CN113631723B

CN113631723B - Application of tuberculosis marker in tuberculosis diagnosis and efficacy evaluation

Info

Publication number: CN113631723B
Application number: CN201980093953.8A
Authority: CN
Inventors: 谢建平; 古寅钟; 龚真; 陈娟
Original assignee: Southwest University
Current assignee: Southwest University
Priority date: 2019-03-29
Filing date: 2019-03-29
Publication date: 2023-11-14
Anticipated expiration: 2039-03-29
Also published as: WO2020198990A1; CN113631723A

Abstract

The invention relates to application of tuberculosis markers BATF2, UBE2L6, VAMP5 and/or SERPING1 in tuberculosis diagnosis and efficacy evaluation. In particular, the invention provides the application of tuberculosis markers BATF2, UBE2L6, VAMP5 and/or SERPING1 genes, mRNA, cDNA, protein or detection reagents thereof, and the application is used for preparing diagnostic reagents or kits for diagnosing tuberculosis. Research shows that the tuberculosis marker can be used as a marker for diagnosing tuberculosis and evaluating the treatment effect of tuberculosis, and has high sensitivity and specificity.

Description

Application of tuberculosis marker in tuberculosis diagnosis and efficacy evaluation

Technical Field

The present invention relates to the field of medicine and diagnostics. More particularly, the invention relates to the use of tuberculosis markers BATF2, UBE2L6, VAMP5 and/or SERPING1 in tuberculosis diagnosis and efficacy assessment.

Background

Tuberculosis is a major infectious disease that endangers human health and is one of the ten leading causes of death worldwide. According to World Health Organization (WHO) 2018 global tuberculosis report, about 1000 tens of thousands of people worldwide have tuberculosis in 2017, and 160 tens of thousands of people die from the disease.

Tuberculosis is long in treatment course, long in infection period and is transmitted through respiratory tract. At present, tuberculosis patients easily delay the disease request, increase the treatment burden and cause the spread of tuberculosis by the side surface due to the lack of an efficient and accurate diagnosis method. Early treatment was found to be critical in preventing and treating tuberculosis.

Diagnosis of tuberculosis mainly depends on the traditional acid-fast staining smear and culture technology of tubercle bacillus. However, the former has poor sensitivity, and the latter takes a long time, and is not satisfactory for rapid diagnosis of tuberculosis. Immunological and molecular diagnosis techniques, imaging methods are convenient and rapid, but the sensitivity of diagnosis is still to be improved. Therefore, developing a sensitive and efficient tuberculosis diagnostic reagent has important significance for preventing and treating tuberculosis.

Thus, there is an urgent need in the art to develop specific markers and detection methods that can be used for early diagnosis of tuberculosis with high sensitivity and high specificity.

Disclosure of Invention

The invention aims to provide a specific marker for early diagnosis of tuberculosis with high sensitivity and high specificity and a detection method.

In a first aspect the present invention provides the use of the tuberculosis marker BATF2, UBE2L6, VAMP5 and/or SERPING1 gene, mRNA, cDNA, protein, or detection reagents thereof for the preparation of a diagnostic reagent or kit for (a) diagnosing tuberculosis and/or (b) evaluating the effect of tuberculosis treatment.

In another preferred embodiment, the tuberculosis comprises tuberculosis.

In another preferred embodiment, the detection reagent comprises:

(a) Specific antibodies, specific binding molecules for tuberculosis markers BATF2, UBE2L6, VAMP5 and/or SERPING 1; and/or

(b) Primers or primer pairs, probes or chips (e.g., nucleic acid chips or protein chips) for specifically amplifying mRNA or cDNA of tuberculosis markers BATF2, UBE2L6, VAMP5 and/or SERPING 1.

In another preferred embodiment, the diagnosis comprises an early diagnosis, an auxiliary diagnosis, or a combination thereof.

In another preferred embodiment, the tuberculosis marker BATF2, UBE2L6, VAMP5 and/or SERPING1 gene, mRNA, cDNA or protein is derived from a mammal.

In another preferred embodiment, the mammal includes a human and a non-human mammal, preferably a primate (e.g., human).

In another preferred embodiment, the tuberculosis marker BATF2, UBE2L6, VAMP5 and/or SERPING1 gene, mRNA, cDNA or protein is of human origin.

In another preferred embodiment, the detection is for an ex vivo sample.

In another preferred embodiment, the ex vivo sample comprises: a blood sample, a serum sample, a tissue sample, a body fluid sample, or a combination thereof.

In another preferred embodiment, the sample is a mononuclear cell sample isolated from peripheral blood.

In another preferred embodiment, the detection is performed by detecting the expression level of BATF2, UBE2L6, VAMP5 and/or SERPING1 genes in peripheral blood mononuclear cells.

In another preferred embodiment, the detection reagent is coupled to or carries a detectable label.

In another preferred embodiment, the detectable label is selected from the group consisting of: chromophores, chemiluminescent groups, fluorophores, isotopes or enzymes.

In another preferred embodiment, the antibody is a monoclonal antibody or a polyclonal antibody.

In another preferred embodiment, the diagnostic reagent comprises an antibody, a primer, a probe, a sequencing library, a nucleic acid chip (e.g., a DNA chip), or a protein chip.

In another preferred embodiment, the nucleic acid chip comprises a substrate and specific oligonucleotide probes spotted on the substrate, wherein the specific oligonucleotide probes comprise probes specifically binding to tuberculosis markers BATF2, UBE2L6, VAMP5 and/or SERPING1 polynucleotides (mRNA or cDNA).

In another preferred embodiment, the protein chip comprises a substrate and specific antibodies spotted on the substrate, wherein the specific antibodies comprise specific antibodies of anti-tuberculosis markers BATF2, UBE2L6, VAMP5 and/or SERPING 1.

In another preferred embodiment, the use further comprises for predicting the therapeutic effect (prognosis) of a patient suffering from tuberculosis.

In another preferred embodiment, when the expression level of the marker gene is reduced to the level of normal population, it is suggested that the tuberculosis treatment effect is better or the tuberculosis is substantially cured.

In another preferred embodiment, the reagents are PCR primer pairs.

