Tuberculosis molecular marker combination and application thereof

Abstract

The invention belongs to the field of biological medicine, and relates to a tuberculosis molecular marker combination and application thereof. Specifically, the invention relates to a molecular marker combination, which comprises (A) nucleic acid for coding TAP1 protein, and one or more of nucleic acid for coding GBP5 protein, nucleic acid for coding UBE2L6 protein and nucleic acid for coding SERPING1 protein; or (B) a TAP1 protein, and one or more selected from the group consisting of a GBP5 protein, a UBE2L6 protein and an SERPING1 protein. The molecular marker combination can effectively diagnose the active tuberculosis, effectively distinguish the active tuberculosis from bacterial pneumonia, or distinguish the active tuberculosis from latent tuberculosis infection and normal control, and has high sensitivity and specificity.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to a molecular marker for tuberculosis and its use, and specifically to a molecular marker combination for distinguishing active pulmonary tuberculosis from bacterial pneumonia and its use. Background Art

[0002] Tuberculosis (TB) is a serious infectious disease that threatens human health. According to the World Health Organization, there are nearly 10 million new cases and over 1 million deaths worldwide each year. Mycobacterium tuberculosis is the primary pathogen of TB, and humans are its sole host. Infection can cause lesions in the lungs and multiple tissues and organs throughout the body. Because TB is a chronic respiratory infection, its course progresses through latent tuberculosis infection (LTBI) and active pulmonary tuberculosis (ATB). Currently, TB diagnosis faces numerous challenges, including the distinction between ATB and LTBI. LTBI patients are asymptomatic, but the lifetime risk of developing ATB is 5%-15%, and this risk may be even higher for immunocompromised individuals. Furthermore, in clinical practice, confusion between ATB and other lung infections is common, particularly when no etiological evidence is available for ATB. For example, nearly 10% of cases of bacterial pneumonia (PN) are secondary diagnosed as ATB. The two share similar symptoms and imaging features, often interfering with diagnosis and hindering the development of effective treatment plans. Inappropriate use of chemotherapy drugs can also lead to the emergence of drug-resistant tuberculosis (Grossman, Hsueh et al. 2014). Currently, there is no convenient and feasible clinical diagnostic method to distinguish between the two. Therefore, there is an urgent need to develop new differential diagnostic methods for ATB, and the first priority is to identify new diagnostic markers.

[0003] In recent years, high-throughput sequencing technologies have been used to explore changes in the host's transcriptional profile and responses during infection and disease, promising new diagnostic markers from the host's perspective. Neutrophils are important immune cells in the human body and the primary phagocytes in the blood. They are the body's first line of defense against pathogen invasion and are typically the first to arrive at sites of inflammation following infection. Their composition and number are closely linked to the onset of tuberculosis.

[0004] GBP5 (Guanylate-binding protein 5) is a member of the interferon (IFN)-induced GTPase family and plays a key role in innate immunity, protecting against a variety of bacterial, viral, and protozoan pathogens (based on similarity analysis). GBP5 can hydrolyze GTP, but unlike other family members, it does not produce guanylate (GMP). Following infection, this GTPase is recruited to pathogen-containing vesicles or escaped bacteria and acts as a positive regulator of inflammasome assembly by promoting the release of inflammasome ligands by bacteria (based on similarity analysis). Once pathogens are released from the cytoplasm, GBP5 promotes the recruitment of proteases with bacterial cell-lytic functions, releasing ligands that can be detected by inflammasomes, such as lipopolysaccharide (LPS), which can activate the atypical CASP4 / CASP11 inflammasome, or double-stranded DNA (dsDNA), which can activate the AIM2 inflammasome.

[0005] UBE2L6 (Ubiquitin / ISG15-conjugating enzyme E2 L6) is a member of the E2 ubiquitin-conjugating enzyme family. Its primary structure is highly similar to that of the enzyme encoded by the UBE2L3 gene. UBE2L6 catalyzes the covalent attachment of ubiquitin or ISG15 to other proteins and plays a role in E6 / E6-AP-induced p53 / TP53 ubiquitination. UBE2L6 also promotes the ubiquitination and subsequent proteasomal degradation of FLT3.

[0006] SERPING1 (Plasma protease C1 inhibitor) is a protein found in human plasma. Its primary function is to inhibit the activity of certain proteases (particularly C1 protease), thereby regulating the immune system and preventing excessive inflammatory responses. SERPING1 plays an important role in regulating the complement system and preventing autoimmune responses. SERPING1 forms a proteolytically inactive complex with C1r or C1s proteases, potentially playing a key role in regulating important physiological pathways, including complement activation, coagulation, fibrinolysis, and kinin generation. The protein is also a very potent FXIIa inhibitor, inhibiting chymotrypsin and kallikrein.

[0007] TAP1 (Antigen peptide transporter 1) is an ABC transporter associated with antigen presentation. TAP1 forms a complex with TAP2, mediating the unidirectional translocation of peptide antigens from the cytoplasm to the endoplasmic reticulum (ER), where they are loaded onto MHC class I (MHCI) molecules.

[0008] There is still a need to develop new technical means to quickly diagnose suspected cases of clinically active pulmonary tuberculosis. Summary of the Invention

[0009] After in-depth research and creative work, the inventors used RNA-seq sequencing analysis of neutrophils to discover differentially expressed genes with upregulated expression levels that are closely related to the state of active pulmonary tuberculosis. From these genes, they screened out the gene combination GUST (abbreviation of the combination, consisting of the first letters of GBP5, UBE2L6, SERPING1 and TAP1) that can distinguish ATB from PN, ATB from LTBI, and ATB from normal controls (HC). Furthermore, the inventors tested the expression levels of candidate genes in target cells in the validation group (ATB, PN, LTBI and HC clinical samples: real-time quantitative analysis of neutrophil-expressed genes) and confirmed that the expression levels of the genes in the combination were significantly increased in ATB. At the same time, the inventors also evaluated through statistical analysis that the new tuberculosis diagnostic marker GUST combination can effectively distinguish ATB from PN, and the GUST combination can effectively distinguish ATB from PN, ATB from LTBI, and ATB from HC, and has the potential to be used as a molecular identifier or molecular marker. The following invention is thus provided:

[0010] One aspect of the present invention relates to a molecular marker combination comprising:

[0011] (A) a nucleic acid encoding a TAP1 protein, and one or more selected from a nucleic acid encoding a GBP5 protein, a nucleic acid encoding a UBE2L6 protein, and a nucleic acid encoding a SERPING1 protein; or

[0012] (B) TAP1 protein, and one or more selected from GBP5 protein, UBE2L6 protein, and SERPING1 protein.

