阅读说明：本技术 提高菌阴结核感染检测敏感性的标志物组合及应用 (Marker combination for improving detection sensitivity of bacterial scrotum infection and application thereof ) 是由 李荣秀 时晓慧 于 2019-11-15 设计创作，主要内容包括：本发明提供了提高菌阴结核感染检测敏感性的标志物组合及应用,具体地,本发明提供了两种蛋白组合物,所述的蛋白组合物可以用于检测结核分枝杆菌感染,一种用于检测结核分支杆菌和非结核分支杆菌的低负荷感染和菌体检测阴性的人群和高风险人群筛查的敏感性,另一种用于临床诊断结核分支杆菌所需的特异性。两种指标蛋白组合可以满足不同应用场景下对结核分支杆菌和非结核分支杆菌敏感性和特异性的不同需求。本发明还提供了现场快速检测的方法,可用于疾病控制机构、偏远欠发达地区卫生机构、和家庭对疑似结核感染的现场快速发现。(The invention provides a marker combination for improving the detection sensitivity of mycobacterium tuberculosis negative nodule infection and application thereof, and particularly provides two protein compositions, wherein the protein compositions can be used for detecting mycobacterium tuberculosis infection, one protein composition can be used for detecting the sensitivity of screening of low-load infection and thallus detection negative crowds and high-risk crowds of mycobacterium tuberculosis and nontuberculous mycobacteria, and the other protein composition can be used for clinically diagnosing the specificity required by the mycobacterium tuberculosis. The combination of the two index proteins can meet different requirements on the sensitivity and specificity of mycobacterium tuberculosis and nontuberculous mycobacterium under different application scenes. The invention also provides a method for on-site rapid detection, which can be used for on-site rapid discovery of suspected tuberculosis infection for disease control institutions, health institutions in remote underdeveloped areas and families.)

1. A protein composition, comprising the following proteins:

(a1)1 and above proteins selected from the group consisting of: rv0440, Rv0577, Rv0685, Rv1094, Rv1468c, Rv2031c, Rv2831, Rv 3048;

(a2)1 and above derived proteins selected from the group consisting of: a protein having a sequence selected from the group consisting of substitution, deletion and addition of 1 or more amino acid residues of any one of the proteins (a 1);

(a3)1 or more specific binding partner proteins selected from the group consisting of: 1 or more proteins that specifically bind to any one of (a1) and (a 2); and/or

(a4)1 and above specific amino acid sequence selected from the group consisting of: 1 or more amino acid sequences comprising the entire sequence or partial sequence of any one of the proteins (a1), (a2) and (a 3).

2. The protein composition of claim 1, wherein the protein composition (a1) is a mycobacterium tuberculosis protein and a nontuberculous mycobacterial protein.

3. The protein composition of claim 1 as a marker for assessing susceptibility to infection by mycobacterium tuberculosis and nontuberculous mycobacteria.

4. The protein composition of claim 1, comprising 3, 4, 5, 6, 7, or 8 proteins.

5. A protein composition, comprising the following proteins:

(b1)1 and above proteins selected from the group consisting of: three or more mycobacterium tuberculosis proteins selected from Rv1575, Rv1773c, Rv1980, Rv2073c, Rv3117, Rv3400, Rv3874, and Rv3875, Rv1514c, Rv1964, Rv1985c, Rv2649, Rv3872, Rv3878 and Rv3402 c;

(b2)1 and above derived proteins selected from the group consisting of: a protein having a sequence selected from the group consisting of substitution, deletion and addition of 1 or more amino acid residues of any one of the proteins (b 1);

(b3)1 or more specific binding partner proteins selected from the group consisting of: 1 or more proteins that specifically bind to any one of (b1) and (b 2); and/or

(b4)1 and above specific amino acid sequence selected from the group consisting of: 1 or more amino acid sequences comprising the entire sequence or partial sequence of any one of the proteins (b1), (b2) and (b 3).

6. The protein composition of claim 5, wherein the protein composition (b1) is a Mycobacterium tuberculosis protein.

7. The protein composition according to claim 5, which is used as a marker for evaluating specificity of Mycobacterium tuberculosis.

8. The protein composition of claim 5, comprising 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 proteins.

9. A kit comprising three or more markers selected from the group consisting of:

(a) the protein composition of claim 1; and

(b) the protein composition of claim 5.

10. A method for detecting mycobacterium tuberculosis protein, comprising the steps of:

(I) providing an isolated tissue sample to be tested, placing the isolated tissue sample and the kit according to claim 9 into the same system, and fully reacting;

(II) isolating the protein binding fragment bearing the isolation tag from the test system using the isolation tag in the kit; and

(III) measuring the fluorescence signal of the detection system for separating the protein binding fragment with the separation label.

Technical Field

The invention belongs to the field of health medical treatment, and particularly relates to a marker combination for improving the detection sensitivity of bacterial scrotal tuberculosis infection and application thereof, and more particularly relates to a marker combination for improving the detection sensitivity and clinical diagnosis specificity of bacterial scrotal tuberculosis infection and application thereof.

