Biomarker combination, detection kit and preparation method thereof

Abstract

The invention relates to a biomarker combination comprising an NF-kappa B activation marker and a nucleic acid probe or primer for determining the mRNA expression levels of one or more genes of BCL2A1, CCL3, G0S2, IL1beta and TNF alpha, the expression levels of at least three of said genes being significantly increased relative to a reference control; the invention also relates to a detection kit, which comprises a reagent for detecting the NF-kappa B activation marker and a nucleic acid probe or primer for determining the gene combination expression level of the BCL2A1, CCL3, G0S2, IL1beta and TNF alpha mRNA. In addition, the invention relates to a method for preparing the detection kit by using a computer program. According to the invention, the activation state of an NF-kappa B signal channel is combined with the expression characteristics of specific inflammation-related genes for the first time, and a stable and consistent ASD molecular diagnosis system is constructed.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the field of molecular diagnostics and detection technology of neuropsychiatric diseases, and particularly to a combination of biomarkers, a detection kit, and a method for preparing the detection kit using a computer program for autism spectrum disorder (ASD) based on the activation status of the NF-κB signaling pathway and the expression characteristics of specific inflammation-related genes. Background Technology

[0002] Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder characterized by impairments in social interaction, communication, and repetitive, stereotyped behaviors. Its pathogenesis is highly heterogeneous, involving multiple factors including genetics, immunity, inflammation, and environment. Currently, the clinical diagnosis of ASD mainly relies on behavioral scale assessments and long-term observation by physicians, such as the Autism Diagnostic Observation Schedule (ADOS) and the Autism Diagnostic Interview-Revised (ADI-R). However, these methods have drawbacks, including strong diagnostic subjectivity, reliance on experience, delayed diagnosis, and difficulty in achieving early screening and risk stratification.

[0003] Recent studies have shown that immune abnormalities and chronic inflammatory responses play a crucial role in the development and progression of acute respiratory syndrome (ASD). Among these, the NF-κB signaling pathway, a core pathway for inflammatory responses and immune regulation, can lead to persistent high expression of various inflammatory factors and immune-related genes through abnormal activation. However, current technologies lack a stable combination of biomarkers and standardized detection protocols based on the systemic molecular characteristics of the NF-κB signaling pathway for the early, objective, and quantifiable diagnosis of ASD.

[0004] Therefore, developing a molecular detection method for ASD based on a clear molecular mechanism and with high consistency and stability, along with a matching reagent kit, is of great scientific significance and clinical application value for achieving early screening, accurate detection, and risk stratification assessment of ASD. Summary of the Invention

[0005] The purpose of this invention is to provide a combination of biomarkers for autism spectrum disorder (ASD) based on objective molecular biological indicators, a detection kit, and a method for preparing the detection kit using a computer program. This overcomes the shortcomings of existing technologies for ASD diagnosis, which primarily rely on behavioral assessments, are highly subjective, and struggle with early screening and risk stratification. By detecting the activation status of the NF-κB signaling pathway and the expression characteristics of specific inflammation-related genes, early, accurate, minimally invasive, or non-invasive diagnosis of ASD can be achieved.

[0006] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0007] 1. A biomarker combination for the diagnosis of ASD, characterized in that it comprises an NF-κB activation biomarker and nucleic acid probes or primers for detecting BCL2A1, CCL3, GOS2, IL1β and TNFα mRNA, including the following detection indicators: activation status of the NF-κB signaling pathway, and expression levels of one or more genes among BCL2A1, CCL3, GOS2, IL1β and TNFα, wherein the expression levels of at least three of the genes are significantly increased relative to a reference control.

[0008] Based on the above combination of biomarkers, it is used for: (1) Obtain biological samples from the subject, wherein the biological samples are samples obtained before or after childbirth, preferably peripheral blood, umbilical cord blood, serum, plasma, saliva or other body fluid samples; (2) Detect the activation status of the NF-κB signaling pathway in the biological sample, the activation status including but not limited to the nuclear translocation level of NF-κB p65, DNA binding activity or downstream transcriptional activity; (3) Detect the mRNA expression level of one or more of the following genes in the biological sample: BCL2A1, CCL3, G0S2, IL1β and TNFα; (4) Compare the detection results of steps (2) and (3) with the normal control reference values. When the NF-κB signaling pathway is in an abnormally activated state and the expression level of one or more of the above genes is significantly increased, the subject is determined to have the risk of ASD or be diagnosed with ASD.

