CN112391460A - Biomarker group for sepsis, sepsis judgment method and kit - Google Patents

Abstract

The invention discloses a biomarker group for sepsis, a sepsis judgment method and a sepsis judgment kit, wherein the biomarker group consists of new biomarkers different from the existing RNA markers, and when sepsis diagnosis is carried out, the score of the expression level of the biomarker group of a tested person is judged through an established biomarker system of the biomarker group, so that sepsis patients are identified and layered, and clues are provided for solving the main pathophysiological mechanism and the potential targeted treatment way of the single patient.

Description

Biomarker group for sepsis, sepsis judgment method and kit

Technical Field

The invention relates to a biomarker group for sepsis, a sepsis judgment method and a sepsis kit, in particular to a combination of biomarkers for assisting sepsis diagnosis or treatment, a sepsis judgment method using the biomarker group and a corresponding kit, and belongs to the technical field of biological medical treatment.

Background

Sepsis is defined as the presence of Systemic Inflammatory Response Syndrome (SIRS) with a known or suspected source of infection. However, from the clinical manifestations, SIRS is not completely sepsis, but is also aseptic inflammation that occurs with non-specific reactions of other non-infectious lesions such as trauma, surgery, and thrombosis, but it is difficult to clinically distinguish systemic inflammation caused by non-infectious sources, such as tissue trauma. A large body of data shows that severe sepsis and shock are one of the leading causes of death worldwide, with over 1500 million deaths per year, and that sepsis is a dysregulated immune response due to invasive infection of host tissues and organs by pathogenic microorganisms, so that in clinical work, timely diagnosis and treatment of sepsis is critical to reducing mortality, increasing the risk of death once delayed. Currently, clinical diagnostic methods for sepsis, such as microbiological culture assays, are known, although the gold standard for determining the cause of infection, only 30% to 40% of the blood cultures are positive in sepsis cases, while in one third of the (clinically defined) cases sepsis is confirmed, false negative, and furthermore, since infection can be caused by multiple pathogens, the presence of the pathogen does not prove the presence of the disease. Therefore, there is an urgent need to develop a highly effective identification method that can be used to distinguish sepsis from inflammation of non-infectious origin.

With the rapid development of molecular biology, methods of using biomarkers for auxiliary diagnosis in the fields of tumors, cancers, and the like are becoming common, but in contrast, the use of biomarkers in the treatment of sepsis is still in the beginning. It is known that clinical use of biomarkers for diagnosis can reduce the inappropriate use of antibiotics and can be used in the management of antibiotics. Therefore, the development of biomarkers indicative of sepsis infection and prognosis to help predict risk profiles for sepsis patients has been an urgent need. Approximately 100 clinical trials are currently recruiting patients to study biomarkers of sepsis. To date, no sufficiently reliable biomarker has been found to diagnose sepsis or predict prognosis, and only PCT (precision cell therapy technology) is used in some medical centers to guide antibiotic treatment in critically ill patients. Because of the short time between stimulation and induction of PCT (detectable after 4 hours, peak at 6 hours), the long half-life of PCT, 25-30 hours, is a widely studied biomarker in sepsis. Recent meta-analysis showed that PCT had a sensitivity of 0.77 (0.72-0.81) and specificity of 0.79 (0.74-0.84) for sepsis and non-infectious systemic inflammatory response syndrome. PCT values at levels between 0.1 and 0.5 ng/mL indicate the need for antimicrobial therapy, but no consensus has been reached internationally regarding the correct threshold for PCT. In addition, PCT levels correlate with the severity of the condition in patients with severe pneumonia, while a decrease in PCT levels correlates with an increase in survival, which limits the clinical use of PCT. This limitation is largely due to the complex pathophysiology of sepsis and it seems unlikely that a single biomarker could provide accurate information about the driver of host response disorders in patients with sepsis.

