions of clinicians and adjust the threshold in combination with clinical experience. Based on quality control: Ensure that the set threshold meets the quality control standards to ensure the accuracy and reliability of the results. Based on standardized guidelines: Refer to relevant standardized guidelines or recommendations of professional organizations, which can provide guidelines for setting thresholds. Regular review and update: Thresholds should be reviewed and updated regularly to ensure that they remain in line with the latest clinical practices and data.

[0281] Taking all the above factors into consideration, clinical laboratory diagnostic experts can develop reasonable thresholds to ensure the accuracy of laboratory test results and the effectiveness of clinical diagnosis.

[0282] The present invention detects the expression of ADGRE3 protein in neutrophils of healthy non-inflammatory population by flow cytometry, calculates the mean fluorescence intensity (MFI) of ADGRE3 protein in each sample and obtains the average value of the mean fluorescence intensity (MFI) of ADGRE3 protein in healthy population, detects the mean fluorescence intensity (MFI) of neutrophil ADGRE3 protein in patients under the same conditions, and divides the mean fluorescence intensity (MFI) of ADGRE3 protein in neutrophil ADGRE3 protein in patients by the average value of the mean fluorescence intensity (MFI) of ADGRE3 protein in healthy population to obtain the expression index (nADGRE3) of neutrophil ADGRE3 protein in patients, that is, nADGRE3=MFI of neutrophil ADGRE3 protein in patients / MFI of neutrophil ADGRE3 protein in healthy control. Therefore, nADGRE3 of healthy control population is close to or equal to 1. On this basis, the sensitivity and specificity of nADGRE3 in infection and non-infection inflammatory response were statistically analyzed, and the optimal cut-off value was 0.8361. When the above method is used, when it is greater than 0.8361, it can be considered healthy or non-systemic inflammation (local inflammation or non-inflammatory disease), and when the measured value is less than 0.8361, it can be considered as systemic inflammation. Calculating the neutrophil ADGRE3 protein expression index (nADGRE3) is only a form of threshold setting and sample classification. Threshold setting and sample classification can also be performed according to the ADGRE3 mRNA copy number in the sample, ADGRE3 protein content (mass or concentration) or any other known method, and the diagnostic classification results obtained are the same.

[0283] The above is a method for setting the threshold value between healthy people and systemic inflammation, but it can be understood that due to different objects of comparison, the threshold value is also set differently. For example, when both are systemic inflammation, when comparing the severity, the threshold value is set to compare the severity of different types. If you want to distinguish, the values ​​of the same type tested generally need to reach a significant difference (P less than 0.05, or extremely significant difference P less than 0.01, at least P value less than 0.05, any corresponding level can be used as a specific value of a threshold). The unit is based on different test methods, and what is reflected is the difference in the level or content of the marker of the present invention between the comparison objects. The unit of the threshold value can be any unit, such as MFI, the number of copies, the concentration value of the protein, or any other unit that indirectly represents the content in the sample.

[0284] Regarding the determination of the content of ADGRE3 mRNA or ADGRE3 mRNA fragments and the setting of thresholds, the detection methods and detection instruments used by various laboratories and testing institutions are currently very different, and the data processing, especially the standardization algorithm, is also different, so it is difficult to specify an exact threshold; however, the significant difference in the content of ADGRE3 mRNA between samples of patients with systemic inflammation and healthy controls is objectively present, and any technician in this field can analyze samples of healthy controls and patients with systemic inflammation according to the detection methods and instruments and data analysis algorithms they use, so as to obtain a suitable threshold for diagnosing systemic inflammation, and the above all belong to the protection scope of the present invention. Similarly, the threshold of ADGRE3 mRNA protein has the same situation.

[0285] Early diagnosis of systemic inflammation, differentiation of infection types, prognosis, disease monitoring, antibiotic medication guidance, etc. Common laboratory indicators

[0286] (1) C-reactive protein (CRP): C-reactive protein (CRP) is a protein synthesized in the liver. Its level usually increases during systemic inflammation and tissue damage. The increase in CRP can be a non-specific response of the body to infection, inflammation, tissue damage, malignant tumors, etc.

[0287] In clinical practice, CRP is often used as an indicator to assess the degree of inflammation and monitor disease activity. In infectious diseases, CRP levels usually increase as inflammation persists, and gradually decrease after inflammation is relieved or treatment is effective. Therefore, CRP can help doctors assess the severity of infection, guide the formulation of treatment plans, and monitor the effectiveness of treatment.

[0288] It should be noted that although CRP is a commonly used inflammatory marker, it is not a highly specific indicator, because many other conditions can also lead to elevated CRP levels, such as autoimmune diseases, cardiovascular diseases, tumors, etc. Therefore, when using CRP for clinical judgment, it is necessary to conduct a comprehensive assessment based on the patient's clinical manifestations, other laboratory test results, imaging examinations and other comprehensive information.

[0289] (2) Procalcitonin (PCT): PCT is a precursor protein that is usually elevated in infection and inflammatory responses. In systemic inflammatory responses and infections, procalcitonin levels usually increase significantly. Procalcitonin levels are often used clinically to assess the severity of infection, guide decisions on antibiotic treatment, and monitor patient efficacy.

[0290] The increase of procalcitonin in infection and inflammation may appear earlier than other inflammatory markers, so it has certain value in early diagnosis and prognosis assessment. In clinical practice, procalcitonin is often used to distinguish bacterial infection from non-bacterial infection and guide the decision of antibiotic use. It is especially important in critically ill patients and is a specific indicator for diagnosing bacterial infection.

[0291] It should be noted that procalcitonin levels are affected by many factors, including infection type, infection site, and patient's immune status. Therefore, when using procalcitonin for clinical judgment, it is necessary to combine the patient's clinical manifestations, other laboratory test results, imaging examinations and other comprehensive information for evaluation and decision-making.

[0292] (3) CD64 infection index is a newly discovered specific indicator for bacterial infection in recent years. CD64 is a cell surface marker that is usually present on monocytes and neutrophils. CD64 expression increases significantly during bacterial infection and inflammation, so it is used as a potential marker for bacterial infection.

[0293] The CD64 infection index (CD64 index) is a method to assess the severity of infection by measuring the expression level of CD64 on the surface of monocytes or neutrophils. During the infection process, the expression level of CD64 on the surface of neutrophils usually increases significantly, so the increase in the CD64 infection index may indicate the presence and severity of bacterial infection.

[0294] The CD64 infection index is used clinically to help distinguish bacterial infections from non-bacterial infections and to assess the severity of infection. However, it should be noted that the CD64 infection index is not a tool for diagnosing infection alone, but requires a comprehensive assessment based on the patient's clinical manifestations, other laboratory test results, and imaging examinations. In addition, the CD64 infection index may have certain variability in different laboratories and studies, so it needs to be interpreted with caution in clinical applications.

[0295] (4) CD169: Also known as Siglec-1, it is a membrane glycoprotein belonging to the protein family Siglec. CD169 is mainly expressed on the surface of dendritic cells and macrophages, and specifically binds to the sugar structure of extracellular viral particles to promote viral phagocytosis and clearance. Therefore, CD169 plays an important role in infection detection. Studies have shown that after viral infection, the expression level of CD169 will increase, indicating that CD169 may be an early indicator of viral infection. In hepatitis virus and HIV infection, the expression of CD169 is related to the spread and clearance of the virus. Therefore, detecting the expression level of CD169 in viral infection can help understand the extent and severity of viral infection.

