CN117448435A - Kit for diagnosing active tuberculosis based on STEAP4 fluorescence quantitative PCR and application thereof - Google Patents
Kit for diagnosing active tuberculosis based on STEAP4 fluorescence quantitative PCR and application thereof Download PDFInfo
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- 101000880402 Homo sapiens Metalloreductase STEAP4 Proteins 0.000 title claims abstract description 59
- 201000008827 tuberculosis Diseases 0.000 title claims abstract description 57
- 208000036981 active tuberculosis Diseases 0.000 title claims abstract description 51
- 102100037654 Metalloreductase STEAP4 Human genes 0.000 title claims abstract description 40
- 238000003753 real-time PCR Methods 0.000 title abstract description 20
- 230000014509 gene expression Effects 0.000 claims abstract description 41
- 210000005259 peripheral blood Anatomy 0.000 claims abstract description 22
- 239000011886 peripheral blood Substances 0.000 claims abstract description 22
- 239000003550 marker Substances 0.000 claims abstract description 14
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- 238000003745 diagnosis Methods 0.000 claims abstract description 9
- 239000000126 substance Substances 0.000 claims description 14
- 108020004414 DNA Proteins 0.000 claims description 12
- 102000053602 DNA Human genes 0.000 claims description 10
- 108020004682 Single-Stranded DNA Proteins 0.000 claims description 10
- 238000012545 processing Methods 0.000 claims description 4
- 238000009007 Diagnostic Kit Methods 0.000 claims description 2
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 abstract description 14
- 238000004458 analytical method Methods 0.000 abstract description 3
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- 239000002299 complementary DNA Substances 0.000 description 6
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- 238000012257 pre-denaturation Methods 0.000 description 2
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- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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Abstract
The invention discloses a kit for diagnosing active tuberculosis based on STEAP4 fluorescence quantitative PCR and application thereof. The kit comprises reagents and/or instruments for detecting the relative expression level of STEAP4 genes in peripheral blood of a person to be detected. The invention detects the relative expression quantity of STEAP4 genes in PBMCs of patients with active tuberculosis and healthy controls by adopting fluorescent quantitative PCR, and discovers that: the relative expression levels of STEAP4 in PBMCs of patients with active tuberculosis were significantly higher than those of healthy controls. The analysis result of the working characteristic curve of the subject shows that STEAP4 is important in the aspect of active tuberculosis diagnosis as a marker.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a kit for diagnosing active tuberculosis based on STEAP4 fluorescence quantitative PCR and application thereof.
Background
Tuberculosis is a disease caused by infection with mycobacterium tuberculosis.
There are many reasons for the current status of tuberculosis epidemic, of which the lack of specific, effective active tuberculosis diagnostic techniques is of paramount importance. Failure to diagnose early, on the one hand, delays the condition, increases the treatment cost and mortality; on the other hand, the infection source cannot be effectively controlled, and the tuberculosis is spread. Therefore, developing specific and effective active tuberculosis diagnostic reagent has important significance for tuberculosis prevention and treatment.
Disclosure of Invention
The invention aims to solve the technical problem of how to specifically and effectively diagnose active tuberculosis.
In order to solve the technical problems, the invention firstly provides a substance for detecting the expression level of STEAP4 genes or a new application of STEAP4 serving as a marker.
The invention provides a substance for detecting STEAP4 gene expression level or application of STEAP4 serving as a marker in preparing a product for diagnosing or assisting in diagnosing active tuberculosis or diagnosing or assisting in diagnosing active tuberculosis.
The invention also provides an application of the substance for detecting the STEAP4 gene expression level or the STEAP4 serving as a marker in preparing a product for detecting or assisting in detecting whether a detected person is an active tuberculosis patient or detecting or assisting in detecting whether the detected person is the active tuberculosis patient.
The invention also provides application of a substance for detecting the STEAP4 gene expression level or STEAP4 serving as a marker in preparing a product for distinguishing or assisting in distinguishing active tuberculosis patients from healthy subjects (normal subjects), or distinguishing or assisting in distinguishing active tuberculosis patients from healthy subjects (normal subjects).
In any of the above applications, the STEAP4 gene expression level is the STEAP4 gene relative expression level in the peripheral blood of the subject.
Further, the substance for detecting the STEAP4 gene expression level is a reagent and/or an instrument for detecting the STEAP4 gene relative expression level in peripheral blood of a person to be detected.
Still further, the reagent includes a primer pair consisting of a single-stranded DNA molecule shown in sequence 1 and a single-stranded DNA molecule shown in sequence 2.
The apparatus comprises a fluorescent quantitative PCR apparatus, in particular a fluorescent quantitative PCR apparatus480 II fluorescent quantitative PCR instrument.
Further, the relative expression level is the relative expression level of the STEAP4 gene reference internal reference gene.
The reference gene is specifically GAPDH gene.
The reagents also included primer pairs consisting of a single-stranded DNA molecule shown in sequence 3 and a single-stranded DNA molecule shown in sequence 4 for amplifying the GAPDH gene.
The calculation method of the relative expression quantity comprises the following steps: using the cDNA of the person to be detected as a template, adopting the primer pair to carry out real-time fluorescence quantitative PCR, and then using 2 -ΔCt And (5) calculating by a method. The subject cDNA may be cDNA of PBMCs isolated from the peripheral blood of the subject.
The real-time fluorescence quantitative PCR system can adopt KAPA TM The rapid quantitative PCR kit is prepared as follows: 2 XGreen Master Mix 10. Mu.L, forward primer (concentration 10. Mu.M) 0.4. Mu.L, reverse primer (concentration 10. Mu.M) 0.4. Mu.L, cDNA (5-20 ng) 2. Mu.L, make up the system to 20. Mu.L with nuclease-free water. The forward primer and the reverse primer are a single-stranded DNA molecule shown in a sequence 1 and a single-stranded DNA molecule shown in a sequence 2 for amplifying STEAP4 genes or a single-stranded DNA molecule shown in a sequence 3 and a single-stranded DNA molecule shown in a sequence 4 for amplifying GAPDH genes, respectively.
The reaction conditions of the real-time fluorescent quantitative PCR are as follows: 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.
In order to solve the technical problems, the invention also provides an active tuberculosis diagnosis kit.
The active tuberculosis diagnostic kit provided by the invention comprises the substance for detecting the STEAP4 gene expression level.
The active tuberculosis diagnosis kit provided by the invention further comprises a data processing device; the data processing device is internally provided with a module: the module has the following functions: judging whether the tested person is an active tuberculosis patient according to the relative expression quantity of STEAP4 genes in peripheral blood of the tested person: if the relative expression level of STEAP4 gene in the peripheral blood of the tested person is higher than 0.01296, the tested person is or is suspected to be an active tuberculosis patient; if the relative expression level of STEAP4 gene in peripheral blood of the subject is equal to or lower than 0.01296, the subject is not or suspected to be an active tuberculosis patient.
In any one of the above applications or kits, the STEAP4 gene sequence is shown as sequence 5 in the sequence table.
In any of the above applications or kits, the active tuberculosis is caused by a mycobacterium tuberculosis infection, and tuberculosis lesions occur in the lung tissue, trachea, bronchus and chronic infectious diseases of pleura.
The present invention detects the relative expression level of STEAP4 in PBMCs of patients with active tuberculosis and healthy controls by using fluorescence quantitative PCR: the relative expression levels of STEAP4 in PBMCs of patients with active tuberculosis were significantly higher than those of healthy controls. The analysis result of the working characteristic curve of the subject shows that STEAP4 is important in the aspect of active tuberculosis diagnosis as a marker.
Drawings
FIG. 1 shows the relative expression levels of STEAP4 in PBMCs of patients with active tuberculosis and healthy controls by real-time fluorescent quantitative PCR.
FIG. 2 is a graph of the diagnostic value of the ROC curve analysis STEAP4 gene expression level to distinguish patients with active tuberculosis from healthy controls.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Active tuberculosis in the following examples refers to a chronic infectious disease caused by infection with Mycobacterium tuberculosis, which occurs in lung tissue, trachea, bronchi and pleura.
Example 1 use of STEAP4 as a marker in the diagnosis of active tuberculosis
1. Experimental materials and methods
1. Obtaining peripheral blood samples
Peripheral blood samples of subjects: peripheral blood samples were collected from 63 active tuberculosis patients and 57 healthy controls (normal controls). The specific obtaining method of the peripheral blood sample is as follows: 2-3mL of peripheral blood of 63 patients with active tuberculosis and 57 healthy controls (informed consent) are respectively extracted, placed in an EDTA-containing anticoagulation blood collection tube, and turned upside down for 5-6 times (the purpose is to mix the anticoagulation agent and the peripheral blood uniformly), so as to obtain a peripheral blood sample.
2. Isolation of Peripheral Blood Mononuclear Cells (PBMCs)
Mononuclear Cells (PBMCs) are isolated from a peripheral blood sample from a subject. The specific steps are as follows (the following steps are completed within 6 h): collecting 2mL of anticoagulated peripheral blood of a subject, and firstly carrying out 1 by using a basic culture medium RPMI-1640: 1, then subjecting to density gradient centrifugation (Ficoll, cytiva, USA) with lymphocyte separation liquid, collecting cells of the intermediate mononuclear cell layer, and washing twice with RPMI-1640 medium to obtain PBMCs.
3. RNA extraction
Using TRIzol TM Reagent (Invitrogen, USA) extracts RNA from PBMCs. The method comprises the following specific steps: 1mL TRIzol and 0.2mL chloroform were added to each PBMCs sample, vigorously shaken for 15 seconds, and left at room temperature for 3 minutes; 12000g, centrifuging at 4deg.C for 15 min, separating upper colorless aqueous phase, adding 0.5mL isopropanol, mixing for several times, and precipitating at room temperature for 10 min; 12000g, centrifuging at 4 ℃ for 10 minutes, discarding the supernatant, and adding 1mL of water without nuclease containing 75% ethanol to wash RNA precipitate; 7500g, 4℃for 10 minutes, the supernatant was discarded, and the RNA pellet was left to stand on ice for air-drying (5-10 minutes) and dissolved in 20. Mu.L of nuclease-free water.
4. cDNA Synthesis
Using PrimeScript TM RT reagent Kit with gDNA Eraser (TaKaRa Biotechnology, japan) was subjected to RNA reverse transcription to obtain cDNA. The method comprises the following specific steps: firstly removing genome DNA, wherein a reaction system is as follows: 5X gDNA Eraser Buffer. Mu.L, gDNA Eraser 1. Mu.L, RNA 0.5. Mu.g, make up the system to 10. Mu.L with nuclease-free water; the reaction conditions are as follows: 42℃for 2 min. And then reverse transcription is carried out, wherein the reaction system is as follows: 10. Mu.L of the reaction solution from which the genomic DNA was removed, 1. Mu.L of RT Enzyme Mix I, 1. Mu.L of RT Primer Mix, 5X Primer Script Buffer. Mu.L, and the system was made up to 20. Mu.L with nuclease-free water; the reverse transcription conditions were: 37 ℃, 15 minutes, 85 ℃ and 5 seconds.
5. Primer sequences and synthesis
The primer sequences shown in Table 1 were synthesized by Shanghai Biotechnology Co., ltd and purified by HPLC.
TABLE 1 primer sequences
| Primer name | Sequence (5 '-3') |
| STEAP4-F | GGCTTTGGGAATACTTGGGTT (sequence 1) |
| STEAP4-R | TGGACAAATCGGAACTCTCTCC (sequence 2) |
| GAPDH-F | TGTTGCCATCAATGACCCCT (sequence 3) |
| GAPDH-R | TCGCCCCACTTGATTTTGGA (sequence 4) |
6. Real-time fluorescent quantitative PCR
KAPA from Kapa Biosystems TM The rapid quantitative PCR kit performs real-time fluorescent quantitative PCR.
The real-time fluorescent quantitative PCR reaction system is as follows: 2 XGreen Master Mix 10. Mu.L, forward primer (concentration 10. Mu.M) 0.4. Mu.L, reverse primer (concentration 10. Mu.M) 0.4. Mu.L, cDNA 2. Mu.L, make up the system with nuclease-free water to 20. Mu.L. The forward primer and the reverse primer are STEAP4-F and STEAP4-R respectively, and GAPDH gene is used as an internal reference gene.
Using the company RocheThe 480 II fluorescent quantitative PCR instrument carries out fluorescent quantitative PCR detection, and the reaction conditions are as follows: 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 -ΔCt The relative expression level was calculated.
7. Statistical analysis
Statistical analysis was performed using GraphPad Prism 5.
2. Experimental results
1. STEAP4 expression level detection results
The relative expression levels of STEAP4 in the PBMCs of 63 active tuberculosis patients and 57 healthy controls were examined using RT-PCR.
The results are shown in FIG. 1, which shows: the relative expression level of STEAP4 in active tuberculosis patients was significantly higher than in healthy controls (p < 0.0001).
2. STEAP4 has high diagnostic value for active tuberculosis diagnosis.
Subject work profile analysis was performed using GraphPad Prism 5 based on the relative expression levels of STEAP4 in active tuberculosis patients and healthy control PBMCs.
As a result, the area under the ROC curve was 0.931, p <0.0001, sensitivity was 80.95%, and specificity was 91.23%, as shown in FIG. 2.
The above results indicate that: STEAP4 as a marker has important value in the diagnosis of active tuberculosis and in distinguishing active tuberculosis patients from healthy persons.
In practical use, active tuberculosis may be diagnosed according to the following criteria: if the relative expression level of STEAP4 gene in peripheral blood of the tested person is higher than 0.01296, the tested person is or is suspected to be an active tuberculosis patient; if the relative expression level of STEAP4 gene in the peripheral blood of the subject is equal to or lower than 0.01296, the subject is not or suspected to be an active tuberculosis patient.
The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.
Claims (10)
1. The substance for detecting STEAP4 gene expression level or the application of STEAP4 as a marker in the preparation of products for diagnosing or assisting in diagnosing active tuberculosis.
2. The substance for detecting STEAP4 gene expression level or STEAP4 is used as a marker in diagnosis or auxiliary diagnosis of active tuberculosis.
3. The application of a substance for detecting STEAP4 gene expression level or STEAP4 as a marker in the preparation of a product for detecting or assisting in detecting whether a subject is an active tuberculosis patient.
4. The substance for detecting STEAP4 gene expression level or STEAP4 as a marker is used for detecting or assisting in detecting whether the detected person is an active tuberculosis patient.
5. The application of a substance for detecting STEAP4 gene expression level or STEAP4 as a marker in the preparation of a product for distinguishing or assisting in distinguishing patients with active tuberculosis from healthy subjects.
6. The use of a substance that detects the expression level of STEAP4 gene or STEAP4 as a marker for distinguishing or aiding in distinguishing patients with active tuberculosis from healthy subjects.
7. Use according to any one of claims 1-6, characterized in that: the substance for detecting the STEAP4 gene expression quantity is a reagent and/or an instrument for detecting the STEAP4 gene relative expression quantity in peripheral blood of a person to be detected.
8. The use according to claim 7, characterized in that: the reagent comprises a primer pair consisting of a single-stranded DNA molecule shown in a sequence 1 and a single-stranded DNA molecule shown in a sequence 2.
9. An active tuberculosis diagnostic kit comprising a substance for detecting the expression level of STEAP4 gene.
10. The kit of claim 9, wherein: the kit further comprises a data processing device; the data processing device is internally provided with a module: the module has the following functions: judging whether the tested person is an active tuberculosis patient according to the relative expression quantity of STEAP4 genes in peripheral blood of the tested person: if the relative expression level of STEAP4 gene in the peripheral blood of the tested person is higher than 0.01296, the tested person is or is suspected to be an active tuberculosis patient; if the relative expression level of STEAP4 gene in peripheral blood of the subject is equal to or lower than 0.01296, the subject is not or suspected to be an active tuberculosis patient.
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| CN202210878150.7A CN117448435A (en) | 2022-07-25 | 2022-07-25 | Kit for diagnosing active tuberculosis based on STEAP4 fluorescence quantitative PCR and application thereof |
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