Background

According to the world health organization data, tuberculosis remains the most deadly killer of infectious diseases in the world, ranking top 10 in all causes of death. The number of worldwide deaths due to tuberculosis is estimated to be about 157 ten thousand, and about 3.7 thousand in China. China still belongs to one of 30 tuberculosis high-load countries all over the world, the number of newly-released patients per year is 88.9 ten thousand, and the incidence rate is 63/10 ten thousand. The number of people with latent tuberculosis infection in 2017 is about 17 hundred million, accounting for 23 percent of the population; . By using a tuberculin skin test detection technology, about 3.6 million people in China are infected with mycobacterium tuberculosis, and nearly one third of the population is under the threat of tuberculosis; the conclusion of the general investigation for detecting the specific antigen in the mycobacterium tuberculosis RD region to stimulate the T cell to generate the gamma-interferon is 18.8% [ the current situation of latent infection of mycobacterium tuberculosis in Li Xiangwei, Jinqi, Heepii and China, the New infectious disease electronic journal of 3 RD year 2017 and 146-15 ]. Since the latently infected population is a large pool of potential patients, tuberculosis occurs in the lifetime of 5% to 10% of latently infected individuals. In addition, Mycobacterium tuberculosis in latently infected people is a mobile source of infection.

The most commonly used methods for the clinical detection of Mycobacterium Tuberculosis (MTB) are smear, culture, T-SPOT, DNA and RNA detection. Quick and cheap sputum smear, simple operation and low detection sensitivity (>10³CFU/ml), only 50-75% of active tuberculosis patients can be detected, the omission ratio is as high as 25-50%, dead bacteria cannot be distinguished, and Mycobacterium Tuberculosis (MTB) and nontuberculous mycobacteria (NTM) cannot be distinguished. The culture method can improve detection sensitivity, but the detection period is as long as 2-8 weeks, and the treatment time window is delayed. Missed detection of mycobacterium tuberculosis (latent) infected people and long detection period all increase the chance of transmitting mycobacterium tuberculosis to a contact person. T-SPOT, DNA and RNA detection methods all require complex instrumentation and specialized operators, and present a number of difficulties for use in remote areas. In addition, the existing detection method is difficult to consider the contradiction between detection sensitivity and specificity.

Therefore, there is a need in the art for a rapid and convenient method for detecting mycobacterium tuberculosis infection, which has high sensitivity for detecting mycobacterium tuberculosis and nontuberculous mycobacteria low-load and bacterial-negative patients, and has high specificity for clinically diagnosing mycobacterium tuberculosis infection.

Disclosure of Invention

The invention aims to provide a rapid and convenient mycobacterium tuberculosis infection detection method which has high sensitivity for detecting the infection of mycobacterium tuberculosis and nontuberculous mycobacteria under low load and bacterial yin patients and has high specificity for clinically diagnosing the infection of mycobacterium tuberculosis.

In a first aspect of the present invention, there is provided a protein composition, characterized in that it comprises the following proteins:

(a1)1 and above proteins selected from the group consisting of: rv0440, Rv0577, Rv0685, Rv1094, Rv1468c, Rv2031c, Rv2831, Rv 3048;

(a2)1 and above derived proteins selected from the group consisting of: a protein having a sequence selected from the group consisting of substitution, deletion and addition of 1 or more amino acid residues of any one of the proteins (a 1);

(a3)1 or more specific binding partner proteins selected from the group consisting of: 1 or more proteins that specifically bind to any one of (a1) and (a 2); and/or

(a4)1 and above specific amino acid sequence selected from the group consisting of: 1 or more amino acid sequences comprising the entire sequence or partial sequence of any one of the proteins (a1), (a2) and (a 3).

In another preferred example, the protein composition (a1) is a mycobacterium tuberculosis and non-mycobacterium tuberculosis protein.

In another preferred embodiment, the protein composition is used as a marker for assessing susceptibility to infection by Mycobacterium tuberculosis and nontuberculous mycobacteria.

In another preferred embodiment, the protein composition comprises 3, 4, 5, 6, 7, or 8 proteins.

In another preferred embodiment, the plurality of proteins comprises 3, 4, 5, 6, 7, or 8 proteins selected from (a 1).

In another preferred embodiment, the protein composition comprises proteins Rv0577 and Rv2831 and Rv3048, and/or their corresponding derivatives, specific binding partner proteins, specific amino acid sequences.

In another preferred embodiment, the protein composition comprises Rv0577 and Rv1094 and Rv2831, and/or their corresponding derivative proteins, specific binding partner proteins, specific amino acid sequences.

In another preferred embodiment, the protein composition comprises Rv2031c and Rv1094 and Rv3048, and/or their corresponding derivative proteins, specific binding partner proteins, specific amino acid sequences.

In another preferred embodiment, the protein composition comprises Rv0440 and Rv2831 and Rv3048, and/or their corresponding derivative proteins, specific binding partner proteins, specific amino acid sequences.