[0013] In some embodiments of the present invention, the molecular marker combination comprises:

[0014] (1) nucleic acid encoding TAP1 protein, nucleic acid encoding GBP5 protein, nucleic acid encoding UBE2L6 protein, and nucleic acid encoding SERPING1 protein;

[0015] (2) nucleic acid encoding TAP1 protein, nucleic acid encoding GBP5 protein, and nucleic acid encoding UBE2L6 protein;

[0016] (3) nucleic acid encoding TAP1 protein, nucleic acid encoding GBP5 protein, and nucleic acid encoding SERPING1 protein;

[0017] (4) nucleic acid encoding TAP1 protein, nucleic acid encoding UBE2L6 protein, and nucleic acid encoding SERPING1 protein;

[0018] (5) nucleic acid encoding TAP1 protein and nucleic acid encoding GBP5 protein;

[0019] (6) nucleic acid encoding TAP1 protein and nucleic acid encoding SERPING1 protein;

[0020] (7) nucleic acid encoding TAP1 protein and nucleic acid encoding UBE2L6 protein;

[0021] (8) TAP1 protein, GBP5 protein, UBE2L6 protein and SERPING1 protein;

[0022] (9) TAP1 protein, GBP5 protein and UBE2L6 protein;

[0023] (10) TAP1 protein, GBP5 protein and SERPING1 protein;

[0024] (11) TAP1 protein, UBE2L6 protein and SERPING1 protein;

[0025] (12) TAP1 protein and GBP5 protein;

[0026] (13) TAP1 protein and SERPING1 protein; or

[0027] (14) TAP1 protein and UBE2L6 protein.

[0028] In some embodiments of the present invention, the molecular marker combination, wherein,

[0029] The amino acid sequence of TAP1 protein is shown in SEQ ID NO: 1.

[0030] The amino acid sequence of GBP5 protein is shown in SEQ ID NO: 2.

[0031] The amino acid sequence of UBE2L6 protein is shown in SEQ ID NO: 3, and / or

[0032] The amino acid sequence of the SERPING1 protein is shown in SEQ ID NO:4.

[0033] In some embodiments of the present invention, the molecular marker combination, wherein,

[0034] The nucleic acid encoding TAP1 protein, the nucleic acid encoding GBP5 protein, the nucleic acid encoding UBE2L6 protein, and the nucleic acid encoding SERPING1 protein are independently DNA or independently RNA;

[0035] Preferably, the nucleic acid encoding TAP1 protein, the nucleic acid encoding GBP5 protein, the nucleic acid encoding UBE2L6 protein, and the nucleic acid encoding SERPING1 protein are all DNA or RNA;

[0036] Preferably,

[0037] The sequence of the nucleic acid encoding TAP1 protein is shown in SEQ ID NO: 5,

[0038] The sequence of the nucleic acid encoding the GBP5 protein is shown in SEQ ID NO: 6,

[0039] The sequence of the nucleic acid encoding the UBE2L6 protein is shown in SEQ ID NO: 7, and / or

[0040] The sequence of the nucleic acid encoding the SERPING1 protein is shown in SEQ ID NO:8.

[0041] In some embodiments of the present invention, the molecular marker combination is a molecular marker combination for tuberculosis;

[0042] Preferably, it is used to diagnose active pulmonary tuberculosis (ATB), differentiate active pulmonary tuberculosis from bacterial pneumonia (PN), differentiate active pulmonary tuberculosis from latent tuberculosis infection (LTBI), or differentiate active pulmonary tuberculosis from healthy people (HC).

[0043] Another aspect of the present invention relates to the use of any one of the molecular marker combinations of the present invention or a reagent for detecting the molecular marker combination in the preparation of a medicament for diagnosing ATB, distinguishing ATB from PN, distinguishing ATB from LTBI, or distinguishing ATB from HC.

[0044] In some embodiments of the present invention, the reagents for the use described herein comprise specific primers for each nucleic acid in the molecular marker combination, or antibodies that specifically bind to each protein in the molecular marker combination; preferably, the antibodies are linked to a detectable label, such as a radioisotope, a fluorescent substance, a colored substance, or an enzyme. The specific primers can be designed and synthesized according to methods known to those skilled in the art.

[0045] In some embodiments of the present invention, the use, wherein the reagent comprises a specific primer for each nucleic acid in (1) to (7) above, or comprises an antibody that specifically binds to each protein in (8) to (14) above; preferably, the antibody is linked to a detectable label, such as a radioactive isotope, a fluorescent substance, a colored substance or an enzyme.

[0046] In some embodiments of the present invention, the use, wherein the relative expression level of each nucleic acid in the molecular marker combination is detected by real-time fluorescence quantitative PCR (qPCR);

[0047] Preferably, the relative expression level of each nucleic acid in any one of (1) to (7) is detected by qPCR.

[0048] In some embodiments of the present invention, the use, wherein the MYO1F gene, ACTB gene or GAPDH gene is used as an internal reference gene;

[0049] Preferably, the MYO1F gene is used as an internal reference gene.

[0050] In some embodiments of the present invention, the use described, wherein the sample to be detected is a peripheral blood sample, a PBMC sample or a neutrophil sample; preferably, the neutrophil sample is a peripheral blood neutrophil sample; more preferably, the sample is a cDNA sample reverse-transcribed after total RNA is extracted from peripheral blood neutrophils.

[0051] In some embodiments of the present invention, the use, wherein,

[0052] The relative expression levels of nucleic acids encoding TAP1 protein, GBP5 protein, UBE2L6 protein, and SERPING1 protein are calculated to determine whether the sample is ATB, PN, LTBI, or HC.

[0053] In some embodiments of the present invention, the use, wherein,

[0054] Calculate P by the following formula TB :

[0055]

[0056] in:

[0057] gbp5, ube2l6, serping1, and tap1 represent the relative expression levels of nucleic acid encoding GBP5 (e.g., GBP5 gene), nucleic acid encoding UBE2L6 (e.g., UBE2L6 gene), nucleic acid encoding SERPING1 (e.g., SERPING1 gene), and nucleic acid encoding TAP1 (e.g., TAP1 gene), respectively;

[0058] The cut-off value is 0.361;

[0059] When P TB When P is greater than 0.361, the sample is diagnosed as ATB. TBWhen it is less than or equal to 0.361, the sample is diagnosed as PN, LTBI, or HC.

[0060] In some embodiments of the present invention, the use, wherein,

[0061] Calculate P by the following formula TB :

[0062]

[0063] in:

[0064] gbp5, ube2l6, serping1, and tap1 represent the relative expression levels of GBP5, UBE2L6, SERPING1, and TAP1 genes, respectively;

[0065] The cut-off value is 0.361;

[0066] When P TB When P is greater than 0.361, the sample is diagnosed as ATB. TB When it is less than or equal to 0.361, the sample is diagnosed as PN, LTBI, or HC.

[0067] In some embodiments of the present invention, the use, wherein the level of the molecular marker combination of any one of the present invention in the subject is compared with the cut-off value of the receiver operating characteristic curve (ROC curve), wherein the receiver operating characteristic curve is a receiver operating characteristic curve of the level of the molecular marker combination of any one of the present invention versus ATB and PN, ATB and LTBI, or ATB and HC, wherein:

[0068] If the level of the subject's molecular marker combination is greater than the cutoff value, it is judged as ATB;

[0069] If the level of the molecular marker combination of the subject is less than or equal to the cutoff value, the subject is judged to be PN, LTBI, or HC;

[0070] Preferably, the number of ATB samples, and PN samples, LTBI samples, or HC samples used to draw the working curve is independently greater than or equal to 10, greater than or equal to 20, greater than or equal to 30, greater than or equal to 50, greater than or equal to 80, greater than or equal to 100, greater than or equal to 200, or greater than or equal to 500, 10-1000, 20-500, 30-300, 30-200, 20-40, or 50-100.

