Serum sncRNA markers for diagnosing mycobacterium tuberculosis infection and application of serum sncRNA markers

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a group of serum sncRNA markers for diagnosing mycobacterium tuberculosis infection and application of the serum sncRNA markers. And the sncRNA marker combination is a combination of any one of tsRNA-iMet-CAT, 28S-rsRNA.3 and snsRNA.1 or a combination of any two or three of the tsRNA-iMet-CAT, the 28S-rsRNA.3 and the snsRNA.1. The three markers are of great significance to early diagnosis, prediction and / or detection of mycobacterium tuberculosis infection, and meanwhile, a universal clinical mycobacterium tuberculosis infection detection product, such as a kit for clinical diagnosis of mycobacterium tuberculosis infection, can be developed and can be used for clinical auxiliary diagnosis of mycobacterium tuberculosis infection. And a research direction is provided for preparing medicines for treating mycobacterium tuberculosis infection.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a set of serum sncRNA markers for diagnosing Mycobacterium tuberculosis infection and their applications. Background Technology

[0002] Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (MTB), with pulmonary tuberculosis being the most common form, posing an extremely high public health burden. According to the World Health Organization's (WHO) 2023 Global TB Report, an estimated 10.6 million new TB cases and 1.3 million deaths occurred globally in 2022, making TB the leading cause of death from a single pathogen. Currently, common diagnostic methods for active TB include: sputum smears and cultures: while highly specific, their sensitivity is limited by sample quality and the culture period is long (2-8 weeks); imaging studies (such as chest X-rays and CT scans): these are highly auxiliary but lack specificity and struggle to differentiate between active and inactive lesions; immunological tests: tuberculin skin test (TST): easily interfered with by BCG inoculation; interferon release assay (IGRA): while possessing some sensitivity, it cannot distinguish between latent infection (LTBI) and active disease.

[0003] In recent years, with the development of high-throughput sequencing and liquid biopsy technologies, researchers have discovered a variety of disease-related non-coding RNAs (ncRNAs) in serum, such as microRNAs (miRNAs), tRNA-derived small RNAs (tsRNAs), and long non-coding RNAs (lncRNAs). These non-coding RNAs exhibit highly reproducible expression differences across various disease states and are increasingly considered to be promising new molecular biomarkers.

[0004] However, no studies have been reported on the use of serum sncRNA in the diagnosis of Mycobacterium tuberculosis. Serum sncRNA has great development potential and broad application prospects as a diagnostic biomarker for Mycobacterium tuberculosis in liquid biopsy. Summary of the Invention

[0005] The purpose of this invention is to provide a set of serum sncRNA markers for the diagnosis of Mycobacterium tuberculosis infection and their applications, so as to achieve effective diagnosis of Mycobacterium tuberculosis infection.

[0006] Therefore, the present invention provides the following technical solution.

[0007] The first aspect of the present invention provides a set of serum sncRNA biomarkers for diagnosing Mycobacterium tuberculosis infection, said sncRNA biomarker combination being any one of tsRNA-iMet-CAT, 28S-rsRNA.3 and snsRNA.1 or a combination of any two or three of said biomarkers.

[0008] In a preferred embodiment of the present invention, the sncRNA marker combination is a combination of three markers: tsRNA-iMet-CAT, 28S-rsRNA.3, and snsRNA.1.

[0009] In a preferred embodiment of the present invention, the nucleotide sequence of the tsRNA-iMet-CAT is shown in SEQ ID NO:1, the nucleotide sequence of the 28S-rsRNA.3 is shown in SEQ ID NO:2, and the nucleotide sequence of the snsRNA.1 is shown in SEQ ID NO:3.

[0010] In a preferred embodiment of the present invention, the sncRNA biomarker is derived from the blood of the subject.

[0011] A second aspect of the invention provides the use of the sncRNA biomarker combination as described above in the preparation of formulations for the detection, screening and / or diagnosis of Mycobacterium tuberculosis infection.

[0012] A third aspect of the invention provides the use of a reagent for detecting the levels of the combination of sncRNA markers as described above in the preparation of products for detecting, screening, and / or diagnosing Mycobacterium tuberculosis infection.

[0013] In a preferred embodiment of the present invention, the reagent includes reagents suitable for at least one of the following methods: fluorescent dye method, digital PCR, resonance light scattering method, real-time quantitative PCR, sequencing or biomolecular mass spectrometry.

[0014] In a preferred embodiment of the present invention, the product is a chip, a reagent kit, a test strip, or an analysis platform.

[0015] A fourth aspect of the present invention provides a kit for Mycobacterium tuberculosis infection, comprising reagents for detecting sncRNA markers as described above, optionally, the detection being a quantitative detection of sncRNA marker levels in the plasma of a subject.

[0016] In a preferred embodiment of the present invention, the reagent includes reagents suitable for at least one of the following methods: fluorescent dye method, digital PCR, resonance light scattering method, real-time quantitative PCR, sequencing or biomolecular mass spectrometry.

[0017] In a preferred embodiment of the present invention, the kit comprises: (1) Primers capable of reverse transcription of tsRNA-iMet-CAT, 28S-rsRNA.3 or snsRNA.1, wherein the nucleotide sequences of the primers are shown in SEQ ID NO: 4-6 respectively; (2) Primers capable of PCR amplification of cDNA corresponding to tsRNA-iMet-CAT, 28S-rsRNA.3 or snsRNA.1, wherein the nucleotide sequences of the primers are shown in SEQ ID NO: 7-12 respectively.