In another preferred embodiment, the primer pair is selected from the group consisting of: a primer pair for amplifying BATF2, a primer pair for amplifying UBE2L6, a primer pair for amplifying VAMP5, a primer pair for amplifying SERPING1, or a combination thereof.

In another preferred example, the primer pair for amplifying BATF2 is SEQ ID No.:1 and 2; the primer pair used to amplify UBE2L6 is SEQ ID No.:3 and 4; the primer pair used to amplify VAMP5 is SEQ ID No.:5 and 6; primer pairs for amplifying SERPING1 are SEQ ID No.:7 and 8.

In a second aspect of the invention, a kit is provided, said kit comprising a detection reagent for detecting the genes, mRNA, cDNA, proteins, or combinations thereof, of the tuberculosis markers BATF2, UBE2L6, VAMP5 and/or SERPING 1.

In another preferred embodiment, the detection reagent comprises a detection reagent for detecting 2, 3 or 4 markers of the tuberculosis markers BATF2, UBE2L6, VAMP5 and SERPING 1.

In another preferred embodiment, the kit contains tuberculosis markers BATF2, UBE2L6, VAMP5 and/or SERPING1 genes, mRNA, cDNA and/or protein as a control or quality control.

In another preferred embodiment, the kit further comprises a label or instructions stating that the kit is used for (a) diagnosing tuberculosis, and/or (b) evaluating the effect of tuberculosis treatment.

In another preferred embodiment, the reagents are PCR primer pairs.

In another preferred embodiment, the label or description refers to the following: if the geometric mean of the transcript levels of the BATF2, UBE2L6, VAMP5 and SERPING1 genes in the PBMCs of the test subject is greater than 0.0009426436, the test subject may be preliminarily predicted to be an active tuberculosis patient, and if the geometric mean of the transcript levels of the BATF2, UBE2L6, VAMP5 and SERPING1 genes in the PBMCs of the test subject is less than or equal to 0.0009426436, the test subject is an inactive tuberculosis patient.

In another preferred embodiment, the label or description refers to the following:

if the concentration C1 of the tuberculosis markers BATF2, UBE2L6, VAMP5 and/or SERPING1 of the detected subject is significantly higher than the control reference value C0, the probability of tuberculosis occurrence of the subject is higher than that of the normal population.

In another preferred embodiment, the control reference value C0 is the concentration of the tuberculosis marker BATF2, UBE2L6, VAMP5 and/or SERPING1 in the same sample in the normal population, the concentration of the tuberculosis marker BATF2, UBE2L6, VAMP5 and/or SERPING1 in the same sample in the non-tuberculosis lung disease patient population, or the concentration of the tuberculosis marker BATF2, UBE2L6, VAMP5 and/or SERPING1 in the same sample of the test subject itself prior to receiving the tuberculosis drug treatment.

if the concentration Z1 of the tuberculosis markers BATF2, UBE2L6, VAMP5 and/or SERPING1 in the detected cells is significantly higher than the control reference value Z0, the probability of tuberculosis occurrence of the detected subject is higher than that of the normal cells.

In another preferred embodiment, the detected cells are mononuclear cells from blood, and the "normal cells" are cells of the same class as the detected cells.

In another preferred embodiment, the control reference value Z0 is the concentration of the tuberculosis markers BATF2, UBE2L6, VAMP5 and/or SERPING1 in the general cells, or the concentration of the tuberculosis markers BATF2, UBE2L6, VAMP5 and/or SERPING1 in the normal cells of the same kind.

In another preferred embodiment, the tuberculosis marker concentration comprises a protein concentration, an mRNA concentration, or a combination thereof.

In another preferred embodiment, the term "significantly higher" means that the ratio of C1/C0 or Z1/Z0 is not less than 2, preferably not less than 5, more preferably not less than 10, most preferably not less than 20.

In another preferred embodiment, the kit is used for diagnosing tuberculosis.

when the expression level of each marker gene (or a combination thereof) is reduced to a level in normal population, it is suggested that the tuberculosis treatment effect is better or the tuberculosis is substantially cured.

In a third aspect of the present invention, there is provided a detection method comprising the steps of:

(a) Providing a detection sample, wherein the detection sample is a mononuclear cell sample separated from peripheral blood;

(b) Detecting the expression level of BATF2, UBE2L6, VAMP5 and/or SERPING1 genes in the mononuclear cells in the detection sample, and marking the expression level as C1; and

(c) The tuberculosis markers BATF2, UBE2L6, VAMP5 and/or SERPING1 concentration C1 were compared to a control reference value C0.

In another preferred embodiment, the sample is from a test object.

In another preferred embodiment, the subject is a human.

In another preferred embodiment, if the tuberculosis marker BATF2, UBE2L6, VAMP5 and/or SERPING1 concentration C1 is significantly higher than the control reference value C0, the subject has a greater chance of developing tuberculosis than the normal population.

In another preferred embodiment, the control reference value C0 is the concentration of the tuberculosis markers BATF2, UBE2L6, VAMP5 and/or SERPING1 in the same sample in the normal population.

In another preferred embodiment, the control reference value C0 is the concentration of the tuberculosis markers BATF2, UBE2L6, VAMP5 and/or SERPING1 in the same sample in a population of non-tuberculosis pulmonary patients.

In another preferred embodiment, when the expression level C1 of each marker gene (or a combination thereof) decreases to the control reference value C0, it is suggested that the therapeutic effect of tuberculosis is better or that tuberculosis is substantially cured.

In another preferred embodiment, the method further comprises comparing the expression level C1 with C2, wherein the C2 is the concentration of the tuberculosis marker BATF2, UBE2L6, VAMP5 and/or SERPING1 in the same sample of the test subject itself prior to receiving the tuberculosis drug treatment.

In a fourth aspect of the invention, there is provided a method of diagnosing tuberculosis comprising the steps of:

a) Providing a test sample from a subject, said test sample selected from the group consisting of: a blood sample, a serum sample, or a combination thereof;

b) Detecting the concentration of mRNA and/or protein of tuberculosis markers BATF2, UBE2L6, VAMP5 and/or SERPING1 in the mononuclear cells in the test sample, and marking the concentration as C1; and

c) Comparing the concentration C1 of mRNA and/or protein of the tuberculosis marker BATF2, UBE2L6, VAMP5 and/or SERPING1 with a control reference value C0,

wherein the concentration C1 of mRNA and/or protein of tuberculosis markers BATF2, UBE2L6, VAMP5 and/or SERPING1 in the sample is significantly higher than a control reference value C0, and the probability of tuberculosis of the object to be detected is higher than that of the common crowd.