[0071] In some embodiments of the present invention, the use, wherein the levels of GBP5 gene, UBE2L6 gene, SERPING1 gene and TAP1 gene are compared with the cutoff value of the receiver operating characteristic curve, wherein the receiver operating characteristic curve is the receiver operating characteristic curve between the levels of GBP5 gene, UBE2L6 gene, SERPING1 gene and TAP1 gene for ATB and PN, ATB and LTBI or ATB and HC, wherein:

[0072] If the levels of the GBP5 gene, UBE2L6 gene, SERPING1 gene, and TAP1 gene of the subject were greater than the cutoff value, they were diagnosed with ATB;

[0073] If the levels of the GBP5 gene, UBE2L6 gene, SERPING1 gene, and TAP1 gene of the subject were less than or equal to the cutoff value, they were judged to be PN, LTBI, or HC;

[0074] Preferably, the number of ATB samples, and PN samples, LTBI samples, or HC samples used to draw the working curve is independently greater than or equal to 10, greater than or equal to 20, greater than or equal to 30, greater than or equal to 50, greater than or equal to 80, greater than or equal to 100, greater than or equal to 200, or greater than or equal to 500, 10-1000, 20-500, 30-300, 30-200, 20-40, or 50-100.

[0075] Yet another aspect of the present invention relates to a method selected from the group consisting of:

[0076] Methods for diagnosing ATB, methods for differentiating ATB from PN, methods for differentiating ATB from LTBI, or methods for differentiating ATB from HC,

[0077] The method comprises the step of detecting the level of any one of the molecular marker combinations of the present invention in the sample to be tested.

[0078] In some embodiments of the present invention, the method, wherein the reagent for detecting the molecular marker combination comprises a specific primer for each nucleic acid in the molecular marker combination, or comprises an antibody that specifically binds to each protein in the molecular marker combination; preferably, the antibody is linked to a detectable label, such as a radioactive isotope, a fluorescent substance, a colored substance or an enzyme.

[0079] In some embodiments of the present invention, the method, wherein the relative expression level of each nucleic acid in the molecular marker combination is detected by real-time fluorescence quantitative PCR (qPCR);

[0080] Preferably, the relative expression level of each nucleic acid in any one of (1) to (7) is detected by qPCR.

[0081] In some embodiments of the present invention, the method, wherein the MYO1F gene, ACTB gene or GAPDH gene is used as an internal reference gene;

[0082] Preferably, the MYO1F gene is used as an internal reference gene.

[0083] In some embodiments of the present invention, the method described herein, wherein the sample to be detected is a peripheral blood sample, a PBMC sample or a neutrophil sample; preferably, the neutrophil sample is a peripheral blood neutrophil sample; more preferably, the sample is a cDNA sample reverse-transcribed after total RNA is extracted from peripheral blood neutrophils.

[0084] In some embodiments of the present invention, the method, wherein,

[0085] The relative expression levels of nucleic acids encoding TAP1 protein, GBP5 protein, UBE2L6 protein, and SERPING1 protein are calculated to determine whether the sample is ATB, PN, LTBI, or HC.

[0086] In some embodiments of the present invention, the method, wherein,

[0087] Calculate P by the following formula TB :

[0088]

[0089] in:

[0090] gbp5, ube2l6, serping1, and tap1 represent the relative expression levels of nucleic acid encoding GBP5 (e.g., GBP5 gene), nucleic acid encoding UBE2L6 (e.g., UBE2L6 gene), nucleic acid encoding SERPING1 (e.g., SERPING1 gene), and nucleic acid encoding TAP1 (e.g., TAP1 gene), respectively;

[0091] The cut-off value is 0.361;

[0092] When P TB When P is greater than 0.361, the sample is diagnosed as ATB. TB When it is less than or equal to 0.361, the sample is diagnosed as PN, LTBI, or HC.

[0093] In some embodiments of the present invention, the method, wherein,

[0094] Calculate P by the following formula TB :

[0095]

[0096] in:

[0097] gbp5, ube2l6, serping1, and tap1 represent the relative expression levels of GBP5, UBE2L6, SERPING1, and TAP1 genes, respectively;

[0098] The cut-off value is 0.361;

[0099] When P TB When P is greater than 0.361, the sample is diagnosed as ATB. TB When it is less than or equal to 0.361, the sample is diagnosed as PN, LTBI, or HC.

[0100] In some embodiments of the present invention, the method, wherein the level of the molecular marker combination of any one of the present invention in a subject is compared with a cut-off value of a receiver operating characteristic curve (ROC curve), wherein the receiver operating characteristic curve is a receiver operating characteristic curve of the level of the molecular marker combination of any one of the present invention versus ATB and PN, ATB and LTBI, or ATB and HC, wherein:

[0101] If the level of the subject's molecular marker combination is greater than the cutoff value, it is judged as ATB;

[0102] If the level of the molecular marker combination of the subject is less than or equal to the cutoff value, the subject is judged to be PN, LTBI, or HC;

[0103] Preferably, the number of ATB samples, and PN samples, LTBI samples, or HC samples used to draw the working curve is independently greater than or equal to 10, greater than or equal to 20, greater than or equal to 30, greater than or equal to 50, greater than or equal to 80, greater than or equal to 100, greater than or equal to 200, or greater than or equal to 500, 10-1000, 20-500, 30-300, 30-200, 20-40, or 50-100.

[0104] In some embodiments of the present invention, the method, wherein the levels of the GBP5 gene, the UBE2L6 gene, the SERPING1 gene, and the TAP1 gene are compared with the cutoff value of the receiver operating characteristic curve, wherein the receiver operating characteristic curve is a receiver operating characteristic curve of the GBP5 gene, the UBE2L6 gene, the SERPING1 gene, and the TAP1 gene levels for ATB and PN, ATB and LTBI, or ATB and HC, wherein:

[0105] If the levels of the GBP5 gene, UBE2L6 gene, SERPING1 gene, and TAP1 gene of the subject were greater than the cutoff value, they were diagnosed with ATB;

[0106] If the levels of the GBP5 gene, UBE2L6 gene, SERPING1 gene, and TAP1 gene of the subject were less than or equal to the cutoff value, they were judged to be PN, LTBI, or HC;

[0107] Preferably, the number of ATB samples, and PN samples, LTBI samples, or HC samples used to draw the working curve is independently greater than or equal to 10, greater than or equal to 20, greater than or equal to 30, greater than or equal to 50, greater than or equal to 80, greater than or equal to 100, greater than or equal to 200, or greater than or equal to 500, 10-1000, 20-500, 30-300, 30-200, 20-40, or 50-100.

[0108] This study uses RNA-seq technology to analyze the transcriptome of peripheral blood neutrophils, a key human immune cell. By comparing patients with clinically active pulmonary tuberculosis with patients with bacterial pneumonia, patients with latent tuberculosis, and a normal control group, a set of differentially expressed genes closely associated with active pulmonary tuberculosis status was identified, which can be used to distinguish active pulmonary tuberculosis from bacterial pneumonia. On this basis, a real-time quantitative analysis system was established that can use changes in peripheral blood neutrophil gene expression to distinguish between active tuberculosis and pulmonary pneumonia. The differential diagnostic ability of this gene combination system was further evaluated using clinical samples, confirming that the GUST combination in this system can serve as a differential diagnostic marker for active pulmonary tuberculosis.

[0109] In the present invention, unless otherwise specified, the term "normal control group" or "healthy control group" refers to healthy people with inactive tuberculosis, non-latent infection, and non-bacterial pneumonia, also referred to as healthy people (HC).