In another preferred embodiment, the protein composition comprises Rv0577 and Rv1094 and Rv3048 and R0440, and/or their corresponding derivative proteins, specific binding partner proteins, specific amino acid sequences.

In another preferred embodiment, the protein composition comprises Rv0577 and Rv2831 and R0440 and Rv2031c, and/or their corresponding derivative proteins, specific binding partner proteins, specific amino acid sequences.

In another preferred embodiment, the protein composition comprises Rv0440 and Rv0577 and Rv0685 and Rv1094, and/or their corresponding derivative proteins, specific binding partner proteins, specific amino acid sequences.

In another preferred embodiment, the protein composition comprises proteins Rv0440 and Rv0577 and Rv1094 and Rv2831, and/or their corresponding derivatives, specific binding partner proteins, specific amino acid sequences.

In another preferred embodiment, the protein composition comprises proteins Rv0577 and Rv1094 and Rv2031c and Rv3048, and/or their corresponding derivative proteins, specific binding partner proteins, specific amino acid sequences.

In another preferred embodiment, the protein composition comprises proteins Rv0440 and Rv0577 and Rv1094 and Rv2031 c.

In another preferred embodiment, the protein composition comprises proteins Rv0440 and Rv2831 and Rv3048 and Rv0577 and Rv2031c and Rv1094, and/or their corresponding derivatives, specific binding partner proteins, specific amino acid sequences.

In a second aspect of the present invention, there is provided a protein composition, characterized in that it comprises the following proteins:

(b1)1 and above proteins selected from the group consisting of: three or more mycobacterium tuberculosis proteins selected from Rv1575, Rv1773c, Rv1980, Rv2073c, Rv3117, Rv3400, Rv3874, and Rv3875, Rv1514c, Rv1964, Rv1985c, Rv2649, Rv3872, Rv3878 and Rv3402 c;

(b2)1 and above derived proteins selected from the group consisting of: a protein having a sequence selected from the group consisting of substitution, deletion and addition of 1 or more amino acid residues of any one of the proteins (b 1);

(b3)1 or more specific binding partner proteins selected from the group consisting of: 1 or more proteins that specifically bind to any one of (b1) and (b 2); and/or

(b4)1 and above specific amino acid sequence selected from the group consisting of: 1 or more amino acid sequences comprising the entire sequence or partial sequence of any one of the proteins (b1), (b2) and (b 3).

In another preferred embodiment, the protein composition (b1) is a mycobacterium tuberculosis protein.

In another preferred embodiment, the protein composition is used as a marker for evaluating specificity of Mycobacterium tuberculosis.

In another preferred embodiment, the protein composition comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 proteins.

In another preferred embodiment, the protein composition comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 proteins selected from (b 1).

In another preferred embodiment, the protein composition comprises 3, 4, 5, 6, 7, or 8 proteins selected from (b 1).

In another preferred embodiment, the protein composition comprises proteins Rv1773c and Rv1980 and Rv3874 and Rv3875, and/or their corresponding derivatives, specific binding partner proteins, specific amino acid sequences.

In another preferred embodiment, the protein composition comprises Rv1575 and Rv1773c and Rv3874 and Rv3875, and/or their corresponding derivative proteins, specific binding partner proteins, specific amino acid sequences.

In another preferred embodiment, the protein composition comprises Rv1514c and Rv1964 and Rv1985c and Rv2649 and Rv3402c and Rv3874, and/or their corresponding derivative proteins, specific binding partner proteins, specific amino acid sequences.

In another preferred embodiment, the protein composition comprises Rv1575 and Rv1773c and Rv3874 and Rv3875, and/or their corresponding derivative proteins, specific binding partner proteins, specific amino acid sequences.

In another preferred embodiment, the protein composition comprises Rv1980 and Rv3117 and Rv3874 and Rv3878, and/or their corresponding derivatives, specific binding partner proteins, specific amino acid sequences.

In another preferred embodiment, the protein composition comprises Rv2073c and Rv3402c and Rv3874 and Rv3878, and/or their corresponding derived proteins, specific binding partner proteins, specific amino acid sequences.

In another preferred embodiment, the protein composition comprises Rv2073c and Rv3400 and Rv3874 and Rv3878, and/or their corresponding derived proteins, specific binding partner proteins, specific amino acid sequences.

In another preferred embodiment, the protein composition comprises Rv3117 and Rv3400 and Rv3874 and Rv3875, and/or their corresponding derivative proteins, specific binding partner proteins, specific amino acid sequences.

In another preferred embodiment, the protein composition comprises proteins Rv3872 and Rv3874 and Rv3875 and Rv3878, and/or their corresponding derivative proteins, specific binding partner proteins, specific amino acid sequences.

In another preferred embodiment, the protein composition comprises Rv1985c and Rv3402c and Rv3874 and Rv3878, and/or their corresponding derivative proteins, specific binding partner proteins, specific amino acid sequences.

