CN107267659B - Application of product for detecting TRIM gene and/or protein level - Google Patents

Abstract

The present invention provides the use of a product for detecting TRIM gene and/or protein levels, which is used in the preparation of a product for identifying, diagnosing and/or screening active tuberculosis. When the product for detecting TRIM gene and/or protein level of the present invention is used to prepare a product for identifying, diagnosing and/or screening active tuberculosis, the product can distinguish latent infection patients and active tuberculosis patients, and the specimen is easy to obtain, and has the advantages of Fast, efficient, high sensitivity, high accuracy, relatively simple operation, easy to promote the characteristics.

Description

Use of Products for Detecting TRIM Gene and/or Protein Levels

technical field

The present invention relates to the field of biotechnology, in particular to the use of a product for detecting TRIM gene and/or protein levels.

Background technique

Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis infection. Although the fight against tuberculosis has never stopped, it still poses a serious threat to global public health. According to the latest report of the World Health Organization, in 2015, there were about 10.4 million new cases of tuberculosis in the world, and about 1.8 million people died of tuberculosis. The number of new cases in China was 918,000, and the number of deaths was 38,000. The total number of cases ranked first in the world. Four, the epidemic situation is very serious. In recent years, with the widespread emergence of drug-resistant tuberculosis and multi-drug-resistant tuberculosis, coupled with HIV co-infection, the control of tuberculosis is facing severe challenges. One of the important measures to control the epidemic of tuberculosis is to cut off the source of infection, and active tuberculosis patients are the main source of infection. Therefore, early and accurate diagnosis of active tuberculosis patients and timely treatment can effectively reduce the spread of tuberculosis. .

At present, the clinical methods for diagnosing tuberculosis all have significant defects, such as acid-fast staining of sputum smear - low sensitivity; traditional Mycobacterium tuberculosis culture method - complicated operation, low positive rate and time-consuming; automated rapid culture method - expensive equipment; chest X-ray testing - characteristic changes of pulmonary tuberculosis are not obvious and cannot be detected early and timely; tuberculin skin test - cannot distinguish natural infection from BCG vaccination; IFN-r release assays (IGRAs) - cannot distinguish latent infection patients and active tuberculosis patients; GeneXpert MTB/RIF method - genetic testing directly from sputum specimens, there is no significant increase in sensitivity, and there are certain false negative and false positive rates. Serological antibody testing—specificity and sensitivity are suboptimal. Therefore, there is an urgent need to develop new diagnostic markers and establish rapid, efficient and sensitive diagnostic methods.

After human infection with Mycobacterium tuberculosis, only about 5% to 10% of people will eventually develop active tuberculosis, and most people are in a state of latent infection without clinical symptoms. However, about 5% to 10% of people with latent infection, when their immunity is weakened, will reactivate Mycobacterium tuberculosis and develop active tuberculosis. After Mycobacterium tuberculosis infects the body, the immune status of host cells plays a crucial role in the pathogenesis of tuberculosis. Among them, the regulators in the immune response, such as cytokines, chemokines, cell surface receptors, etc., has always been the focus of research. In recent years, studies have found that the three-domain protein (TRIM) family can act as a host restriction factor and participate in the innate immune response, which has attracted much attention.

The TRIM protein family is composed of many members and exists in almost all multicellular animals. It acts as an immunomodulatory protein and E3 ubiquitin ligase and participates in the body's innate immune response. With the deepening of research, it has been found that the TRIM protein family can not only maintain the normal physiological functions of the body, such as cell growth, cell differentiation, membrane repair, signaling pathways and apoptosis, but also participate in the regulation of various diseases. In addition, TRIM proteins have characteristic expression profiles in different human diseases, which have important hints for the development, diagnosis or prognosis of diseases. For example, Kosaka et al. found that TRIM29 is up-regulated in gastric cancer patients and can be used as a marker to indicate lymph node metastasis. Yamada et al. found that TRIM44 is upregulated in patients with testicular germ cell tumors (TGCT) and is expected to be a new prognostic factor. In addition, Nenasheva et al. compared the transcriptomes of pluripotent stem cells from induced Parkinson's (PD) patients and healthy people and found that the expression levels of TRIM6 and TRIM24 genes were significantly different in the two groups, suggesting that these genes can be used for disease development and immune system. Dysregulated warning genes. However, there are still many unknowns in the current research on the expression and regulation of TRIM gene in tuberculosis. If the characteristic TRIM gene related to tuberculosis can be found, it may be possible to prepare products for tuberculosis prevention, diagnosis and treatment. New targets and ideas.