From the current state of the art, it has been found that a combination of multiple biomarkers can provide unique sepsis signatures with the hope of providing information for a particular pathological abnormality and/or prognosis, which, of course, is a highly desirable approach to targeted therapy. In addition, published patent documents describe the use of RNA as a marker for the diagnosis of sepsis, allowing the differentiation between infectious and non-infectious diseases. For example, there is a method for distinguishing infection-positive (sepsis) from infection-negative (systemic inflammation) using an RNA marker described in patent document CN105981026A (biomarker identification method and apparatus and kit therefor, 2016.09.28), which is composed of 4 genes (CEACAM 4, LAMP1, PLA2G7 and PLAC 8) and is used for distinguishing sepsis from non-infectious systemic inflammatory response syndrome. The rapid molecular detection method is a first clinical diagnosis tool based on RNA, which utilizes the whole blood of a patient to diagnose, and scores the patient based on the expression quantity of the four RNAs for characterizing whether the patient is infected with sepsis; AUC was 0.89 (range: 0.85-0.93). In addition, patent document CN107532214A (2018.01.02) also describes a method for diagnosing sepsis in a subject, which mainly comprises 11 gene biomarkers (CEACAM 1, ZDHHC19, C9orf95, GNA15, BATF, C3AR1, KIAA1370, TGFBI, MTCH1, RPGRIP1, and HLA-DPB 1), and the expression level of the gene biomarkers can be used to distinguish sepsis patients from sterile inflammation patients, and AUC is 0.87 (range: 0.70-0.98).

With the continuous development of biomedical technology, in the clinical practice process of applying biomarkers to sepsis diagnosis and treatment, we find that the research on biomarkers generally focuses on identification of infectious and non-infectious critical diseases and sepsis prognosis, however, a large number of clinical reports show that different patients have different clinical manifestations due to differences of physiological mechanisms, and particularly in the clinical application of sepsis, not only rapid and efficient identification of sepsis but also diagnosis according to identification results are needed to give feasible targeted therapy, so that new biomarkers for sepsis identification are urgently needed from the purposes of more understanding or judging main pathophysiological mechanisms of different patients and obtaining potential targeted therapy approaches thereof.

Disclosure of Invention

The invention aims to provide a biomarker group for sepsis, which consists of new biomarkers different from the existing RNA markers, can provide a quick and efficient basis for the identification of sepsis, and a large amount of clinical data show that the preparation of the biomarker group for the identification of sepsis can approach 80%.

Another object of the present invention is to provide a method for sepsis diagnosis using the biomarker panel, which can be used to stratify patients with sepsis according to biochemical and/or immunological characteristics when performing sepsis diagnosis, and provide clues for understanding the main pathophysiological mechanisms and potential targeted therapeutic approaches of individual patients by establishing an index system of the biomarker panel.

Another object of the present invention is to provide a kit for sepsis judgment, which can control the detection time within 4 hours by using the detection method of RNA marker; compared with a microbial culture method, the method greatly shortens the detection time (at least 24 hours) and strives for valuable treatment time for patients.

The invention is realized by the following technical scheme: a biomarker panel for sepsis comprising the biomarkers TLR5 (Toll-like receptor 5), CD59 (protection), clu (clusterin), FGL2 (Fibrinogen-like 2), IL7R (Interleukin-7 receptor) and CPVL (viral-like) extracted from a biological sample.

The biological sample is selected from whole blood, buffy coat, plasma, serum, peripheral blood mononuclear cells, rod shaped nuclear cells, neutrophilic granulocytes, monocytes or T cells.

A method for judging sepsis by using a biomarker panel, measuring the expression levels of the biomarkers TLR5, CD59, CLU, FGL2, IL7R and CPVL in a biological sample, establishing an index system by using the expression level of the biomarkers of an uninfected subject and the expression level of the biomarkers of a sepsis patient, and judging whether the subject suffers from sepsis or not by using the index system.

The method for measuring the expression amount of the biomarker comprises microarray analysis, polymerase chain reaction, reverse transcriptase polymerase chain reaction, Northern blotting or gene expression series analysis.

Further comprising calculating a score for the expression level of the biomarker according to the following formula:

T-SCORE = log₂ （TLR5/FGL2 + CD59/IL7R + CLU/CPVL）。

the index system is as follows:

infection with sepsis: T-SCORE is more than or equal to 13;

suspected sepsis infection: T-SCORE is more than 6 and less than 13;

non-infectious inflammation: T-SCORE is less than or equal to 6.