[0296] (5) HLA-DR: HLA-DR is an MHC II molecule in human leukocyte antigens, mainly expressed on the surface of antigen presenting cells (such as dendritic cells, macrophages, etc.), and participates in regulating T cell immune responses. In infectious diseases, the expression level of HLA-DR can change, mainly manifested in the following two situations:

[0297] Increase: Increased expression of HLA-DR usually occurs in the early stages of infection and is an immune response of the body to infection. During infection, immune cells release pro-inflammatory factors after being stimulated, which stimulate antigen-presenting cells to express HLA-DR, thereby inducing T cell immune responses. Increased HLA-DR can also be used as a biomarker to assess the early diagnosis and severity of infection.

[0298] Reduction: The reduction in HLA-DR expression often occurs in the late stages of infection and is an inhibitory response to the body's immune function. In the days or weeks after infection, immune cells are stimulated and activated for a long time, which can easily lead to abnormal function of antigen presenting cells and immune exhaustion. At this time, the expression of HLA-DR usually decreases, indicating that the body's immune response is suppressed. The reduction of HLA-DR can also be used as a biomarker to assess the state of immunosuppression after infection.

[0299] (6) Heparin-binding protein (HBP): It is a granule protein derived from neutrophils, mainly stored in azurophilic granules, and a small part is stored in secretory vesicles. HBP is a member of the serine protease family and has no protease activity. Its structure consists of a single-chain protein of 222 amino acids, containing 8 cysteine ​​residues and glycosylation sites at the 100th, 114th or 145th aspartic acid residues. Its structure is similar to that of neutrophil elastin, with a homology of 45%.

[0300] It has a high affinity for lipopolysaccharide lipid A. Neutrophils release HBP when they are activated and degranulate. When an acute bacterial infection occurs, the concentration of HBP in the blood can increase significantly within 1-2 hours, while HBP does not increase or increases slightly during viral infection. HBP is also a pathogenic factor. When the patient receives effective treatment, the HBP concentration drops rapidly. Therefore, detecting the HBP content in the patient's blood can assist in the diagnosis and prediction of acute bacterial infections, the determination of the severity of infections, and the monitoring of the efficacy of antibiotics. It is also an indicator for the specific diagnosis of bacterial infections.

[0301] (7) White blood cell count (WBC): White blood cells are part of the immune system and their number usually increases during an inflammatory response. An increase in the white blood cell count can reflect the presence and severity of inflammation.

[0302] (8) Neutrophil-to-Lymphocyte Ratio (NLR): NLR is the ratio of neutrophil count to lymphocyte count and can be used as an indicator of inflammatory response and prognosis. A high NLR is usually associated with a poor prognosis.

[0303] (9) Platelet count: Platelets also change during inflammatory responses. Low platelet counts may be associated with systemic inflammatory responses and increased risk of bleeding.

[0304] (10) Serum lactate level: Elevated lactate level may reflect tissue hypoxia and metabolic disorders, and is associated with severe infection and systemic inflammatory response.

[0305] The above indicators can be used as a reference for evaluating the prognosis of patients with systemic inflammatory response, but a comprehensive evaluation is required based on multiple information such as clinical manifestations and imaging examinations.

[0306] Specific markers for diagnosing bacterial inflammation include but are not limited to CD64, PCT, and HBP. The above markers are specific to bacterial inflammation (inflammatory response caused by bacterial infection). When an individual has bacterial inflammation, the measured values ​​of CD64, PCT, and HBP indicators rise. When an individual does not have inflammation or has non-bacterial inflammation (such as viral inflammation, inflammation caused by autoimmune disease), the measured values ​​of CD64, PCT, and HBP indicators will not rise or rise insignificantly; some patients with non-bacterial inflammation will also have an increase in CD64, PCT, and HBP indicators, and the reason is usually that as their non-bacterial inflammation develops, bacterial infection occurs. Therefore, in some ways, all specific indicators that can be used to diagnose bacterial infection can be combined with the markers of the present invention to diagnose and distinguish whether it is systemic inflammation caused by bacterial infection, such as distinguishing systemic inflammation caused by bacterial infection or viral infection, or distinguishing bacterial infection from non-bacterial infection (for example, systemic inflammation caused by viral infection or autoimmune disease, organ transplant rejection reaction and systemic inflammation caused by).

[0307] For example, refer to Fig.32 When using flow cytometry, we set the threshold of 0.8361. In patients with systemic infections caused by viruses, our markers can achieve a detection rate of 88.42%. Among these detection rates, the detection rate of specific bacterial indicators is very low, indicating that the indicators of bacterial infection, especially the specific indicators, are basically unchanged among these patients (PCT and nCD64) ( Fig.32 The same situation occurs with systemic inflammation caused by autoimmune diseases or organ transplant rejection ( Fig.33). On the contrary, in patients with bacterial systemic inflammation, the marker can reach a detection rate of 97.68%, and in these patients with systemic inflammation, bacterial-specific indicators have also increased significantly, at least relative to the threshold (PCT and nCD64), with detection rates of 93% and 98%, respectively.

[0308] When we used this theory to conduct a reverse clinical experiment, we randomly mixed and blindly tested 100 healthy people (local infection or healthy people), 100 patients with systemic inflammation caused by viral infection, 100 patients with systemic inflammation caused by autoimmune disease or organ transplant rejection, and 100 patients with systemic inflammation caused by bacterial infection, using the same threshold and flow cytometry for simultaneous testing. Fig.31 The various markers in the test, including the protein markers of the present invention, have obtained a high degree of complexity with the actual situation, distinguishing between healthy and systemic patients. 295 patients were determined to be systemic inflammation (test rate was 98%). Among these patients, 90 patients had elevated PCT and CD64 and other specific cells, indicating that 90 patients had systemic inflammation caused by bacterial infection, while the PCT and CD64 indicators of the other 205 patients did not change significantly. It can be understood that the markers of the present invention combined with some bacterial infection-specific markers can be used to distinguish between bacterial infection and non-bacterial infection in patients with systemic inflammation. Non-bacterial infection includes viral infection, autoimmune disease or systemic inflammation caused by organ transplantation. After the distinction is made, other clinical indicators or markers can be combined to distinguish between viral infection, autoimmune disease, or systemic inflammation caused by organ transplantation.

[0309] Therefore, the present invention discovered that nADGRE3 can be combined with markers for diagnosing bacterial inflammation to classify patients with systemic inflammation or diagnose lesions. When it is known that a patient's nADGRE3 is reduced, markers for diagnosing bacterial inflammation are detected. If the measured value is higher or lower than that of a healthy population, the patient is diagnosed with bacterial systemic inflammation; otherwise, the patient is diagnosed with non-bacterial systemic inflammation. The patient can be further classified (classified as viral systemic inflammation or non-infectious systemic inflammation) based on clinical information, medical history and other laboratory tests.

[0310] Diagnosis and testing

[0311] The "diagnosis or detection" of the present invention refers to the detection or analysis of biomarkers in a sample, or the content of a target biomarker, such as an absolute content or a relative content, and then the presence or amount of the target marker is used to indicate whether the individual providing the sample may have or suffer from a certain disease, or the possibility of having a certain disease. The meanings of diagnosis and detection here are interchangeable.