Therefore, it is urgent to study the TRIM gene related to tuberculosis infection, to find new diagnostic markers, and to establish a more rapid, efficient and sensitive method for the identification, diagnosis and screening of active tuberculosis.

SUMMARY OF THE INVENTION

One aspect of the present invention is to provide the use of a product for detecting TRIM gene and/or protein levels in view of the lack of a more rapid, efficient and sensitive method for identifying, diagnosing and/or screening tuberculosis in the prior art.

In order to solve the above-mentioned technical problems, the technical scheme provided by the present invention is:

Use of a product for detecting TRIM gene and/or protein levels in the preparation of a product for identifying, diagnosing and/or screening active tuberculosis.

Preferably, in one embodiment of the present invention, the above-mentioned TRIM gene is one or more selected from TRIM35, TRIM47, TRIM65 or TRIM68.

Preferably, in one embodiment of the present invention, the above-mentioned TRIM protein is a protein expressed by one or more genes selected from TRIM35, TRIM47, TRIM65 or TRIM68.

In the present invention, any suitable product for detecting TRIM gene and/or protein level can achieve the purpose of the present invention, including but not limited to quantitative and/or qualitative products. But preferably, in an embodiment of the present invention, the product for detecting the level of TRIM gene and/or protein in the sample is a product for quantitatively detecting the level of TRIM gene and/or protein.

More preferably, the product for detecting the TRIM gene and/or protein level in the sample is one or more selected from gene amplification primers, probes, gene chips, antibodies, protein chips or antibodies.

More preferably, the above are PCR primers for amplifying one or more genes in TRIM35, TRIM47, TRIM65 or TRIM68.

Further preferably, the PCR primers are one or several pairs of PCR primers selected from SEQ ID No. 1-8.

The above-mentioned PCR primers are preferably real-time quantitative PCR (qRT-PCR) primers.

Preferably, in one embodiment of the present invention, the inventors use the following method to screen the target target:

1. Provide a set of target TRIM genes, including TRIM35, TRIM47, TRIM65 and TRIM68 genes;

2. Collect peripheral blood of active tuberculosis patients, latent tuberculosis infected patients and healthy people, lyse peripheral blood red blood cells, extract RNA, and save for future use;

3. Optimize the experimental conditions, design primers for the target genes, and use qRT-PCR to detect the expression levels of TRIM35, TRIM47, TRIM65 and TRIM68 genes in the three groups of people;

4. The 2- ^ΔΔCt method was used to analyze the data, and the TRIM35, TRIM47, TRIM65 and TRIM68 genes were used to identify the diagnostic effect of active tuberculosis by decision tree analysis.

Another aspect of the present invention is to provide a kit for identifying, diagnosing and/or screening active tuberculosis, the kit comprising a product for detecting the level of TRIM gene and/or protein in a sample.

Preferably, the above-mentioned kit includes one or more products selected from gene amplification primers, probes, gene chips, antibodies, protein chips or antibodies.

More preferably, the above kit includes PCR primers for amplifying one or more genes in TRIM35, TRIM47, TRIM65 or TRIM68.

Further preferably, the above-mentioned kit includes one or several pairs of PCR primers selected from SEQ ID No. 1-8.

The above-mentioned PCR primers are preferably qRT-PCR primers.

Generally, the above-mentioned kit also includes any necessary components, such as kit instructions and the like.

Another aspect of the present invention provides the use of the above-mentioned kit in the preparation of identification, diagnosis and/or screening of active tuberculosis.

Beneficial effects of the present invention:

Because IFN-r release assays (IGRAs) have the characteristics of rapidity and high sensitivity, they are widely used in clinical practice at present, but the biggest defect of this method is that it cannot distinguish latent infection patients and active tuberculosis patients, and the biggest advantage of the present invention is that make up for this defect.

In addition, the specimen of the present invention is easy to obtain, has the characteristics of rapidity, high efficiency, high sensitivity, high accuracy, relatively simple operation, and easy popularization.

Description of drawings

Figure 1 is a scatter plot of the relative expression levels of TRIM genes in patients with active tuberculosis, latent infection and healthy controls, in which Figure 1A is TRIM 35, Figure 1B is TRIM 47, Figure 1C is TRIM65, and Figure 1D is TRIM 68 , where TB is active tuberculosis patients, LTBI is latent infection, and HC is healthy control;

Figure 2 is a schematic diagram of the decision tree analysis of the TRIM gene, in which Figure 2A is TRIM 35, Figure 2B is TRIM 47, Figure 2C is TRIM 65, Figure 2D is TRIM 68, where N is the number of samples, TB is active tuberculosis patients, LTBI for latent infection.

sequence description

SEQ ID No.1-2 are respectively the upstream and downstream PCR primer sequences of TRIM 35 in the present invention;

SEQ ID No.3-4 are respectively the upstream and downstream PCR primer sequences of TRIM 47 in the present invention;

SEQ ID No.5-6 are respectively the upstream and downstream PCR primer sequences of TRIM 65 in the present invention;

SEQ ID Nos. 7-8 are respectively the upstream and downstream PCR primer sequences of TRIM 68 in the present invention.