[0110] In the present invention, the term "False Discovery Rate" (FDR) is a necessary parameter used in expression profile analysis. It is the expected value of the ratio of the number of false rejections (rejection of a true (null) hypothesis) to the number of rejected null hypotheses. FDR has the following advantages: (1) its value can be flexibly adjusted. As a control indicator for the error rate of hypothesis testing, its control value can be flexibly selected as needed, while the value of traditional hypothesis testing (FWER) is relatively fixed, usually set at 0.05; (2) FDR has a clear meaning and can be used as an evaluation indicator for screened differential variables, while FWER is mainly used to control Type I error.

[0111] The term "log2Fold change" refers to the log2 value of the difference in gene expression levels between two groups of samples. In order to better display the difference in fold, it is difficult to display very large and very small fold differences together without converting them.

[0112] The term "FPKM" (Fragments Per Kilobase of exon model per Million mapped fragments) refers to the number of fragments per kilobase of transcript per million mapped reads. The FPKM value is used to measure and calculate the expression level of each gene in all samples in different groups. The calculation formula is as follows:

[0113]

[0114] Where N represents the total number of fragments uniquely aligned to the entire reference genome, C represents the number of fragments uniquely aligned to the exons of the gene, and L represents the total exon length (number of bases) of the gene. The FPKM algorithm corrects for both total data volume and gene length and can be used for subsequent transcriptome differential expression analysis.

[0115] The term "Lasso model regression analysis" is a linear model that implements feature selection and regularization by introducing an L1 regularization term. The objective function of Lasso regression is to minimize the following expression:

[0116]

[0117] in:

[0118] ·y i is the target value of the i-th sample.

[0119] ·x ij is the jth eigenvalue of the i-th sample.

[0120] β j is the coefficient of the j-th feature.

[0121] n is the sample size.

[0122] m is the number of features.

[0123] λ is the regularization parameter that controls the strength of the penalty term.

[0124] The term "cut off value" or "cutoff value" or "threshold value" refers to a critical value set by a classifier to distinguish between normal and abnormal prediction results.

[0125] The GBP5 gene, UBE2L6 gene, SERPING1 gene, and TAP1 gene are also referred to herein as G gene, U gene, S gene, and T gene, respectively.

[0126] GBP5 protein, UBE2L6 protein, SERPING1 protein and TAP1 protein are also referred to herein as G protein, U protein, S protein and T protein, respectively.

[0127] In the present invention, unless otherwise specified, the term "relative expression level" refers to the expression level relative to that of an internal reference gene (eg, MYO1F gene, ACTB gene, or GAPDH gene).

[0128] The level of the molecular marker combination can be the concentration, content or expression level of each molecular marker, such as the relative expression level.

[0129] Advantageous Effects of the Invention

[0130] The molecular marker combination of the present invention can effectively diagnose ATB, effectively distinguish ATB from PN, and distinguish ATB from LTBI and HC, with high sensitivity and specificity.

[0131] This invention can be used in the following situations, including but not limited to: 1. Clinically suspected cases of ATB that cannot be distinguished from PN using existing testing methods; 2. ATB screening in healthy individuals; or 3. Determining whether close contacts of individuals with LTBI positive cases have ATB. Currently, there are no available differential diagnostic methods for any of these three situations. This invention provides a novel tuberculosis diagnostic marker that meets the World Health Organization's standards for host diagnostic markers and has significant clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0132] Figures 1A to 1C :ATB and HC( Figure 1A ), ATB and LTBI ( Figure 1B ) and ATB and PN( Figure 1CThe composition of differentially expressed genes between groups ( ). The vertical and horizontal axes are the significance of gene expression differences and the fold difference between groups, respectively. Red dots represent upregulated differentially expressed genes, blue dots represent downregulated differentially expressed genes, and green dots represent target-related genes.

[0133] Figures 2A to 2D :Detection of candidate target gene GBP5( Figure 2A )、UBE2L6( Figure 2B )、SERPING1( Figure 2C ) and TAP1( Figure 2D ) expression levels.

[0134] Figure 3 : ROC analysis of GUST, a differential diagnostic marker for ATB.

[0135] The partial sequences involved in the present invention are as follows:

[0136] 1. TAP1 protein

[0137] MASSRCPAPRGCRCLPGASLAWLGTVLLLLADWVLLRTALPRIFSLLVPTALPLLRVWAVGLSRWAVLWLGACGVLRATVGSKSENAGAQGWLAALKPLAAALGLALPGLALFRELISWGAPGSADSTRLLHWGSHPTAFVVSYAAALPAAALWHKLGSLWVPGGQGGSGNPVRRLLGCLGSETRRLSLFLVLVVLSSLGEMAIPFFTGRLTDWILQDGSADTFTRNLTLMSILTIASAVLEFVGDGIYNNTMGHVHSHLQGEVFGAVLRQETEFFQQNQTGNIMSRVTEDTSTLSDSLSENLSLFLWYLVRGLCLLGIMLWGSVSLTMVTLITLPLLFLLPKKVGKWYQLLEVQVRESLAKSSQVAIEALSAMPTVRSFANEEGEAQKFREKLQEIKTLNQKEAVAYAVNSWTTSISGMLLKVGILYIGGQLVTSGAVSSGNLVTFVLYQMQFTQAVEVLLSIYPRVQKAVGSSEKIFEYLDRTPRCPPSGLLTPLHLEGLVQFQDVSFAYPNRPDVLVLQGLTFTLRPGEVTALVGPNGSGKSTVAALLQNLYQPTGGQLLLDGKPLPQYEHRYLHRQVAAVGQEPQVFGRSLQENIAYGLTQKPTMEEITAAAVKSGAHSFISGLPQGYDTEVDEAGSQLSGGQRQAVALARALIRKPCVLILDDATSALDANSQLQVEQLLYESPERYSRSVLLITQHLSLVEQADHILFLEGGAIREGGTHQQLMEKKGCYWAMVQAPADAPE(SEQ ID NO:1)

[0138] 2. GBP5 protein

[0139] MALEIHMSDPMCLIENFNEQLKVNQEALEILSAITQPVVVVAIVGLYRTGKSYLMNKLAGKNKGFSVASTVQSHTKGIWIWCVPHPNWPNHTLVLLDTEGLGDVEKADNKNDIQIFALALLLSSTFVYNTVNKIDQGAIDLLHNVTELTDLLKARNSPDLDRVEDPADSASFFPDLVWTLRDFCLGLEIDGQLVTPDEYLENSLRPKQGSDQRVQNFNLPRLCIQKFFPKKKCFIFDLPAHQKKLAQLETLPDDELEPEFVQQVTEFCSYIFSHSMTKTLPGGIMVNGSRLKNLVLTYVNAISSGDLPCIENAVLALAQRENSAAVQKAIAHYDQQMGQKVQLPMETLQELLDLHRTSEREAIEVFMKNSFKDVDQSFQKELETLLDAKQNDICKRNLEASSDYCSALLKDIFGPLEEAVKQGIYSKPGGHNLFIQKTEELKAKYYREPRKGIQAEEVLQKYLKSKESVSHAILQTDQALTETEKKKKEAQVKAEAEKAEAQRLAAIQRQNEQMMQERERLHQEQVRQMEIAKQNWLAEQQKMQEQQMQEQAAQLSTTFQAQNRSLLSELQHAQRTVNNDDPCVLL(SEQ ID NO:2)

