Variable splicing marker for tuberculosis diagnosis, screening method and application

Abstract

The invention discloses a variable splicing marker for tuberculosis diagnosis, a screening method and application, and relates to the technical field of marker screening, and the technical key points are as follows: the variable splicing marker is AIM2-Intron3-IR; by combining bioinformatics analysis, in-vitro experiments and clinical queue verification analysis of a large amount of high-throughput sequencing data, variable splicing markers closely related to tuberculosis are mined and determined, and a high-efficiency new tool is provided for tuberculosis diagnosis.

Description

Technical Field

[0001] The present invention relates to the technical field of marker screening, and in particular to a variable splicing marker for tuberculosis diagnosis, a screening method and an application thereof. Background Art

[0002] The high incidence of tuberculosis requires early diagnosis. Currently, only 63% of patients have laboratory evidence, indicating that existing diagnostic capabilities cannot meet laboratory requirements and that new markers need to be developed urgently.

[0003] Host-derived transcriptome markers have been shown to reflect the occurrence and development of tuberculosis, and have low detection costs and short cycles, making them a new type of tuberculosis marker that has attracted widespread attention. Existing transcriptome markers are mainly screened based on "changes in expression levels", ignoring the impact of internal heterogeneity of genes. Variable splicing treats a gene as multiple different transcripts, which can reflect changes in the composition of different transcripts, transcript expression, and the ratio between the above transcripts. This shows that variable splicing can reflect the occurrence and development of tuberculosis from multiple angles and has the characteristics of being a highly sensitive marker.

[0004] To this end, the present invention aims to provide a variable splicing marker for tuberculosis diagnosis, a screening method and application to solve the above problems. Summary of the invention

[0005] The purpose of the present invention is to solve the above-mentioned problems and provide a variable splicing marker, screening method and application for diagnosing tuberculosis. The present invention mines and determines the variable splicing markers closely related to tuberculosis by combining bioinformatics analysis of a large amount of high-throughput sequencing data, in vitro experiments and clinical cohort validation analysis, thereby providing a new and efficient tool for predicting the progression and diagnosis of tuberculosis.

[0006] In order to achieve the above object, the technical solution of the present invention is as follows:

[0007] The invention provides a variable splicing marker for diagnosing tuberculosis, the variable splicing marker being AIM2-intron3-IR.

[0008] A screening method and application of variable splicing markers for tuberculosis diagnosis, the screening method comprising the following steps:

[0009] S1. We used bioinformatics to analyze 1706 raw data sets from GEO, SRA, and EBI databases, and used IRFinder (v 1.3.1) to analyze and compare the differential intron retention events IR in blood leukocytes between healthy controls and TB-infected groups. We performed differential analysis on IR events, and used splicing percentages to quantify alternative splicing events.

[0010] The results showed that AIM2-intron3-IR was significantly upregulated in the TB group, so the AIM2-intron3-IR intron retention event is a potential diagnostic marker for TB;

[0011] S2. Verify the results:

[0012] (1) Total RNA was extracted from 100 healthy controls and 100 TB patients using the Tizol method;

[0013] (2) Real-time fluorescence quantitative PCR was used to verify the candidate events. The PCR data copied by the instrument were analyzed by 2 -△△Ct The abundance of intron retention events was evaluated using PSI.

[0014] (3) The analysis results showed that the PSI value of AIM2-intron3-IR events in the TB infection group was significantly higher than that in the normal group, and the difference was statistically significant (P<0.001);

[0015] The alternative splicing marker AIM2-intron3-IR is used to predict tuberculosis progression.

[0016] In this program,

[0017] Compared with the prior art, this solution has the following beneficial effects:

[0018] 1. The discovery of alternative splicing in the present invention allows researchers to reveal subtle changes in transcription levels at a more detailed level, which can reflect changes in transcript expression, structure, and ratios between transcripts, and reflect disease-related signals from multiple angles. This feature determines that alternative splicing has the potential to be a highly sensitive marker;

[0019] 2. The present invention has clearly identified AIM2-intron3-IR as a key splicing event in the onset of tuberculosis by integrating bioinformatics analysis of a large amount of sequencing data, in vitro experiments and clinical cohort validation studies. By combining candidate markers and electronic medical records, a TB diagnostic model can be constructed using a variety of machine learning algorithms, which can further improve the diagnostic performance of tuberculosis and is an effective supplement to existing etiological detection;

[0020] 3. The variable splicing events involved in the present invention can be detected by qPCR in clinical practice. qPCR has a short detection cycle, low cost and relatively high accuracy. Therefore, this model is easier to promote in poor areas with a high incidence of tuberculosis;

[0021] 4. The sample type used for detecting variable shearing in the present invention is peripheral blood, which is relatively less affected by sampling factors (compared to sputum samples commonly used for tuberculosis), and is easy to obtain, store and transport. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a diagram showing the difference analysis results of the AIM2-intron3-IR events in this published embodiment;

[0023] Figure 2 This is a schematic diagram of expression changes in the in vitro infection model of the AIM2-intron3-IR event in an embodiment of the present invention;

[0024] Figure 3 It is a schematic diagram of clinical cohort verification of AIM2-intron3-IR events in an embodiment of the present invention. DETAILED DESCRIPTION

[0025] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0026] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present invention can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0027] Example:

[0028] The present invention provides an alternative splicing marker AIM2-intron3-IR for diagnosing tuberculosis. A screening method and application of an alternative splicing marker for diagnosing tuberculosis, the screening method comprising the following steps:

[0029] S1. Identify the AIM2-intron3-IR intron retention event as a potential diagnostic marker for TB

[0030] Firstly, 1706 original data sets from GEO, SRA and EBI databases were analyzed using bioinformatics technology, and IRFinder (v 1.3.1) was used to analyze and compare the differential intron retention events in blood leukocytes of healthy control group and TB infection group.

[0031] Further differential analysis of significantly altered IR events was performed, and alternative splicing events were quantified using splicing percentage. The results showed that AIM2-intron3-IR was significantly upregulated in the TB group. Figure 1 As shown, A is a violin plot showing the quantitative value of intron 3 retention of the AIM2 gene between the two groups.

[0032] S2. Verification of AIM2-intron3-IR intron retention events using in vitro infection model

[0033] To further clarify the relationship between the AIM2-intron3-IR intron retention event and the occurrence and development of TB, its dynamic changes under different infection degrees were observed. In THP-1 cells, the attenuated inactivated strain H37Ra (HKMT) was used to construct a TB infection model. qPCR results showed that under different treatment concentrations, the retention of intron 3 of the AIM2 gene showed different degrees of upregulation and was related to the infection time. Figure 2 As shown, AB is the phase change of the retention up-regulation ratio of intron 3 of AIM2 gene in HKMT infection model detected by RT-PCR; * indicates compared with the control group, *, P<0.05, **, P<0.01, ***, P<0.001, n=3 / group.

[0034] S3. Validation of AIM2-intron3-IR intron retention events in clinical cohorts

[0035] To further evaluate the value of AIM2-intron3-IR in the diagnosis of tuberculosis, this study included two groups of clinical research subjects, a healthy control group and a confirmed tuberculosis patient group, with 100 cases in each group, for a total of 200 cases. The Trizol method was used to extract total RNA from peripheral blood leukocytes, and then fluorescent quantitative PCR was used to detect AIM2-intron3-IR events, and PSI was used to quantify splicing events. The results showed that the number of AIM2-intron3-IR events in the tuberculosis patient group (TB) increased significantly, and the difference was statistically significant. Figure 3 As shown, Figure 3 The ratio of intron 3 retention events of AIM2 gene in healthy control group and TB patient group was detected by RT-PCR. * indicates that compared with the control group, ****, P<0.0005, n=3 / group.

[0036] The above specific embodiments are merely explanations of the present invention and are not limitations of the present invention. After reading this specification, those skilled in the art may make modifications to the embodiments without any creative contribution as needed. However, such modifications are protected by the patent law as long as they are within the scope of the claims of the present invention.

Claims

1. An alternative splicing marker for tuberculosis diagnosis, characterized in that: The variable splicing marker is AIM2-intron3-IR.

2. The screening method and application of an alternative splicing marker for tuberculosis diagnosis according to claim 1, characterized in that: The screening method comprises the following steps: S1. 1706 original data sets from GEO, SRA and EBI databases were analyzed by bioinformatics, and IRFinderv1.3.1 was used to analyze and compare the intron retention events IR between the healthy control group and the TB infection group. S2. We performed differential analysis on IR events and used the percentage of splicing to quantify alternative splicing events, referred to as PSI. The results showed that AIM2-intron3-IR was significantly upregulated in the TB group, so AIM2-intron3-IR intron retention events are potential diagnostic markers for TB. S3. Verify the results: (1) Total RNA was extracted from 100 healthy controls and 100 TB patients using the Tizol method; (2) Real-time fluorescence quantitative PCR was used to verify the candidate events. The abundance of intron retention events was evaluated using PSI. (3) The analysis results showed that the PSI value of AIM2-intron3-IR events in the TB infection group was significantly higher than that in the normal group, and the difference was statistically significant (P<0.001); The alternative splicing marker AIM2-intron3-IR is used to predict tuberculosis progression.

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