[0018] In a preferred embodiment of the present invention, the kit further includes dNTP / AMV reverse transcriptase and buffer, MgCl2, DEPC water and Taq DNA polymerase.

[0019] A fifth aspect of the invention provides a method for assessing whether a drug can prevent or treat Mycobacterium tuberculosis infection, comprising using any one, two, or three of tsRNA-iMet-CAT, 28S-rsRNA.3, and snsRNA.1 as sncRNA markers to assess the efficacy of the drug.

[0020] A sixth aspect of the invention provides a method for screening compounds capable of preventing or treating Mycobacterium tuberculosis infection, comprising evaluating the efficacy of the compounds using any one, two, or three of tsRNA-iMet-CAT, 28S-rsRNA.3, and snsRNA.1 as sncRNA markers.

[0021] By employing the above technical solution, the present invention has at least the following advantages: This invention isolates serum from sex- and age-matched patients with Mycobacterium tuberculosis infection and healthy controls, extracts RNA, and performs high-throughput sequencing analysis. Through preliminary screening with small samples and large-scale clinical validation, a set of sncRNA biomarkers associated with Mycobacterium tuberculosis infection—tsRNA-iMet-CAT, 28S-rsRNA.3, and snsRNA.1—was obtained. Studies have demonstrated that these three biomarkers are of great significance for the early diagnosis, prediction, and / or detection of Mycobacterium tuberculosis infection. Furthermore, this invention provides a basis for developing a universally applicable clinical detection product for Mycobacterium tuberculosis infection, such as a kit for the clinical diagnosis of Mycobacterium tuberculosis infection, which can be used for auxiliary clinical diagnosis of Mycobacterium tuberculosis infection and provides research directions for the preparation of drugs to treat Mycobacterium tuberculosis infection. The biomarker combination screened according to the method of this invention has high sensitivity and specificity, overcoming the shortcomings of current diagnostic methods for Mycobacterium tuberculosis infection. The biomarkers of this invention can achieve early diagnosis of Mycobacterium tuberculosis infection.

[0022] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below. Attached Figure Description

[0023] Figure 1 (A) Volcano plot of differential sncRNA expression in serum between the tuberculosis group and the normal group; (B) Heatmap of differential sncRNA expression in serum between the tuberculosis group and the normal group; Figure 2 Differential expression analysis of serum sncRNA in internal validation set samples; Figure 3 ROC curves for three serum differentially expressed sncRNAs in the internal validation set samples; Figure 4 Differential expression analysis of three serum sncRNAs in the external validation set samples; Figure 5 ROC curves for three serum differentially expressed sncRNAs in the external validation set samples. Detailed Implementation

[0024] To make the technical means, creative features, achieved objectives, and effects of this invention readily understandable, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0025] Mycobacterium tuberculosis is a major pathogenic factor causing pulmonary tuberculosis, seriously endangering patients' lives, health, and quality of life. To address this issue, this invention provides a set of serum sncRNA biomarkers for diagnosing Mycobacterium tuberculosis infection. The sncRNA biomarker combination is any one of tsRNA-iMet-CAT, 28S-rsRNA.3, and snsRNA.1, or a combination of any two or three of these biomarkers.

[0026] It should be noted that the sequence information of tsRNA-iMet-CAT, 28S-rsRNA.3, and snsRNA.1 in this invention is shown in the table below: tsRNA-iMet-CAT belongs to the tRNA-derived small RNA (tsRNA) family, produced by enzymatic cleavage of mature tRNA or tRNA precursors, and is typically 15-40 nt in length. iMet-CAT may originate from a specific cleavage fragment of initiating methionine tRNA (tRNAiMet). Its function is similar to miRNA: it targets mRNA through AGO protein binding, regulating translation repression or mRNA stability18; it regulates protein synthesis, such as by binding to ribosomal components to inhibit translation initiation, or by interfering with translation factors through the formation of RNA G tetramers13; and it is associated with stress responses: its expression is upregulated under pathological conditions such as liver injury, potentially participating in cell protection mechanisms. Related studies have shown that it can serve as a potential biomarker for diseases such as liver cancer; for example, serum tRF-Gln-TTG-006 has been shown to be used for early diagnosis. However, no studies have yet demonstrated a correlation between tsRNA-iMet-CAT and Mycobacterium tuberculosis.

[0027] 28S-rsRNA.3 is a small ribosomal RNA-derived RNA (rsRNA), produced by the cleavage of 28S rRNA, and is approximately 20-30 nt in length. 28S rRNA is a core component of the large subunit of the eukaryotic ribosome. It may be involved in rRNA quality control: clearing abnormal or damaged 28S rRNA fragments to maintain ribosome function; and regulating gene expression: similar to other rsRNAs, it may affect translation efficiency by binding to mRNA or proteins. Currently, there is limited research on the specific functions of 28S-rsRNA.3, but rsRNA as a whole is considered to play a regulatory role in cancer and neurodegenerative diseases. However, no studies have yet demonstrated a correlation between 28S-rsRNA.3 and Mycobacterium tuberculosis infection.