[0140] 3. UBE2L6 protein

[0141] MMASMRVVKELEDLQKKPPPYLRNLSSDDANVLVWHALLLPDQPPYHLKAFNLRISFPPEYPFKPPMIKFTTKIYHPNVDENGQICLPIISSENWKPCTKTCQVLEALNVLVNRPNIREPLRMDLADLLTQNPELFRKNAEEFTLRFGVDRPS(SEQ ID NO:3)

[0142] 4. SERPING1 protein

[0143] MASRLTLLTLLLLLLAGDRASSNPNATSSSSQDPESLQDRGEGKVATTVISKMLFVEPILEVSSLPTTNSTTNSATKITANTTDEPTTQPTTEPTTQPTIQPTQPTTQLPTDSPTQPTTGSFCPGPVTLCSDLESHSTEAVLGDALVDFSLKLYHAFSAMKKVETNMAFSPFSIASLLTQVLLGAGENTKTNLESILSYPKDFTCVHQALKGFTTKGVTSVSQIFHSPDLAIRDTFVNASRTLYSSSPRVLSNNSDANLELINTWVAKNTNNKISRLLDSLPSDTRLVLLNAIYLSAKWKTTFDPKKTRMEPFHFKNSVIKVPMMNSKKYPVAHFIDQTLKAKVGQLQLSHNLSLVILVPQNLKHRLEDMEQALSPSVFKAIMEKLEMSKFQPTLLTLPRIKVTTSQDMLSIMEKLEFFDFSYDLNLCGLTEDPDLQVSAMQHQTVLELTETGVEAAAASAISVARTLLVFEVQQPFLFVLWDQQHKFPVFMGRVYDPRA(SEQ ID NO:4)

[0144] 5. Nucleic acid sequence encoding the TAP1 protein

[0145]

[0146] 6. Nucleic acid sequence encoding GBP5 protein

[0147]

[0148] 7. Nucleic acid sequence encoding UBE2L6 protein

[0149] ATGATGGCGAGCATGCGAGTGGTGAAGGAGCTGGAGGATCTTCAGAAGAAGCCTCCCCCATACCTGCGGAACCTGTCCAGCGATGATGCCAATGTCCTGGTGTGGCACGCTCTCCTCCTACCCGACCAACCTCCCTACCACCTGAAAGCCTTCAACCTGCGCATCAGCTTCCCGCCGGAGTATCCGTTCAAGCCTCCCATGATCAAATTCACAACCAAGATCTACCACCCCAACGTGGACGAGAACGGACAGATTTGCCTGCCCATCATCAGCAGTGAGAACTGGAAGCCTTGCACCAAGACTTGCCAAGTCCTGGAGGCCCTCAATGTGCTGGTGAATAGACCGAATATCAGGGAGCCCCTGCGGATGGACCTCGCTGACCTGCTGACACAGAATCCGGAGCTGTTCAGAAAGAATGCCGAAGAGTTCACCCTCCGATTCGGAGTGGACCGGCCCTCCTAA(SEQ ID NO:7)

[0150] 8. Nucleic acid sequence encoding SERPING1 protein

[0151]

[0152] 9.MYO1F protein

[0153]

[0154] 10. Nucleic acid sequence encoding MYO1F protein

[0155] DETAILED DESCRIPTION

[0156] The embodiments of the present invention will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0157] Example 1: Study on molecular markers of active pulmonary tuberculosis in hosts

[0158] (1) Inclusion and exclusion criteria for research subjects

[0159] The diagnostic criteria for active pulmonary tuberculosis (ATB) patients included in this study were based on the "Health Industry Standard of the People's Republic of China (WS288-2017) Diagnosis of Pulmonary Tuberculosis" and included positive pathogen detection of sputum or bronchoalveolar lavage fluid specimens (at least one of the three tests, smear / culture / nucleic acid test, was positive), no previous history of tuberculosis (no old tuberculosis lesions on medical interview and chest X-ray examination), and first-time anti-tuberculosis treatment for less than 7 days.

[0160] The latent tuberculosis infection group (LTBI) refers to patients with no history of tuberculosis, no tuberculosis-related clinical symptoms, normal chest X-rays but positive interferon-γ release test.

[0161] Healthy controls (HC) with inactive pulmonary tuberculosis / non-latent infection were defined as those with no history of tuberculosis, no tuberculosis-related clinical symptoms, negative interferon-γ release test, and normal chest X-ray.

[0162] Bacterial pneumonia is a community-acquired pneumonia. The blood routine test indicators of patients with bacterial pneumonia, such as the total white blood cell count and neutrophil percentage, show a significant increase. Clinical symptoms and chest X-ray results support the diagnosis of bacterial pneumonia.

[0163] The study subjects were aged 18 to 65 years. Pregnant or lactating women, patients with malignant tumors, those with immune system disorders or receiving immunotherapy, and those infected with the human immunodeficiency virus were excluded. This study was approved by the Ethics Committee of the Institute of Pathogenic Biology, Chinese Academy of Medical Sciences. After obtaining informed consent from the subjects, the present inventors drew peripheral blood from them for subsequent experiments. Information on the included samples is shown in Table 1. The 125 samples in Table 1 served as the discovery group.

[0164] Table 1: Information of the enrolled samples

[0165]

[0166] (II) Transcriptome analysis of peripheral blood neutrophils

[0167] 1. Peripheral Blood Neutrophil Isolation and Enrichment

[0168] 2 ml of peripheral venous blood was collected from participants using a heparin anticoagulant tube, and neutrophils were sorted out from the venous blood. The specific steps are as follows:

[0169] 1) Take 1 ml of anticoagulated blood, add 20 ml of ACK red blood cell lysis buffer, incubate at 2°C-8°C for 3-5 minutes, wait until the liquid becomes clear, centrifuge at 2000 rpm for 5 minutes, and discard the supernatant;

[0170] 2) Resuspend the leukocyte pellet in 10 ml of PBS, centrifuge at 2000 rpm for 10 minutes, discard the supernatant, and resuspend the leukocytes in 100 μL of PBS;

[0171] 3) Add 3 μL of anti-CD45APC, 3 μL of anti-CD3PE, and 2 μL of anti-CD64FICT (BD biosciences) and incubate at 2-8°C in the dark for 30 min;

[0172] 4) After incubation, add 3 ml of PBS, mix thoroughly, transfer to a 5 ml flow cytometry tube, centrifuge at 2000 rpm for 5 minutes, and carefully discard the supernatant.

[0173] 5) Add 400 μL PBS to resuspend the cells and store in a dark place at 2-8°C until use.

[0174] 6) Dilute RNase inhibitor (40 U / μL) 20-fold with RNase-free water. Add 8 μL of the diluted RNase inhibitor to a 1.5 ml protein low-adsorption centrifuge tube to collect the sorted cells.

[0175] 7) Debug the flow cytometer, set the sorting mode to "Purity", set the number of sorted cells to 150, and sort the cells obtained in step 5) using the set flow cytometer;

[0176] 8) The sorted cells were immediately centrifuged into an RNase inhibitor solution at the bottom of the tube for use in the following transcriptome library preparation or stored at -80°C.

[0177] 2. Transcriptome Library Construction and Sequencing

[0178] cDNA libraries were prepared using the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Takara). RNA-Seq sequencing libraries were constructed using the Nextera XT DNA Library Preparation Kit (Illumina). First-strand cDNA synthesis, addition of Illumina adapters and indexes, and RNA-Seq library purification using AMPure magnetic beads were performed according to the kit's instructions. Final RNA-Seq library amplification and purification using AMPure magnetic beads were then performed to obtain RNA-Seq libraries, which were then sequenced after passing quality control on an Agilent 2100 instrument.