Also included is the process of measurement and determination after observation of the subject suspected of being sepsis infected.

The non-infectious inflammation is a systemic inflammatory response syndrome, an autoimmune disorder, a traumatic injury or surgery.

A kit for sepsis determination comprising reagents or microarrays for measuring the expression levels of the biomarkers TLR5, CD59, CLU, FGL2, IL7R, and CPVL in a biological sample.

The reagents include primers or probes for amplifying the biomarkers.

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

(1) the invention provides clinical basis for treating the patients with the biological infection by detecting the expression level of each biomarker TLR5, CD59, CLU, FGL2, IL7R and CPVL in the biomarker group to judge whether the subjects have the sepsis infection.

(2) The invention forms a new biomarker group by each biomarker, determines the score of the expression quantity of the biomarker group by a formula, and compared with an index system, the score can be used for distinguishing and judging multiple degrees or states of sepsis infection, suspected sepsis infection and non-infectious inflammation, thereby providing convenience for clinical application.

(3) The biomarker group is expected to be popularized and applied to judging or guiding main pathogens of sepsis patients in the subsequent clinical test process, and lays a foundation for providing more reliable and targeted therapy for the follow-up process.

(4) A large amount of clinical data show that the accuracy of the method is particularly outstanding when people in China are taken as subjects to carry out measurement and judgment.

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto.

Example 1:

this example relates to a biomarker panel for sepsis.

Human being is used as a biological sample collector, and the biological sample may be whole blood, buffy coat, plasma, serum, peripheral blood mononuclear cells, rod-shaped nuclear cells, neutrophil, monocyte or T cells, and collected by a known method such as whole blood collection. Extracting biomarkers TLR5, CD59, CLU, FGL2, IL7R and CPVL from the biological sample to form a biomarker group.

The extraction method of the biomarker group can adopt a known method such as extraction of total RNA of animal tissues and the like.

Example 2:

this example relates to a biomarker panel for sepsis.

The biomarker panel may be RNA fragments of the biomarkers TLR5, CD59, CLU, FGL2, IL7R and CPVL amplified as described in example 1. For example, amplified RNA fragments as shown in table 1 below.

Example 3:

this example relates to a method for sepsis determination using a biomarker panel.

According to the score of the expression level of the biomarker of the measured subject, the sepsis infection, the suspected sepsis infection and the non-infectious inflammation can be distinguished by judging through the established index system, and the accuracy of an expected model is close to 80 percent by conjecturing with the known clinical data and the currently-performed clinical test.

First, measuring the expression levels of the biomarkers TLR5, CD59, CLU, FGL2, IL7R and CPVL in a biological sample, such as microarray analysis, polymerase chain reaction, reverse transcriptase polymerase chain reaction, Northern blot or serial analysis of gene expression, can be used to measure the biomarkers in the biological sample. The expression levels of the biomarkers TLR5, CD59, CLU, FGL2, IL7R and CPVL are different for different sepsis infections, and particularly when the expression levels of the biomarkers are compared for patients with noninfected concentration symptoms, the expression levels of the biomarkers TLR5, CD59 and CLU are shown to be increased, and the expression levels of the biomarkers FGL2, IL7R and CPVL are shown to be decreased, so that the expression levels of the biomarkers in the biomarker group in response to each biomarker need to be scored, and the specific score can be calculated according to the following formula:

T-SCORE = log₂ （TLR5/FGL2 + CD59/IL7R + CLU/CPVL），

wherein T-SCORE represents the SCORE of the expression of the biomarker panel.

After obtaining the score of the expression quantity of the biomarker group in the biological sample, comparing the score with the established index system, judging whether the test subject suffers from sepsis or not according to the score, and providing a basis for clinical diagnosis.

In this embodiment, the established marker system can be obtained by collating a large amount of experimental data, for example, by measuring the expression levels of the biomarkers in the uninfected subjects and the expression levels of the biomarkers in the sepsis patients by the same method, respectively obtaining the scores of the expression levels of the biomarker groups in the uninfected patients and the scores of the expression levels of the biomarker groups in the sepsis patients, and then combining a large number of clinical experiments and analyses, the corresponding marker system can be established as follows:

infection with sepsis: T-SCORE is more than or equal to 13;

suspected sepsis infection: T-SCORE is more than 6 and less than 13;

non-infectious inflammation: T-SCORE is less than or equal to 6.