In another preferred embodiment, the subject includes human and non-human mammals.

In another preferred embodiment, the general population includes a healthy population.

In another preferred embodiment, the subject is a patient suffering from a pulmonary disorder.

In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

In another preferred embodiment, the term "significantly higher" means that the ratio of C1 to C0 is no less than 2, preferably no less than 5, more preferably no less than 10, most preferably no less than 20 or no less than 50.

In another preferred embodiment, the quantitative PCR is used to detect the amounts of the BATF2, UBE2L6, VAMP5 and SERPING1 genes, and the data processing is performed, where the data processing determines whether the test object is a tuberculosis patient according to the amounts of the BATF2, UBE2L6, VAMP5 and SERPING1 genes of the test object.

In a fifth aspect of the present invention, there is provided a method of evaluating the effect of treatment of tuberculosis, comprising the steps of:

(a) Providing a test sample which is a single nuclear cell sample isolated from peripheral blood of a subject, wherein the subject is a patient during or after treatment for tuberculosis;

(c) Comparing the concentration C1 of the tuberculosis markers BATF2, UBE2L6, VAMP5 and/or SERPING1 with a control reference value C0, wherein if the concentration C1 of the tuberculosis marker is reduced to the control reference value C0, the tuberculosis treatment effect is better or the tuberculosis is basically cured.

In another preferred embodiment, the "concentration C1 decreases to a control reference value C0" means that the ratio C1/C0 is 0.8-1.2.

In another preferred embodiment, the method further comprises: comparing the expression level C1 with C2, wherein the C2 is the concentration of tuberculosis markers BATF2, UBE2L6, VAMP5 and/or SERPING1 in the same sample of the test subject itself prior to receiving the tuberculosis drug treatment.

In a sixth aspect of the invention there is provided the use of a tuberculosis marker BATF2, UBE2L6, VAMP5 and/or SERPING1 gene, mRNA, cDNA or protein as a marker for diagnosing tuberculosis and/or evaluating the effect of tuberculosis treatment.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

FIG. 1 shows the expression levels of BATF2, UBE2L6, VAMP5 and SERPING1 in tuberculosis group (tuberculosis patient) and healthy control group (normal control) detected by real-time fluorescent quantitative PCR.

Fig. 2 shows an analysis of ROC curves.

FIGS. 3A and 3B show that the VAMP5 gene is expressed in Active tuberculosis patients (Active) significantly higher than other groups, and the expression level gradually decreases during the course of treatment, respectively.

FIGS. 4A and 4B show, respectively, that the UBE2L6 gene is expressed in significantly higher amounts in patients with active tuberculosis than in other groups, and that the expression levels gradually decrease during the course of treatment.

FIGS. 5A and 5B show that the SERPING1 gene expression level is significantly higher in active tuberculosis patients than in other groups, and gradually decreases during treatment.

FIGS. 6A and 6B show that the expression level of BATF2 gene is significantly higher in patients with active tuberculosis than in other groups, respectively, and gradually decreases during treatment.

Figures 7A and 7B show that the geometric mean of the 4 genes is significantly higher in active tuberculosis patients than in other groups, respectively, and gradually decreases during treatment.

In fig. 3-7, it includes: healthy people (control), active before treatment, active under treatment (treatment), active after recovery and recovery after treatment (recovery).

Detailed Description

The present inventors have conducted extensive and intensive studies and, for the first time, unexpectedly found a class of tuberculosis markers present in blood, including BATF2, UBE2L6, VAMP5 and/or SERPING1. The tuberculosis marker is specifically highly expressed in mononuclear cells in blood of a tuberculosis patient, and thus can be used as a specific marker for tuberculosis diagnosis (especially early diagnosis and/or auxiliary diagnosis). The experimental results show that compared with normal people, the expression level of the tuberculosis markers BATF2, UBE2L6, VAMP5 and/or SERPING1 in tuberculosis patients is higher than 10 times (about 60-100 times) on average, and is earlier than other most detectable indexes, and the tuberculosis markers can be used as markers for diagnosing tuberculosis. The present invention has been completed on the basis of this finding.

Terminology

The term "sample" or "specimen" as used herein refers to a material that is specifically associated with a subject from which particular information about the subject can be determined, calculated, or inferred. The sample may be composed in whole or in part of biological material from the subject.

As used herein, the term "expression" includes the production of mRNA from a gene or gene portion, and includes the production of a protein encoded by RNA or gene portion, and also includes the presence of a detection substance associated with expression. For example, cDNA, binding of a binding ligand (e.g., an antibody) to a gene or other oligonucleotide, protein or protein fragment, and chromogenic portions of the binding ligand are included within the term "expressed". Thus, an increase in half-pel density on immunoblots, such as Western blots, is also within the term "expression" based on biological molecules.

As used herein, the term "reference value" or "control reference value" refers to a value that is statistically relevant to a particular result when compared to the result of an analysis. In a preferred embodiment, the reference value is determined based on a statistical analysis of mRNA expression and/or protein expression of the comparative tuberculosis markers bat 2, UBE2L6, VAMP5 and/or SERPING1 and study of known clinical results. Some of these studies are shown in the examples section herein. However, the studies from the literature and the user experience of the methods disclosed herein can also be used to produce or adjust the reference value. Reference values may also be determined by considering conditions and results that are particularly relevant to the patient's medical history, genetics, age and other factors.

Tuberculosis markers BATF2, UBE2L6, VAMP5 and/or SERPING1

As used herein, the term "tuberculosis marker of the present invention" refers to one or more markers selected from the group consisting of: BATF2, UBE2L6, VAMP5 and SERPING1.