[0179] The libraries that passed the quality inspection were sequenced using the Illumina Novaseq 6000 platform. The sequencing strategy was paired-end sequencing, and the length was approximately 150 bp.

[0180] 3. Transcriptome Data Analysis

[0181] Data quality control and alignment analysis: Trimmomatic software was used to perform quality filtering on the raw data, including removing adapter sequences (introduced during library construction), low-quality sequences (caused by errors in the sequencer itself), and shorter sequences (sequences <30 bp) in the sequencing reads.

[0182] Gene expression statistics and standardized analysis: This study used Cufflinks software and FPKM (Fragments Per Kilobase of exon model per Million mapped fragments) to calculate gene expression. Genes with FPKM values ​​greater than 100 in at least five samples were retained for subsequent analysis.

[0183] Differentially expressed gene analysis and candidate molecular marker screening: Pairwise comparisons were performed using the DESeq2 algorithm to calculate fold change (FC) in gene expression. P-values ​​and false discovery rates (FDR) were then calculated for significance analysis. In this study, differentially expressed genes (DEGs) in the neutrophil transcriptome were screened for FPKM > 100, p < 0.05, and |log2FC| > 0.5.

[0184] The four groups of samples were compared pairwise to calculate the fold change (FC) to screen differentially expressed genes, and the p-value (p-value) and false discovery rate (FDR) were calculated for significance analysis. The inventors detected a total of 3595 genes constituting the neutrophil expression profile in samples from the latent infection, active pulmonary tuberculosis group, and healthy control group. Figures 1A to 1C As shown in Figure 2, 444 genes with significant differences between ATB and HC sample groups were screened, including 239 up-regulated genes and 205 down-regulated genes ( Figure 1A ); There were 257 genes with significant differences between ATB and LTBI sample groups, including 146 up-regulated genes and 111 down-regulated genes ( Figure 1B ); There were 181 genes with significant differences between ATB and PN sample groups, including 104 up-regulated genes and 77 down-regulated genes ( Figure 1C ).

[0185] (III) Screening and determination of candidate molecular markers

[0186] Among the differentially expressed genes, we further screened for highly expressed DEGs in ATB using the intra-group mean and inter-group mean of the statistical parameters (satisfying both ATB mean / HC mean ≥ 1 and ATB mean / PN mean ≥ 1). Furthermore, we considered inter-group differences with a significant p-value < 0.05, intra-group standard deviation, and the coverage of differentially expressed genes in the samples, resulting in the selection of 15 DEGs. The results of the screened DEGs are shown in Table 2.

[0187] Table 2: Expression levels, significance of differences, and fold differences of candidate differentially expressed genes in neutrophils in ATB, PN, and HC

[0188]

[0189]

[0190] Lasso regression was used to predict DEGs between the ATB and PN, and ATB and HC groups. A gene panel of GBP5, UBE2L6, SERPING1, and TAP1 was identified, which can be used to differentiate ATB from PN. Subsequently, real-time quantitative PCR was performed on samples from the validation cohort to evaluate the effectiveness of the gene panel in distinguishing ATB from PN.

[0191] Example 2: Establishment of a qPCR Detection System for Clinical Samples and Verification of ATB Molecular Markers

[0192] Through transcriptomic analysis, the inventors identified differentially expressed neutrophil gene combinations that can identify active pulmonary tuberculosis. Based on this, they established a real-time qPCR quantitative analysis system for clinical testing and tested its effectiveness in differential diagnosis using clinical samples.

[0193] 1. Research subjects and groups

[0194] In this study, 106 test subjects were initially selected (including 17 patients in the ATB group, 31 patients in the LTBI group, 30 patients in the HC group, and 28 patients in the PN group). The inclusion criteria were the same as those for the discovery group (with no overlap between the samples and the discovery group), as shown in Table 3.

[0195] Table 3: Demographic data of the test group samples

[0196]

[0197]

[0198] 2. Experimental Methods

[0199] 1) Whole blood sample processing and magnetic cell separation and enrichment: Following the instructions for commercial reagents, pipette 0.8 ml of mixed whole blood into a 5 ml flow cytometry tube. Add 1.6 ml of 4°C pre-chilled separation buffer at a ratio of 1:2 to the tube and mix thoroughly by pipetting. Add CD15+ magnetic beads (Invitrogen, US) and quickly add an aliquot of magnetic beads to the diluted blood. Securely cap the tube and proceed with incubation and cell collection.

[0200] 2) Total RNA extraction: Total RNA was extracted from magnetic bead-sorted neutrophils using the RNeasy Plus Mini Kit (Qiagen, Germany). For detailed procedures, see the experimental manual.

[0201] 3) cDNA synthesis: Total cellular RNA was collected and synthesized using SuperScript TM IV VILO TM Master Mix (Invitrogen, US) was used for reverse transcription to obtain cDNA of cell samples.

[0202] 4) Gene Expression Detection: Real-time fluorescence quantitative PCR (TaqMan system) is used to detect the actual expression of the target gene in the host. Using the cDNA obtained in step 3) as a template, specific primer pairs or internal reference primer pairs are added to perform real-time quantitative PCR. The amplification constants of each gene and the internal reference gene in each sample-derived template are obtained, and the relative expression of each target gene is calculated.

[0203] a) Prepare reaction system 1 and reaction system 2

[0204] Reaction system 1 (target gene): 20 μL, polymerase chain reaction Fast Advanced Master Mix, target gene Assay primer, sample cDNA and nuclease-free water.

[0205] Reaction system 2 (reference gene): 20 μL, consisting of Fast Advanced Master Mix, Assay primer, sample cDNA and nuclease-free water.

[0206] b) Real-time quantitative PCR detection:

[0207] The reaction systems prepared in step a) were TM Real-time quantitative PCR was performed on a 6and 7Flex real-time fluorescence quantitative PCR instrument (Applied Biosystems, US). -ΔΔCt The relative expression level of the target gene in each template was calculated using the following reaction conditions: 50°C for 2 min, pre-denaturation at 5°C for 3 min, 95°C for 1 s, 60°C for 20 sec, 40 cycles, and fluorescence signal acquisition during the extension phase.

[0208] The present inventors found that MYO1F has a more stable expression in neutrophils than the universal internal reference gene, and therefore it is used as the internal reference gene in this detection system.

[0209] MYO1F was used as the internal reference gene, and qPCR analysis was performed on the four target genes of neutrophils in the test group samples. -ΔΔCt The algorithm statistically analyzed the expression levels of the target genes. Quantitative analysis of target gene expression and statistical differences between groups were performed. Kruskal-Wallis one-way analysis of variance was used for statistical analysis among the four groups, and Dunn's test was used for pairwise comparisons among multiple groups.

[0210] 3. Experimental Results

[0211] The results are as follows Figures 2A to 2D The results showed that GBP5 gene ( Figure 2A )、UBE2L6 gene ( Figure 2B )、SERPING1 gene ( Figure 2C ) and TAP1 genes ( Figure 2D) were significantly elevated in the ATB group. There were statistically significant differences between the ATB and PN groups, the ATB and LTBI groups, and the ATB and HC groups (p<0.0014, p<0.0001). There were no significant differences between the LTBI and HC groups. Overall, the qPCR results were generally consistent with the transcriptome sequencing data in Example 1, indicating that the RNA-seq analysis results were accurate and reliable.

