CN-121992097-A - Biomarker combination, detection kit and preparation method thereof

Abstract

The present invention relates to biomarker combinations comprising NF- κb activation markers and nucleic acid probes or primers for determining mRNA expression levels of one or more of BCL2A1, CCL3, G0S2, il1β and tnfα, the expression levels of at least three of the genes being significantly increased relative to a reference control, and detection kits 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, G0S2, il1β and tnfα mRNA as described above, and in addition to methods for preparing the detection kits as described above using computer programs. The invention combines the activation state of NF- κB signal path with the expression characteristic of specific inflammation related gene for the first time, and constructs a stable and consistent ASD molecular diagnosis system.

Inventors

• YAO DACHUN

Assignees

• 姚大纯
• Seawire咨询与贸易有限公司

Dates

Publication Date

20260508

Application Date

20260214

Claims (10)

1. 1. A biomarker combination comprising an NF- κb activation marker and a nucleic acid probe or primer for detecting mRNA of BCL2A1, CCL3, G0S2, il1β and tnfα.
2. 2. The biomarker combination according to claim 1, wherein the NF- κb activation marker is used to detect the activation status of NF- κb signaling pathway in a biological sample derived from a subject, the activation status comprising nuclear translocation level, DNA binding activity or downstream transcriptional activity of NF- κ B p.
3. 3. The biomarker combination according to claim 2, wherein the activation state 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 fluorescent quantitative PCR or transcriptional activity analysis methods.
4. 4. The biomarker combination according to claim 2, wherein the biological sample is selected from peripheral blood, umbilical cord blood, serum, plasma or saliva.
5. 5. The biomarker combination according to claim 1, wherein the nucleic acid probes or primers are used to determine mRNA expression levels of one or more of BCL2A1, CCL3, G0S2, il1β and tnfα, and the expression levels of at least three of the genes are significantly increased relative to a reference control.
6. 6. The biomarker combination according to claim 5, wherein the expression levels of the five genes are increased.
7. 7. The biomarker combination according to any of claims 1 to 6, wherein gene expression is determined by reverse transcription-polymerase chain reaction, digital PCR or RNA sequencing.
8. 8. The biomarker combination according to claim 4, wherein NF- κb activation status and mRNA expression levels of BCL2A1, CCL3, G0S2, IL1 β and tnfa are detected in a biological sample of the umbilical cord blood.
9. 9. A detection kit comprising reagents for detecting the NF- κB activation marker and nucleic acid probes or primers for determining the gene combination expression levels of BCL2A1, CCL3, G0S2, IL1 β and TNFα mRNA according to any one of claims 1 to 8, preferably the detection kit comprises nucleic acid extraction reagents, reverse transcription reagents, amplification reagents, standards, internal control and instructions for use.
10. 10. A method for preparing a detection kit according to claim 9 by means of a computer program, characterized in that it comprises the following steps: (1) Inputting NF- κb activation data of the NF- κb activation marker and the gene combination expression data into a computer program; (2) Such that the expression levels of at least three genes in the gene combination are significantly increased relative to a reference control.

Description

Biomarker combination, detection kit and preparation method thereof Technical Field The invention belongs to the technical field of biomedicine, in particular relates to the technical field of molecular diagnosis and mental nerve disease detection, and especially relates to a biomarker combination and a detection kit for autism spectrum disorder (Autism Spectrum Disorder, ASD) based on NF- κB signal channel activation state and specific inflammation related gene expression characteristics, and a method for preparing the detection kit by using a computer program. Background Autism Spectrum Disorder (ASD) is a complex neuro-developmental disorder characterized mainly by social communication disorder, communication disorder and repetitive notch plate behavior, whose pathogenesis is highly heterogeneous involving multiple factors such as genetics, immunity, inflammation and environment. Currently, the clinical diagnosis of ASD mainly depends on the evaluation of behavioural scale and the long-term observation of professional doctors, such as the method of autism diagnosis observation scale (Autism Diagnostic Observation Schedule, abbreviated as ADOS) ‌, autism diagnosis interview scale revision (Autism Diagnostic Interview-Revised, abbreviated as ADI-R) ‌, and the like, and the method has the defects of strong subjectivity of diagnosis, experience dependence, lag of diagnosis time point, difficulty in realizing early screening, risk stratification and the like. Recent studies have shown that immune abnormalities and chronic inflammatory responses play an important role in the development and progression of ASD. The NF- κB signal channel is used as the core channel of body inflammatory reaction and immune regulation, and its abnormal activation can induce the continuous high expression of several inflammatory factors and immune related genes. However, the prior art has lacked stable biomarker combinations and standardized detection schemes based on NF- κb signaling pathway systematic molecular characteristics that can be used for early, objective and quantifiable diagnosis of ASD. Therefore, the ASD molecular detection method and the kit based on a definite molecular mechanism and with high consistency and high stability are developed, and the method has important scientific significance and clinical application value for realizing early screening, accurate detection and risk layering evaluation of ASD. Disclosure of Invention The invention aims to provide a biomarker combination and a detection kit for Autism Spectrum Disorder (ASD) based on objective molecular biological indexes and a method for preparing the detection kit by using a computer program, so as to overcome the defects that ASD diagnosis mainly depends on behavioral assessment, has strong subjectivity, is difficult to realize early screening, risk layering assessment and the like in the prior art. By detecting the activation state of NF- κB signal channel and the expression characteristics of specific inflammation related genes, early, accurate, minimally invasive or noninvasive diagnosis of ASD is realized. In order to achieve the aim of the invention, the invention adopts the following technical scheme: 1. A biomarker combination for ASD diagnosis comprising NF- κb activation markers and nucleic acid probes or primers for detecting BCL2A1, CCL3, G0S2, il1β and tnfα mRNA, comprising detection indicators of the activation state of the NF- κb signaling pathway and the expression level of one or more of BCL2A1, CCL3, G0S2, il1β and tnfα genes, and the expression level of at least three of said genes is significantly increased relative to a reference control. The biomarker combination according to the above, for: (1) Obtaining a biological sample of a subject, the biological sample being a sample obtained either prenatally or postnatally, preferably a peripheral blood, umbilical cord blood, serum, plasma, saliva or other body fluid sample; (2) Detecting an activation state of an NF- κb signaling pathway in the biological sample, including but not limited to nuclear translocation levels, DNA binding activity, or downstream transcriptional activity of NF- κ B p 65; (3) Detecting in the biological sample mRNA expression levels of one or more of BCL2A1, CCL3, G0S2, il1β, and tnfα; (4) Comparing the detection results of step (2) and step (3) with a normal control reference value, and determining that the subject is at risk of or diagnosed with ASD when the NF- κb signaling pathway is abnormally activated and the expression level of one or more of the genes is significantly increased. The biomarker combination according to the above, wherein the activation state of NF- κb signaling pathway is detected by immunological methods, molecular biological methods or combinations thereof, including but not limited to immunoblotting, immunofluorescence, enzyme-linked immunosorbent assay (Enzyme-Linked Immunosorbent Assay, ELISA for short), chromatin immunoprecipitati