[0312] Relationship between markers or biomarkers and diseases: markers and biomarkers have the same meaning in the present invention. Relationship here means that the appearance or change in the content of a certain biomarker in a sample has a direct correlation with a specific disease, such as a relative increase or decrease in the content, indicating whether it is a local infection in an inflammatory patient, a healthy person, or a systemic inflammation, and classifying systemic inflammation and the causes of systemic inflammation, such as infectious systemic inflammation or non-infectious systemic inflammation.

[0313] Test reagents

[0314] Any reagent or method capable of testing ADGRE3 mRNA or protein can be applied to the present invention, such as nucleic acid amplification reagents, in situ hybridization, nucleic acid and protein fusion technology, gene editing technology, etc., which can be used to test the level or quantity of ADGRE3 gene expression. Reagents for protein testing include any reagent that can test the gene protein, such as antibody reagents based on immune principles, which can specifically bind to the protein or protein fragments. In some ways, the antibody specifically binds to the extramembrane protein or protein fragment of the protein, or the extramembrane amino acid sequence or peptide fragment. The test method can be a lateral flow reagent strip, or it can be directly tested based on liquid mass spectrometry, or it can be tested based on the principles of flow cytometry, ELISA, etc.

[0315] Antibody

[0316] The antibody used in the present invention can be any antibody that can specifically bind to the analyte in the sample. The antibody that can be used as a binding agent can be any antibody known to those skilled in the art. "Antibody" can be an immunoglobulin molecule and an antigen-binding portion of an immunoglobulin molecule, i.e., a molecule containing an antigen-binding site that specifically binds to an analyte, an analyte analog, or a ligand ("immunoreaction"). This term also includes derivatives of antibodies in which the binding ability is retained, and also includes any protein containing a binding domain that is homologous or largely homologous to the binding domain of an immunoglobulin. These proteins may be derived from natural substances or may be partially or completely synthesized. An antibody may be monoclonal or polyclonal. An antibody may be a member of any immunoglobulin type, including any human and mammalian immunoglobulin type: IgG, IgM, IgA, IgD, IgG, and IgE. "Antibody fragment" is a derivative of an antibody or a portion of an antibody that is less than the full length. Antibody fragments can retain at least one significant site of the binding ability of a full-length antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, scFv, Fv, dsFv dimer, and Fd fragments. Antibody fragments can be generated in any manner. For example, antibody fragments can be generated by enzymatic or chemical cleavage of an intact antibody, or by recombination from a gene encoding a portion of an antibody sequence. In other words, antibody fragments can be partially or completely recombinantly produced. Antibody fragments can be any single-chain antibody fragment. In other words, antibody fragments can contain multiple peptide chains linked to each other, for example, by disulfide bonds. Antibody fragments can also be any multimolecular complex. A functional antibody fragment generally contains at least about 50 amino acids, while more antibody fragments generally contain at least about 200 amino acids. Single-chain Fvs (scFvs) are recombinant antibody fragments that consist only of a variable light chain (VL) and a variable heavy chain (VH) covalently bound to each other as polypeptide chains. One of the VL and VH has an amino terminal region. The length and composition of the polypeptide chain are variable, and its length can bridge the two variable domains and has no serious effect on the arrangement of atoms. The polypeptide chain is usually mainly composed of glycine and serine residues, with some glutamic acid and lysine residues scattered to increase its solubility. "Dimer" refers to a dimer of single-chain Fvs. The monomers of the dimer usually contain shorter peptide chains than most single-chain Fvs, and they show a tendency to form dimers.

[0317] An "Fv" fragment consists of a VH and a VL domain non-covalently linked to each other. The term "dsFv" as used herein refers to an Fv that contains an intermolecular disulfide bond that stabilizes the VH-VL pair. A "F(ab')" fragment is a fragment of an antibody that is essentially the same as the fragment obtained by digesting an immunoglobulin (usually IgG) with pepsin at pH 4.0-4.5. This fragment can also be recombinantly synthesized. A "Fab'" fragment is an antibody fragment that is essentially the same as the fragment obtained by reducing the disulfide bonds that link the two heavy chains on a F(ab') fragment. A Fab' fragment can also be recombinantly synthesized. A "Fab" fragment is an antibody fragment that is essentially the same as the fragment obtained by digesting an immunoglobulin (usually IgG) with papain. A Fab fragment can also be recombinantly synthesized. The heavy chain fragment on the Fab fragment is the Fd fragment. DETAILED DESCRIPTION

[0318] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0319] Example 1 Discovery of ADGRE3 as a biomarker for diagnosing systemic inflammation

[0320] The present invention initially aimed to find genes that play an important role in the pathogenesis of sepsis, and analyzed and compared the differences in gene expression between sepsis patients and patients with non-infectious systemic inflammation in public databases, such as Figure 1 As shown in the results, the ADGRE3 gene was found to be significantly downregulated, and it was believed that this gene could be used as a marker to distinguish patients with sepsis from patients with non-infectious systemic inflammation. Later, the changes in gene expression profiles between sepsis patients and healthy controls were analyzed, such as Figure 2 As shown, it was found that the ADGRE3 gene was significantly downregulated in patients with sepsis, and the changes in ADGRE3 gene expression were extremely significant.

[0321] Then, we singled out the ADGRE3 gene and verified the changes in the expression of this gene in different infection types in the public data; the specific results are as follows Figure 3-Figure 11 shown.

[0322] For example, Figure 3As shown, we analyzed 16 healthy-control samples (from public database markers, healthy people in physical examinations), 23 samples from patients with systemic inflammation caused by bacterial infection, and 28 samples from patients with systemic inflammation caused by viral infection. We found that the expression of this gene in patients with systemic inflammation caused by bacterial or viral infection was significantly decreased compared with that in healthy controls, indicating that this gene can be used to distinguish health from systemic inflammation caused by viral or bacterial infection. The AUC values ​​are: the AUC value for distinguishing health from viral infection is 0.9063; the AUC value for distinguishing health from bacterial infection is 0.9402.

[0323] like Figure 4 As shown in the figure, we analyzed 33 healthy control samples, 18 samples of patients with systemic inflammation caused by Gram-positive bacteria infection, and 19 samples of patients with systemic inflammation caused by H1N1 virus infection, and found that the expression of this gene in patients with systemic inflammation caused by Gram-positive bacteria infection or H1N1 virus infection was significantly decreased compared with healthy controls, and the AUC value of differentiation was: 0.8215. Among them, the expression of ADGRE3 gene in patients with systemic inflammation caused by Gram-positive bacteria infection was significantly decreased compared with patients with systemic infection caused by H1N1 virus infection.

[0324] like Figure 5 As shown, we analyzed 26 healthy control samples and 93 samples of patients with systemic inflammation caused by dengue virus infection and found that the expression of this gene in patients with systemic inflammation caused by dengue virus infection was significantly decreased compared with healthy controls, and the AUC value for differentiation was 0.8156.

[0325] like Figure 6 As shown, we analyzed 18 healthy control samples, 18 samples of patients with systemic inflammation caused by asymptomatic new coronavirus infection, and 11 samples of patients with symptomatic infection that developed into systemic inflammation. We found that the expression of the gene was significantly decreased in patients with systemic inflammation caused by asymptomatic new coronavirus infection and patients with symptomatic infection that developed into systemic inflammation compared with healthy controls. The AUC value for distinguishing between health and new coronavirus infection was 0.9502. This shows that the expression of this gene can confirm health and systemic inflammation caused by new coronavirus infection regardless of whether there are symptoms. However, there is no significant difference between systemic inflammation caused by asymptomatic new coronavirus infection and systemic inflammation developed from symptomatic infection.