[0028] snsRNA.1, or small nuclear RNA or snRNA, is a class of small RNA molecules found in the nucleus of eukaryotic cells. The average length of snRNA is approximately 150 nucleotides. snsRNA.1 is a small RNA derived from snRNA, originating from small fragments (typically 18–35 nt in length) of mature snRNA, produced by cleavage by Dicer or other nucleases. However, no studies have yet demonstrated a correlation between snsRNA.1 and Mycobacterium tuberculosis infection.

[0029] It should be noted that the three sncRNAs mentioned above can serve not only as early diagnostic biomarkers for Mycobacterium tuberculosis infection, but also as screening biomarkers and diagnostic products for Mycobacterium tuberculosis infection. They may also become targets for drug treatment.

[0030] Any one of these biomarkers can be used individually as a basis for diagnosing Mycobacterium tuberculosis infection. However, since a single biomarker may have a certain possibility of misdiagnosis, a more preferred approach to improve the accuracy and reliability of diagnosis is to combine these three biomarkers as markers for Mycobacterium tuberculosis infection.

[0031] Preferably, combining tsRNA-iMet-CAT, 28S-rsRNA.3, and snsRNA.1 can improve the sensitivity and specificity of diagnosing Mycobacterium tuberculosis infection. This combined strategy helps reduce the possibility of misdiagnosis and improves the diagnostic accuracy of Mycobacterium tuberculosis infection, providing a more reliable basis for patient treatment and management.

[0032] Therefore, although a single biomarker has some diagnostic value, the combination of three biomarkers is still preferred as the basis for diagnosing Mycobacterium tuberculosis infection to ensure the accuracy and reliability of the diagnostic results.

[0033] In another embodiment of the present invention, the use of the above-described combination of three sncRNA biomarkers in the preparation of a formulation for detecting, screening and / or diagnosing Mycobacterium tuberculosis infection is provided.

[0034] In another embodiment of the invention, a reagent for detecting the combination of the three sncRNA markers described above is provided for use in the preparation of products for detecting, screening, and / or diagnosing Mycobacterium tuberculosis infection.

[0035] Since the aforementioned biomarkers for Mycobacterium tuberculosis infection may be a single biomarker or a combination of multiple biomarkers, the corresponding diagnostic kits or detection devices can also take many forms. For example, a diagnostic kit or detection device targeting a single biomarker may be a product specifically designed to detect any one of tsRNA-iMet-CAT, 28S-rsRNA.3, and snsRNA.1. More exemplaryly, a combination of diagnostic products containing multiple biomarkers may also be used, i.e., a product that simultaneously detects tsRNA-iMet-CAT, 28S-rsRNA.3, and snsRNA.1. Exemplary products include, but are not limited to, chips, kits, test strips, or analytical platforms.

[0036] This diverse design allows diagnostic products to have a wider range of applications and greater flexibility, enabling the selection of appropriate testing protocols based on specific clinical needs and diagnostic requirements. However, whether it's a single-marker diagnostic kit or a combination of multiple markers, it is expected to provide relatively reliable and accurate diagnostic results.

[0037] The reagents included in the specific kits of this invention can be customized according to the detection method for each biomarker. This means that for the three biomarkers tsRNA-iMet-CAT, 28S-rsRNA.3, and snsRNA.1, specific methods and reagents suitable for their detection can be selected.

[0038] The reagents included in the specific kits of this invention can be customized according to the detection method for each sncRNA biomarker. This means that for the two different sncRNA biomarkers, tsRNA-iMet-CAT, 28S-rsRNA.3, and snsRNA.1, specific methods and reagents suitable for their detection can be selected.

[0039] Since these three biomarkers are all known substances, there are already widely available and reliable detection methods. For example, immunological methods, such as enzyme-linked immunosorbent assay (ELISA) and immunofluorescence analysis, can be used to detect the levels of these biomarkers. Molecular biological techniques, such as polymerase chain reaction (PCR) and in situ hybridization, can also be used to detect the gene expression levels of these biomarkers. Additionally, mass spectrometry can also be employed.

[0040] Based on the extensive experience and mature methods of existing technologies, the most suitable method for detecting the aforementioned biomarkers can be selected by referring to existing detection techniques and reagents. Then, according to the selected detection method, corresponding diagnostic kits or detection devices can be designed in accordance with existing technical standards and procedures.

[0041] ELISA is a commonly used method for detecting biomarkers. It utilizes the binding of specific antibodies to the biomarker to be detected, followed by an enzyme reaction to generate a measurable signal. ELISA has high sensitivity and specificity and can be applied to the detection of single or multiple biomarkers.

[0042] Immunofluorescence analysis utilizes fluorescently labeled antibodies to bind to the biomarker to be detected, and then uses a fluorescence microscope or fluorometer to detect the intensity of the fluorescence signal, thereby quantitatively analyzing the amount of biomarker present.

[0043] Mass spectrometry analysis can accurately quantify biomarkers, including methods such as proton nuclear magnetic resonance (NMR) and mass spectrometry (MS), which can provide very precise information on molecular structure and relative abundance.

[0044] PCR technology can be used to detect transcribed RNA as a biomarker. The RNA or fragments to be detected are amplified by PCR, and then quantitatively analyzed using methods such as gel electrophoresis or real-time quantitative PCR.