[0212] Example 3: Determination of differential diagnosis between ATB and PN

[0213] In the clinical detection system, the more detection targets there are, the better the effect of distinguishing different types of disease-related samples. However, in actual applications, too many detection genes will lead to an increase in workload, as well as difficulty in operation and quality control. Therefore, optimizing the multi-gene combination and taking into account both diagnostic effect and operability are the only way to screen disease diagnostic markers. In the present invention, the four genes analyzed by qPCR real-time quantitative analysis showed differences between the groups of ATB and PN, ATB and LTBI, or ATB and HC in active pulmonary tuberculosis samples. SPSS binary logistic regression analysis was used to calculate and statistically analyze the combined expression levels of the four genes, and finally a detection system that uses GUST to predict the probability of ATB occurrence was screened out, which can well distinguish ATB and PN samples.

[0214] The present inventors used regression analysis to detect the gene expression of neutrophils in samples, and calculated the expression of each GUST to obtain the predicted probability P for predicting the possibility of ATB. TB (Predicting probability of tuberculosis), based on statistically estimated P TB The threshold is used to determine the possibility that the sample is ATB.

[0215] P TB The calculation formula is:

[0216]

[0217] Among them, P TB To determine whether the sample is ATB, the numerical range is 0-1; the power value of the natural logarithm is calculated by multiplying the regression coefficient of the corresponding gene by the gene expression level. gbp5, ube2l6, serping1 and tap1 represent the expression levels of GBP5 gene, UBE2L6 gene, SERPING1 gene and TAP1 gene, respectively. After substituting them into the formula, P is calculated. TB The cutoff value for the sample prediction of ATB is 0.361. TB When >0.361, the sample is ATB, P TBWhen <=0.361, it is PN.

[0218] Example 4: Evaluation of GUST diagnostic effect

[0219] The expression levels of the four genes in ATB and PN samples (Table 3) were quantitatively analyzed using the previously established qPCR detection system, and the predicted value P was calculated by the combined expression level. TB The cut-off value can be used to determine whether the sample is ATB. At the same time, the identification can also effectively distinguish ATB from HC, ATB from LTBI.

[0220] (I) Evaluation of the effectiveness of combined GUST expression and single gene expression as differential diagnostic indicators for ATB and PN

[0221] For the peripheral blood samples of patients, magnetic cell separation technology and qPCR detection method were used to obtain the real-time expression of 4 target genes, and the expression of P TB The formula was used to calculate the combined gene prediction value of individual samples, and the cutoff value was used to determine whether they were ATB or PN. The prediction results of each sample were compared with the actual diagnosis results, and the identification effect was evaluated using the area under the ROC curve (AUC value) and the sensitivity and specificity values. Figure 3 As shown in Table 4, the ROC value of the marker for distinguishing ATB and PN samples was 0.973 (0.93-1.01, p = 0), with a significant difference, the Youden index was 0.870, and when the specificity was 0.929, the sensitivity was 0.941. From the parameters in Table 4, the ROC values ​​of single genes for distinguishing ATB and PN were all greater than 0.65, with significant differences (p < 0.05), while the prediction value of the combined expression of the four genes was P < 0.05. TB The efficiency of differentiating ATB from PN was significantly improved, with both the AUC value (area under the receiver operating characteristic (ROC) curve) and Youden index superior to those of a single gene. With a sensitivity of 0.941, GUST showed significantly better specificity than a single gene.

[0222] Table 4: Evaluation of the GUST identification mark on the identification effect of ATB and PN

[0223]

[0224] Remark:

[0225] a. According to non-parametric assumptions;

[0226] b. Null hypothesis: True area = 0.5.

[0227] (II) Evaluation of the effectiveness of the combined expression of two genes and three genes as indicators for differential diagnosis of ATB and PN

[0228] The present inventors calculated the combined expression levels of any two or three of the four genes for prediction and identification, where G, U, S, and T represent GBP5, UBE2L6, SERPING1, and TAP1, respectively. Gene combinations are represented by abbreviations. For example, GUS represents the combined marker for GBP5, UBE2L6, and SERPING1, and so on. As shown in Table 5, considering all characteristic parameters, the area under the receiver operating characteristic (ROC) curve, Youden index, specificity, and sensitivity of the four-gene combination were improved overall, indicating that the four-gene combination is superior to any combination of two or three genes in identification.

[0229] Table 5: ROC values, specificity, and sensitivity analysis of any combination of GUST genes for differentiating ATB from PN

[0230]

[0231]

[0232] Remark:

[0233] a. According to non-parametric assumptions;

[0234] b. Null hypothesis: True area = 0.5.

[0235] Example 5: Verification of the differential diagnosis effect of GUST (1)

[0236] The present inventors mixed and renumbered all samples from the validation group (ATB, PN, HC, and LTBI groups; see Table 3) (as test group 1). The GUST expression level for each sample was calculated according to the formula in Example 3. ATB was determined based on the cutoff value (0.361). The predicted results were then grouped and compared with the actual samples. The ATB positivity rate in each group was calculated to assess the accuracy of combining GUST expression levels to identify ATB. The statistical results are shown in Table 6.

[0237] Table 6: P according to the diagnosis of active pulmonary tuberculosis TB The value prediction sample is the data statistics of ATB

[0238]

[0239] As shown in Table 6, the positive detection rate of this diagnostic marker in the ATB group was 94.1%, the false positive rate in the PN group was 7.1%, and the false positive rates in the LTBI group and the HC group were 9.7% and 6.7%, respectively. This demonstrates the high specificity of this marker in the differential diagnosis of ATB and PN, and its ability to effectively assist in the diagnosis of active pulmonary tuberculosis, demonstrating its excellent clinical value.

[0240] Example 6: Verification of the differential diagnosis effect of GUST (2)

[0241] To verify the effect of the differential diagnosis marker, 105 hospital clinical samples were collected as a parallel test group (test group 2), including 27 HC, 30 LTBI, 21 ATB and 27 PN. The inclusion criteria were the same as before; as shown in Table 7. The expression levels of G, U, S, and T genes in each sample in the group were obtained using the same qPCR detection method as test group 1, and SPSS binary regression statistical analysis was performed. With reference to Example 3, the expression levels of GUST were calculated, and ATB was identified in random mixed samples. It was not only possible to accurately identify ATB from ATB and PN mixed samples, but also with an AUC value of 0.959 (0.913-1.006, p < 0.000), a sensitivity of 0.905, a specificity of 0.889, and a Youden index of 0.794. At the same time, the marker can also accurately identify ATB samples from ATB and LTBI, ATB and HC, ATB and LTBI plus HC, and ATB and LTBI plus PN plus HC. The discrimination effects, AUC values, sensitivity and specificity parameters of different groups are shown in Table 8.

[0242] Table 7: Demographic data of test group 2 samples

[0243]

[0244] Table 8: Evaluation of the effectiveness of differential diagnosis using GUST targets

[0245]

[0246]

[0247] Remark:

[0248] a. According to non-parametric assumptions;

[0249] b. Null hypothesis: True area = 0.5.