[0326] like Figure 7As shown in the figure, we analyzed 31 healthy control samples and 107 samples of patients with systemic inflammation caused by RSV (respiratory syncytial virus) virus infection and found that the expression of this gene was significantly decreased compared with the healthy controls, and the AUC value of differentiation was 0.8936. However, as treatment measures were taken, the expression of this gene returned to normal healthy levels in samples from RSV systemic inflammation to recovery. This shows that the expression of this gene can predict the effect of recovery, such as monitoring or testing the effect of using treatment measures to restore systemic inflammation.

[0327] like Figure 8 As shown in the figure, we analyzed 37 healthy control samples and 11 samples of patients with systemic inflammation caused by cytomegalovirus infection and found that the expression of this gene was significantly decreased compared with that of healthy controls, and the AUC value for differentiation was 0.9459.

[0328] like Fig. 9 As shown, we analyzed 6 healthy control samples and 11 samples of patients with systemic inflammation caused by Epstein-Barr virus infection and found that the expression of this gene was significantly decreased compared with that of healthy controls, and the AUC value for differentiation was 0.9091.

[0329] The above-mentioned H1N1, dengue fever, COVID, RSV, cytomegalovirus and Epstein-Barr virus infections are typical systemic inflammations caused by viral infections. It can be inferred that the expression of the ADGRE3 gene in patients with systemic inflammation caused by viral infections showed an extremely significant decrease, and the expression of the ADGRE3 gene can be used to distinguish healthy people from patients with systemic inflammation caused by viral infections.

[0330] like Fig.10 As shown, we analyzed 30 healthy control samples and 94 samples from patients with systemic inflammation caused by liver transplant rejection, and found that the expression of this gene in patients with systemic inflammation caused by liver transplant rejection showed a very significant decrease, and the AUC value for distinction was 0.7220.

[0331] For non-infectious systemic inflammation and sepsis, Fig.11 As shown in Figure 2, the expression of this gene is significantly downregulated between sepsis patients and healthy people, and is also significantly downregulated compared to non-infectious systemic inflammation. This indicates that this gene can be used to distinguish between healthy and sepsis people (its AUC value is 0.920, as shown in Figure 2). Fig.12 ) Alternatively, it can also be used as a marker to distinguish between non-infectious systemic inflammatory symptom (SIRS) and sepsis, with an AUC value of 0.864 (e.g. Fig.13 ).

[0332] The above results are obtained through analysis of public data. Through the actual clinical samples obtained in our hospital, we further verified whether the expression of this gene or the protein content can be used in the clinical diagnosis of systemic inflammation, as well as the treatment or prognosis of systemic inflammation, or the classification of systemic inflammation.

[0333] Example 2 Differences in ADGRE3 mRNA transcription levels between healthy subjects and patients with viral infection

[0334] ADGRE3 mRNA was specifically amplified by PCR to detect the transcription level of leukocyte ADGRE3 mRNA in the clinical samples (blood) of each patient. The specific method is as follows:

[0335] 1. RNA extraction from peripheral blood leukocytes

[0336] 1. Centrifuge the blood in the anticoagulant tube at 4°C, 3000rpm for 10min, and draw out the serum into a 1.5ml EP tube for storage (discard the serum if it is not needed);

[0337] 2. Add 3 ml PBS to the anticoagulant tube, centrifuge at 3000 rpm for 10 min at 4°C, and discard the supernatant, being careful not to touch the middle white cell layer;

[0338] 3. Prepare 1× red blood cell lysis buffer (5ml:50ml), add 4ml 1× red blood cell lysis buffer to the anticoagulant tube, and lyse for 15 minutes (10× red blood cell lysis buffer formula: 41.45g ammonium chloride and 42.00g sodium bicarbonate dissolved in 500mL sterile water, pH 7.2-7.4; Note: 10× red blood cell lysis buffer should be diluted with pure distilled water);

[0339] 4. After lysis, centrifuge at 4°C, 2500 rpm for 5 min, discard the supernatant, resuspend the leukocyte pellet in 1 ml PBS and transfer it to the 1.5 ml EP tube prepared in advance;

[0340] Centrifuge at 5.4°C, 2500 rpm for 5 min, discard the supernatant with a pipette, and add 1 ml of TRIZOL (TriQuick Reagent total RNA extraction reagent, Beijing Solebow Technology Co., Ltd., China).

[0341] 6. Add 200 μL of chloroform to a 1.5 ml EP tube, mix by inversion and shake vigorously for 10-15 seconds, and let stand at room temperature for 5 minutes;

[0342] 7.4℃, centrifuge at 120000rpm for 15min (the centrifuge was precooled at 4℃ in advance);

[0343] 8. Gently pipette the top aqueous phase (the middle layer is white precipitate, the bottom layer is red organic layer) into a new 1.5mL EP tube without RNAse, add an equal volume of isopropanol, mix by inverting, and let stand at room temperature for 5 minutes;

[0344] 9.4℃, centrifuge at 120000rpm for 30min (the centrifuge was precooled at 4℃ in advance);

[0345] 10. Aspirate and discard the supernatant, add 1 mL of 75% ethanol, and mix well (75% ethanol should be prepared and used immediately, and RNase-free water should be used to dilute anhydrous ethanol);

[0346] 11.4°C, 120,000 rpm, centrifuge for 10 min;

[0347] 12. Aspirate and discard the supernatant, dry at room temperature for 5-10 minutes until the white precipitate becomes transparent, and then add an appropriate amount of DEPC water (20-50 μL) to dissolve the RNA precipitate;

[0348] 2. Reverse Transcription

[0349] 1. Perform reverse transcription according to the instructions of the HiFiScript cDNA Synthesis Kit (CW2569M, Kangwei Century Biotechnology Co., Ltd., China). Dissolve the relevant reagents on ice in advance, take 500ng of the RNA extracted in the previous step and prepare the reaction system according to the following reverse transcription system, with a total reaction volume of 20μL; the reverse transcription reaction system is shown in Table 1;

[0350] Table 1: Reverse transcription reaction system

[0351]

[0352]

[0353] 2. Briefly centrifuge to mix the reaction system, and perform reverse transcription reaction on a common PCR instrument at 42°C for 15 min and 85°C for 5 min;

[0354] 3. After the reaction is completed, take out the product, centrifuge briefly, and cool it on ice;

[0355] 3. qPCR

[0356] 1. Perform qPCR reaction according to the instructions of NovoStart SYBR qPCR SuperMix Plus Kit (E096-01B, Shanghai Jinan Technology Co., Ltd., China). Dissolve the template cDNA, primers and related reagents on ice in advance, prepare the reaction system according to the following reverse transcription system, and the total reaction volume is 20 μL; the qPCR reaction system is shown in Table 2;

[0357] Table 2: qPCR reaction system

[0358] Reagents volume 2x Ultra SYBR mix 10μL Forward Primer,10 Reversal 0.5μL Reverse Primer, 10 Reversals 0.5μL cDNA 9μL

[0359] 2. Briefly centrifuge to mix the reaction system, and perform the reaction in a fluorescent quantitative PCR instrument. The reaction conditions are: pre-deformation at 95℃ for 1min, then denaturation at 95℃ for 20s, extension at 60℃ for 1min, for a total of 40 cycles. The melting curve parameters are: 95℃ for 15s, 60℃ for 1min, 95℃ for 15s, and 60℃ for 15s.