[0045] Protein chip technology can detect multiple biomarkers simultaneously. It works by immobilizing various antibodies on the chip surface, which then bind to proteins in the sample to be tested (e.g., tissue), and finally detecting the biomarkers through fluorescent or radioactively labeled signals.

[0046] For example, considering detection efficiency and convenience, the kit can be a PCR kit.

[0047] In one example, the PCR kit includes: (1) Primers capable of reverse transcription of tsRNA-iMet-CAT, 28S-rsRNA.3 or snsRNA.1, wherein the nucleotide sequences of the primers are shown in SEQ ID NO: 4-6 respectively; (2) Primers capable of PCR amplification of cDNA corresponding to tsRNA-iMet-CAT, 28S-rsRNA.3 or snsRNA.1, wherein the nucleotide sequences of the primers are shown in SEQ ID NO: 7-12 respectively.

[0048] In a preferred embodiment, the kit also includes dNTP / AMV reverse transcriptase and buffer, MgCl2, DEPC water, and Taq DNA polymerase.

[0049] In another embodiment of the present invention, the application of the above-mentioned sncRNA biomarker in Mycobacterium tuberculosis infection for non-pathological diagnostic or therapeutic purposes is provided. Non-pathological diagnostic or therapeutic Mycobacterium tuberculosis infection can be for scientific research, non-medical commercial testing or detection, etc.

[0050] For example, sncRNA biomarkers of Mycobacterium tuberculosis infection can be used to study disease mechanisms, pathophysiological processes, and potential therapeutic targets. By analyzing changes in these biomarkers during disease progression, we can gain a deeper understanding of the disease's development and metastasis mechanisms, providing a more comprehensive understanding for disease prevention and treatment.

[0051] Biomarkers also play a crucial role in drug development. They serve as indicators for drug efficacy evaluation, helping researchers assess the safety and effectiveness of new drugs. By monitoring the impact of drugs on biomarkers, potential drug toxicity and adverse reactions can be detected early, guiding the adjustment and optimization of drug dosage.

[0052] Biomarkers can also be used in individual health management and preventive medicine. By regularly monitoring specific biomarkers, changes in an individual's health status can be detected in a timely manner, the risk of disease can be predicted, and corresponding health management measures can be taken, such as adjusting lifestyle, dietary habits, or drug treatment, to maintain health and slow disease progression.

[0053] Biomarkers can be used to assess the impact of lifestyle on health. By monitoring changes in these biomarkers, the extent to which different lifestyles affect health can be assessed, and individuals can be guided to take appropriate lifestyle interventions, such as weight loss, smoking cessation, and increased physical activity, thereby improving health status and reducing disease risk.

[0054] This invention also provides a method for assessing Mycobacterium tuberculosis infection status in subjects. The key to this method is detecting the levels of tsRNA-iMet-CAT, 28S-rsRNA.3, and snsRNA.1 in the subject's blood.

[0055] First, the implementation of this method involves sample collection and processing. Extracting samples from the subjects' blood requires adherence to strict standard operating procedures to ensure sample quality and integrity.

[0056] Secondly, for the obtained samples, appropriate techniques and reagents can be used to detect the levels of tsRNA-iMet-CAT, 28S-rsRNA.3, and snsRNA.1 in the subjects' Mycobacterium tuberculosis infection. These detection methods can be immunological techniques, such as ELISA and immunofluorescence analysis, or molecular biological techniques, such as PCR and in situ hybridization, or mass spectrometry. These methods can accurately quantify the levels of certain biomarkers in the subjects' tissues.

[0057] After obtaining the biomarker test results, they need to be compared and analyzed with relevant data from healthy patients. Based on these comparisons, the subject's status of Mycobacterium tuberculosis infection can be determined. If the level of a certain biomarker or combination of biomarkers is significantly higher than the normal range, it can be inferred that the subject is highly likely to have Mycobacterium tuberculosis infection.

[0058] The reliability and accuracy of this method depend on the sensitivity and specificity of the selected detection method, as well as the determination of the reference range or threshold. Therefore, when implementing this method, it is necessary to strictly control experimental conditions and combine clinical experience and data analysis for the interpretation and diagnosis of results. Furthermore, the detection results of any single biomarker should be carefully evaluated and interpreted to avoid uncertainty in diagnostic results due to the potential for misinterpretation by a single biomarker.

[0059] This invention provides a method for pre-assessing whether a drug can prevent or treat Mycobacterium tuberculosis infection, which includes using tsRNA-iMet-CAT, 28S-rsRNA.3 and snsRNA.1 biomarkers to assess the drug's effectiveness in order to achieve the goal of precision medicine.

[0060] The implementation of this method requires starting with experimental design and sample collection. During the design phase of a study or clinical trial, the drug, treatment regimen, and evaluation indicators to be assessed must be clearly defined. Simultaneously, the selection criteria for subjects need to be determined, and appropriate biological samples, such as plasma, tissue, or cell samples, need to be collected for subsequent experimental analysis.

[0061] Once the biological sample is obtained, appropriate techniques and reagents can be used to detect the level of the selected biomarker. These detection methods can be immunological techniques, such as ELISA and immunofluorescence analysis, molecular biological techniques, such as PCR and in situ hybridization, or mass spectrometry. These methods allow for the accurate quantification of biomarker levels in the subject's biological sample.

