CN115927582A - Sepsis diagnostic reagent kit based on detecting ITM2A gene expression level - Google Patents

Abstract

The invention discloses a novel sepsis molecular marker ITM2A and application thereof. The invention also discloses an application of the detection reagent of the ITM2A gene or protein in the preparation of a sepsis diagnostic kit. The primer for the real-time fluorescent quantitative PCR detection of the ITM2A gene has high specificity and sensitivity, and can be used for high-efficiency diagnosis of clinical sepsis.

Description

Sepsis diagnostic reagent kit based on detecting ITM2A gene expression level

Technical Field

The invention relates to the field of clinical molecular diagnosis, in particular to a novel sepsis molecular marker ITM2A and application thereof.

Background

Sepsis is a systemic inflammatory response syndrome caused by invasion of pathogenic microorganisms such as bacteria into the body, usually causes functional damage to organs which are life threatening, and the major damaged organs include: lung, blood circulation system, urinary system, abdominal cavity, etc. Worldwide, sepsis (sepsis) has a fatality rate of 25% -30%, and if the patient still has symptoms of shock, i.e., septic shock, the fatality rate of the patient will reach 40% -50%.

Some existing sepsis markers such as IL-6 (interleukin-6), CRP (C-reactive protein) and PCT (procalcitonin) have the problems of low specificity and low sensitivity in early sepsis diagnosis. Therefore, finding highly sensitive and specific molecular markers and determining suitable detection methods for them are critical to the diagnosis and treatment of sepsis.

Disclosure of Invention

In order to solve the technical problems, the invention collects and performs credit generation analysis on related data of a public gene database, and finds that the expression of the ITM2A gene in the blood of a sepsis patient is obviously lower than that of a healthy control group; clinical samples are further collected and verified by a real-time fluorescent quantitative PCR method, and the obtained result is consistent with the student's information analysis result.

Specifically, the invention provides application of a detection reagent of ITM2A gene or protein in preparing a sepsis diagnostic kit.

In certain embodiments, the detection reagents for the ITM2A gene include primers for real-time fluorescent quantitative PCR detection of ITM 2A.

In certain embodiments, the detection reagent for the ITM2A protein comprises an ITM 2A-specific antibody.

In certain embodiments, the primers for ITM2A real-time fluorescent quantitative PCR detection PCR comprise: human-ITM2A forward primer 5 'CGCGGCAAGACGTGGAG-3' (SEQ ID NO: 1); human-ITM2A reverse primer 5-.

In a second aspect, the present invention provides a sepsis diagnostic kit comprising a detection system for the amount of ITM2A mRNA expression in a sample to be tested.

In certain embodiments, the kit further comprises an isolation system for RNA extraction of the sample to be tested.

In certain embodiments, the kit further comprises a mathematical model and threshold for determining the high/low cutoff criteria for ITM2A expression level.

In certain embodiments, the test sample is human Peripheral Blood Mononuclear Cells (PBMCs).

In certain embodiments, the separation system comprises a separation fluid for separating PBMCs from peripheral blood, such as lymphocyte separation fluid, trizol reagent, and the like; reagents for extracting and isolating RNA from PBMC such as chloroform, isopropanol, absolute ethanol, DEPC water, etc.

In certain embodiments, the ITM2A gene detection system comprises reagents for reverse transcription of RNA, reagents for PCR amplification of cDNA, primers, and the like.

In certain embodiments, the detection system comprises an ITM2A gene expression detection reagent.

In certain embodiments, the detection reagents include primers for ITM2A real-time fluorescent quantitative PCR.

In certain embodiments, the primer sequences for ITM2A real-time fluorescent quantitative PCR are: human-ITM2A forward primer 5 'CGCGGCAAGACGTGGAG-3' (SEQ ID NO: 1); human-ITM2A reverse primer 5-.

In certain embodiments, the kit further comprises a detection primer for internal reference GAPDH and optionally instructions.

In certain embodiments, the detection primer sequence of GAPDH is: human-GAPDH forward primer 5 'AGCCACATCGCTCAGACAC-3' (SEQ ID NO: 3); human-GAPDH reverse primer 5 'GCCCAATACGACCAAAATCC-3' (SEQ ID NO: 4).