[0360] 3. Result analysis: The mRNA expression of β-Actin / GAPDH was used as the standard control, and the mRNA expression level of the target gene was calculated using the 2-ΔΔCt method. ADGRE3 (EMR3) fluorescence quantitative PCR primers:

[0361] Forward primer (5'->3'): TCCAGCACGTGAAGATGACC

[0362] Reverse primer (5'->3'): ACGTGTATCAGGAAGCAGCC

[0363] The ADGRE3 mRNA transcription levels in leukocytes of healthy subjects (20 cases) (the “healthy subjects” here and below were selected by our hospital based on the standard that all physical examination items were normal), patients with systemic infection of influenza A and B viruses (39 patients with systemic inflammation), and patients with systemic infection of the new coronavirus (20 patients with systemic inflammation) were measured. The results are as follows Fig.14 As shown, there are extremely significant differences in the leukocyte ADGRE3 mRNA transcription levels between patients with systemic infection with influenza A and B viruses and patients with systemic infection with the new coronavirus and healthy controls, further suggesting that ADGRE3 mRNA can be used as a biomarker for diagnosing infection and for diagnosing systemic inflammation caused by viral infection.

[0364] Example 3 ADGRE3 mRNA transcript levels in healthy subjects and non-infectious systemic inflammatory response syndrome patients The Difference

[0365] Non-infectious systemic inflammatory response syndrome patients were tested, and the leukocyte ADGRE3 mRNA transcription levels of healthy subjects (15 cases), patients with systemic inflammation caused by systemic lupus erythematosus (27 cases), patients with systemic inflammation caused by rheumatoid arthritis (25 cases), patients with systemic inflammation caused by vasculitis (10 cases), and patients with systemic inflammation caused by ankylosing spondylitis (15 cases) were tested and compared. Fig.15As shown, the ADGRE3 mRNA transcription levels in leukocytes of healthy people are significantly different from those of patients with systemic inflammatory syndrome of the above four autoimmune diseases, suggesting that this marker can be used to distinguish healthy people from patients with non-infectious systemic inflammatory response syndrome.

[0366] Example 4 Differences in ADGRE3 mRNA transcription levels between healthy subjects and patients with sepsis

[0367] The ADGRE3 mRNA transcription levels in leukocytes of healthy subjects (20 cases) and patients with sepsis (96 patients with systemic inflammation) were tested and compared. Fig.16 As shown, it is proved again that there is a significant difference between the leukocyte ADGRE3 mRNA transcription level of healthy people and sepsis patients. This marker can be used to distinguish healthy people from sepsis patients.

[0368] Example 5 ADGRE3 protein expression levels in healthy subjects and patients with systemic inflammation caused by viral infection The Difference

[0369] The experiments in Examples 2 to 4 show that the leukocyte ADGRE3 mRNA transcription level can diagnose systemic inflammation, including viral infection, non-infectious systemic inflammatory response syndrome, and sepsis; this example further studies the ADGRE3 protein expression level, and the method for detecting the neutrophil ADGRE3 protein expression level is as follows:

[0370] Take a sample containing 1×10 6 Add 2ul of mouse anti-human ADGRE3 antibody labeled with different fluorescent dyes to the EDTA or sodium citrate anticoagulated peripheral blood of leukocytes, shake and mix, incubate in the dark for 20 minutes at room temperature, add 2ml of red blood cell lysis liquid and shake and mix, incubate in the dark for 15 minutes. Centrifuge at 2000rpm for 3 minutes, remove the supernatant and resuspend the cells, add 2ml phosphate buffer or saline and centrifuge at 2000rpm for 3 minutes, discard the supernatant and add phosphate buffer to resuspend the cell pellet, and analyze it on a flow cytometer (Beckman DxFLEX). During the flow cytometric analysis, neutrophils, monocytes and lymphocytes were gated according to the differences in FSC and SSC, and the average fluorescence intensity of neutrophil ADGRE3 was calculated. The specific results are as follows:

[0371] The ADGRE3 protein expression levels of neutrophils in healthy subjects (26 cases), patients infected with influenza A and B viruses (89 cases with systemic inflammation), patients infected with the new coronavirus (22 cases with systemic inflammation), patients with dengue fever (5 cases with systemic inflammation), and patients infected with the new bunyavirus (5 cases with systemic inflammation) were detected. The results are as follows Fig.17As shown, the expression level of ADGRE3 protein in neutrophils of healthy people is significantly different from that in patients infected with the above viruses, and the expression level of ADGRE3 protein in neutrophils decreases significantly after infection with each virus. This suggests that the expression level of ADGRE3 protein in neutrophils can distinguish healthy people from patients with systemic inflammation caused by viral infection.

[0372] The expression of ADGRE3 in neutrophils of healthy non-inflammatory population was detected by flow cytometry. The mean fluorescence intensity (MFI) of ADGRE3 in each sample was calculated and the average value of the mean fluorescence intensity (MFI) of ADGRE3 in healthy population was obtained. The mean fluorescence intensity (MFI) of ADGRE3 in patient neutrophils was detected under the same conditions. The mean fluorescence intensity (MFI) of ADGRE3 in patient neutrophils was divided by the average value of the mean fluorescence intensity (MFI) of ADGRE3 in healthy population to obtain the patient neutrophil ADGRE3 expression index (nADGRE3), i.e., nADGRE3 = patient neutrophil ADGRE3 MFI / healthy control neutrophil ADGRE3 MFI.

[0373] The ratio of different mean fluorescence intensity values ​​was taken as the threshold. The sample with nADGRE3 greater than the threshold was diagnosed as a healthy person, and less than the threshold was diagnosed as a patient with systemic inflammation caused by viral infection (including systemic inflammation caused by influenza A and B virus infection, new coronavirus infection, dengue fever and new bunyavirus infection). The sensitivity and specificity (sensitivity = number of true positives / (number of true positives + number of false negatives)*100%; specificity = number of true negatives / (number of true negatives + number of false positives))*100%) are shown in Table 3. The ROC curve is drawn as shown in Table 3. Fig.18 As shown, the calculated AUC value was 0.9654, indicating that the ADGRE3 protein expression level can diagnose systemic inflammation caused by viral infection.

[0374] Table 3: Neutrophil ADGRE3 protein expression level and diagnosis of viral infection and systemic inflammation

[0375] nADGRE3 expression level cutoff Sensitivity (%) Specificity (%) 0.7559 75.51 96.15 0.7733 80.00 96.15 0.7912 84.29 96.15 0.8072 87.14 96.15 0.8092 87.14 92.31 0.8209 88.57 88.46 0.8361 91.43 88.46

[0376] Based on the above results, the expression level of ADGRE3 protein in neutrophils of healthy people (26 cases), patients with mild viral infection (36 cases with systemic inflammation), severe patients (20 cases with systemic inflammation) and critically ill patients (12 cases with systemic inflammation) was further tested. Fig.19As shown, there are extremely significant differences in the expression levels of neutrophil ADGRE3 protein in healthy people, patients with mild viral infection, severe patients and critical patients. As the severity of the disease increases, the expression level of neutrophil ADGRE3 protein decreases, suggesting that the expression level of ADGRE3 protein can be used as a basis for classifying patients with systemic inflammation caused by viral infection according to the severity of their condition.