[0250] Although specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and substitutions may be made to those details in light of all the teachings disclosed herein, and such modifications are within the scope of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

1. Molecular marker combination, including: (A) a nucleic acid encoding a TAP1 protein, and one or more selected from a nucleic acid encoding a GBP5 protein, a nucleic acid encoding a UBE2L6 protein, and a nucleic acid encoding a SERPING1 protein; or (B) TAP1 protein, and one or more selected from the group consisting of GBP5 protein, UBE2L6 protein and SERPING1 protein.

2. The molecular marker combination according to claim 1, comprising: (1) nucleic acid encoding TAP1 protein, nucleic acid encoding GBP5 protein, nucleic acid encoding UBE2L6 protein and nucleic acid encoding SERPING1 protein; (2) nucleic acid encoding TAP1 protein, nucleic acid encoding GBP5 protein and nucleic acid encoding UBE2L6 protein; (3) nucleic acid encoding TAP1 protein, nucleic acid encoding GBP5 protein and nucleic acid encoding SERPING1 protein; (4) nucleic acid encoding TAP1 protein, nucleic acid encoding UBE2L6 protein, and nucleic acid encoding SERPING1 protein; (5) nucleic acid encoding TAP1 protein and nucleic acid encoding GBP5 protein; (6) nucleic acid encoding TAP1 protein and nucleic acid encoding SERPING1 protein; (7) nucleic acid encoding TAP1 protein and nucleic acid encoding UBE2L6 protein; (8) TAP1 protein, GBP5 protein, UBE2L6 protein and SERPING1 protein; (9) TAP1 protein, GBP5 protein and UBE2L6 protein; (10) TAP1 protein, GBP5 protein and SERPING1 protein; (11) TAP1 protein, UBE2L6 protein and SERPING1 protein; (12) TAP1 protein and GBP5 protein; (13) TAP1 protein and SERPING1 protein; or (14) TAP1 protein and UBE2L6 protein.

3. The molecular marker combination according to any one of claims 1 to 2, wherein: The amino acid sequence of TAP1 protein is shown in SEQ ID NO:

1. The amino acid sequence of GBP5 protein is shown in SEQ ID NO:

2. The amino acid sequence of UBE2L6 protein is shown in SEQ ID NO: 3, and / or The amino acid sequence of SERPING1 protein is shown in SEQ ID NO:

4.

4. The molecular marker combination according to any one of claims 1 to 3, wherein: The nucleic acid encoding TAP1 protein, the nucleic acid encoding GBP5 protein, the nucleic acid encoding UBE2L6 protein, and the nucleic acid encoding SERPING1 protein are independently DNA or independently RNA; Preferably, the nucleic acid encoding TAP1 protein, the nucleic acid encoding GBP5 protein, the nucleic acid encoding UBE2L6 protein and the nucleic acid encoding SERPING1 protein are all DNA or all RNA; Preferably, The sequence of the nucleic acid encoding TAP1 protein is shown in SEQ ID NO: 5, The sequence of the nucleic acid encoding the GBP5 protein is shown in SEQ ID NO:6, The sequence of the nucleic acid encoding the UBE2L6 protein is shown in SEQ ID NO: 7, and / or The sequence of the nucleic acid encoding the SERPING1 protein is shown in SEQ ID NO:

8.

5. The molecular marker combination according to any one of claims 1 to 4, which is a molecular marker combination for tuberculosis; Preferably, it is used to diagnose active pulmonary tuberculosis (ATB), to differentiate active pulmonary tuberculosis from bacterial pneumonia (PN), to differentiate active pulmonary tuberculosis from latent tuberculosis infection (LTBI), or to differentiate active pulmonary tuberculosis from healthy persons (HC).

6. Use of the molecular marker combination according to any one of claims 1 to 5 or a reagent for detecting the molecular marker combination in the preparation of a drug for diagnosing ATB, distinguishing ATB from PN, distinguishing ATB from LTBI, or distinguishing ATB from HC.

7. The use according to claim 6, wherein The reagents include specific primers for each nucleic acid in the molecular marker combination, or antibodies that specifically bind to each protein in the molecular marker combination; preferably, the antibodies are linked to detectable markers, such as radioisotopes, fluorescent substances, colored substances or enzymes.

8. The use according to any one of claims 6 to 7, wherein The relative expression level of each nucleic acid in the molecular marker combination is detected by real-time fluorescence quantitative PCR (qPCR); Preferably, the relative expression level of each nucleic acid in any one of (1) to (7) is detected by qPCR; Preferably, the MYO1F gene, ACTB gene or GAPDH gene is used as an internal reference gene.

9. The use according to any one of claims 6 to 8, wherein The sample to be tested is a peripheral blood sample, a PBMC sample or a neutrophil sample; preferably, the neutrophil sample is a peripheral blood neutrophil sample; more preferably, the sample is a cDNA sample obtained by reverse transcription after total RNA is extracted from peripheral blood neutrophils.

10. The use according to any one of claims 6 to 9, wherein Calculate the relative expression levels of nucleic acid encoding TAP1 protein, nucleic acid encoding GBP5 protein, nucleic acid encoding UBE2L6 protein, and nucleic acid encoding SERPING1 protein to determine whether the sample is ATB, PN, LTBI, or HC; Preferably, P is calculated by the following formula TB : in: gbp5, ube2l6, serping1 and tap1 represent the relative expression levels of nucleic acid encoding GBP5 (e.g., GBP5 gene), nucleic acid encoding UBE2L6 (e.g., UBE2L6 gene), nucleic acid encoding SERPING1 (e.g., SERPING1 gene), and nucleic acid encoding TAP1 (e.g., TAP1 gene), respectively; The cut off value is 0.361; When P TB When P is greater than 0.361, the sample is diagnosed as ATB. TB When it is less than or equal to 0.361, the sample is diagnosed as PN, LTBI, or HC.

11. The use according to any one of claims 6 to 10, wherein The level of the molecular marker combination of any one of claims 1 to 5 of the subject is compared with the cutoff value of the receiver operating characteristic curve, wherein the receiver operating characteristic curve is a receiver operating characteristic curve of the level of the molecular marker combination of any one of claims 1 to 5 for ATB and PN, ATB and LTBI or ATB and HC, wherein: If the level of the subject's molecular marker combination is greater than the cutoff value, it is judged as ATB; If the level of the subject's molecular marker combination was less than or equal to the cutoff value, it was judged as PN, LTBI, or HC; Preferably, the number of the ATB samples, and the HC samples, or the LTBI samples, or the PN samples used to draw the working curve is independently greater than or equal to 10, greater than or equal to 20, greater than or equal to 30, 20-40, or 50-100.

12. The use according to any one of claims 6 to 10, wherein The levels of GLRX gene, FCGR1A gene, IFITM1 gene and UBE2L6 gene were compared with the cutoff values ​​of the receiver operating characteristic curve, which was the receiver operating characteristic curve between the levels of GLRX gene, FCGR1A gene, IFITM1 gene and UBE2L6 gene and ATB and PN, ATB and LTBI or ATB and HC, wherein: If the levels of the subject's GLRX gene, FCGR1A gene, IFITM1 gene, and UBE2L6 gene were greater than the cutoff value, it was judged as ATB; If the levels of the subject's GLRX gene, FCGR1A gene, IFITM1 gene, and UBE2L6 gene were less than or equal to the cutoff value, the subject was judged to be PN, LTBI, or HC; Preferably, the number of ATB samples, and PN samples, LTBI samples, or HC samples used to draw the working curve is independently greater than or equal to 10, greater than or equal to 20, greater than or equal to 30, 20-40, or 50-100.

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