Detailed ways

The invention discloses the use of the product for detecting the TRIM gene and/or protein level, and those skilled in the art can learn from the content of this article and appropriately improve the process parameters to achieve. It should be specially pointed out that all similar substitutions and modifications are obvious to those skilled in the art, they are all considered to be included in the present invention, and relevant persons can obviously do so without departing from the content, spirit and scope of the present invention. The content described herein can be modified or appropriately changed and combined to realize and apply the technology of the present invention.

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the laboratory operation steps such as cell biology and molecular biology used in this paper are routine steps widely used in the corresponding fields.

In order to make those skilled in the art better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to specific embodiments.

In this specific embodiment, the term "active tuberculosis patient" refers to a confirmed tuberculosis patient who has clinical symptoms of tuberculosis, positive smear or culture, abnormal chest X-ray, and has not started or just started systemic chemotherapy.

The term "latent infection" refers to those with no clinical complaints, positive tuberculin skin test (TST) test and T-SPOT test, and negative chest X-ray.

The term "healthy controls" refers to those with no clinical complaints, negative TST and T-SPOT tests, and negative chest X-rays.

All of the above groups excluded diabetes, HIV infection and other autoimmune diseases.

The above population comes from the Beijing Chest Hospital affiliated to Capital Medical University.

In this specific embodiment, the inventors divided the samples into three groups: active tuberculosis group (TB, 39 cases), latent infection group (LTBI, 29 cases) and healthy control group (HC, 33 cases).

Example 1 Preparation of peripheral blood nucleated cells

2 mL of peripheral blood was collected from each subject on an empty stomach in a blood collection tube anticoagulated with heparin sodium. Nucleated cells were prepared within 6 h using erythrocyte lysate (solarbio, product number R1010). The specific steps are as follows:

1 According to the volume ratio of fresh whole blood: erythrocyte lysate 1:3, add 6 mL of erythrocyte lysate, and mix by vortexing or inversion gently.

2 Place on ice for 15 minutes, vortex gently to mix twice, the solution should be clear and transparent after the red blood cells are lysed.

3 Collect cells: centrifuge at 450 × g for 10 min at 4°C to pellet white blood cells, and discard the supernatant.

4 Add 2 volumes (4 mL) of erythrocyte lysate to the leukocyte pellet to resuspend the leukocytes.

5. Collect cells: centrifuge at 450 × g for 10 min at 4°C to precipitate leukocytes, carefully and thoroughly aspirate the supernatant.

6 Add 1 mL of TRIZOL reagent (Invitrogen, product number: 15596) to lyse the cells, then transfer to a centrifuge tube and store at -80°C for subsequent RNA extraction.

Example 2 Application of qRT-PCR method to detect the expression changes of TRIM gene

1. RNA extraction

The cell lysate obtained in Example 1 was thawed at room temperature, 0.2 mL of chloroform was added to each tube, and vigorously shaken up and down by hand for 15 s, resulting in a pink turbid liquid without stratification, and allowed to stand at room temperature for 3 min. Centrifuge at 12000 r/min, 4°C for 15 min, take out the centrifuge tube, and the sample is divided into three layers: a colorless upper water phase, a middle white layer and a pink lower organic phase. Carefully pipette the colorless supernatant water phase to a new centrifuge tube, add 1/2 TRIZOL volume (500mL) of isopropanol to the obtained supernatant water phase, mix gently, and let stand at room temperature for 10min. Centrifuge at 12000 r/min at 4°C for 10 min, discard the supernatant, add 1 mL of 75% ethanol (prepared with RNase-free water), and mix gently. Centrifuge at 7500 r/min for 5 min at 4 °C, carefully aspirate the supernatant, dry the precipitate at room temperature for 5 min, add 30 μL of RNase-free water to dissolve the RNA precipitate, and store at -80 °C until use.

2. Reverse Transcription

Using the PrimeScriptTMRT reagent Kit of TAKARA (Cat. No.: RR037Q), take 2 μg of the RNA obtained in 1) and carry out reverse transcription, and the total system is 20 μL. The preparation of the reaction system was carried out on ice. In order to ensure the accuracy of the preparation of the reaction solution and reduce the errors caused by sub-packaging, the reaction solution should be prepared in a slightly larger volume than the actual amount, and finally the RNA sample was added. The system is as follows:

For the prepared reaction system, perform the following reactions on the PCR instrument: 37°C, 15min; 85°C, 5s; after the reaction, add 40 μL ddH ₂ O to each cDNA sample, mix well, and store at 4°C.