In another preferred embodiment, the protein composition comprises Rv1980 and Rv3872 and Rv3874 and Rv3878, and/or their corresponding derivative proteins, specific binding partner proteins, specific amino acid sequences.

In another preferred embodiment, the protein composition comprises Rv1773c and Rv3117 and Rv3874 and Rv3875, and/or their corresponding derivatives, specific binding partner proteins, specific amino acid sequences.

In another preferred embodiment, the protein composition comprises Rv1980 and Rv1985c and Rv3874 and Rv3875, and/or their corresponding derivative proteins, specific binding partner proteins, specific amino acid sequences.

In another preferred embodiment, the protein composition comprises Rv1773c and Rv3400 and Rv3874 and Rv3875, and/or their corresponding derivative proteins, specific binding partner proteins, specific amino acid sequences.

In another preferred embodiment, the protein composition comprises Rv1575 and Rv3117 and Rv3874 and Rv3875, and/or their corresponding derivatives, specific binding partner proteins, specific amino acid sequences.

In another preferred embodiment, the protein composition comprises Rv1575 and Rv1773c and Rv1980 and Rv3400 and Rv3874 and Rv3875, and/or their corresponding derivatives, specific binding partner proteins, specific amino acid sequences.

In another preferred embodiment, the protein composition comprises Rv1773c and Rv1980 and Rv2073c and Rv3117 and Rv3400 and Rv3874 and Rv3875, and/or their corresponding derivatives, specific binding partner proteins, specific amino acid sequences.

In a third aspect of the present invention, there is provided a kit comprising three or more markers selected from the group consisting of:

(a) a mycobacterium tuberculosis and nontuberculous mycobacterial protein composition according to the first aspect of the invention; and

(b) a mycobacterium tuberculosis protein composition according to the second aspect of the invention.

In another preferred example, the markers are Rv0440 and Rv0577 and Rv0685 and Rv 1094; and Rv3872 and Rv3874 and Rv3875 and Rv3878, and/or their corresponding derivative proteins, specific binding partner proteins, specific amino acid sequences.

In another preferred example, the markers are Rv0577 and Rv1094 and Rv2831 and Rv 3048; and Rv1980 and Rv1985c and Rv3874 and Rv3875, and/or their corresponding derivative proteins, specific binding partner proteins, specific amino acid sequences.

In another preferred embodiment, the markers are Rv0440 and Rv0577 and Rv1094 and Rv 2831; and Rv1575 and Rv1773c and Rv3874 and Rv3875, and/or their corresponding derivative proteins, specific binding partner proteins, specific amino acid sequences.

In another preferred example, the markers are Rv0577 and Rv1094 and Rv2031c and Rv 3048; and combinations of Rv1773c and Rv3400 and Rv3874 and Rv3875, and/or their corresponding derivative proteins, specific binding partner proteins, specific amino acid sequences.

In another preferred embodiment, the marker is a combination of Rv0440 and Rv0577 and Rv1094 and Rv2031c and Rv1575 and Rv3117 and Rv3874 and Rv3875, and/or their corresponding derivative proteins, specific binding partner proteins, specific amino acid sequences.

In another preferred embodiment, the separation tag specifically binds to one or more markers in the kit.

In another preferred embodiment, the separation tag is previously or in use associated with a corresponding label.

In another preferred embodiment, the kit is used for evaluating the sensitivity and specificity of tuberculosis.

In another preferred example, the kit comprises an ELISA immunoassay kit and a semi-dry chromatography kit.

In a fourth aspect of the present invention, there is provided a method for detecting a protein of Mycobacterium tuberculosis, comprising the steps of:

(I) providing an isolated tissue sample to be tested, placing the isolated tissue sample and the kit of the third aspect of the invention into the same system, and fully reacting;

(II) isolating the protein binding fragment bearing the isolation tag from the test system using the isolation tag in the kit; and

(III) measuring the fluorescence signal of the detection system for separating the protein binding fragment with the separation label.

In another preferred example, the detecting of the method includes: qualitative detection and quantitative detection.

In another preferred example, the method further comprises performing a parallel control experiment on a blank sample, wherein the blank sample does not contain mycobacterium tuberculosis protein, and the measured fluorescence signal is A0; if the signal A1/A0>1 measured in the step (III), the mycobacterium tuberculosis protein is considered to be in the sample to be detected; and (5) if the signal A1/A0 measured in the step (III) is less than or equal to 1, determining that the mycobacterium tuberculosis protein is not contained in the sample to be detected.

In another preferred example, in the determination of the fluorescence signal in the step (III), the determination is performed by a microplate reader.

In a fifth aspect of the invention, there is provided use of the kit of the third aspect of the invention, for detection of infection by mycobacterium tuberculosis.

In another preferred embodiment, the mycobacterium tuberculosis infection comprises a pulmonary infection, and/or an extrapulmonary infection.