[0062] Next, the medication or treatment regimen needs to be administered to the subject and carried out according to the predetermined plan. During treatment, the subject's biological samples need to be monitored regularly, and the levels of the selected biomarkers need to be repeatedly tested. This allows for timely understanding of the drug's therapeutic effect and whether the expected treatment goals have been achieved.

[0063] Finally, the therapeutic effect of the drug can be evaluated and determined based on the obtained test results. If the level of the selected biomarker changes significantly and shows a clear difference compared to before treatment, it can be inferred that the drug may have the potential to prevent or treat Mycobacterium tuberculosis infection. Conversely, if the biomarker level does not change significantly or does not achieve the expected effect, it may be necessary to re-evaluate the treatment plan or try other treatment strategies.

[0064] This invention provides a method for screening compounds that can prevent or treat Mycobacterium tuberculosis infection, which includes using tsRNA-iMet-CAT, 28S-rsRNA.3 and snsRNA.1 as biomarkers of Mycobacterium tuberculosis infection to evaluate the efficacy of the compounds in order to achieve the purpose of precision medicine.

[0065] First, the implementation of this method requires the establishment of a suitable experimental system and model. Cell lines or animal models infected with Mycobacterium tuberculosis can be used to simulate the physiological process of Mycobacterium tuberculosis infection and to evaluate the effects of compounds on it.

[0066] Secondly, appropriate biomarkers should be selected to evaluate the efficacy of compounds. tsRNA-iMet-CAT, 28S-rsRNA.3, and snsRNA.1, as biomarkers of Mycobacterium tuberculosis infection, can reflect the biological processes and pathological states of Mycobacterium tuberculosis infection. Therefore, they can be used as biomarkers to screen compounds and evaluate their intervention effects on Mycobacterium tuberculosis infection.

[0067] Next, a compound screening experiment is conducted. The compounds to be screened are added to cells or animals, and their effects on tsRNA-iMet-CAT, 28S-rsRNA.3, and snsRNA.1 in Mycobacterium tuberculosis infection are observed. Appropriate techniques and reagents, such as immunological and molecular biological techniques, can be used to detect the levels of these markers. Based on the test results, the potential therapeutic effect of the compounds on Mycobacterium tuberculosis infection can be preliminarily assessed.

[0068] After obtaining preliminary screening results, further validation and confirmation experiments can be conducted. By using more refined and rigorous experimental design and procedures, the impact of compounds on biomarker levels can be verified, and their therapeutic efficacy against Mycobacterium tuberculosis infection can be further evaluated.

[0069] Finally, based on the experimental results, further research and development will be conducted on compounds with potential therapeutic effects. This includes further evaluation of their pharmacological properties, toxic side effects, pharmacokinetics, and other drug attributes, as well as preclinical and clinical trials.

[0070] The present invention will further demonstrate the selection principle, process and effect of the above-mentioned biomarkers through a specific embodiment, so as to enable those skilled in the art to understand the essence of the present invention.

[0071] The specific process for marker screening in this invention includes: (1) Collect compliant serum specimens according to standard procedures (SOP) and collect complete clinical medical record information for each sample.

[0072] (2) Screening analysis of differential expression profiles of serum sncRNAs: Patients with tuberculosis branch infection and healthy volunteers matched for their gender and age were screened. The expression profiles of sncRNAs in serum were analyzed by sequencing to screen out differentially expressed sncRNAs and to verify them in multiple stages using clinical samples.

[0073] (3) Real-time quantitative PCR was performed on the sncRNAs screened above to identify sncRNAs associated with tuberculosis branch infection.

[0074] (4) Develop a diagnostic kit for tuberculosis branch infection based on the sncRNAs screened above.

[0075] Example 1: Data Sources and Grouping The dataset used for biomarker screening and validation in this invention comprises three independent datasets: a training set, an internal validation set, and an external validation set. All samples were sourced from Nanjing Gulou Hospital, Changzhou Third People's Hospital, and Zhenjiang Third People's Hospital. All tuberculosis patients were individually assessed and enrolled by two physicians with intermediate professional titles. All subjects or their legal guardians were fully informed and signed written consent forms. This invention has been approved by the Institutional Review Committee of Nanjing University School of Medicine Affiliated Gulou Hospital.

[0076] 1. Specific inclusion criteria: ① Sputum smear acid-fast staining showed positive for Mycobacterium tuberculosis; ② Culture of Mycobacterium tuberculosis in sputum or other body fluids (such as bronchoalveolar lavage fluid, pleural effusion); ③GeneXpert MTB / RIF test was positive for Mycobacterium tuberculosis.

[0077] 2. Specific exclusion criteria: Subjects will be excluded if they meet any of the following criteria: ① Combined with a confirmed nontuberculous mycobacterial (NTM) infection; ② Comorbid active lung diseases (such as bacterial pneumonia, fungal infection, lung cancer); ③ Comorbid severe immunosuppression (such as HIV infection, receiving immunosuppressive therapy); ④ Those who have received anti-tuberculosis treatment for more than two weeks.

[0078] 3. Specific grouping information is as follows: 3.1 Development Cohort: Divided into a training set and an internal validation set. All patients in this cohort were clinically diagnosed with pulmonary tuberculosis at Nanjing Drum Tower Hospital, collected from May 2023 to January 2025. A total of 59 pulmonary tuberculosis patients and 45 healthy controls were ultimately included, divided into: Training set: 18 patients with pulmonary tuberculosis and 8 healthy controls; Internal validation set: 41 patients with pulmonary tuberculosis and 37 healthy controls.