[0377] This also shows that for some patients, although they all have systemic inflammation, the severity is different. The ADGRE3 protein expression level of the present invention can also be used to divide patients with systemic inflammation into different severity levels, which is conducive to taking appropriate treatment measures in time. Of course, it can also be used as a monitoring marker for changes in severity to monitor or warn of the disease.

[0378] Example 6 Differences in ADGRE3 protein expression levels between healthy subjects and patients with non-infectious systemic inflammation

[0379] To further investigate the differences in ADGRE3 protein expression levels between healthy people and patients with non-infectious systemic inflammation, the ADGRE3 protein expression levels of neutrophils in patients with autoimmune diseases were studied. The ADGRE3 protein expression levels of neutrophils in healthy people (20 cases), patients with systemic inflammation caused by systemic lupus erythematosus (27 cases, including 14 patients in the stable phase and 13 patients in the active phase), patients with systemic inflammation caused by rheumatoid arthritis (31 cases, including 15 patients in the stable phase and 16 patients in the active phase), patients with systemic inflammation caused by vasculitis (12 cases), patients with systemic inflammation caused by ankylosing spondylitis (16 cases), and patients with systemic inflammation caused by autoimmune hepatitis (6 cases) were detected. The results are as follows: Fig. 20 As shown in the figure, except for patients with stable rheumatoid arthritis, there are extremely significant differences in the expression levels of neutrophil ADGRE3 protein between healthy people and the above-mentioned other systemic inflammatory patients, suggesting that the expression level of ADGRE3 protein can diagnose autoimmune diseases.

[0380] It is worth noting that among the above-mentioned patients with systemic inflammation, there were extremely significant differences in the expression levels of neutrophil ADGRE3 protein between patients in the stable phase and those in the active phase of systemic lupus erythematosus and rheumatoid arthritis, suggesting that the expression level of ADGRE3 protein can diagnose autoimmune diseases and that ADGRE3 can be used to distinguish between the stable and active phases of systemic lupus erythematosus and rheumatoid arthritis.

[0381] Non-infectious systemic inflammation also includes systemic inflammation caused by transplant organ rejection. Healthy people (16 cases), patients with systemic inflammation caused by kidney transplant rejection (6 cases) and patients with systemic inflammation caused by bone marrow transplant rejection (8 cases) were selected to measure the expression level of neutrophil ADGRE3 protein. Fig.21 As shown, the expression level of neutrophil ADGRE3 protein in patients with systemic inflammation caused by kidney transplantation and bone marrow transplantation rejection was significantly lower than that in healthy people, suggesting that the expression level of neutrophil ADGRE3 protein can distinguish healthy people from patients with systemic inflammation caused by transplant organ rejection.

[0382] For other types of non-infectious systemic inflammation, due to the similar types of inflammation, the expression level of neutrophil ADGRE3 protein is theoretically similar to the systemic inflammation caused by the above-mentioned autoimmune diseases and transplant organ rejection. Therefore, the expression level of neutrophil ADGRE3 protein can distinguish between healthy people and non-infectious systemic inflammation.

[0383] The nADGRE3 of the sample was calculated according to the method of Example 5, and the ratio of different average fluorescence intensity values ​​was intercepted as the threshold. The sample with nADGRE3 greater than the threshold was diagnosed as a healthy person, and less than the threshold was diagnosed as a patient with non-infectious systemic inflammation. The sensitivity and specificity are shown in Table 4. The ROC curve is plotted as shown in Table 4. Fig. 22 As shown, the calculated AUC value was 0.9145, indicating that the ADGRE3 protein expression level can diagnose patients with non-infectious systemic inflammation.

[0384] Table 4: Neutrophil ADGRE3 protein expression levels and diagnosis of patients with non-infectious systemic inflammation

[0385] nADGRE3 expression level cutoff Sensitivity (%) Specificity (%) 0.8363 73.68 96.15 0.8555 75.00 92.31 0.8887 78.95 92.31 0.8983 81.58 92.31 0.9129 82.89 92.31 0.9362 84.21 88.46 0.9429 85.53 84.62

[0386] Example 7 Differences in ADGRE3 protein expression levels between healthy subjects and sepsis patients

[0387] The expression level of ADGRE3 protein in neutrophils of patients with sepsis was studied. The results of the determination of ADGRE3 protein expression in neutrophils of healthy subjects (23 cases) and patients with sepsis (97 cases) were as follows: Fig.23 As shown, there is a very significant difference in the expression level of ADGRE3 protein in neutrophils between healthy people and patients with sepsis, suggesting that ADGRE3 can be used to diagnose sepsis.

[0388] The nADGRE3 of the sample was calculated according to the method of Example 5, and the ratio of different average fluorescence intensity values ​​was intercepted as the threshold. The sample with nADGRE3 greater than the threshold was diagnosed as a healthy person, and less than the threshold was diagnosed as a sepsis patient. The sensitivity and specificity are shown in Table 5. The ROC curve is drawn as shown in Table 5. Fig.24As shown, the calculated AUC value was 0.9973, indicating that the ADGRE3 protein expression level can diagnose sepsis.

[0389] Table 5: Neutrophil ADGRE3 protein expression level and diagnosis of sepsis

[0390]

[0391]

[0392] Healthy subjects (23 cases), patients with sepsis caused by Gram-positive bacteria (33 cases), patients with sepsis caused by Gram-negative bacteria (44 cases), and patients with sepsis caused by fungal infections (10 cases) were selected to detect the expression level of ADGRE3 protein in neutrophils. The results are as follows: Fig.25 As shown, the expression levels of ADGRE3 protein in neutrophils of healthy subjects were significantly different from those of patients with the above three types of sepsis, suggesting that ADGRE3 can be used to diagnose different types of sepsis.

[0393] To further investigate the correlation between ADGRE3 protein expression and the severity of sepsis, the ADGRE3 protein expression in neutrophils was detected in healthy subjects (23 cases), sepsis patients (48 cases), and septic shock patients (19 cases). Fig.26 As shown, there are extremely significant differences in the expression levels of neutrophil ADGRE3 protein among healthy people, sepsis patients, and septic shock patients, and as the severity of sepsis increases, the expression level of neutrophil ADGRE3 protein decreases, suggesting that the expression level of neutrophil ADGRE3 protein can be used to divide sepsis patients according to the severity of the disease.

[0394] According to the survival and death of sepsis patients, the expression level of neutrophil ADGRE3 protein in sepsis survivors (n=50) and sepsis deaths (n=18) during the illness period was retrospectively analyzed. Fig. 27 As shown, there was a very significant difference in the expression level of neutrophil ADGRE3 protein between sepsis survivors and patients who died of sepsis, suggesting that the expression level of neutrophil ADGRE3 protein can also be used to predict the risk of death in sepsis patients.

[0395] The nADGRE3 of the sample was calculated in the manner of Example 5, and the ratio of different mean fluorescence intensity values ​​was taken as the threshold. When the nADGRE3 of the sample was greater than the threshold, it was predicted that the death risk of the sepsis patient was low, and when it was less than the threshold, it was predicted that the death risk of the sepsis patient was high. The predicted death risk was compared with the actual sepsis survivors and deaths, and the sensitivity and specificity were calculated as shown in Table 6. The ROC curve was drawn as shown in Table 6. Fig.28As shown, the calculated AUC value was 0.8374, indicating that the expression level of ADGRE3 protein can predict the risk of death in patients with sepsis.