In another preferred example, the use further comprises: detection of suspected mycobacterium tuberculosis infection, screening for prophylactic mycobacterium tuberculosis infection in non-medical facilities, and/or home preventative mycobacterium tuberculosis infection self-screening.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Detailed Description

The inventor of the invention discovers level difference of 2 groups of specific antibody proteins in human and animal blood of healthy people and different infection degrees of mycobacterium tuberculosis for the first time, and prepares an ELISA (enzyme-linked immunosorbent assay) kit and a field rapid detection kit by utilizing the relatively independent property of the 2 groups of protein indexes in the aspects of sensitivity and specificity, so that the defects that the existing tuberculosis detection reagent has low sensitivity in high-risk people infection screening and cannot give consideration to both sensitivity and specificity can be overcome. On this basis, the inventors have completed the present invention.

Description of the terms

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the term "comprising" or "includes" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….

As used herein, the term "protein" refers to a protein purified by extraction from the supernatant and the cells of a Mycobacterium tuberculosis culture, or a recombinant protein prepared by recombinant gene technology, according to the present invention.

Mycobacterium

The mycobacteria include Mycobacterium tuberculosis (tuberculosis) and nontuberculous mycobacteria (nontuberculous mycobacteria). Nontuberculous mycobacteria are other than Mycobacterium tuberculosis, Mycobacterium bovis and Mycobacterium leprae, and are also called atypical mycobacteria (atypical mycobacteria) which have characteristics different from those of Mycobacterium tuberculosis, such as sensitivity to acid and alkali; the medicine is relatively tolerant to common anti-tubercle bacillus medicines; the growth temperature is not as stringent as for M.tuberculosis; the bacillus subtilis is mostly present in the environment, is a conditional pathogen and can cause tuberculosis-like lesion; the drug resistance to common tuberculosis drugs including isoniazid, streptomycin and the like is increased due to the L-type of the tuberculosis after treatment, and some tuberculosis drugs are not cured after years of treatment. The L type is not easy to sensitize lymphocytes due to the lipid deficiency of cell walls, and the tuberculin test can be negative. Therefore, the kit also has important clinical value for detecting the infection of nontuberculous mycobacteria.

Genetic loci

As used herein, the terms "Rv 0440", "Rv 0577", "Rv 0685", "Rv 1094", "Rv 1468 c", "Rv 1575", "Rv 1773 c", "Rv 1980", "Rv 2031 c", "Rv 2073 c", "Rv 2831", "Rv 3048", "Rv 3117", "Rv 3400", "Rv 3874", "Rv 3875", "Rv 1514 c", "Rv 1964", "Rv 1985 c", "Rv 2649", "Rv 3402 c", "Rv 3872", "Rv TCC 3878" are sequential Locus Names of the whole genome of the Mycobacterium tuberculosis standard strain Mycobacterium tuberculosis (strain Na 27294/H37Rv) (Ordered Locus Names, https:// myofiber. flu. disc). The cloned genes mentioned herein are all derived from Mycobacterium tuberculosis (strain ATCC27294/H37 Rv). All amino acid sequences were derived from the UNIPROT protein database (https:// www.uniprot.org /), and the gene sequences were derived from the gene database GeneBank (https:// www.ncbi.nlm.nih.gov/nuccore /).

The profile of each locus herein is as follows, and the sequence information is shown in table 1:

rv0440, 60kDa chaperone 2, otherwise known to include 65kDa antigen, antigen a, GroEL protein 2, heat shock protein 65, protein Cpn 60-2; the gene name: groEL2, groEL-2, groL2, hsp65, mtc 28; participate in the maintenance of other functional protein conformations of the mycobacterium tuberculosis under stress conditions. The amino acid sequence is shown as SEQ ID No. 1.

Rv0577, glyoxalase glyyxylase CFP32, alias 27kDa antigen CFP32, gene name: cfp32, cfp30B, TB 27.3. Participate in the metabolic pathway of pyruvaldehyde detoxification, induce the maturation of dendritic cells, and increase the expression of cell surface molecular markers CD80, CD86, MHC I and II and the synthesis and secretion of inflammatory cytokines (TNF-alpha, IL-6, IL-1beta and IL-12p 70). The amino acid sequence is shown as SEQ ID No. 2.

Rv0685, elongation factor Tu, EF-Tu for short, gene name tuf; promotes binding of GTP-dependent aminoacyl-tRNA to the ribosomal A site during protein synthesis. The amino acid sequence is shown as SEQ ID No. 3.

Rv1094 acyl dehydrogenase DesA2, gene name: desA 2; is involved in the metabolism of fatty acids and lipids of mycobacteria. The amino acid sequence is shown as SEQ ID No. 4.

Rv1468c, PE-PGRS family protein PE _ PGRS29, Gene name: PE _ PGRS29, the amino acid sequence is shown as SEQID No. 5.

Rv1575, presumed to be the PhiRv1 phage protein, gene name: rv 1575. The amino acid sequence is shown as SEQ ID No. 6.

Rv1773c, presumed to be a transcriptional regulator protein, gene name: rv1773c, and the amino acid sequence is shown in SEQ ID No. 7.