When a specific test subject is judged, a biological sample is obtained, the score of the expression level of the biomarker group in the biological sample is measured, the score is compared with the existing index system, and if sepsis infection is caused by the comparison result, the antibiotic treatment can be performed by combining clinical diagnosis; if the comparison result is non-infectious inflammation, judging whether the inflammation is the inflammation caused by systemic inflammatory response syndrome, autoimmune diseases, traumatic injury or operation or not by combining the clinical diagnosis condition, and giving corresponding treatment; if the comparison result is sepsis infection in turn, observation can be carried out, and measurement and judgment can be carried out again after 1-2 hours of observation by combining clinical diagnosis conditions, and if the result is sepsis infection, treatment is carried out according to the sepsis infection conditions.

Example 4:

this example relates to a kit for sepsis determination.

The kit comprises reagents for measuring the expression levels of the biomarkers TLR5, CD59, CLU, FGL2, IL7R and CPVL in a biological sample, which may be primers or probes for amplifying the biomarkers, and instructions for use, see example 2.

In the specific measurement, after a biological sample is obtained, the sample is extracted according to the kit instruction, and the expression quantity of the biomarker is quantified by using the reagent. Then, the test sample is scored according to the scoring method described in example 3, and the disease condition is judged.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and all simple modifications and equivalent variations of the above embodiments according to the technical spirit of the present invention are included in the scope of the present invention.

SEQUENCE LISTING

<110> Sichuan Sheng Meisida Biotech Co., Ltd

<120> biomarker group for sepsis and sepsis determination method and kit

<130> 2019

<160> 18

<170> PatentIn version 3.3

<210> 1

<211> 20

<212> DNA

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<213> RNA fragment after FGL2 amplification

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Claims (10)

1. A biomarker panel for sepsis, characterized by: comprising the biomarkers TLR5, CD59, CLU, FGL2, IL7R and CPVL extracted from a biological sample.

2. The biomarker panel for sepsis according to claim 1, characterized in that: the biological sample is selected from whole blood, buffy coat, plasma, serum, peripheral blood mononuclear cells, rod shaped nuclear cells, neutrophilic granulocytes, monocytes or T cells.

3. A method of determining sepsis using a biomarker panel, comprising: measuring the expression levels of the biomarkers TLR5, CD59, CLU, FGL2, IL7R and CPVL in the biological sample, establishing an index system by using the expression level of the biomarkers in an uninfected subject and the expression level of the biomarkers in a sepsis patient, and judging whether the subject suffers from sepsis or not by using the index system.

4. A method of determining sepsis using a biomarker panel according to claim 3, wherein: the method for measuring the expression amount of the biomarker comprises microarray analysis, polymerase chain reaction, reverse transcriptase polymerase chain reaction, Northern blotting or gene expression series analysis.

5. A method of determining sepsis using a biomarker panel according to claim 3, wherein: further comprising calculating a score for the expression level of the biomarker according to the following formula:

T-SCORE = log₂ （TLR5/FGL2 + CD59/IL7R + CLU/CPVL）。

6. a method of using a biomarker panel for sepsis determination according to claim 5, wherein: the index system is as follows:

infection with sepsis: T-SCORE is more than or equal to 13;

suspected sepsis infection: T-SCORE is more than 6 and less than 13;

non-infectious inflammation: T-SCORE is less than or equal to 6.

7. A method of determining sepsis using a biomarker panel according to claim 6, wherein: also included is the process of measurement and determination after observation of the subject suspected of being sepsis infected.

8. A method of determining sepsis using a biomarker panel according to claim 6, wherein: the non-infectious inflammation is a systemic inflammatory response syndrome, an autoimmune disorder, a traumatic injury or surgery.

9. A kit for sepsis determination, characterized by: includes reagents or microarrays for measuring the expression levels of the biomarkers TLR5, CD59, CLU, FGL2, IL7R, and CPVL in a biological sample.

10. A kit for sepsis determination according to claim 9, characterized in that: the reagents include primers or probes for amplifying the biomarkers.