[0009] Based on the above combination of biomarkers, the activation status of the NF-κB signaling pathway is detected by immunological methods, molecular biological methods, or combinations thereof. These methods include, but are not limited to, Western blotting, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), chromatin immunoprecipitation (ChIP), real-time quantitative PCR, or transcriptional activity analysis.

[0010] 1. Based on the above combination of biomarkers, the expression level of the aforementioned gene is detected by reverse transcription-polymerase chain reaction (RT-qPCR), digital PCR, or RNA sequencing.

[0011] 2. A diagnostic kit for autism spectrum disorder, characterized in that it comprises: (1) Detection reagents for detecting the activation state of the NF-κB signaling pathway; (2) Detection reagents for detecting the mRNA expression levels of one or more genes among BCL2A1, CCL3, G0S2, IL1β and TNFα; The test kit may also include nucleic acid extraction reagents, reverse transcription reagents, amplification reagents, standards, internal controls, and instructions for use.

[0012] 3. A method for preparing the above-mentioned test kit using a computer program, characterized in that it comprises the following steps: (1) Input the NF-κB activation data of the NF-κB activation marker and the gene combination expression data into a computer program; (2) The expression levels of at least three genes in the gene combination are significantly increased relative to the reference control.

[0013] Compared with the prior art, the present invention has the following beneficial effects: First, this invention is the first to combine the activation state of the NF-κB signaling pathway with the expression characteristics of specific inflammation-related genes, thereby constructing a stable and consistent molecular diagnostic system for ASD. Second, this invention uses objective and quantifiable molecular indicators as diagnostic criteria, avoiding the subjectivity and lag of traditional behavioral assessment methods, and improving the accuracy and repeatability of ASD diagnosis. Third, this invention is applicable to various biological samples before or after childbirth, supports minimally invasive or non-invasive testing methods, and is beneficial for early screening, accurate diagnosis and risk stratification assessment of ASD. Fourth, the biomarker combination and diagnostic kit provided by this invention have good standardization and clinical operability, and are suitable for widespread application. Attached Figure Description

[0014] To more clearly illustrate the technical solution of the present invention, the present invention will be further described below with reference to the accompanying drawings. It should be understood that the following drawings are only used to illustrate preferred embodiments of the present invention and do not constitute a limitation on the scope of protection of the present invention.

[0015] Figure 1 This diagram illustrates the screening and analysis of differentially expressed genes in umbilical cord blood and peripheral blood of subjects with autism spectrum disorder (ASD) and normal controls, showing the expression characteristics of downstream genes of the NF-κB signaling pathway in different sample sources.

[0016] Figure 2 This is a schematic diagram showing the detection results of NF-κB signaling pathway activation in umbilical cord blood samples, illustrating the characteristics of enhanced NF-κB activation and elevated expression of related inflammatory genes in ASD subjects.

[0017] Figure 3 This is a schematic diagram showing the detection results of NF-κB signaling pathway activation status indicators in peripheral blood samples, illustrating the differences between ASD subjects and normal controls.

[0018] Figure 4 This is a schematic diagram showing the activation of the NF-κB signaling pathway and changes in the expression of related genes in the peripheral blood of newborn offspring in an animal model induced by adverse prenatal valproic acid exposure.

[0019] Figure 5 This diagram illustrates the changes in expression and transcriptional regulation of downstream genes in the NF-κB signaling pathway in peripheral blood mononuclear cells under different intervention conditions, specifically the effects of antioxidants and NF-κB inhibitors on prenatal valproic acid-induced NF-κB signaling pathway activation.

[0020] Figure 6 This is a schematic diagram showing the changes in the activation status of the peripheral blood NF-κB signaling pathway under different intervention conditions, specifically the effects of antioxidants and NF-κB inhibitors on prenatal valproic acid-induced NF-κB activation.

[0021] Figure 7 This is a schematic diagram illustrating the correlation between neurobehavioral indicators and molecular detection results in animal models, specifically a diagram illustrating autism-like behavior induced by prenatal valproic acid exposure and its correlation with the NF-κB signaling pathway.

[0022] Figure 8This is an overall schematic diagram of the molecular mechanism and diagnostic principle of autism spectrum disorder as described in this invention. It shows the technical idea of ​​oxidative stress inducing the activation of the NF-κB signaling pathway and leading to the abnormal expression of specific inflammation-related genes, which can be used for ASD diagnosis and risk assessment. That is, the schematic diagram of the molecular mechanism and diagnostic principle of adverse prenatal exposure inducing oxidative stress, activating the NF-κB signaling pathway and leading to autism-like phenotype as described in this invention.