Rv1980, immunogenic protein MPT64, gene name: mpt64, the amino acid sequence is shown in SEQ ID No. 8.

Rv2031c, Alpha-crystallin Acr, alternative name: 14kDa antigen, 16kDa antigen, HSP 16.3, Nox 16; the gene name: hspX; has molecular chaperone function and can inhibit the thermal denaturation of alcohol dehydrogenase strongly. The amino acid sequence is shown as SEQ ID No. 9.

Rv2073c, redox protein, gene name: rv2073 c. The amino acid sequence is shown as SEQ ID No. 10.

Rv2831, acyl-CoA dehydrogenase Crotonase, EchA16, gene name: echA 16. Is involved in the metabolism of fatty acids and lipids of mycobacteria. The amino acid sequence is shown as SEQ ID No. 11.

Rv3048, nucleoside diphosphate reductase beta subunit nrdF2, alternative name: the small subunit of ribonucleotide reductase R2-2; the gene name: nrdF 2. Catalyzes the reaction for synthesizing deoxynucleoside by taking nucleoside as a raw material and participates in a DNA replication biological pathway. The amino acid sequence is shown as SEQ ID No. 12.

Rv3117, presumably thiosulfate thiotransferase, alternative name: a rhodanese-like protein; the gene name: cysA1, cysA. The amino acid sequence is shown as SEQ ID No. 13.

Rv3400, an unidentified protein, gene name: rv 3400; the amino acid sequence is shown as SEQ ID No. 14.

Rv3874, ESAT-6-like early protein EsxB, alias 10kDa culture filtrate protein CFP-10, and secretory antigen protein MTSA-10; the gene name: esxB, the amino acid sequence is shown in SEQ ID No. 15.

The Rv3875, 6kd early-stage secretes antigen protein, namely ESAT-6; the gene name: esxA, the amino acid sequence is shown as SEQ ID No. 16.

Rv1514c, a glycosyltransferase, gene name: rv1514c, the amino acid sequence is shown in SEQ ID No. 17.

Rv1964, a conserved membrane protein having phospholipid transfer activity, gene name: ybE 3A, and the amino acid sequence is shown in SEQ ID No. 18.

Rv1985c, an HTH-type transcription regulator, gene name: rv1985c, and the amino acid sequence is shown in SEQ ID No. 19.

Rv2649, a transposase, necessary for transposition of transposable element IS 6110. The amino acid sequence is shown as SEQ ID No. 20.

Rv3402c, a transaminase, gene name: rv3402c, and the amino acid sequence is shown as SEQ ID No. 21.

Rv3872, PE family immunomodulatory protein PE35, gene name: PE35, the amino acid sequence is shown in SEQ ID No. 22.

Rv3878, ESX-1 secretion-related protein EspJ, Gene name: espJ, the amino acid sequence is shown in SEQ ID No. 23.

TABLE 1

Variants of the proteins described herein and shown in table 1, said variants comprising: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that hybridizes under high or low stringency conditions with DNA encoding an antibody of the invention, and polypeptides or proteins obtained using antisera to an antibody of the invention.

In the present invention, "a derived protein having a sequence of one or more amino acid residues of the protein described in (i)" refers to a polypeptide formed by substituting up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with amino acids having similar or similar properties, as compared with the amino acid sequence of the protein described in Table 1 of the present invention. The polypeptides resulting from these amino acid substitutions are preferably produced by performing amino acid substitutions according to Table 2.

TABLE 2

Initial residue(s)	Representative substitutions	Preferred substitutions
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Lys；Arg	Gln
Asp(D)	Glu	Glu
			Cys(C)	Ser	Ser
Gln(Q)	Asn	Asn
			Glu(E)	Asp	Asp
Gly(G)	Pro；Ala	Ala
			His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu；Val；Met；Ala；Phe	Leu
			Leu(L)	Ile；Val；Met；Ala；Phe	Ile
Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Leu；Val；Ile；Ala；Tyr	Leu
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Ser	Ser
Trp(W)	Tyr；Phe	Tyr
			Tyr(Y)	Trp；Phe；Thr；Ser	Phe
Val(V)	Ile；Leu；Met；Phe；Ala	Leu

Sensitivity and specificity

Sensitivity is the ratio of the number of patients with disease detected in the test to the total number of patients (true positive rate), and is used to indicate how much the index is likely to be used for diagnosing the disease without missed diagnosis.

Specificity is the ratio of the number of patients detected to the total number of patients without a disease (false positive rate), and is the index of how much chance of not misdiagnosing a disease is given.

An ideal clinical test marker should have 100% diagnostic specificity and 100% sensitivity, and virtually nonexistent. When diagnosing disease with a single index, the relationship between sensitivity and specificity can be represented by the ROC curve: the sensitivity of detection is increased and the specificity is inevitably reduced, and vice versa. The area under the ROC curve was used to determine the diagnostic efficacy of the index. There is a need to determine the optimal diagnostic value based on the significance and value of specific disease missed and misdiagnosis to the patient and the public's life health, balancing sensitivity and specificity.