In the present invention, the terms "tuberculosis marker protein of the present invention", "polypeptide of the present invention", or "BATF2, UBE2L6, VAMP5 and/or SERPING1 polypeptide" are used interchangeably and refer to a polypeptide having any one or more of the four tuberculosis markers BATF2, UBE2L6, VAMP5 and SERPING1 of the present invention. The proteins of the invention may or may not contain an initiating methionine. Furthermore, the term also includes full length tuberculosis markers BATF2, UBE2L6, VAMP5 and/or SERPING1 and fragments thereof. In the present invention, the tuberculosis marker protein includes its complete amino acid sequence, its secretory protein, its mutant and its functionally active fragment.

BATF2, basic leucine zipper transcriptional factor ATF-like, basic leucine zipper transcription factor 2 (BATF 2), belongs to the family of AP-1 transcription factors. BATF2, by binding to AP-1, inhibits AP-1 mediated transcription of target genes, thereby affecting the ability of cells to proliferate, invade, etc. Alternatively, BATF2 induces apoptosis in tumor cells by inhibiting the activity of nuclear factor KB (NF-xB) in the case of p53 mediation. In addition, BATF2 can also inhibit lung adenocarcinoma epithelial cell interstitium (EMT) by activating GSK-3p and 13-catenin signaling pathway, thereby promoting tumor invasion and metastasis.

UBE2L6, ubiquitin/ISG15-conjugating enzyme E L6, E2 Ubiquitin ligase. UBE2L6 catalyzes the covalent binding of ubiquitin or ISG15 to other proteins. Play a role in E6-AP-induced ubiquitination of p 53. Promoting ubiquitination and subsequent proteasome degradation of FLT 3.

VAMP5, vesicle-associated membrane protein 5, vesicle-associated membrane protein 5.VAMP5 belongs to a member of the SNARE superfamily, involved in the constitutive exocytosis process, and is involved in the transport and fusion of vesicles. Is a component of the secretory vesicle, myotube and vacuole structures.

SERPING1, P ] plasma protease C1 inhibitor, human plasma protease C1 inhibitor. By inhibiting C1r or C1s protease activity, the C1 complement activation pathway is regulated. In addition, there are critical roles in the clotting, fibrinolysis and kininogenesis processes. Is a high-efficiency FXIIIa inhibitor.

Gene accession numbers of the tuberculosis markers BATF2, UBE2L6, VAMP5 and SERPING1 of the present invention are shown in the following table.

Gene name	NCBI numbering
		UBE2L6	NM_004223.3
BATF2	NM_138456.3
		SERPING1	NM_001032295.1
VAMP5	NM_006634.2

In the present invention, the terms "tuberculosis marker gene", "tuberculosis marker polynucleotide" are used interchangeably and refer to the nucleotide sequences of tuberculosis markers BATF2, UBE2L6, VAMP5 and/or SERPING 1.

It is understood that substitution of nucleotides in the codon is acceptable when encoding the same amino acid. It is further understood that nucleotide substitutions are also acceptable when conservative amino acid substitutions are made by the nucleotide substitutions.

In the present invention, the protein of the present invention also includes conservative variants thereof, which means that up to 10, preferably up to 8, more preferably up to 5, most preferably up to 3 amino acids are replaced by amino acids of similar or similar nature, as compared to the amino acid sequence of the protein of the present invention, to form a polypeptide. These conservatively variant polypeptides are preferably generated by amino acid substitutions according to Table A.

Table A

In case of obtaining amino acid fragments of the nuclear disease markers BATF2, UBE2L6, VAMP5 and/or SERPING1, a nucleic acid sequence encoding the same can be constructed therefrom, and a specific probe can be designed according to the nucleotide sequence. The full-length nucleotide sequence or a fragment thereof can be obtained by PCR amplification, recombinant methods or artificial synthesis. For the PCR amplification method, primers can be designed according to the disclosed tuberculosis markers BATF2, UBE2L6, VAMP5 and/or SERPING1 nucleotide sequences, especially open reading frame sequences, and amplified to obtain the relevant sequences using a commercially available cDNA library or a cDNA library prepared according to a conventional method known to those skilled in the art as a template. When the sequence is longer, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.

Furthermore, the sequences concerned, in particular fragments of short length, can also be synthesized by artificial synthesis. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them.

At present, it is entirely possible to obtain the DNA sequences encoding the proteins of the invention (or fragments, derivatives thereof) by chemical synthesis. The DNA sequence may then be introduced into a variety of existing DNA molecules (e.g., vectors) and cells known in the art.

The polynucleotide sequences of the present invention may be used to express or produce recombinant tuberculosis markers BATF2, UBE2L6, VAMP5 and/or SERPING1 polypeptides by conventional recombinant DNA techniques.

Specific antibodies

In the present invention, the terms "antibody of the present invention" and "antibody specific for an anti-tuberculosis marker" are used interchangeably.

The invention also includes polyclonal and monoclonal antibodies, particularly monoclonal antibodies, specific for the tuberculosis markers BATF2, UBE2L6, VAMP5 and/or SERPING1 polypeptides. The invention also includes antibodies that bind to the gene products of the modified or unmodified forms of the human tuberculosis markers BATF2, UBE2L6, VAMP5 and/or SERPING 1.

The invention includes not only intact monoclonal or polyclonal antibodies, but also antibody fragments having immunological activity, such as Fab' or (Fab) 2 fragments; antibody heavy chain; an antibody light chain; genetically engineered single chain Fv molecules (Ladner et al, U.S. Pat. No.4,946,778); or chimeric antibodies, such as antibodies having murine antibody binding specificity but retaining antibody portions derived from humans.

Antibodies of the invention may be prepared by various techniques known to those skilled in the art. For example, purified human tuberculosis markers BATF2, UBE2L6, VAMP5 and/or SERPING1 gene products or antigenic fragments thereof can be administered to animals to induce polyclonal antibody production. Similarly, cells expressing the human tuberculosis markers BATF2, UBE2L6, VAMP5 and/or SERPING1 proteins or antigenic fragments thereof can be used to immunize animals to produce antibodies. The antibodies of the invention may also be monoclonal antibodies. Such monoclonal antibodies can be prepared using hybridoma technology

Antibodies against human tuberculosis markers BATF2, UBE2L6, VAMP5 and/or SERPING1 proteins can be used in immunohistochemical techniques to detect human tuberculosis markers BATF2, UBE2L6, VAMP5 and/or SERPING1 proteins in a sample, especially a tissue sample or blood sample. Since the tuberculosis markers BATF2, UBE2L6, VAMP5 and/or SERPING1 protein exist in a single nuclear cell in a blood sample, the expression amount thereof can be a detection object.