[0023] Figure 9 This is a graph illustrating the ROC curve for the diagnostic performance of the joint scoring model. Detailed Implementation

[0024] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings. Those skilled in the art should understand that the following embodiments are only used to illustrate the technical solutions of the present invention and should not be construed as limiting the scope of protection of the present invention. Equivalent substitutions or modifications made to the technical solutions of the present invention without departing from the technical concept of the present invention should all fall within the scope of protection of the present invention. Implementation Method 1: A Molecular Diagnostic Method for ASD Based on NF-κB Signaling Pathway and Specific Gene Expression Characteristics

[0025] like Figures 1 to 3 As shown, this embodiment provides a molecular diagnostic method for identifying autism spectrum disorder, which includes the following steps:

[0026] (1) Sample acquisition

[0027] Biological sample 1 is obtained from the subject. Biological sample 1 can be a prenatal or postnatal sample, preferably umbilical cord blood, peripheral blood, serum, plasma, or saliva. Studies have found that if the sample volume is lower than the minimum required for routine molecular testing (e.g., less than 200 μL of blood sample), RNA extraction efficiency will decrease significantly, thus affecting detection sensitivity; while if the sample volume is significantly higher than the upper limit of detection requirements (e.g., more than 5 mL of blood sample), sampling trauma and operational costs will increase, which is not conducive to clinical promotion.

[0028] (2) Detection of the activation state of the NF-κB signaling pathway

[0029] After processing biological sample 1, the activation state 2 of the NF-κB signaling pathway was detected. Activation state 2 includes one or more of the following: nuclear translocation level of the NF-κB p65 subunit, DNA binding activity, or downstream transcriptional activity. Figure 2 and Figure 3 As shown, the nuclear localization ratio of NF-κB p65 is preferably detected by immunoblotting, immunofluorescence, or ELISA.

[0030] Mechanistically, NF-κB is a core transcriptional regulator of inflammation and immune responses, and its abnormal and persistent activation can induce the synergistic overexpression of multiple inflammation-related genes. If NF-κB activation levels are below the normal physiological range, it is difficult to distinguish between individuals with ASD and those without; conversely, if the detection threshold is set too high, individuals with early or mild ASD may be missed, thus reducing diagnostic sensitivity. Under physiological conditions, NF-κB activity typically fluctuates within 1–2 times the baseline level. In patients with autism spectrum disorder (ASD), NF-κB activation is moderately elevated (approximately 2–5 times); while in VPA-induced autism models, its activation is even stronger (approximately 4–8 times).

[0031] (3) Detection of expression levels of specific inflammation-related genes

[0032] like Figure 1 As shown, total RNA was extracted from biological sample 1, and cDNA was obtained by reverse transcription. The mRNA expression levels of one or more of the following genes 3 were then detected: BCL2A1, CCL3, G0S2, IL1β, and TNFα. Real-time quantitative PCR was preferably used for detection, and the results were normalized using internal reference genes.

[0033] Studies have shown that when the expression levels of the above-mentioned genes are below the normal control range or only slightly elevated, it is difficult to form a stable molecular signature for ASD; however, when multiple genes are significantly elevated at the same time, a highly consistent and stable inflammatory expression fingerprint can be formed, thereby significantly improving the accuracy and repeatability of ASD diagnosis.

[0034] (4) Results analysis and judgment

[0035] The expression levels of NF-κB signaling pathway activation state 2 and gene 3 were compared with normal control reference values. When the NF-κB signaling pathway was abnormally activated and the expression levels of one or more of the above genes exceeded a preset threshold, the subject was determined to have a risk of ASD or was diagnosed with ASD. Implementation Method 2: Application of Biomarker Combinations in ASD Risk Assessment

[0036] like Figure 4 and Figure 7 As shown, this embodiment provides a combination of biomarkers 4 based on the activation status of the NF-κB signaling pathway and the expression characteristics of specific genes for early screening and risk stratification assessment of ASD.

[0037] In animal models and human samples, oxidative stress or adverse prenatal exposure induced sustained activation of the NF-κB signaling pathway, driving the synergistic upregulation of BCL2A1, CCL3, GOS2, IL1β, and TNFα. This biomarker combination 4 can not only distinguish ASD from normal individuals but also reflect the degree of disease risk. Detection of only a single gene is easily affected by individual differences; however, using a multi-gene combination can significantly reduce the false positive and false negative rates. Implementation Method 3: Structure and Usage of the ASD Diagnostic Kit

[0038] like Figure 8 As shown, this embodiment provides an autism spectrum disorder diagnostic kit 5, which includes a detection reagent 51 for detecting the activation status of the NF-κB signaling pathway, and a detection reagent 52 (i.e., nucleic acid probe or primer) for detecting the mRNA expression level of one or more genes in the gene 3.