[0396] Table 6: Neutrophil ADGRE3 protein expression level and risk of death in sepsis patients

[0397] nADGRE3 expression level cutoff Sensitivity (%) Specificity (%) 0.2596 61.11 88.89 0.2822 72.22 85.19 0.2943 72.22 74.07 0.3185 77.78 74.07 0.3605 83.33 70.37 0.4570 88.89 59.26

[0398] According to the improvement, no improvement (still no improvement on the 7th day) or death of sepsis patients, the expression level of neutrophil ADGRE3 protein in sepsis patients on each day after diagnosis was retrospectively analyzed. The day of diagnosis was recorded as day 0. The results are as follows: Fig.29 As shown, the neutrophil ADGRE3 protein expression level of the patient group with improved sepsis is always higher than that of the patient group that has not improved or died, and the neutrophil ADGRE3 protein expression level of the patient group with improved sepsis has increased significantly over time, especially from the 3rd day to the 5th day, while the neutrophil ADGRE3 protein expression level of the patient group that has not improved or died shows a downward trend during this period. Therefore, the neutrophil ADGRE3 protein expression level can be used to predict whether sepsis will improve, especially the change of ADGRE3 protein expression from the 3rd day to the 5th day can be used to predict whether the condition of sepsis patients will improve in the future.

[0399] Example 8 The expression level of ADGRE3 protein in neutrophils of healthy people and patients without systemic inflammation is not There is a significant difference

[0400] Example 1 By analyzing public data, ADGRE3 gene expression has high specificity in diagnosing systemic inflammation. Therefore, there should be no significant difference in the ADGRE3 protein content between healthy people and patients with non-systemic inflammation. To verify this, the neutrophil ADGRE3 protein expression level of healthy people (15 cases) and patients with local inflammation (including 10 hepatitis B patients, 6 hepatitis C patients, and 7 hepatitis E patients) was measured. The results are as follows: Fig.30 As shown, there was no significant difference in the expression level of ADGRE3 protein in neutrophils between healthy people and patients with local inflammation.

[0401] Therefore, the neutrophil ADGRE3 protein expression level can be used to diagnose systemic inflammation without being interfered by local inflammation, verifying its high specificity.

[0402] Example 9 Neutrophil ADGRE3 protein combined with CD64, PCT and CRP in differentiating inflammation types application

[0403] PCT (procalcitonin), CRP (C-reactive protein), IL-6 and the expression of CD64 infection index (nCD64 infection index) of peripheral blood neutrophils are currently commonly used clinical biomarkers for bacterial infection and inflammatory response. In this study, flow cytometry was used to simultaneously detect the mean fluorescence intensity (MFI) of ADGRE3 in peripheral blood neutrophils of healthy controls (n=42), patients with systemic inflammatory response to known infectious pathogens (viruses, bacteria and fungi) (n=218), patients with autoimmune diseases (n=92) and patients with transplant rejection (n=14). The mean fluorescence intensity (MFI) of ADGRE3 in peripheral blood neutrophils of healthy controls was used as a control to calculate the relative expression of ADGRE3 in peripheral blood neutrophils of patients with systemic inflammatory response (nADGRE3), that is, nADGRE3 = (MFI of neutrophils ADGRE3 in patients) / (MFI of neutrophils ADGRE3 in healthy controls). MFI), the optimal cutoff value of nADGRE3 was set as 0.8361 through statistical analysis, and nADGRE3<0.8907 was determined as a systemic inflammatory response; at the same time, the detection data of serum PCT, CRP, IL-6 and nCD64 infection index of patients were collected and improved, and the changes of nADGRE3, nCD64, PCT, CRP and IL-6 in viral infection, sepsis and non-infectious systemic inflammatory response were statistically compared; the changes of nADGRE3, nCD64, PCT, CRP and IL-6 and the corresponding inflammation types (systemic inflammatory response of viral infection, sepsis and non-infectious systemic inflammatory response including autoimmune diseases and transplant rejection) are shown in Table 7.

[0404] Table 7: Application of neutrophil nADGRE3 combined index in distinguishing inflammation types

[0405]

[0406]

[0407] We compared neutrophil nADGRE3 with existing clinical inflammation indicators and found that this indicator has a very high sensitivity for systemic inflammation. More patients with systemic inflammation have reduced neutrophil nADGRE3, so more patients with systemic inflammation can be diagnosed based on laboratory test results of reduced neutrophil nADGRE3. However, the existing clinical inflammation indicators have low sensitivity in patients with viral infection systemic inflammation and non-infectious systemic inflammation, and it is very likely that normal test results will lead to misdiagnosis. Therefore, the sensitivity of neutrophil nADGRE3 for systemic inflammation exceeds that of existing clinical indicators, which shows that neutrophil nADGRE3 has a high sensitivity in diagnosing systemic inflammation, so the accuracy of diagnosis is higher.

[0408] At the same time, when diagnosing systemic inflammation, the existing clinical inflammation indicators are not specific and cannot rule out the interference of local inflammation. Therefore, it is often necessary to combine imaging for comprehensive diagnosis. For example, a patient has local inflammation in multiple places, but it does not actually fall into the category of systemic inflammation (this is often the case with patients with autoimmune diseases). If imaging indicates inflammation in multiple places, the patient may be misclassified as having systemic inflammation when combined with existing clinical inflammation indicators. However, neutrophil nADGRE3 has a high enough specificity that it can be diagnosed without combining imaging. When neutrophil nADGRE3 decreases, the patient is specifically diagnosed with systemic inflammation, which can rule out the interference of local inflammation. Therefore, compared with existing clinical inflammation indicators, neutrophil nADGRE3 has a higher specificity for diagnosing systemic inflammation, so the accuracy of diagnosis is higher.

[0409] Patients with systemic inflammation were classified according to the etiology into patients with systemic inflammatory response caused by bacterial infection, patients with systemic inflammatory response caused by viral infection, and patients with systemic inflammatory response caused by autoimmune diseases and transplant rejection. They were classified according to nADGRE3 and other inflammatory indicators (CRP, PCT, IL-6, and nCD64), respectively. Fig.31 , Fig.32 and Fig.33 As shown;

[0410] Fig.31 The results showed that among patients with systemic inflammatory response caused by bacterial infection, the proportion of patients with decreased ADGRE3 protein expression level was 96.51%, among patients with decreased ADGRE3 protein expression level, the proportion of patients with increased CRP was 92.86%, the proportion of patients with increased PCT was 93.02%, the proportion of patients with increased IL-6 was 96.42%, and the proportion of patients with increased nCD64 was 98.81%;

[0411] Fig.32 The results showed that among patients with systemic inflammatory response caused by viral infection (a type of non-bacterial systemic inflammatory response), the proportion of patients with decreased ADGRE3 protein expression level was 88.42%, among patients with decreased ADGRE3 protein expression level, the proportion of patients with increased CRP was 48.59%, the proportion of patients with increased PCT was 0.93% (only 1 case), the proportion of patients with increased IL-6 was 64.48%, and the proportion of patients with increased nCD64 was 8.41% (only 9 cases);