SARS and cold are exemplified. SARS is a disease with extremely strong spread and infection, one missing diagnosis has the chance of infecting dozens of people, and once any person is infected, the cure rate is low and the survival chance is small. Therefore, the missed diagnosis has great risks to the individual life of the patient and the life of the public in society, the sensitivity needs to reach 100 percent, the missed diagnosis cannot be performed for one position even if the missed diagnosis is 100 percent, and the specificity is not required. Although the diseases such as cold are also strong in spreading infection, the disease can be self-healed generally for about 1 week even if the disease is infected, the personal life and other lives of patients are not greatly threatened by missed diagnosis, and the requirement on sensitivity can be properly reduced.

Common experimental techniques and methods

Enzyme linked immunosorbent assay (ELISA) color development technology

Preparing the protein concentration of the mycobacterium tuberculosis of the invention to be 1-5 mu g/mL by using 0.05mmol/L carbonate buffer solution (pH 9.6), coating a 96-pore plate by 100 mu L per pore, and standing overnight at 4 ℃; washing the plate washing machine with 1 XPBST washing buffer for the next time for 5 times; adding 300 μ L of sealing solution containing 3% skimmed milk powder into each well, sealing at 37 deg.C for 2 hr, washing the plate with PBST for 6 times, and drying; mu.L of blocking solution was added to each well using a lining gun, 50. mu.L of sample treatment solution was added to each well, and 50. mu.L of the plasma sample to be tested was added to each well. Incubating for 1h at 37 ℃, and washing a 96-well plate for 6 times; adding pre-prepared HRP-labeled goat anti-human IgG polyclonal antibody 100 mu L diluted by PBST according to 5,000-10,000 times into each hole, incubating for 1h at 37 ℃, and washing a 96-hole plate for 6 times; adding 100 mu L of TMB substrate buffer solution into each hole, and incubating for 10-30 min at 37 ℃; adding 50 mu L of H2SO4 solution with the concentration of 2mol/L into each hole, and stopping the reaction; measuring an Abs value at a wavelength of 450nm of the microplate reader, and judging an experimental result (if the OD450 value of the sample to be detected is greater than the sum of the OD450 mean value of the negative control sample and 3 times of standard deviation thereof, judging that the sample to be detected is positive, and if the OD450 value is less than the sum, judging that the sample to be detected is negative;

enzyme linked immunosorbent assay (ELISA) chemiluminescence technology

Respectively preparing the protein of the mycobacterium tuberculosis of the invention to the concentration of 5 mu g/mL by using 0.05mmol/L carbonate buffer solution (pH 9.6), adding one protein into each 96-well plate, adding 100 mu L of protein into each well, standing overnight at 4 ℃, and washing 5 times by daily 1 XPBST washing buffer solution on a plate washing machine; adding 300 μ L of sealing solution containing 3% skimmed milk powder into each well, sealing at 37 deg.C for 2 hr, washing the plate with PBST for 6 times, and drying; adding 100 mu L of sealing solution into each hole of the row gun, and incubating for 16h at 4 ℃; and (3) drying: drying overnight at the temperature of 18-26 ℃ and the humidity of less than 20%; and (4) vacuumizing and packaging the coated plate by using an aluminum foil bag, and preserving at 2-8 ℃.

50 mul of sample processing liquid is added into each hole of a 96-hole enzyme label plate, then 50 mul of plasma sample to be detected is respectively added, and incubation is carried out for 60min at 37 ℃. The plate was washed 5 times with the washing solution using a plate washer and patted dry on absorbent paper. Adding 100 mu l of HRP-labeled goat anti-human IgG polyclonal antibody diluted by PBST according to 5,000-10,000 times into each hole, and incubating for 1h at 37 ℃; the plate was washed 5 times with the washing solution using a plate washer and patted dry on absorbent paper. Adding 100 mu L of TMB substrate buffer solution into each hole, and incubating for 10-30 min at 37 ℃; adding 50 mu L of H2SO4 solution with the concentration of 2mol/L into each hole, and stopping the reaction; and (5) measuring the light absorption value at the wavelength of 450nm by using an enzyme-labeling instrument, and recording. Judging the experimental result (the OD450 value of the sample to be tested is larger than the sum of the negative control sample OD450 mean value and 3 times of standard deviation thereof, judging the sample to be tested to be positive, and judging the sample to be negative if the value is lower than the value;

semi-dry micro-chromatography rapid detection test paper

The bottom layer of the test paper is used as a support plate, a sample pad, a combination pad, an NC membrane and an absorption pad which are closely connected are sequentially adhered to the support plate, a colloidal gold-labeled or fluorescence-labeled anti-human antibody monoclonal antibody is adsorbed on the combination pad, the Mycobacterium tuberculosis protein is sprayed on the NC membrane to serve as a detection line, and human antibodies (IgG, IgM, IgA and IgE) are sprayed to serve as a quality control line, and the amino acid sequence of the natural Mycobacterium tuberculosis protein is shown in table 1.