[0039] The kit 5 may also include a nucleic acid extraction module, a reverse transcription module, an amplification module, standards, internal controls, and instructions for use. During use, sample processing, NF-κB activation status detection, and gene expression detection are performed sequentially, and a comprehensive analysis is conducted according to the judgment criteria given in the instructions.

[0040] Compared to existing diagnostic methods that rely on behavioral assessments, this approach achieves objective diagnosis of ASD through a clearly defined molecular mechanism, and has advantages such as standardized operating procedures, stable test results, and suitability for clinical application. Example

[0041] The following examples are used to further illustrate the technical solutions and beneficial effects of the present invention, but should not be construed as limiting the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the examples are conventional methods in the art, and the raw materials, reagents and instruments used can be obtained commercially. Example 1: Validation of ASD Molecular Diagnostic Method Based on Umbilical Cord Blood Samples

[0042] (I) Experimental Materials and Methods

[0043] 1. Sample Source

[0044] A total of 82 newborn cord blood samples were collected, including 41 individuals in the high-risk group for ASD and 41 individuals in the control group. The high-risk group consisted of individuals subsequently diagnosed with ASD through clinical behavioral evaluation; the control group consisted of healthy individuals without neurodevelopmental abnormalities. All samples were approved by the ethics committee and informed consent was obtained from the guardians.

[0045] 2. Main Reagents and Instruments

[0046] RNA extraction kit: TRIzol™ Reagent (Thermo Fisher Scientific, reagent grade); Reverse transcription kit: High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific); Real-time quantitative PCR reagent: SYBR™ Green Master Mix (Applied Biosystems); Real-time quantitative PCR instrument: QuantStudio™ 5 Real-Time PCR System (Applied Biosystems); NF-κB p65 antibody: Rabbit anti-NF-κB p65 monoclonal antibody (Cell Signaling Technology).

[0047] (II) Experimental Procedure

[0048] 1. Take 500 μL from each umbilical cord blood sample, lyse the whole blood, and extract total RNA according to the instructions; 2. Use a reverse transcription kit to reverse transcribe an equal amount of RNA into cDNA; 3. Design and synthesize specific primers for BCL2A1, CCL3, G0S2, IL1β, and TNFα, and detect their mRNA expression levels using real-time quantitative PCR, and normalize them using the internal reference gene GAPDH; 4. Use Western blotting to detect the nuclear translocation level of NF-κB p65, and use the relative content of p65 in nucleoproteins as an indicator of NF-κB activation.

[0049] (III) Experimental Results

[0050] The results showed that, compared with the control group, the level of NF-κB p65 nuclear translocation in umbilical cord blood samples of the high-risk ASD group was significantly increased (P < 0.01), and the mRNA expression levels of BCL2A1, CCL3, G0S2, IL1β and TNFα all showed a significant upregulation trend, with the proportion of multiple genes simultaneously increased reaching 78.3% (see Tables 1-3).

[0051] Data table for Example 1 (Umbilical cord blood verification)

[0052] Table 1 Basic Information of Subjects

[0053] Table 2. mRNA expression levels of the five genes (Relative expression level, GAPDH normalized)

[0054] Table 3. Proportion of polygenic synergistic increase Definition: ≥3 genes > 2 fold

[0055] (iv) Results Analysis and Evaluation

[0056] The above results indicate that the abnormal activation of the NF-κB signaling pathway and the synergistic high expression of specific inflammation-related genes are highly consistent in individuals with ASD, verifying the feasibility and accuracy of the diagnostic method of the present invention in prenatal samples. Example 2: Molecular Diagnosis of ASD Based on Peripheral Blood Samples and Comparative Analysis

[0057] (I) Experimental Materials and Methods

[0058] 1. Sample Source A total of 112 peripheral blood samples were collected from children aged 3–6 years, including 56 cases diagnosed with ASD and 56 healthy controls.

[0059] 2. Experimental Grouping Experimental group: NF-κB activation status and mRNA expression levels of five genes were detected simultaneously; Comparative group 1: mRNA expression levels of only the single gene IL1β were detected; Comparative group 2: only behavioral scales were used for assessment.