[0079] 3.2 External validation set: All patients in this cohort were from the Third People's Hospital of Changzhou and the Third People's Hospital of Zhenjiang. The data were collected from September 2023 to December 2024. A total of 71 patients with pulmonary tuberculosis and 70 healthy controls were included.

[0080] Example 2: Experimental Methods and Data Analysis 1 Experimental Methods 1.1 Total RNA was extracted from the serum sample using the Trizol (Invitrogen life technologies) method, specifically including the following steps: (1) Take 300 μL of serum sample into a 1.5 mL centrifuge tube, add 900 μL of lysis buffer RZ, mix well, and obtain a homogenate.

[0081] (2) Place the above mixture at room temperature for 5 min.

[0082] (3) Add 240 μL of chloroform and cel-39 (final concentration 100 pmol) as external reference. Mix well using a vortex mixer and let stand at room temperature for 2-3 min. Then centrifuge at 4 ℃ and 12000 rpm for 10 min. Aspirate about 800 μL of the supernatant (using a 200 μL pipette, aspirate 200 μL of the upper water-soluble layer each time, for a total of 4 times, to avoid aspirating the middle protein layer and the lower organic layer) into a 2 mL centrifuge tube.

[0083] (4) Add 1.5 times the volume of anhydrous ethanol to the collected supernatant solution and mix well.

[0084] (5) Prepare the appropriate number of collection columns. Each set of collection columns includes one upper column and one lower collection tube.

[0085] (6) After mixing the mixed solution obtained in step (4) with a 1 mL pipette, take 800 μL into the collection column, centrifuge at 12000 rpm at room temperature for 15 s, so that all the liquid in the collection column passes through the column, discard the liquid that has passed through the column, and keep the collection column.

[0086] (7) Repeat step (6) until the mixture has completely passed through the column.

[0087] (8) Take 500 μL of Buffer RD (confirm that anhydrous ethanol has been added) and add it to the collection column after the treatment in step (7). Centrifuge at 12000 rpm at room temperature for 15 s to ensure that all the liquid in the collection column passes through the column. Then discard the liquid that has passed through the column and keep the collection column.

[0088] (9) Take 500 μL Buffer RW (confirm that anhydrous ethanol has been added) and add it to the collection column after step (8). Let it stand for 2 minutes, centrifuge at 12000 rpm at room temperature for 15 s, so that all the liquid in the collection column passes through the column. Then discard the liquid that has passed through the column and keep the collection column.

[0089] (10) Repeat step (9).

[0090] (11) Centrifuge the column collected in step (10) at 12000 rpm for 2 min.

[0091] (12) Arrange the corresponding number of 1.5 mL centrifuge tubes, take the column collected in step (11) and place it in a 1.5 mL centrifuge tube, open the cap and let it dry for 1 minute.

[0092] (13) Add 50 μL of RNase-free water to the collection column of step (12) (make sure it is perpendicular to the membrane so that the liquid can be completely wetted by the membrane, rather than accumulating on the plastic ring at the edge of the column), let stand for 2 minutes, centrifuge at 12000 rpm for 2 minutes, discard the collection column, and obtain the RNA solution below.

[0093] 1.2 RNA quality testing: The purity and concentration of RNA were determined by agarose gel electrophoresis and qubit, with the following requirements: A260 / A280 of 1.8-2.0; small RNA library construction usually requires ≥1 ng total RNA.

[0094] 1.3 High-throughput sequencing of RNA.

[0095] ① The above RNA was recovered by PAGE electrophoresis; ② Use next-generation sequencing systems for sequencing and high-throughput gene expression database big data analysis to screen tsRNAs; ③Delete duplicate, mismatched, and poorly expressed sequences, and select sncRNAs with a fold change >10 and P < 0.01 for differential analysis; 1.4 RT-qPCR quantitative detection of sncRNA.

[0096] ④ Extract total RNA from the serum sample using the Trizol method as described in 1.1; ⑤ First, the RNA sample was reverse transcribed using sRNA stem-loop primers synthesized by GenScript and according to the reaction system shown in Table 1 to obtain cDNA; Table 1 Reverse transcription reaction system Experimental procedure: 1) Calculate the reaction system: Calculate the required system volume for reverse transcription reaction based on the sample size.

[0097] 2) Genomic DNA removal: Prepare the following mixture (DEPC water + 5×g DNA buffer + RNA) in an RNase-free centrifuge tube and perform the genomic DNA removal reaction under the following conditions: 42 °C for 2 min.

[0098] 3) First-strand cDNA synthesis: Prepare the following mixture (DEPC water + 10× reverse transcription reaction solution + reverse transcriptase + reaction product from step 2) in an RNase-free centrifuge tube, and carry out the first-strand cDNA synthesis reaction under the following conditions: 25 °C 5 min — 50 °C 15 min — 85 °C 5 min to obtain the cDNA product.

[0099] 4) The obtained product can be used immediately for the qPCR reaction described below, or stored at -20°C and used within six months; for long-term storage, it is recommended to aliquot and store at -70°C; cDNA should avoid repeated freeze-thaw cycles.

[0100] Then, sRNA forward primers synthesized by GenScript were used to perform quantitative PCR on cDNA according to the reaction system shown in Table 2.

[0101] Table 2 PCR reaction system Experimental procedure: 1) Calculate the reaction system: Calculate the amount of system required for the reaction based on the amount of sample to be tested.

[0102] 2) Distribute the PCR mixture: Prepare the following mixture (DEPC water + 2 × ChamQ Universal SYBR qPCR Master Mix + Primer 1 + Primer 2) in RNase-free centrifuge tubes and distribute it at 19 μL / well into the corresponding 96-well PCR plates.

[0103] 3) Add sample: Add 1 μL of cDNA obtained from reverse transcription to the 96-well PCR plate from step 2) and attach the plate.

[0104] 4) Mix well: Place the 96-well PCR plate on a vortex mixer and vortex for about 5 seconds, then centrifuge in a plate centrifuge (1500 rpm, 30 seconds).

[0105] PCR reaction procedure: Place the 96-well plate into the Roche Real-Time PCR instrument, set the program, and perform the qPCR reaction. The control and target marker probe fluorescent labeling type is SYBR, and the fluorescence calibration is set to none. Then, perform the PCR reaction according to the following program: 95℃, 5 min — (95℃, 15 s — 60℃, 1 min) × 40 cycles. In the second step of each cycle, i.e., 60℃, 1 min, collect the fluorescence signal. Finally, add the instrument's default melting curve program.

[0106] ⑥ Detect and compare the differences in sncRNA expression levels in the serum of Mycobacterium tuberculosis infected individuals and healthy controls.

[0107] 2. Data Processing and Analysis All data were statistically analyzed using Excel, Graphpad Prism 8.0, and SPSS 24.0 software. Values ​​are expressed as mean ± SEMs. A p-value < 0.05 was considered statistically significant. When the samples were normally distributed and had equal variance, t-tests or one-way ANOVA were used; when the samples were not normally distributed, U-tests or nonparametric tests were used.

[0108] Example 3: Screening of biomarkers 1. Small sample initial screening Small RNA was extracted and high-throughput sequenced from serum samples of the training set (serum from 18 tuberculosis patients and 8 healthy controls) using the method described in Example 2. Based on the sequencing results, differential expression volcano plots and differential heatmaps were generated. The results are shown below. Figure 1 .

[0109] like Figure 1 As shown, the volcano plot indicates that 23 sncRNAs are highly expressed and 24 sncRNAs are lowly expressed (1A); the differential heatmap shows significant differences in sncRNA expression between the tuberculosis group and the normal group. The six sncRNAs with significantly elevated expression levels in patients with Mycobacterium tuberculosis infection, in order of order, are: 28S-rsRNA.4, tsRNA-iMet-CAT, snsRNA.1, 28S-rsRNA.2, 28S-rsRNA.6, and 28S-rsRNA.3 (1B).

[0110] 2. Larger sample sizes were used to further screen and validate the differentially expressed sncRNAs. Six sncRNAs were extracted from serum samples of the internal validation set (41 tuberculosis patients and 37 healthy controls) using the method described in Example 2, and then quantitatively detected by RT-qPCR for further biomarker screening and analysis. Internal validation was performed by plotting differential expression analysis graphs and receiver operating characteristic (ROC) curves. The results are shown in [Figure 2]. Figure 2 and Figure 3 .

[0111] like Figure 2As shown, the six differentially expressed sncRNAs selected above were first internally validated in samples from Nanjing Drum Tower Hospital. The results showed that, compared with the healthy control group, the expression levels of the six sncRNAs in the patient group—28S-rsRNA.4, tsRNA-iMet-CAT, snsRNA.1, 28S-rsRNA.2, 28S-rsRNA.6, and 28S-rsRNA.3—showed an increasing trend. Among them, the expression levels of three sncRNAs—28S-rsRNA.3, tsRNA-iMet-CAT, and snsRNA.1—were significantly increased, with statistically significant differences. However, the expression levels of the other three sncRNAs—28S-rsRNA.4, 28S-rsRNA.2, and 28S-rsRNA.6—were not statistically different.

[0112] like Figure 3 As shown in the figure, the AUC values ​​of the three sncRNAs were 0.92 (tsRNA-iMet-CAT), 0.74 (28S-rsRNA.3), and 0.88 (snsRNA.1), respectively, and the AUC value of the combination of the three sncRNAs was 0.97.

[0113] The above results indicate that the sensitivity of the three sncRNAs as biomarkers individually is lower than that of the three sncRNAs used in combination as biomarkers. Therefore, the combination of 28S-rsRNA.3, tsRNA-iMet-CAT, and snsRNA.1 as a diagnostic biomarker for Mycobacterium tuberculosis infection has higher sensitivity and stronger specificity.

[0114] Example 4: Validation of the marker The three sncRNAs (tsRNA-iMet-CAT, 28S-rsRNA.3, and snsRNA.1) selected in Example 3 were quantified and differentially analyzed in serum samples from an external validation set (71 patients with pulmonary tuberculosis and 70 healthy controls) using the method described in Example 2. Differential expression plots and receiver operating characteristic (ROC) curves were plotted. The results are shown in [Figure 2]. Figure 4 and 5 .

[0115] like Figure 4 As shown, by further validating the three differentially expressed sncRNAs selected from samples collected at Nanjing Gulou Hospital—28S-rsRNA.3, tsRNA-iMet-CAT, and snsRNA.1—in external validation sets (two infectious disease hospitals, Zhenjiang Third People's Hospital and Changzhou Third People's Hospital), it was found that their expression levels still showed significant differences.

[0116] like Figure 5 As shown in the ROC curves, the AUC values ​​of the three sncRNAs were 0.85 (tsRNA-iMet-CAT), 0.81 (28S-rsRNA.3), and 0.86 (snsRNA.1), respectively, with a combined AUC value of 0.94. Compared to the aforementioned single biomarkers, the combination of 28S-rsRNA.3, tsRNA-iMet-CAT, and snsRNA.1 exhibits better predictive accuracy. These results suggest that the three sncRNAs possess high sensitivity and specificity as biomarkers and can be used as diagnostic markers for pulmonary tuberculosis.

[0117] Example 5: Detection kit for Mycobacterium tuberculosis infection This embodiment provides a detection kit for Mycobacterium tuberculosis infection. The kit is designed to detect the levels of three sncRNAs in the serum of subjects: 28S-rsRNA.3, tsRNA-iMet-CAT, and snsRNA.1, thereby achieving accurate diagnosis of Mycobacterium tuberculosis infection.

[0118] The kit consists of: (1) Primers capable of reverse transcription of tsRNA-iMet-CAT, 28S-rsRNA.3 or snsRNA.1; The reverse transcription primers include the nucleotide sequences shown in Table 3: Table 3 Reverse transcription primers (2) Primers that can perform PCR amplification of cDNA corresponding to tsRNA-iMet-CAT, 28S-rsRNA.3 or snsRNA.1; The PCR amplification primers include the nucleotide sequences shown in Table 4: Table 4 PCR Amplification Primers (3) dNTP / AMV reverse transcriptase and buffer, MgCl2, DEPC water and Taq DNA polymerase.

[0119] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the methods and techniques disclosed above without departing from the scope of the present invention to create equivalent embodiments. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A set of serum sncRNA markers for diagnosing Mycobacterium tuberculosis infection, characterized in that, The sncRNA biomarker combination is any one of tsRNA-iMet-CAT, 28S-rsRNA.3 and snsRNA.1, or a combination of any two or three of these biomarkers.

2. The sncRNA biomarker combination according to claim 1, characterized in that, The sncRNA biomarker combination is a combination of three biomarkers: tsRNA-iMet-CAT, 28S-rsRNA.3, and snsRNA.

1.

3. The sncRNA biomarker combination according to claim 1 or 2, characterized in that, The nucleotide sequence of the tsRNA-iMet-CAT is shown in SEQ ID NO: 1, the nucleotide sequence of the 28S-rsRNA.3 is shown in SEQ ID NO: 2, and the nucleotide sequence of the snsRNA.1 is shown in SEQ ID NO:

3.

4. The use of the sncRNA biomarker combination according to any one of claims 1-3 in the preparation of a formulation for detecting, screening and / or diagnosing Mycobacterium tuberculosis infection.

5. The use of a reagent for detecting the level of a combination of sncRNA biomarkers as described in any one of claims 1-3 in the preparation of products for detecting, screening, and / or diagnosing Mycobacterium tuberculosis infection.

6. The application according to claim 5, characterized in that, The reagents include those suitable for at least one of the following methods: fluorescent dye method, digital PCR, resonance light scattering method, real-time quantitative PCR, sequencing or biomolecular mass spectrometry; The products mentioned are chips, reagent kits, test strips, or analysis platforms.

7. A kit for Mycobacterium tuberculosis infection, characterized in that, Includes reagents for detecting the sncRNA biomarker as described in any one of claims 1-3, optionally, the detection being a quantitative detection of the sncRNA biomarker level in the plasma of a subject; The reagents include those suitable for at least one of the following methods: fluorescent dye method, digital PCR, resonance light scattering method, real-time quantitative PCR, sequencing or biomolecular mass spectrometry.

8. The reagent kit according to claim 7, characterized in that, The kit includes: (1) Primers capable of reverse transcription of tsRNA-iMet-CAT, 28S-rsRNA.3 or snsRNA.1, wherein the nucleotide sequences of the primers are shown in SEQ ID NO: 4-6 respectively; (2) Primers capable of PCR amplification of cDNA corresponding to tsRNA-iMet-CAT, 28S-rsRNA.3 or snsRNA.1, wherein the nucleotide sequences of the primers are shown in SEQ ID NO: 7-12 respectively; (3) dNTP / AMV reverse transcriptase and buffer, MgCl2, DEPC water and Taq DNA polymerase.

9. A method for assessing whether a drug can prevent or treat Mycobacterium tuberculosis infection, characterized in that, This includes using any one, two, or three of tsRNA-iMet-CAT, 28S-rsRNA.3, and snsRNA.1 as sncRNA markers to assess the drug's efficacy.

10. A method for screening compounds capable of preventing or treating Mycobacterium tuberculosis infection, characterized in that, This includes using one, two, or three of tsRNA-iMet-CAT, 28S-rsRNA.3, and snsRNA.1 as sncRNA markers to evaluate the efficacy of compounds.

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