In certain embodiments, as a mathematical model for determining the high/low cutoff criteria for mRNA expression of ITM2A, it is a delta Δ CT calculation formula for quantitative fluorescence PCR. Δ CT is a simplified form of a quantitative calculation formula for fluorescence and is used to compare specific gene expression copy number differences or change ratios between different samples. Ct = -1/lg (1 + Ex) × lgX0+ lgN/lg (1 + Ex), wherein N is the cycle number of the amplification reaction, X0 is the initial template amount, ex is the amplification efficiency, and N is the amount of the amplification product when the fluorescence amplification signal reaches the threshold intensity. Δ Ct (n) = Ct (target gene) -Ct (reference gene); Δ CT (n) =Δct (n) - Δct (1). The larger the initial copy number of the gene, the smaller the Ct value. The initial copy number of a certain gene in the sample can be calculated as long as the Ct value of the certain gene in the sample is obtained. In the present invention, the target gene is ITM2A and the reference gene is human GAPDH gene. The invention selects 0.50 as the threshold value of the kit for diagnosing the sepsis, and the value is not higher than the threshold value to consider that the patient suffers from the sepsis.

In certain embodiments, the mathematical model is the Δ CT calculation for fluorescent quantitative PCR for detection of the ITM2A gene: Δ Ct (n) = Ct (ITM 2A gene) -Ct (reference gene); Δ CT (n) =Δct (n) - Δct (1), where n is the number of cycles of the amplification reaction; when the Δ CT value is not higher than 0.50, sepsis is suggested.

In a third aspect, the invention provides the use of an ITM2A gene activator in the preparation of a medicament for the treatment of sepsis.

Compared with the prior art, the invention has the beneficial effects that:

the invention starts from the blood of sepsis (including adults and children) and healthy people, and researches the relation between the expression of ITM2A and sepsis by combining a real-time fluorescent quantitative PCR (polymerase chain reaction) and an analysis method of chip data, thereby proving that the ITM2A is specifically and lowly expressed in the blood of sepsis patients (including adults and children). ITM2A may be used as a marker for diagnosing patients with sepsis, instructing clinicians to prescribe an appropriate treatment regimen. The invention also provides a fluorescent quantitative PCR kit for diagnosing sepsis, wherein the primer has high specificity and sensitivity.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

Figure 1 shows ITM2A expression in PBMCs (N = 5/group). ^* P<0.05； ^** P<0.01

Fig. 2 shows the integration of three sets of data sets, GSE131761, GSE57065, and GSE 66890. Fig. 2A is a PCA analysis of the three sets of data prior to batch effect removal, and fig. 2B is a PCA analysis of the three sets of data integrated after batch effect removal by the sva-wrapped kabat function. The abscissa is the first principal component and the ordinate is the second principal component.

FIG. 3 shows the expression of ITM2A gene in patients with sepsis in the set of data GSE95233, GSE26440, GSE131761, GSE57065, GSE66890 and GSE 139046. ^* P<0.05； ^** P<0.01， ^*** P<0.001

Detailed Description

The present invention is further described below, and the embodiments described in the present description are only exemplary and do not limit the scope of the present invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and it is intended that all such changes and modifications be considered as within the scope of the invention. Unless otherwise indicated, all reagents, apparatus, devices and methods used in this patent are conventional and commercially available reagents, apparatus, devices and methods in the art.

As used herein, the term "ITM2A Protein", integral Membrane Protein 2A, is a type II transmembrane Protein belonging to the ITM2 family, encoding a Protein sequence of 263 amino acids in length and having a molecular weight of 29.741kDa. The molecular function of ITM2A was annotated in the Gene Ontology database (GO) as amyloid β protein binding (amyloid-beta), and the GO database notes that the major biological processes in which ITM2A is involved are negative regulation of amyloid precursor protein biosynthesis (negative differentiation of amyloid precursor protein) and plasma cell differentiation (plasma cell differentiation).

The detection target of the kit is to measure the mRNA expression level of ITM2A in PBMC of a patient, and when the CT value of ITM2A is not higher than a certain threshold value, sepsis is suggested.

Example 1 bioinformatic data analysis of sepsis ITM2A differential expression

1.1 data set Collection

The raw chip data set in the gene expression repertoire GEO was collected for the following information, where the collected samples were all blood samples.

GSE95233 (adult group): data from a longitudinal study, which included 51 septic shock adults and 22 healthy adult controls, wherein septic shock patients were bled 2 times each, the first bleed was into the ICU and the second bleed was into the ICU on the second or third day. The control group was healthy adults. Of these, 34 survival cases and 17 death cases were observed in septic shock patients.

GSE131761 (adult group): the study included 15 healthy adults as normal controls, 76 patients diagnosed with septic shock and 30 patients with no symptoms of septic shock.

GSE57065 (adult group): longitudinal study data. Inclusion into 28 adult patients who entered ICU due to septic shock, 3 blood collections were obtained per patient: septic shock occurred within 30 minutes, 24 hours, and 48 hours, with 14 patients classified as "low" and the other 14 as "high" by the simplified acute physiological score II (SAPSII) criteria. An additional study was included with 25 healthy adults as a control group.

GSE66890 (adult group): 5 normal adults were included as a control group, 9 patients with septic shock and 13 patients with sepsis without shock symptoms were a case group.

GSE26440 (children group): whole blood expression profile data for 98 septic shock children and 32 healthy children. Of these, 81 septic shock children died and 17 septic shock children survived.

GSE13904 (children group): longitudinal study data. As a control group, 18 healthy children were included, 53 septic shock and 26 septic shock symptom-free children. Wherein, the control group only takes blood on the first day, and septic shock and non-shock children take blood on the first day and the third day.

Chip sequencing is carried out on the collected blood to obtain the gene expression profile of each sample. The gene expression profile refers to the type and abundance of gene expression of a biological sample at a specific time, and can be used for researching pathogenesis of diseases, early diagnosis and prognosis prediction of diseases and the like.

TABLE 1 data set for raw data analysis

1.2 dataset preprocessing

For the Affy microarray platform data set, the data is preprocessed by using an Affy package in an R language, and the data set is subjected to background correction, standardization, log transformation and the like by using a rma function to obtain a probe expression profile. And then performing gene annotation on the probe, namely, the probe corresponds to the gene name, and the processing principle is as follows: deleting a probe if the probe corresponds to a plurality of gene names; deleting a probe if the probe does not have a corresponding gene name; if a plurality of probes correspond to one gene, taking the average value of the probes as the expression value of the gene; if the expression profile processed by the method has missing values or zero values, the missing values or the zero values are filled by using a k-nearest neighbor method, and the k value is selected to be 3. After the above treatment, gene expression profile data, which are row genes, are listed as samples are generated.

For the Agilent microarray platform dataset, the data was preprocessed using the "limma" package in the R language. Background correction and standardization are carried out on the data set by using two functions of backsgroudcorre and normalizeBetwennays in sequence. After obtaining the probe expression profile, mapping the gene, that is, the probe is corresponding to the gene name, and the processing principle is consistent with the steps of the Affy sequencing platform, namely: deleting the probe without the corresponding gene; deleting probes corresponding to a plurality of genes; if a plurality of probes correspond to one gene, taking the average value of the probes as the expression value of the gene; if the expression spectrum has a missing value or a zero value, the k-nearest neighbor method is used for supplementing, and the k value is 3. After the above treatment, gene expression profile data, row genes, are generated and listed as samples.

1.3 differential expression of ITM2A

For the processed gene expression profile data set, the expression condition of each gene among the groups is calculated by using the functions of lmFit, makeContrasts, mutants, eBays and topTable in the R language 'limma' package, and log2FC value is used for expressing the difference expression condition of the gene between the experimental group and the control group, wherein in the common condition, | log2FC | >1 can prompt that the gene is the difference expression gene, log2FC >1 expresses that the gene expression of the experimental group is up-regulated compared with the control group, and log2FC < -1 expresses that the gene expression of the experimental group is down-regulated compared with the control group. In addition, in this study, the Benjamini-Hochberg method was used to correct the p-value in statistics. The corrected p-value (adj.p.val) <0.05, and the difference was considered to be statistically significant.

For the GSE95233 dataset, the ITM2A gene was significantly down-regulated in septic shock surviving patients compared to healthy controls and in septic shock dead patients compared to healthy controls (see table 2).

TABLE 2 expression differences between groups for ITM2A in the GSE95233 data set

In the GSE26440 dataset of the child group, the expression of ITM2A was compared between groups using the differential gene screening method described above, and the results showed that ITM2A was significantly reduced in septic shock survival and healthy control comparisons, as well as in septic shock death and healthy control comparisons. The results were consistent with the results of the GSE95233 (adult group) data analysis. The results are shown in Table 3.

TABLE 3 differences in ITM2A expression between groups in the GSE26440 dataset

For the three sets of data, GSE131761, GSE57065 and GSE66890, these 3 sets were integrated as they contained only septic shock, septic non-shock and healthy controls. For integration across platform datasets, the batch effect removal operation was performed using the Combat function in the "sva" package in the R language. Principal Component Analysis (PCA) of the pre-batch effect and post-batch effect datasets, the results are shown in fig. 2. The integrated gene expression profile was screened for differentially expressed genes using the limma package differential gene selection method. The results show that the ITM2A gene expression is significantly reduced in septic shock and healthy controls, as well as in septic non-shock and healthy control controls.

TABLE 4 differences in ITM2A expression between groups in the GSE131761, GSE57065 and GSE66890 integrated data sets

In the children group data set GSE13904, the differential expression of ITM2A in each group is compared by using the same differential gene screening method, and the result shows that the ITM2A has significantly reduced comparative expression in septic shock and a healthy control group, and has significantly reduced expression in septic non-shock and a healthy control group. The results are consistent with the results of the integration of three sets of data sets, namely the adult group GSE131761, GSE57065 and GSE 66890. See table 5.

TABLE 5 differences in ITM2A expression between groups in the GSE13904 data set

Combining the data sets of the above adult and child groups, it can be concluded that: ITM2A expression in sepsis (shock/no shock, survival/death) was significantly reduced, and the reduction in expression was statistically significant. Therefore, the expression level of ITM2A can be used as a molecular marker for diagnosing early sepsis.

Example 2 real-time fluorescent quantitative PCR detection of ITM2A expression

Blood samples were collected from healthy controls, ICU control patients, sepsis survivors and sepsis dead patients, of which 5 samples were collected from each group. PBMC is separated and extracted by a Ficol gradient centrifugation method. Collecting cells, washing with PBS, adding 1ml Trizol, repeatedly blowing and beating for several times to lyse the cells, transferring to 1.5ml RNase-free tube, adding 500ul chloroform, mixing, standing at room temperature for 10min, centrifuging at 4 deg.C at 12000rpm for 15min, transferring the upper aqueous phase to a new 1.5ml tube, adding equal volume of isopropanol to precipitate RNA, mixing, standing at room temperature for 10min, and centrifuging at 4 deg.C at 12000rpm for 15min. Discarding the supernatant, adding 75% ethanol to wash the precipitate, centrifuging at 12000rpm at 4 ℃ for 5min, discarding the supernatant, adding 20ul DEPC water to dissolve RNA after the precipitate is dried, measuring the RNA concentration by using a Nanodrop, and directly carrying out the next reverse transcription according to the Oliga (dT) method to obtain cDNA.

Adding each reaction component into the cDNA as template according to the reaction system of the instruction book (RR 086A, TAKARA), mixing, and keeping at 95 deg.C for 5min;95 ℃ 10s,60 ℃ 15s,72 ℃ 1min (40 cycles repeated from the second step). And (3) according to the Ct value, taking GAPDH as an internal reference gene, and carrying out relative quantitative calculation on the expression of the target gene in the sample according to a 2-delta Ct method.

The primer sequence information of the fluorescent quantitative PCR is shown below;

Human-ITM2A forward primer 5 'CGCGGCAAGACGTGGAG-3' (SEQ ID NO: 1);

Human-ITM2A reverse primer 5 'CCACTCGCCAGTTTGCCA-3' (SEQ ID NO: 2);

Human-GAPDH forward primer 5 'AGCCACATCGCTCAGACAC-3' (SEQ ID NO: 3);

Human-GAPDH reverse primer 5 'GCCCAATACGACCAAAATCC-3' (SEQ ID NO: 4).

Real-time quantitative PCR results show that the expression level of ITM2A detected by using the primers in a sepsis survival group and a death group is significantly lower than that of a healthy control group and an ICU control group, and meanwhile, the expression level of ITM2A in the ICU control group is significantly lower than that of the healthy control group, and the results show that the sensitivity of ITM2A as a diagnostic marker of sepsis.

Example 3 sepsis diagnostic kit and use thereof

Based on the findings of examples 1 and 2, the present invention designs a kit for sepsis diagnosis, comprising: an RNA extraction and separation system for extracting ITM2A RNA from human peripheral blood mononuclear cells; an ITM2A gene detection system for detecting the mRNA expression level of ITM2A in human peripheral blood mononuclear cells, which comprises an ITM2A gene detection reagent; and the mathematical model and the threshold value used for determining the ITM2A expression high/low boundary standard are internally referenced by GAPDH. When the mRNA expression level of ITM2A in human peripheral blood mononuclear cells is not higher than the threshold, sepsis is suggested.

The ITM2A detection primer reagent and the detection primer for the internal reference GAPDH gene are as described in example 2.

The ITM2A RNA extraction and separation system in the kit comprises: a separation solution for separating PBMCs from peripheral blood, such as lymphocyte separation solution, trizol reagent, etc.; reagents for extracting and isolating RNA from PBMC such as chloroform, isopropanol, absolute ethanol, DEPC water, etc. The ITM2A gene detection system comprises reagents for reverse transcription of RNA, reagents for PCR amplification of cDNA, primers and the like.

As a mathematical model for determining the high/low cut-off criterion of the mRNA expression level of ITM2A, it is a calculation formula of delta-delta CT of fluorescent quantitative PCR. Δ CT is a simplified form of a quantitative calculation formula for fluorescence and is used to compare specific gene expression copy number differences or change ratios between different samples. Ct = -1/lg (1 + Ex): lgX0+ lgN/lg (1 + Ex), wherein N is the cycle number of the amplification reaction, X0 is the initial template amount, ex is the amplification efficiency, and N is the amount of the amplification product when the fluorescence amplification signal reaches the threshold intensity. Δ Ct (n) = Ct (target gene) -Ct (reference gene); Δ CT (n) =Δct (n) - Δct (1). The larger the initial copy number of the gene, the smaller the Ct value. The initial copy number of a certain gene in a sample can be calculated as long as the Ct value of the certain gene in the sample is obtained. In the present invention, the target gene is ITM2A and the reference gene is human GAPDH gene. The invention selects 0.50 as a threshold value of the kit for diagnosing the sepsis, and the value of the threshold value is not higher than the threshold value, so that the patient is considered to suffer from the sepsis.

Table 6 establishes the critical value for Δ CT for ITM2A

Based on this statistical result, it is reasonable to take a Δ CT value of 0.50 as a critical value for sepsis determination. Sepsis is indicated when Δ CT is 0.50 or less.

While there have been shown and described what are at present considered the fundamental principles and essential features of the invention and its advantages, it will be apparent to those skilled in the art that the invention is not limited to the details of the foregoing exemplary embodiments, but is capable of other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.

Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and the description is given here only for clarity, and those skilled in the art should integrate the description, and the embodiments may be combined appropriately to form other embodiments understood by those skilled in the art.

Claims (8)

1. An application of a detection reagent of ITM2A gene or protein in preparing a sepsis diagnostic kit.

2. The use according to claim 1, wherein the detection reagents for the ITM2A gene comprise primers for real-time fluorescent quantitative PCR detection of ITM 2A; the detection reagent of the ITM2A protein comprises an ITM2A specific antibody.

3. The use according to claim 2, wherein the primers for ITM2A real-time fluorescent quantitative PCR detection PCR comprise: human-ITM2A forward primer SEQ ID NO:15'-CGCGGCAAGACGTGGAG-3' (SEQ ID NO: 1); human-ITM2A reverse primer 5 'CCACTCGCCAGTTTGCCA-3' (SEQ ID NO: 2).

4. A sepsis diagnostic kit, comprising a detection system for an amount of mRNA expression of ITM2A in a sample to be tested; optionally, the kit further comprises an isolation system for extracting RNA of a sample to be detected and/or a mathematical model and a threshold value for determining the ITM2A expression level high/low demarcation standard; preferably, the sample to be tested is human peripheral blood mononuclear cells.

5. The kit of claim 4, wherein the detection system comprises an ITM2A gene expression detection reagent; preferably, the detection reagent comprises a primer for ITM2A real-time fluorescence quantitative PCR, and the sequence of the primer is as follows: human-ITM2A forward primer 5 'CGCGGCAAGACGTGGAG-3' (SEQ ID NO: 1); human-ITM2A reverse primer 5 'CCACTCGCCAGTTTGCCA-3' (SEQ ID NO: 2).

6. The kit of claim 4 or 5, wherein the kit further comprises a detection primer for internal reference GAPDH and optionally instructions; preferably, the detection primer sequence of GAPDH is: human-GAPDH forward primer 5 'AGCCACATCGCTCAGACAC-3' (SEQ ID NO: 3); human-GAPDH reverse primer 5 'GCCCAATACGACCAAAATCC-3' (SEQ ID NO: 4).

7. The kit according to claim 4, characterized in that the mathematical model is the calculation formula for Δ CT of the fluorescent quantitative PCR for the detection of the ITM2A gene: Δ Ct (n) = Ct (ITM 2A gene) -Ct (reference gene); Δ CT (n) =Δct (n) - Δct (1), where n is the number of cycles of the amplification reaction; when Δ CT values were not higher than 0.50, sepsis was suggested.

Use of ITM2A gene activator in preparing a medicament for treating sepsis.

Images