Detection method

Based on the high expression of tuberculosis markers BATF2, UBE2L6, VAMP5 and/or SERPING1 in mononuclear cells in blood, the invention also provides a corresponding method for diagnosing tuberculosis.

The present invention relates to diagnostic assays for the quantitative and positional detection of protein or mRNA levels of the human tuberculosis markers BATF2, UBE2L6, VAMP5 and/or SERPING 1. Such tests are well known in the art. The human tuberculosis markers BATF2, UBE2L6, VAMP5 and/or SERPING1 protein level or mRNA level detected in the assay can be used for diagnosing (including aiding in diagnosis) whether tuberculosis exists.

A preferred method is to perform quantitative PCR on mRNA or cDNA.

One preferred method is to quantitatively detect mRNA or cDNA, sequencing.

One preferred method is to quantitatively detect the proteins that are markers of tuberculosis.

Preferably, a method for detecting the presence or absence of a tuberculosis marker protein in a sample is to detect using a specific antibody, which comprises: contacting the sample with tuberculosis markers BATF2, UBE2L6, VAMP5 and/or SERPING1 protein specific antibodies; observing whether an antibody complex is formed, the formation of which indicates the presence of tuberculosis markers BATF2, UBE2L6, VAMP5 and/or SERPING1 proteins in the sample.

The tuberculosis marker protein or the polynucleotide thereof can be used for diagnosing tuberculosis. A part or all of the polynucleotides of the present invention can be immobilized as probes on a microarray or DNA chip for analysis of differential expression of genes in mononuclear cells and for gene diagnosis. Antibodies against tuberculosis markers may be immobilized on a protein chip for detection of tuberculosis markers BATF2, UBE2L6, VAMP5 and/or SERPING1 proteins in a sample.

Based on the studies of the present invention, the expression levels (mRNA levels or protein levels) of BATF2, UBE2L6, VAMP5 and/or SERPING1 genes were significantly increased in tuberculosis patients, and the respective increase amounts were at least 10-fold or more. Thus, BATF2, UBE2L6, VAMP5 and/or SERPING1, alone or in combination, may be used as markers for detection or diagnosis (especially for aiding diagnosis and/or early diagnosis) of tuberculosis. In the detection, if the ratio (C1/C0) of the expression level C1 of the marker gene (i.e., BATF2, UBE2L6, VAMP5 and/or SERPING 1) to the corresponding expression level C0 in the normal population is not less than 2, preferably not less than 5 or not less than 10, more preferably not less than 20, most preferably not less than 50, then the risk of tuberculosis is considered to be increased. In particular, when the expression levels of 2, 3 or 4 markers are comprehensively detected, more accurate detection results can be obtained.

According to the experimental results of the present invention, a preferred method of diagnosing active tuberculosis patients is based on geometric mean of transcript levels. Preferably, the method comprises the following criteria: if the geometric mean of the transcript levels of the BATF2, UBE2L6, VAMP5 and SERPING1 genes in the PBMCs of the test subject is greater than 0.0009426436, the test subject may be preliminarily predicted to be an active tuberculosis patient, and if the geometric mean of the transcript levels of the BATF2, UBE2L6, VAMP5 and SERPING1 genes in the PBMCs of the test subject is less than or equal to 0.0009426436, the test subject is an inactive tuberculosis patient.

Furthermore, given the clear trend of decreasing transcript levels of the individual marker genes (BATF 2, UBE2L6, VAMP5 and/or SERPING 1) during and at the completion of the treatment in patients with active tuberculosis, the therapeutic effect of tuberculosis can be reflected or evaluated by detecting the expression levels of said marker genes. Wherein, when the expression level of each marker gene (or the combination thereof) is reduced to the level of normal population, the treatment effect of tuberculosis is indicated to be better or the tuberculosis is basically cured.

Detection kit

Based on the correlation of tuberculosis markers with tuberculosis, the tuberculosis markers BATF2, UBE2L6, VAMP5 and/or SERPING1 can be used as a diagnosis marker of tuberculosis.

The invention also provides a kit for diagnosing tuberculosis, which comprises a detection reagent for detecting tuberculosis markers BATF2, UBE2L6, VAMP5 and/or SERPING1 genes, mRNA, cDNA, protein or a combination thereof. Preferably, the kit contains an antibody or immunoconjugate of the anti-tuberculosis markers BATF2, UBE2L6, VAMP5 and/or SERPING1 of the invention, or an active fragment thereof; or a primer or primer pair, probe or chip containing mRNA or cDNA specifically amplifying tuberculosis markers BATF2, UBE2L6, VAMP5 and/or SERPING 1.

In another preferred embodiment, the kit further comprises a label or instructions stating that the kit is used for diagnosing tuberculosis and/or evaluating the therapeutic effect of tuberculosis.

The main advantages of the invention include:

(a) BATF2, UBE2L6, VAMP5 and/or SERPING1 are the first tuberculous markers discovered by the present inventors, and can be used for diagnosis including tuberculosis, providing reference for more accurate and early diagnosis.

(b) Tuberculosis markers BATF2, UBE2L6, VAMP5 and/or SERPING1 exist in mononuclear cells in blood, so that the sample and detection can be conveniently performed.

(c) Tuberculosis markers BATF2, UBE2L6, VAMP5 and/or SERPING1 can be used as markers for effectively evaluating the treatment effect of tuberculosis.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.

Materials, reagents, instruments and the like used in the examples are commercially available unless otherwise specified.

The quantitative tests in the following examples were all set up in triplicate and the results averaged.

General method

Real-time fluorescent quantitative PCR

Real-time fluorescent quantitative PCR is an experimental method that applies fluorescent energy technology to the polymerase chain reaction. A fluorescent dye called SYBR Green I was used in this experiment. In a PCR reaction system, SYBR Green I emits a fluorescent signal after being specifically doped into a DNA double strand; while SYBR dye molecules that are not incorporated into the chain do not emit any fluorescent signal. Because this method allows the increase in fluorescence signal to be synchronized with the increase in PCR product, that is, the intensity of fluorescence signal emitted by the fluorescent dye is proportional to DNA yield. Therefore, the initial concentration of the target sequence can be obtained by detecting the fluorescence signal intensity in the PCR process, so that the aim of quantification can be fulfilled.