The preparation method of the semi-dry micro-chromatography rapid detection test paper (test card) specifically comprises the following steps:

(1) preparation of colloidal gold and fluorescently labeled antibody

Diluting rabbit anti-human IgG, IgM, IgA and IgE monoclonal antibodies by using PBS (phosphate buffer solution) with the particle diameter of 25-30 nm, adding the diluted rabbit anti-human IgG, IgM, IgA and IgE monoclonal antibodies into a colloidal gold solution to enable the final concentration of the antibodies to be 15 mu g/mL, removing supernatant through centrifugal purification, using 1% bovine serum albumin BSA as a stabilizer to obtain the rabbit anti-human IgG, IgM, IgA and IgE monoclonal antibodies marked by the colloidal gold, and storing the monoclonal antibodies at 4 ℃ for later use;

the preparation method comprises the following steps of (1) respectively diluting rabbit anti-human IgG, IgM, IgA and IgE monoclonal antibodies by using a nano fluorescent material (the particle diameter is less than 200nm) with PBS (phosphate buffer solution) with the pH value of 8.2 and the concentration of 0.01M, adding the diluted rabbit anti-human IgG, IgM, IgA and IgE monoclonal antibodies into colloidal gold solution to enable the final concentration of the antibodies to be 15 mu g/mL, removing supernatant through centrifugal purification, and using 1% bovine serum albumin BSA as a stabilizer to obtain the rabbit anti-human IgG, IgM, IgA and IgE monoclonal antibodies with nano fluorescent markers, and storing the monoclonal antibodies at the temperature of 4;

(2) preparation of NC film

Respectively and uniformly spraying natural mycobacterium tuberculosis protein and human antibodies (IgG, IgM, IgA and IgE) onto an NC membrane to serve as a detection line and a quality control line, wherein the line width of the detection line and the quality control line is 1mm, the distance between the detection line and the quality control line is 2-5mm, the detection line and the quality control line are positioned at the center of the NC membrane, and then placing the NC membrane at 37 ℃ for drying, and storing for later use after packaging;

(3) preparation of a reagent strip or test card

Adsorbing a colloidal gold-labeled or fluorescence-labeled monoclonal antibody on a binding pad, sequentially keeping the overlapping width of 2mm of a sample pad, the gold-labeled antibody binding pad, an NC membrane and an absorption pad, adhering the sample pad, the gold-labeled antibody binding pad, the NC membrane and the absorption pad to a support plate to obtain a reagent plate, cutting the reagent plate into 50mm multiplied by 10mm to obtain reagent strips, immediately putting the reagent strips into an aluminum foil bag with a drying agent, and sealing the aluminum foil bag; or cutting the reagent plate into 30mm × 10mm, placing into a test card box to obtain the test card, and immediately placing into an aluminum foil bag for sealing.

(4) Use method of rapid detection immune test paper

Dripping a proper amount of sample solution on a sample pad, standing for 5-10 min, judging whether the sample contains the specific antibody of the mycobacterium tuberculosis according to the color development conditions of the detection line and the quality control line, and if the detection line and the quality control line are both shown, obtaining a positive result, wherein the sample contains the specific antibody of the mycobacterium tuberculosis; if the detection line is not displayed and the quality control line is displayed, the result is negative, and the specific antibody of the mycobacterium tuberculosis in the sample is lower than the detection limit of the test strip; if the quality control line is not displayed, the test strip is invalid.

The technical scheme of the invention has the following beneficial effects:

1. the sensitivity and specificity for detecting the mycobacterium tuberculosis infection are divided into 2 groups of protein marker combinations, sensitivity index data can be mainly referred to when screening the population with low load infection and negative thallus detection and the population with high risk, and specificity index data is mainly referred to when in clinical diagnosis, so that different requirements on the sensitivity and the specificity under different application scenes are met.

2. The mycobacterium tuberculosis protein combination used in the invention has good complementarity for antibody reaction of human infection mycobacterium tuberculosis, can overcome the defects of insufficient sensitivity of the existing tuberculosis detection kit, incapability of finding tuberculosis infection in time and effective treatment and control of infection. Not only improves the sensitivity of detection of people without tuberculosis symptoms to people with bacterial yin infection; meanwhile, the specificity of the mycobacterium tuberculosis infection required by accurate judgment in clinical treatment is ensured.

3. The detection test paper prepared by the invention has the advantages of small sample consumption when in use, simple operation, on-site result output within 5-10 min, high detection speed, no need of special instruments and equipment and low detection cost, thereby solving the problem that suspected tuberculosis infected persons need to be confirmed as soon as possible in remote underdeveloped areas lacking professional medical staff.

4. The detection result of the detection test paper can be uploaded to a remote expert system for semi-quantitative data processing except for on-site visual detection of an operator, is more professional, plays an important role in preventing and controlling tuberculosis in remote underdeveloped areas, and has potential popularization and application values.

The invention is further illustrated with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring Harbor laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight.