CN112725434B - Rifampicin-resistant tuberculosis molecular marker, detection reagent and application thereof - Google Patents
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Abstract
The invention provides a rifampicin-resistant tuberculosis molecular marker, a detection reagent and application thereof. Relates to the technical field of biomedicine. The molecular markers comprise TRIM9, TRIM21 and TRIM56 genes and expression products thereof; the TRIM9, TRIM21, and TRIM56 genes are down-regulated in expression in rifampicin resistant tuberculosis patients. The method can identify the rifampicin-resistant tuberculosis patients more quickly, efficiently and sensitively, and can be used for screening rifampicin-resistant tuberculosis.
Description
Technical Field
The invention relates to the technical field, in particular to a rifampicin-resistant tuberculosis molecular marker, a detection reagent and application thereof.
Background
Tuberculosis (TB) is a chronic infectious disease caused by infection with mycobacterium tuberculosis and is one of ten causes of death worldwide. According to the estimation of World Health Organization (WHO), about 996 thousands of new tuberculosis patients worldwide in 2019 are basically maintained at the same level in recent years. Of new tuberculosis patients discovered worldwide in 2019, about 3.3% of new patients and 18% of patients treated with the new tuberculosis patients are resistant to rifampicin. The number of patients with rifampicin-resistant tuberculosis (RR-TB) is estimated to be about 46.5 million, of which multi-drug resistant tuberculosis (MDR-TB) accounts for about 78%. Recent treatment outcome data for MDR/RR-TB patients showed that the global treatment success rate for MDR/RR-TB patients was 57%. Rifampicin-resistant tuberculosis is an important challenge for worldwide tuberculosis control, and early diagnosis and rational treatment of rifampicin-resistant tuberculosis patients are critical to reduce tuberculosis transmission.
Currently, phenotypic culture-based methods remain the primary means of drug susceptibility testing, but are time consuming and require stringent microbiological safety precautions. Although molecular techniques such as Xpert MTB/RIF or linear probes are rapid and efficient, the method still has certain false positive rate and false negative rate, is high in cost and is not popularized in basic level units. Therefore, there is an urgent need to develop new diagnostic markers and establish a rapid, efficient, sensitive and economical diagnostic method.
The natural immune response of the host is crucial to the progression and control of Mycobacterium Tuberculosis (MTB) infection. Rifampicin resistant and sensitive strains are reported to elicit different immune responses upon infection of the body. Therefore, the searching of the regulatory factor which has the function of regulating the natural immune response is beneficial to the early diagnosis of the disease and the searching of a new therapeutic target.
The present invention has been made in view of the above circumstances.
Disclosure of Invention
The invention aims to provide a rifampicin-resistant tuberculosis molecular marker, a detection reagent and application thereof, and the rifampicin-resistant tuberculosis molecular marker can be used for identifying rifampicin-resistant tuberculosis patients more quickly, efficiently and sensitively and can be used for screening rifampicin-resistant tuberculosis.
In order to achieve the purpose, the invention adopts the technical scheme that:
a rifampicin resistant tuberculosis molecular marker comprises TRIM9, TRIM21 and TRIM56 genes and expression products thereof; the TRIM9, TRIM21, and TRIM56 genes are down-regulated in expression in rifampicin resistant tuberculosis patients.
The invention finds 3 main detection targets of rifampicin-resistant tuberculosis from more than 70 TRIM protein families of different members: TRIM9, TRIM21, and TRIM56 genes.
In one embodiment, the molecular marker refers to mRNA or expressed protein of TRIM9, TRIM21, and TRIM56 genes.
The rifampicin-resistant tuberculosis molecular marker is applied to preparation of a reagent for diagnosing and/or screening rifampicin-resistant tuberculosis.
The application of a detection reagent in preparing a product for diagnosing and/or screening rifampicin-resistant tuberculosis is disclosed, wherein the detection reagent is used for detecting TRIM9, TRIM21 and TRIM56 genes and expression products thereof.
In one embodiment, the product comprises a product that detects TRIM9, TRIM21, and TRIM56 genes by RT-PCR, real-time quantitative PCR, immunodetection, in situ hybridization, chip, or high throughput sequencing platform.
In one embodiment, the detection reagent can quantitatively detect the expression amount of TRIM9, TRIM21 and TRIM56 genes in a sample;
preferably, the detection reagent comprises one or more of PCR amplification primer, probe and gene chip;
more preferably, the detection reagents comprise real-time fluorescent quantitative PCR (RT-qPCR) primers for amplifying TRIM9, TRIM21, and TRIM56 genes.
In one embodiment, the detection reagent detects the expression levels of the TRIM9, TRIM21 and TRIM56 genes in blood, preferably the expression levels of the TRIM9, TRIM21 and TRIM56 genes in peripheral blood mononuclear cells.
In one embodiment, the real-time fluorescent quantitative PCR primers for amplifying the TRIM9 gene are shown as SEQ ID No.1 and SEQ ID No. 2;
the real-time fluorescent quantitative PCR primer for amplifying the TRIM21 gene is shown as SEQ ID NO.3 and SEQ ID NO. 4; and
the real-time fluorescent quantitative PCR primer for amplifying the TRIM56 gene is shown as SEQ ID NO.5 and SEQ ID NO. 6.
Application of promoters of TRIM9, TRIM21 and TRIM56 genes and/or expression products thereof in preparation of drugs for treating rifampicin-resistant tuberculosis.
In one embodiment, the promoter includes an agent that promotes the expression of TRIM9, TRIM21, and TRIM56 genes, an agent that promotes the stability of the expression products of TRIM9, TRIM21, and TRIM56 genes, an agent that promotes the activity of the expression products of TRIM9, TRIM21, and TRIM56 genes, and an agent that promotes the function of the expression products of TRIM9, TRIM21, and TRIM56 genes.
In one embodiment, the agent for promoting the expression of TRIM9, TRIM21, and TRIM56 genes and/or expression products thereof includes an agent containing TRIM9, TRIM21, and TRIM56 genes, an agent formed by a vector or host cell carrying TRIM9, TRIM21, and TRIM56 genes, or an agent containing TRIM9, TRIM21, and TRIM56 proteins.
A rifampicin-resistant tuberculosis diagnostic kit comprises real-time fluorescent quantitative PCR primers for detecting the expression quantity of TRIM9, TRIM21 and TRIM56 genes in a sample; wherein, the real-time fluorescent quantitative PCR primer for amplifying the TRIM9 gene is shown as SEQ ID NO.1 and SEQ ID NO. 2; the real-time fluorescent quantitative PCR primer for amplifying the TRIM21 gene is shown as SEQ ID NO.3 and SEQ ID NO. 4; and real-time fluorescent quantitative PCR primers for amplifying the TRIM56 gene are shown as SEQ ID NO.5 and SEQ ID NO. 6.
In one embodiment, the kit further comprises a primer pair for amplifying an internal reference gene, wherein the internal reference gene is GAPDH; preferably, the primers for amplifying the internal reference gene are shown as SEQ ID NO.7 and SEQ ID NO. 8.
In one embodiment, the kit further comprises a reagent for extracting RNA and a reagent for reverse transcription of RNA.
In one embodiment, the present invention performs screening and diagnosis of rifampicin-resistant tuberculosis by:
(1) cracking peripheral red blood cells, extracting total RNA by a Trizol method, and storing for later use;
(2) optimizing experimental conditions, designing primers aiming at target genes (TRIM family genes comprising TRIM9, TRIM21 and TRIM56 genes), and detecting the expression levels of TRIM9, TRIM21 and TRIM56 genes in peripheral venous blood of two groups of people (rifampicin drug-resistant tuberculosis patients and drug-sensitive tuberculosis patients) by adopting an RT-qPCR method;
(3) by means of 2-ΔCtThe method analyzes data, and analyzes relative expression amounts of single TRIM9, TRIM21 and TRIM56 genes and a combination thereof through a Receiver Operating Characteristic (ROC) curve to identify the diagnosis effect of rifampicin-resistant tuberculosis.
Has the advantages that:
the invention uses TRIM9, TRIM21 and TRIM56 genes and expression products thereof as diagnostic markers of rifampicin-resistant tuberculosis for the first time, and can distinguish rifampicin-resistant tuberculosis patients from drug-sensitive tuberculosis patients; and provides a corresponding detection kit, which can better realize the diagnosis and screening of rifampicin-resistant tuberculosis.
1. The diagnosis efficiency is high: the method obviously improves the diagnosis efficiency of rifampicin-resistant tuberculosis by designing a specific primer, adopting real-time fluorescent quantitative PCR detection and fitting the result through a decision tree model; compared with the traditional detection means, the method has good diagnosis sensitivity and strong specificity.
2. Is quick and convenient: the specimen of the invention adopts human peripheral venous blood, and the specimen is easy to obtain. The pretreatment of the specimen is easy, the real-time fluorescence quantitative PCR consumes short time, the operation is simple and convenient, and the detection time is obviously shortened compared with the solid or liquid culture and drug sensitive test which is widely applied clinically at present.
3. The biological safety is good: compared with the culture drug-adding sensitive method, the invention does not need strict microbiological safety precaution measures for detecting the expression quantity of the marker gene.
4. The cost is low: compared with the traditional culture method and the molecular detection technology on the market at present, the invention has lower cost and reduces the treatment burden of patients.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a schematic diagram showing a comparison of the relative expression level of TRIM9 gene in peripheral blood nucleated cells of drug sensitive tuberculosis patients and rifampin-resistant tuberculosis patients according to an embodiment of the present invention;
FIG. 2 is a schematic diagram showing the comparison of the relative expression level of TRIM21 gene in the peripheral blood nucleated cells of drug sensitive tuberculosis patients and rifampicin-resistant tuberculosis patients according to the present invention;
FIG. 3 is a schematic diagram showing the comparison of the relative expression level of TRIM56 gene in the peripheral blood nucleated cells of drug sensitive tuberculosis patients and rifampicin-resistant tuberculosis patients according to the present invention;
FIG. 4 is a schematic diagram of the ROC curves for TRIM9, TRIM21, TRIM56, and compositions provided by an embodiment of the present invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The materials, reagents and the like used in the present invention are commercially available unless otherwise specified. In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art.
The active tuberculosis patients refer to tuberculosis patients who have clinical symptoms of tuberculosis and are positively detected by sputum antacid bacillus smear or culture, pathologically confirmed or confirmed by sputum molecular biology, and include drug sensitive tuberculosis patients and rifampicin drug resistant tuberculosis patients.
Drug sensitive tuberculosis (DS-TB) refers to drug sensitivity test of tuberculosis patients, which suggests sensitivity to first-line antitubercular drugs (isoniazid, rifampin, pyrazinamide, ethambutol, streptomycin). Rifampicin resistant tuberculosis (RR-TB) refers to a drug sensitivity test of tuberculosis patients suggesting resistance to at least rifampicin.
All the people can eliminate tumor, take immune regulation medicine to treat, HIV infection and other autoimmune diseases.
In this embodiment, the inventors divided the samples into two groups: drug-sensitive tuberculosis group (DS-TB, 32 cases) and rifampin-resistant tuberculosis group (RR-TB, 31 cases).
Example 1 preparation of nucleated cells of peripheral blood
(1) 2mL of peripheral blood of each subject was collected on an empty stomach in the morning in a heparin sodium anticoagulated blood collection tube, and nucleated cells were prepared within 6 h.
(2)1 volume of fresh whole blood, 3 volumes of red blood cell lysate were added. In this example, 2ml of fresh whole blood was added to 6ml of red blood cell lysate (solarbio, ID: R1010), and mixed by gentle swirling or inversion.
(3) After being placed on ice for 15 minutes, the mixture is gently vortexed and mixed twice, and the solution should be clear and transparent after the red blood cells are lysed.
(4) Collecting cells: the leukocytes were pelleted by centrifugation at 450 Xg for 10min at 4 ℃ and the supernatant carefully aspirated.
(5) To the white blood cell pellet was added two volumes of red blood cell lysate (4 ml in this example) and the white blood cells were resuspended thoroughly by gentle swirling.
(6) The leukocytes were pelleted by centrifugation at 450 Xg for 10min at 4 ℃ and the supernatant carefully and thoroughly aspirated.
(7) Cells were lysed by adding 1mL of TRIZOL reagent (Invitrogen, ID: 15596), then transferred to a centrifuge tube and stored at-80 ℃ for subsequent RNA extraction.
Example 2 detection of expression Change of TRIM Gene by RT-qPCR
Extraction of RNA
(1) The cell lysate obtained in example 1 was thawed at room temperature, 200. mu.L of chloroform was added to each tube, and the mixture was inverted and mixed for 20 seconds to produce a pink turbid solution without delamination, which was allowed to stand at room temperature for 10 min.
(2)12000g/min, centrifuging for 15min at 4 ℃, taking out a centrifugal tube, and dividing a sample into three layers: a colorless supernatant phase, an intermediate white layer and a pink lower organic phase.
(3) The colorless supernatant was carefully removed and transferred to a new RNase-free centrifuge tube, and isopropanol (500 mL in this example) in a volume of 1/2TRIZOL was added to the supernatant, and the mixture was gently mixed and left at-20 ℃ for 20 min.
(4)12000g/min, 4 ℃ centrifugation for 10min, abandoning the supernatant, adding 1mL 75% ethanol (no RNase water preparation), gently shaking the centrifuge tube, washing the RNA precipitate.
(5)7500g/min, centrifuging at 4 deg.C for 5min, carefully sucking off the supernatant, and drying the RNA precipitate at room temperature for 5 min.
(6) Add 20. mu.L of RNase-free water to dissolve the RNA pellet and reverse transcribe it immediately into cDNA.
2. Reverse transcription
Using TRANS corporation
1. mu.g of RNA obtained from All-in One First-Strand cDNA Synthesis Supermix for qPCR (One-Step gDNAremove, ID: AE341) was reverse-transcribed and the total amount was 20. mu.L. The preparation of the reaction system is carried out on ice, and in order to ensure the accuracy of the preparation of the reaction solution and reduce errors caused by subpackaging, the reaction solution is prepared according to a volume slightly larger than the actual dosage, and finally the RNA sample is added. The system is as follows:
the prepared reaction system carries out the following reactions on a PCR instrument: 42 ℃ for 15 min; after the reaction was completed at 85 ℃ for 5 seconds, the reaction mixture was stored at-20 ℃.
3. Real-time fluorescent quantitative PCR
3.1 primer sequences
3.2 real-time fluorescent quantitative PCR detection of target genes
The cDNA obtained in the above step was used as a template, and StarLighter kit from FOREVER STAR
Green qPCR Mix (Universal, ID: FS-Q1001) detects the expression level of TRIM9, TRIM21 and TRIM56 genes, selects GAPDH as an internal reference gene, and sets a negative control hole without adding a template. The detection was carried out using ABI7500 real-time fluorescent quantitative PCR instrument from Applied Biosystems. The reaction system is as follows:
the reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 20s, annealing at 60 ℃ for 34s, 40 cycles.
3.3 data processing and statistical analysis
Based on the results of real-time fluorescent quantitative PCR, the results were analyzed by ABI7500 v2.3 software using the GAPDH gene as the reference gene and 2-△ctThe method calculates the relative expression quantity of the TRIM gene of the drug sensitive tuberculosis and rifampicin resistant tuberculosis patients relative to respective reference genes.
The difference is statistically different by adopting an independent sample t test analysis method and taking the P value less than 0.05 as the difference. Histograms were generated using GraphPad Prism 8 software, and the results are shown in FIGS. 1-3, where the abscissa represents the different populations and the ordinate represents the relative expression (2)-△ctRepresentation).
The results showed that the expression levels of TRIM9, TRIM21, and TRIM56 genes were significantly lower in RR-TB patients than in DS-TB patients. In FIGS. 1-3, DS-TB is drug sensitive tuberculosis patient, RR-TB is rifampin resistant tuberculosis patient, the horizontal line is mean + -SE, and the symbol indicates P < 0.05.
Example 3 determination method of TRIM Gene for discriminating RR-TB and DS-TB
According to the above experimental results, RR-TB and DS-TB patients can be identified by RT-qPCR. The present invention analyzes the diagnostic efficacy of each and combinations thereof by ROC curve using SPSS 24.0 software, in terms of the relative expression amounts of TRIM9, TRIM21, and TRIM 56. The method comprises the following specific steps:
respectively calculating ROC curves of the relative expression quantity of the single TRIM gene to identify rifampicin-resistant tuberculosis, wherein the ROC curves comprise area under the curve (AUC), sensitivity and specificity, and the results show that the AUC of the single TRIM gene is 0.808, 0.704 and 0.526 respectively; sensitivity was 66.7%, 71.4% and 95.2%, respectively, and specificity was 87.8%, 65.9% and 19.5%, respectively. The result shows that the efficiency of differential diagnosis of rifampicin tuberculosis by relative expression of single TRIM gene is not ideal.
Calculating an ROC curve of the composition of the genes TRIM9, TRIM21 and TRIM56 for identifying rifampicin-resistant tuberculosis by adopting a Logistic stepwise regression model, and constructing a regression equation of a binary Logistic stepwise model of the composition by data calculation and analysis:
logit (p) 1.90-1077.96 × (relative expression of TRIM 9) -26.41 × (relative expression of TRIM 21) +41.54 × (relative expression of TRIM 56)
The sensitivity of ROC curve is 83.3%, specificity is 75.6%, AUC is 0.828 (95% CI is 0.734-0.921) (FIG. 4) has higher diagnostic efficacy.
The TRIM gene is used as a template to design a specific primer for relative quantitative RT-qPCR detection, can be used for rapidly detecting the peripheral blood of rifampicin-resistant tuberculosis patients, has higher diagnosis efficiency, is relatively simple and convenient to operate, and can improve the early diagnosis rate of rifampicin-resistant tuberculosis.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
SEQUENCE LISTING
<110> the university of capital medical department affiliated to the Beijing thoracic hospital; research institute of tuberculosis and breast tumor in Beijing
<120> rifampicin-resistant tuberculosis molecular marker, detection reagent and application thereof
<130> PA20047858
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<170> PatentIn version 3.3
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Claims (9)
1. The application of the detection reagent in preparing products for diagnosing and/or screening rifampicin-resistant tuberculosis is characterized in that the detection reagent is used for detecting the genes and expression products of TRIM9, TRIM21 and TRIM 56.
2. The use according to claim 1, wherein said products comprise products for detection of the TRIM9, TRIM21 and TRIM56 genes by RT-PCR, real-time quantitative PCR, immunodetection, in situ hybridization, chip or high throughput sequencing platform.
3. The use of claim 1, wherein the detection reagent is capable of quantitatively detecting the expression levels of TRIM9, TRIM21, and TRIM56 genes in a sample.
4. The use of claim 1, wherein the detection reagent comprises one or more of a PCR amplification primer, a probe, and a gene chip.
5. The use of claim 1, wherein the detection reagents comprise real-time fluorescent quantitative PCR primers for amplifying TRIM9, TRIM21, and TRIM56 genes.
6. The use according to claim 5, wherein the real-time fluorescent quantitative PCR primers for amplifying TRIM9 gene are shown as SEQ ID No.1 and SEQ ID No. 2;
the real-time fluorescent quantitative PCR primer for amplifying the TRIM21 gene is shown as SEQ ID NO.3 and SEQ ID NO. 4; and the number of the first and second groups,
the real-time fluorescent quantitative PCR primer for amplifying the TRIM56 gene is shown as SEQ ID NO.5 and SEQ ID NO. 6.
7. A rifampicin-resistant tuberculosis diagnostic kit is characterized by comprising real-time fluorescent quantitative PCR primers for detecting the expression quantity of TRIM9, TRIM21 and TRIM56 genes in a sample; wherein, the real-time fluorescent quantitative PCR primer for amplifying the TRIM9 gene is shown as SEQ ID NO.1 and SEQ ID NO. 2; real-time fluorescent quantitative PCR primers for amplifying the TRIM21 gene are shown as SEQ ID NO.3 and SEQ ID NO. 4; and real-time fluorescent quantitative PCR primers for amplifying the TRIM56 gene are shown as SEQ ID NO.5 and SEQ ID NO. 6.
8. The diagnostic kit of claim 7, further comprising a primer pair for amplifying an internal reference gene, wherein the internal reference gene is GAPDH.
9. The diagnostic kit of claim 8, wherein the primers for amplifying the reference gene are shown as SEQ ID No.7 and SEQ ID No. 8.
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