Example 1

BATF2, UBE2L6, VAMP5 and SERPING1 are serum markers for tuberculosis

1.1, study object

In this example, subjects were divided into tuberculosis group and healthy control group

Tuberculosis group includes 15 patients clinically diagnosed as active tuberculosis, without diabetes, tumor and immune related diseases. The ages are 27-63 years old, and the average ages are 37.8 years old, 11 men and 4 women.

Healthy control group: 16 individuals were classified into healthy persons of an age comparable to that of the tuberculosis group. The ages are 20-40 years old, 9 men and 7 women.

1.2 detection of the expression level of BATF2, UBE2L6, VAMP5 and SERPING1 in peripheral blood

For each study object, peripheral blood is collected, peripheral blood mononuclear cells are separated, RNA is extracted and reverse transcribed after culture, and real-time fluorescent PCR quantitative detection is carried out. The method comprises the following steps:

(a) Peripheral blood was collected from each subject in the tuberculosis group and healthy control group.

(b) Peripheral Blood Mononuclear Cells (PBMCs) were isolated: diluting 2ml of collected peripheral blood with basal medium 1640 culture medium, performing density gradient centrifugation with Ficoll (GE Biosciences, USA), collecting cells of middle mononuclear cell layer, washing twice with 1640 culture medium, resuspending cells with serum-free medium AIMV to obtain cell suspension, dripping the cell suspension into 96-well cell culture plate, adding Mycobacterium tuberculosis H37Rv lysate, costimulatory factors CD28 and CD49, 37deg.C, and 5% CO ₂ The incubator was incubated overnight.

(c) RNA extraction and reverse transcription: after step (b) is completed, using TRIzol ^TM Each sample RNA was extracted by Reagent (Invitrogen, USA), and cDNA was obtained by reverse transcription using 20. Mu.l of nuclease-free water as a template after dissolving the RNA.

(d) Real-time fluorescent quantitative PCR: performing real-time fluorescence quantitative PCR detection of the expression levels of BATF2, UBE2L6, VAMP5 and SERPING1 using the cDNA of each sample obtained in step (c) as a template, using a kit KAPA from Kapa Biosystems ^TM The internal reference of the rapid quantitative PCR kit is beta-actin gene, and the primers of BATF2, UBE2L6, VAMP5 and SERPING1 and beta-actin genes are shown in table 1.

TABLE 1 primer sequences

Real-time fluorescent quantitative PCR reaction system: 2 XGreen Master Mix 10. Mu.l, forward primer 0.4. Mu.l, reverse primer 0.4. Mu.l, cDNA 0.2. Mu.l, make up 20. Mu.l with nuclease-free water. The concentration of the forward primer and the reverse primer in the reaction system is 200nM, and the cDNA content in the reaction system is 10ng.

Fluorescent quantitative PCR detection was performed using a LightCycler 480II fluorescent quantitative PCR instrument from roche company.

Reaction conditions: pre-denaturation: 3 minutes at 95 ℃; amplification reaction: the fluorescent signal was detected during the extension phase at 95℃for 5 seconds, 60℃for 30 seconds, 40 cycles. Use 2 ^-△Ct The relative expression level was calculated.

(e) Statistical analysis: for group differences, wilcoxon tests were used for analysis using the R language and ROC curve analysis was performed.

1.3 results

The real-time fluorescent quantitative PCR results show that the expression levels of the BATF2, UBE2L6, VAMP5 and SERPING1 genes are remarkably increased in the tuberculosis group, and the relative expression levels of the BATF2, UBE2L6, VAMP5 and SERPING1 genes in the tuberculosis group are remarkably higher than those in the healthy control group (FIGS. 1A and 1B).

The results showed that there was a significant difference in the expression levels of all 4 genes compared to the expression levels in the normal control:

(a) UBE2L6 and SERPING1 expression level M in tuberculosis patients _Disease And the expression level M in healthy people _Control In contrast, there is about a 16-50 fold difference;

(b) Expression level M of BATF2 in tuberculosis patients _Disease And the expression level M in healthy people _Control Compared with the prior art, the difference is more than or equal to 100 times;

(c) Expression level M of VAMP5 in tuberculosis patients _Disease And the expression level M in healthy people _Control There is a difference of about 50-100 times compared.

(d) For a geometric mean expression based on 4 genes, the geometric mean expression was about 60-100 times higher in patients with active tuberculosis compared to healthy people (normal controls).

The results show that the BATF2, UBE2L6, VAMP5 and/or SERPING1 genes have higher accuracy in diagnosing patients with active tuberculosis.

Subject work profile analysis was performed using the R language based on geometric mean of the relative expression levels of the bat f2, UBE2L6, VAMP5 and SERPING1 genes in tuberculosis group and healthy control group PBMCs.

Subject performance characteristics (receiver operating characteristic, ROC) reflect a balance between sensitivity and specificity, with area under the ROC curve being an important test accuracy indicator, the larger the area under the ROC curve, the greater the diagnostic value of the test.

Sensitivity (true positive rate): the greater the sensitivity, the better the sensitivity, and the ideal sensitivity is 100%.

Specificity (true negative rate): the greater the specificity, the better the ideal specificity is 100%, and the percentage of the actual disease-free which is correctly judged as being disease-free according to the test standard.

The ROC curve is shown in fig. 2. The area under the ROC curve is 0.941 (95% CI: 0.79-1), which demonstrates that diagnosing patients with active tuberculosis based on the expression levels of BATF2, UBE2L6, VAMP5 and SERPING1 genes has a high accuracy.

When the geometric average cut-off value of the transcriptional levels of BATF2, UBE2L6, VAMP5 and SERPING1 genes is taken to be2 ^-10.051 When= 0.0009426436, the sensitivity was 90.0% and the specificity was 92.9%.

Example 2

Changes in transcriptional levels of BATF2, UBE2L6, VAMP5 and SERPING1 genes during treatment of patients with active tuberculosis

In this example, the gene expression of a tuberculosis patient whole blood (whole blood) sample during the course of treatment was studied.

Violin plots show the change in 4 genes during TB treatment, and the rank sum test (Wilcoxon rank sum test) calculates the difference between the two time points.

In this example, 24 tuberculosis patients were examined before treatment (0 month), and the expression of 4 genes was examined for 0.5 (two weeks), 2 months, 6 months and 12 months, respectively.

As shown in fig. 3B, 4B, 5B and 6B, the results indicate the transcriptional level or expression level of the bat f2, UBE2L6, VAMP5 or SERPING1 gene for patients with active tuberculosis:

(a) Expression level M at two weeks of treatment _0.5 And an expression level M at 2 months ₂ There is a significant difference between them;

(b) Expression level M after 6 months of treatment ₆ And the expression level M after 12 months of treatment ₁₂ No obvious difference exists between the two;

(c) And the expression level M before treatment ₀ In contrast, the expression level of each gene (i.e., M) was measured for 2 weeks, 2 months, 6 months and 12 months _0.5 ，M ₂ 、M ₆ 、M ₁₂ ) There are significant differences.

In addition, the change in the expression level of each gene in the nucleated patients before Treatment (Active), during Treatment (Treatment) and after recovery (recovery) was also measured. In healthy persons (Control) as a Control, it was seen that 4 genes were expressed significantly higher in Active tuberculosis patients (Active) than in other groups and decreased during the course of treatment. When the patient recovered, there was no difference (p=0.37) from the healthy human gene expression level (fig. 3A, 4A, 5A and 6A).

In addition, for the geometric mean expression amount based on 4 genes, the change of the overall expression amount before Treatment (Active), during Treatment (Treatment) and after recovery (recovery) was also studied. By taking healthy people (Control) as a Control, the overall expression level of 4 genes is obviously higher than that of other groups in Active tuberculosis patients (Active), and is reduced in the treatment process. When the patient recovered, there was no difference (p=0.37) from the healthy human gene expression level (fig. 7A and 7B).

Example 3

Kit for detecting tuberculosis

Preparing a kit for detecting tuberculosis, the kit comprising:

(a) A container, and a primer specific for a marker of tuberculosis located within the container, comprising:

primer pair for amplification of BATF 2: SEQ ID No.:1 and 2;

primer pair for amplifying UBE2L 6: SEQ ID No.:3 and 4;

primer pair for amplifying VAMP 5: SEQ ID No.:5 and 6; and

primer pair for amplifying SERPING 1: SEQ ID No.:7 and 8;

(b) And a label or instruction stating that the kit is for use in detecting tuberculosis.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Sequence listing

<110> university of southwest

<120> application of tuberculosis marker in tuberculosis diagnosis and efficacy evaluation

<130> P2019-0333

<160> 10

<170> PatentIn version 3.5

<210> 1

<211> 19

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 1

caccagcagc acgagtctc 19

<210> 2

<211> 19

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 2

tgtgcgaggc aaacaggag 19

<210> 3

<211> 15

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 3

cgcgctgtgt cgcgg 15

<210> 4

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 4

gcaggggctc cctgatattc 20

<210> 5

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 5

atgcgtaaca acttcggcaa g 21

<210> 6

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 6

ggccaggttc tgtgtagtct t 21

<210> 7

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 7

gggatgcttt ggtagatttc tcc 23

<210> 8

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 8

gaggatgctc tccaggtttg t 21

<210> 9

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 9

ttccttcctg ggcatggagt cc 22

<210> 10

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 10

tggcgtacag gtctttgcgg 20

Claims

1. Use of the tuberculosis markers BATF2, UBE2L6, VAMP5 and SERPING1 genes, mRNA, cDNA, proteins, or detection reagents thereof, for the preparation of a diagnostic reagent or kit for (a) diagnosing tuberculosis, and/or (b) evaluating the effect of tuberculosis treatment.

2. The use according to claim 1, wherein the tuberculosis is tuberculosis.

3. The use of claim 1, wherein the detection reagent comprises:

(a) Specific antibodies and specific binding molecules for tuberculosis markers BATF2, UBE2L6, VAMP5 and SERPING 1; and/or

(b) Primers or primer pairs, probes or chips for specifically amplifying mRNA or cDNA of tuberculosis markers BATF2, UBE2L6, VAMP5 and SERPING1, wherein the chips are nucleic acid chips or protein chips.

4. The use according to claim 1, wherein the tuberculosis markers BATF2, UBE2L6, VAMP5 and SERPING1 genes, mRNA, cDNA, or proteins are of human origin.

5. The use according to claim 1, wherein the assay is an ex vivo sample assay.

6. The use of claim 5, wherein the ex vivo sample comprises: a blood sample, a serum sample, a tissue sample, a body fluid sample, or a combination thereof.

7. The use according to claim 1, wherein the sample is a mononuclear cell sample isolated from peripheral blood.

8. The use according to claim 1, wherein said detection is of the expression level of the BATF2, UBE2L6, VAMP5 and SERPING1 genes in peripheral blood mononuclear cells.

9. The use according to claim 1, wherein the detection reagent is coupled to or carries a detectable label.

10. The use according to claim 9, wherein the detectable label is selected from the group consisting of: chromophores, chemiluminescent groups, fluorophores, isotopes or enzymes.

11. The use of claim 1, wherein the diagnostic reagent comprises an antibody, a primer, a probe, a sequencing library, a nucleic acid chip, or a protein chip.

12. The use according to claim 1, wherein the diagnostic reagent or kit is for evaluating the therapeutic effect of a patient suffering from tuberculosis.

13. The use according to claim 12, wherein a decrease in the expression level of the marker gene to a level of normal population indicates a better therapeutic effect on tuberculosis or a substantial cure of tuberculosis.

14. The use of claim 1, wherein the reagent is a PCR primer pair.

15. The use of claim 14, wherein the primer pair comprises: a primer pair for amplifying BATF2, a primer pair for amplifying UBE2L6, a primer pair for amplifying VAMP5, and a primer pair for amplifying SERPING 1.

16. The use according to claim 15, wherein the primer pair for amplification of BATF2 is SEQ ID Nos. 1 and 2; the primer pair used to amplify UBE2L6 is SEQ ID nos.: 3 and 4; the primer pair used to amplify VAMP5 is SEQ ID nos. 5 and 6; primer pairs for amplifying SERPING1 are SEQ ID No. 7 and 8.

17. The use according to claim 1, wherein the kit is used in a detection method comprising the steps of:

(b) Detecting the expression amounts of BATF2, UBE2L6, VAMP5 and SERPING1 genes in the mononuclear cells in the detection sample, and marking the expression amounts as C1; and

(c) The tuberculosis markers BATF2, UBE2L6, VAMP5 and SERPING1 concentrations C1 were compared with a control reference value C0.

18. The use of claim 17, wherein the sample is from a test subject.

19. The use of claim 18, wherein the test subject is a human.

20. Use according to claim 1, wherein if the tuberculosis markers BATF2, UBE2L6, VAMP5 and SERPING1 concentration C1 are significantly higher than the control reference value C0, said "significantly higher" means that the ratio C1/C0 is ≡2, said control reference value C0 being the tuberculosis markers BATF2, UBE2L6, VAMP5 and SERPING1 concentration in the same sample in the normal population, or the tuberculosis markers BATF2, UBE2L6, VAMP5 and/or SERPING1 concentration in the same sample in the non-tuberculosis pulmonary patient population.

21. A kit comprising a detection reagent for detecting genes, mRNA, cDNA, proteins, or combinations thereof, of tuberculosis markers BATF2, UBE2L6, VAMP5, and SERPING 1.

22. The kit of claim 21, wherein the detection reagent comprises detection reagents for detecting the tuberculosis markers BATF2, UBE2L6, VAMP5 and SERPING 1.

23. The kit of claim 21, wherein the kit contains tuberculosis markers BATF2, UBE2L6, VAMP5 and SERPING1 genes, mRNA, cDNA and/or protein as a control or quality control.

24. The kit of claim 21, further comprising a label or instructions that prescribe use of the kit for (a) diagnosing tuberculosis, and/or (b) evaluating the effect of tuberculosis treatment.

25. The kit of claim 21, wherein the reagents are PCR primer pairs and the primer pairs comprise: a primer pair for amplifying BATF2, a primer pair for amplifying UBE2L6, a primer pair for amplifying VAMP5, and a primer pair for amplifying SERPING 1.

26. The kit of claim 25, wherein the primer pair for amplifying BATF2 is SEQ ID Nos. 1 and 2; the primer pair used to amplify UBE2L6 is SEQ ID nos.: 3 and 4; the primer pair used to amplify VAMP5 is SEQ ID nos. 5 and 6; primer pairs for amplifying SERPING1 are SEQ ID No. 7 and 8.

27. The kit of claim 24, wherein the label or instructions are as follows: if the geometric mean of the transcript levels of the BATF2, UBE2L6, VAMP5 and SERPING1 genes in the PBMCs of the test subject is greater than 0.0009426436, the test subject may be preliminarily predicted to be an active tuberculosis patient, and if the geometric mean of the transcript levels of the BATF2, UBE2L6, VAMP5 and SERPING1 genes in the PBMCs of the test subject is less than or equal to 0.0009426436, the test subject is an inactive tuberculosis patient; and/or

The labels or descriptions herein are noted as follows:

when the expression level of each marker gene or a combination thereof is reduced to the level in normal population, it is suggested that the tuberculosis treatment effect is better or the tuberculosis is basically cured.

28. The kit of claim 24, wherein the label or instructions are as follows:

if the concentration C1 of tuberculosis markers BATF2, UBE2L6, VAMP5 and SERPING1 of the detected object is significantly higher than the reference value C0, the probability of tuberculosis of the object is larger than that of normal people; the expression "significantly higher" means that the ratio of C1/C0 is greater than or equal to 2, and the control reference value C0 is the concentration of the tuberculosis markers BATF2, UBE2L6, VAMP5 and SERPING1 in the same sample in the normal population, or the concentration of the tuberculosis markers BATF2, UBE2L6, VAMP5 and/or SERPING1 in the same sample in the non-tuberculosis pulmonary patient population.

29. The kit of claim 28, wherein the control reference value C0 is the concentration of the tuberculosis markers bat 2, UBE2L6, VAMP5 and SERPING1 in the same sample in the normal population, or the concentration of the tuberculosis markers bat 2, UBE2L6, VAMP5 and/or SERPING1 in the same sample in the non-tuberculosis pulmonary patient population.

30. The kit of claim 24, wherein the label or instructions are as follows:

if the concentration Z1 of tuberculosis markers BATF2, UBE2L6, VAMP5 and SERPING1 in the detected cells is significantly higher than the reference value Z0, the probability of tuberculosis of the detected objects is high; the expression "significantly higher" means that the ratio of Z1/Z0 is not less than 2; the control reference value Z0 is the concentration of tuberculosis markers BATF2, UBE2L6, VAMP5 and SERPING1 in normal similar cells.

31. The kit of claim 30, wherein the cells to be detected are mononuclear cells from blood.

32. The kit of claim 30, wherein the control reference value Z0 is the concentration of tuberculosis markers BATF2, UBE2L6, VAMP5 and SERPING1 in the normal homogeneous cells.

33. The kit of claim 28, wherein the concentration of the tuberculosis marker comprises a protein concentration, an mRNA concentration, or a combination thereof.

34. The kit of claim 28, wherein "significantly higher" means that the ratio of C1/C0 is ≡2.

35. The kit of claim 28, wherein "significantly higher" means that the ratio of C1/C0 is ≡5.

36. The kit of claim 28, wherein "significantly higher" means that the ratio of C1/C0 is ≡10.

37. The kit of claim 28, wherein "significantly higher" means that the ratio of C1/C0 is ≡20.

38. The kit of claim 24, wherein the label or instructions are as follows:

39. The kit of claim 30, wherein "significantly higher" means that the ratio of Z1/Z0 is ≡2.

40. The kit of claim 30, wherein "significantly higher" means that the ratio of Z1/Z0 is ≡5.

41. The kit of claim 30, wherein "significantly higher" means that the ratio of Z1/Z0 is ≡10.

42. The kit of claim 30, wherein "significantly higher" means that the ratio of Z1/Z0 is ≡20.