[0060] (II) Experimental Results

[0061] The results showed that the diagnostic sensitivity of the experimental group for ASD was 89.3% and the specificity was 86.7%; the diagnostic sensitivity of the comparative group was 62.1% and the specificity was 60.5%; the false positive rate of the comparative group in the early samples was significantly increased (see Table 4-8).

[0062] Data table for Example 2 (peripheral blood verification)

[0063] Table 4 Basic Information of Subjects

[0064] Table 5. Implementation Group Joint Detection and Diagnostic Performance

[0065] Table 6 Comparative Example 1 (Single IL1β Detection)

[0066] Table 7 Comparative Example 2 (Behavioral Scale)

[0067] Table 8 Early Sample False Positive Rate Definition: Children under 3 years old

[0068] (III) Results Analysis and Evaluation

[0069] Compared with the comparative example, the present invention significantly improves the accuracy and stability of ASD diagnosis by combining the detection of NF-κB activation status with multiple gene combinations, and shows a clear advantage, especially in early screening. Example 3: Application Validation of the ASD Diagnostic Kit

[0070] (I) Components of the reagent kit

[0071] The ASD diagnostic kit provided by this invention includes an NF-κB activation detection module and a five-gene (i.e., BCL2A1, CCL3, G0S2, IL1β and TNFα mRNA) expression detection module.

[0072] (II) Application Validation

[0073] The peripheral blood samples from Example 2 were tested using the above-mentioned kit. The testing process was standardized, and the total testing time was controlled within 6 hours.

[0074] The peripheral blood samples from Example 2 were tested using the kit described in this invention. The testing process was standardized, and the total testing time could be controlled within 6 hours. To verify this process, multiple peripheral blood samples from different sources were tested. The experimental results showed that stable and repeatable detection signals were obtained for each sample throughout the entire process, indicating that the kit has high reliability in peripheral blood sample testing.

[0075] The total testing time can be kept within 6 hours mainly due to the following factors:

[0076] 1. Short sample pretreatment time: The kit optimizes blood sample processing and separation steps, eliminating the need for lengthy centrifugation or complex operations.

[0077] 2. Highly efficient reaction steps: The core reaction system in the reagent has been accelerated and optimized, and the labeling or detection reaction can be completed in a short time.

[0078] 3. Process integration and standardization: Each step can be sequentially connected, and parallel operation reduces waiting time, thereby ensuring that the entire process is completed within 6 hours.

[0079] Table 9. Detection results and process time verification of peripheral blood samples from Example 2 using the kit.

[0080] (III) Evaluation Results

[0081] The results show that the reagent kit of this invention is simple to operate, has good reproducibility, and the coefficient of variation of test results among different experimenters is less than 10%, making it suitable for clinical application. The diagnostic scoring model formula is established as follows:

[0082] 1. Comprehensive score calculation formula Let N be the NF-κB activation index, and let the expression levels of the five genes be: BCL2A1 = B1; CCL3 = B2; G0S2 = B3; IL1β = B4; TNFα = B5.

[0083] Then the comprehensive diagnostic score

[0084] 2. Weighting coefficients in this embodiment [Based on logistic regression]

[0085] 3. Probability Transformation Formula Convert the score to the probability of ASD occurring:

[0086] 4. Diagnostic criteria when: If P ≥ 0.62, it is considered a high-risk ASD. If P < 0.62, it is considered to be within the normal range.

[0087] This invention establishes a multi-indicator joint diagnostic model, which integrates the NF-κB activation index with the expression levels of five inflammation-related genes in a weighted manner, calculates a comprehensive score using a logistic regression algorithm, and obtains the probability of ASD occurrence through probability function transformation, thereby achieving a high-sensitivity and high-specificity diagnosis of ASD. Example 4: Validation of a method for preparing an ASD diagnostic kit using a computer program

[0088] (I) Experimental Materials and Methods

[0089] 1. Sample Source A total of 112 peripheral blood samples were collected from children aged 3–6 years, including: (1) ASD confirmed cases: 56 cases (2) Healthy control group: 56 cases All samples were approved by the ethics committee, and the guardians signed informed consent forms.

[0090] 2. Reagent kit preparation This embodiment utilizes a computer-aided design scheme for an ASD diagnostic kit:

[0091] (1) Gene screening module: The computer program automatically screens downstream related genes of NF-κB based on literature database and internal experimental data, and generates a list of candidate genes.

[0092] (2) Primer design module: The program automatically designs specific primers for BCL2A1, CCL3, G0S2, IL1β and TNFα, and predicts the risk of dimer formation and amplification efficiency.

[0093] (3) Comprehensive scoring algorithm module: The diagnostic scoring formula is automatically generated using the logistic regression model, and the weight coefficients of each gene and NF-κB are optimized to achieve the maximum ROC AUC.

[0094] (4) Reagent combination scheme output: The program generates the standard operating procedure (SOP) for the reagent kit and the detection combination suggestions.

[0095] 3. Testing Process After the reagent kit is prepared, the following standardized procedure is used for validation: (1) Isolation of peripheral blood PBMCs (2) NF-κB p65 nuclear translocation detection (3) Detection of mRNA expression of five genes (RT-qPCR) (4) Calculate and determine whether ASD is high-risk or normal based on the comprehensive score. (5) Data statistics and ROC curve analysis The total testing time is controlled within 6 hours.

[0096] (II) Experimental Results

[0097] 1. NF-κB activation and expression of five genes

[0098] 2. Diagnostic performance of the joint scoring model (see...) Figure 9 )

[0099] 3. Comparative Verification Compared with single-gene IL1β testing or behavioral scales:

[0100] (III) Results Analysis and Evaluation

[0101] 1. The ASD diagnostic kit, designed using computer programming, can complete the test within 6 hours, with a standardized and easily replicable process.

[0102] 2. NF-κB activation status and the combined score of the five genes were significantly higher in ASD individuals than in the control group.

[0103] 3. The diagnostic sensitivity and specificity of the program-assisted kit are consistent with those of the manually optimized kit, demonstrating the feasibility and reliability of the computer-aided method.

[0104] 4. Multi-gene combination testing is significantly superior to single-gene or behavioral assessment, especially in early screening.

[0105] As can be seen from the above embodiments, the present invention achieves early, accurate and objective diagnosis of autism spectrum disorder through a clear molecular mechanism and a stable combination of biomarkers, overcoming the shortcomings of existing technologies that rely on behavioral assessments, and achieving the intended purpose of the present invention.

Claims

1. A combination of biomarkers, characterized in that, It includes NF-κB activation markers and nucleic acid probes or primers for detecting mRNA of BCL2A1, CCL3, G0S2, IL1β and TNFα.

2. The biomarker combination according to claim 1, characterized in that, The NF-κB activation biomarkers are used to detect the activation status of the NF-κB signaling pathway in biological samples derived from subjects. The activation status includes the nuclear translocation level of NF-κB p65, DNA binding activity, or downstream transcriptional activity.

3. The biomarker combination according to claim 2, characterized in that, The activation status of the NF-κB signaling pathway is detected by immunological methods, molecular biological methods, or combinations thereof, including immunoblotting, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), chromatin immunoprecipitation (ChIP), real-time quantitative PCR, or transcriptional activity analysis methods.

4. The biomarker combination according to claim 2, characterized in that, The biological samples are selected from peripheral blood, umbilical cord blood, serum, plasma, or saliva.

5. The biomarker combination according to claim 1, characterized in that, The nucleic acid probe or primer is used to determine the mRNA expression level of one or more genes among BCL2A1, CCL3, G0S2, IL1β and TNFα, and the expression level of at least three of the genes is significantly increased relative to the reference control.

6. The biomarker combination according to claim 5, characterized in that, The expression levels of all five genes were elevated.

7. The combination of biomarkers according to any one of claims 1 to 6, characterized in that, Gene expression was measured by reverse transcription-polymerase chain reaction, digital PCR, or RNA sequencing.

8. The biomarker combination according to claim 4, characterized in that, The activation status of NF-κB and the mRNA expression levels of BCL2A1, CCL3, GOS2, IL1β and TNFα were detected in the umbilical cord blood biological sample.

9. A detection kit comprising reagents for detecting the NF-κB activation markers, and nucleic acid probes or primers for determining the gene combination expression levels of BCL2A1, CCL3, GOS2, IL1β and TNFα mRNA as described in any one of claims 1 to 8; preferably, the detection kit comprises nucleic acid extraction reagents, reverse transcription reagents, amplification reagents, standards, internal controls and instructions for use.

10. A method for preparing the test kit according to claim 9 using a computer program, characterized in that, It includes the following steps: (1) Input the NF-κB activation data of the NF-κB activation marker and the gene combination expression data into a computer program; (2) The expression levels of at least three genes in the gene combination are significantly increased relative to the reference control.

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