[0412] Fig.33It showed that among patients with systemic inflammatory reactions caused by autoimmune diseases and transplant rejection (a type of non-bacterial systemic inflammatory patients), the proportion of patients with decreased ADGRE3 protein expression levels was 79.25%, and some patients took immunosuppressive drugs or hormones, which led to increased ADGRE3 protein expression levels and were classified as normal; among patients with decreased ADGRE3 protein expression levels, the proportion of patients with increased CRP was 61.90%, the proportion of patients with increased PCT was 1.19% (only 1 case), the proportion of patients with increased IL-6 was 53.57%, and the proportion of patients with increased nCD64 was 8.33% (only 7 cases);

[0413] In patients with systemic inflammatory responses caused by viral infection, autoimmune disease, and transplant rejection, the proportion of elevated PCT and nCD64 in those patients with decreased ADGRE3 protein expression levels was extremely low. This is because PCT and nCD64 are markers for diagnosing bacterial inflammation, and are specific for the diagnosis of bacterial systemic inflammation in systemic inflammation. Individual cases of elevated PCT and nCD64 were accompanied by bacterial infection. In addition, markers for diagnosing bacterial inflammation also include HBP (heparin-binding protein), etc.

[0414] Therefore, the neutrophil ADGRE3 protein expression level can be combined with the markers for diagnosing bacterial inflammation to perform classification diagnosis or lesion diagnosis on patients with systemic inflammation, and distinguish bacterial systemic inflammation from non-bacterial systemic inflammation; specifically: the ADGRE3 protein expression level and the markers for diagnosing bacterial inflammation are detected. If the ADGRE3 protein expression level decreases and the content of the markers for diagnosing bacterial inflammation changes at the same time, bacterial systemic inflammation is diagnosed; if the ADGRE3 protein expression level decreases and the content of the markers for diagnosing bacterial inflammation is normal, non-bacterial systemic inflammation is diagnosed.

[0415] "Change" in the content of markers for diagnosing bacterial inflammation, that is, the detection value of the marker does not fall within the detection value range of healthy people, includes both high and low situations, both of which indicate the occurrence of bacterial inflammation; "normal" content of markers for diagnosing bacterial inflammation, that is, the detection value of the marker is within the detection value range of healthy people, indicating that bacterial inflammation has not occurred.

[0416] Fig.34A flowchart for diagnosing whether patients suspected of systemic inflammatory reaction have systemic inflammation, and for classifying patients with systemic inflammation into bacterial systemic inflammation or non-bacterial systemic inflammation. First, the nADGRE3 of the patient's neutrophils is detected and calculated, and compared with the threshold value of 0.8361. If it is greater than the threshold, the systemic inflammatory reaction is excluded, and if it is less than the threshold, it is diagnosed as a patient with systemic inflammation. Then, the nCD64, PCT, HBP and other indicators for diagnosing bacterial systemic inflammation are tested for the confirmed patients with systemic inflammation. If they are elevated, it is considered that the patient has systemic inflammation caused by bacterial infection or systemic inflammation accompanied by bacterial infection. If the indicator test value is normal, viral infection or non-infectious systemic inflammation is considered, and further confirmation can be combined with the patient's clinical information and other laboratory tests. For example, if the patient is found to have a history of organ transplantation, it is considered whether it is systemic inflammation caused by rejection of the transplanted organ.

[0417] All patents and publications mentioned in the present specification indicate that these are public technologies in the field and can be used in the present invention. All patents and publications cited here are also listed in the references, just as each publication is specifically cited separately. The present invention described here can be implemented in the absence of any one or more elements, one or more restrictions, and such restrictions are not specifically stated here. For example, in each example here, the terms "comprising", "substantially consisting of..." and "consisting of..." can be replaced by the other two terms of one of the two. The so-called "one" here only means "one", but does not exclude that it only includes one, and can also mean that it includes more than two. The terms and expressions used here are descriptive and not limited by them. There is no intention to indicate that these terms and explanations described in this book exclude any equivalent features, but it can be known that any appropriate changes or modifications can be made within the scope of the present invention and the claims. It can be understood that the embodiments described in the present invention are some preferred embodiments and features, and any person skilled in the art can make some changes and variations based on the essence of the present invention, and these changes and variations are also considered to belong to the scope of the present invention and the scope limited by the independent claims and the appended claims.

Claims

1. Use of a reagent for detecting the expression level of ADGRE3 protein in a sample in the preparation of a reagent for diagnosing systemic inflammation.

2. The use according to claim 1, characterized in that The diagnosis is whether or not systemic inflammation is present or the extent of systemic inflammation.

3. The use according to claim 1, characterized in that The reagent for diagnosing systemic inflammation is used to detect ADGRE3 protein in an individual sample.

4. The use according to claim 3, characterized in that The sample includes any one or more of peripheral blood, urine, pus, body fluid, spinal cord, and fresh tissue.

5. The use according to claim 1, characterized in that The systemic inflammation includes any one or more of bacterial infection, viral infection, and fungal infection.

6. The use according to claim 5, characterized in that The viral infection includes any one or more of influenza virus infection, new coronavirus infection, dengue virus infection, new bunyavirus infection, respiratory syncytial virus infection, cytomegalovirus and Epstein-Barr virus.

7. The use according to claim 1, characterized in that The systemic inflammation includes any one or more of systemic inflammation caused by autoimmune diseases, transplant organ rejection, and tissue damage.

8. The use according to claim 7, characterized in that The autoimmune diseases include any one or more of systemic lupus erythematosus, rheumatoid arthritis, vasculitis, ankylosing spondylitis, and autoimmune hepatitis.

9. The use according to claim 1, characterized in that The systemic inflammation includes sepsis.

10. The use according to claim 9, characterized in that The sepsis is caused by any one or more of lung infection, biliary tract infection, urinary tract infection, digestive tract infection, bloodstream infection, reproductive tract infection, abdominal cavity infection, central nervous system infection, skin and soft tissue infection, and suppurative osteomyelitis.

11. The use according to claim 9, characterized in that The sepsis includes any one or more of gram-positive bacteria infection sepsis, gram-negative bacteria infection sepsis, fungal infection sepsis, and viral infection sepsis.

12. Use of a reagent for detecting the expression level of ADGRE3 protein in a sample in the preparation of a reagent for distinguishing patients with systemic inflammation caused by viral infection into mild patients, severe patients, and critical patients.

13. Use of a reagent for detecting the expression level of ADGRE3 protein in a sample in the preparation of a reagent for diagnosing whether a sepsis patient is a septic shock patient.

14. Use of a reagent for detecting the expression level of ADGRE3 protein in a sample in the preparation of a reagent for predicting the risk of death in sepsis patients.

15. The use according to claim 14, characterized in that The use also includes: if the ADGRE3 protein level in the sample on the 5th day after the individual is diagnosed with the disease is higher than the ADGRE3 protein level in the sample on the 3rd day, it is predicted that the individual's sepsis will be improved; otherwise, it is predicted that the individual's sepsis will not be improved or the individual will die.

16. The use according to any one of claims 1 to 15, characterized in that The ADGRE3 protein is obtained by translating any one of the mRNAs selected from XM_054322398.1, XM_047439546.1, XM_011528374.3, NM_001289158.2, NM_001289159.2, and NM_032571.

5.

17. The use according to any one of claims 1 to 15, wherein the agent comprises an antibody.

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