3. Real-time PCR

3.1 Primer sequences

3.2 Real-time fluorescence quantitative PCR detection of target genes

Premix Ex TaqTMII试剂盒(TAKARA，货号：RR820Q)检测TRIM35、TRIM47、TRIM65和TRIM68基因的表达水平，同时选用GAPDH为内参基因，同时设置不加模板的阴性对照孔。使用ABI7500实时荧光定量PCR仪进行检测。反应体系如下：Using the cDNA obtained in 2) as a template, adopt

Premix Ex TaqTMII kit (TAKARA, Cat. No.: RR820Q) was used to detect the expression levels of TRIM35, TRIM47, TRIM65 and TRIM68 genes. GAPDH was selected as the internal reference gene, and negative control wells without template were set. The detection was carried out using ABI7500 real-time fluorescence quantitative PCR instrument. The reaction system is as follows:

The reaction conditions were: pre-denaturation at 95 °C for 40 s, 1 cycle; denaturation at 95 °C for 5 s, annealing at 60 °C for 34 s, 40 cycles.

3.3 Data processing and statistical analysis

According to the results of qRT-PCR, ABI7500software v2.0.6 was used to analyze the results. Taking GAPDH gene as the internal reference gene, the expression levels of latent infected patients and healthy controls relative to active tuberculosis patients were calculated by the 2- ^△△Ct method. . One way ANOVA combined with Bonferroni correlation coefficient analysis method was used, and the threshold was set at P<0.05, which was statistically significant. The results are shown in Figure 1A-D, where the abscissa represents different populations, the ordinate represents the relative expression level, and the larger the ordinate value, the higher the expression level.

The results showed that the expression levels of TRIM35, TRIM47, TRIM65 and TRIM68 genes in active tuberculosis patients were significantly lower than those in latent infection and healthy controls. (In Figure 1A-D, TB is active tuberculosis patients, LTBI is latent infection, HC is healthy control, the horizontal line is the mean ± standard error, where ns means p>0.05, * means p=0.01-0.05, ** means p<0.01, *** means p<0.001, **** means p<0.0001.)

Example 3 TRIM gene is used to determine the method of diagnosing active tuberculosis

According to the experimental results of Example 2, active tuberculosis patients and latent tuberculosis infection patients can be diagnosed by qRT-PCR. The present invention specifically provides four independent diagnostic determination methods, respectively using the relative expression multiples of TRIM35, TRIM47, TRIM65 and TRIM68 to determine the optimal threshold of each gene through ROC curve analysis. details as follows:

When the relative expression multiple of TRIM35 is used, if the relative expression multiple of TRIM35 is less than 1.939, the test result is positive, that is, active tuberculosis is determined; if it is greater than or equal to 1.939, the test result is negative, which is determined to be latent tuberculosis infection. Among the 68 samples in this example (including 39 patients with active tuberculosis and 29 patients with latent infection), the diagnostic sensitivity of this method was 92.31%, the specificity was 96.55%, and the discrimination accuracy was 94.12% (Fig. 2A).

When the relative expression multiple of TRIM47 is used, if the relative expression multiple of TRIM47 is less than 2.059, the test result is positive, that is, active tuberculosis is determined; if it is greater than or equal to 2.059, the test result is negative, which is determined to be latent tuberculosis infection. Among the 68 samples in this example (including 39 patients with active tuberculosis and 29 patients with latent infection), the diagnostic sensitivity of this method was 94.87%, the specificity was 96.55%, and the discrimination accuracy was 95.59% (Fig. 2B).

When the relative expression multiple of TRIM65 is used, if the relative expression multiple of TRIM65 is less than 2.567, the test result is positive, that is, active tuberculosis is determined; if it is greater than or equal to 2.567, the test result is negative, which is determined to be latent tuberculosis infection. In the 68 samples in this example (including 39 patients with active tuberculosis and 29 patients with latent infection), the diagnostic sensitivity and specificity of this method were 100%, and the discrimination accuracy was 100% (Figure 2C). .

When the relative expression multiple of TRIM68 is used, if the relative expression multiple of TRIM68 is less than 2.516, the test result is positive, that is, active tuberculosis is determined; if it is greater than or equal to 2.516, the test result is negative, which is determined to be latent tuberculosis infection. Among the 68 samples in this example (including 39 patients with active tuberculosis and 29 patients with latent infection), the diagnostic sensitivity of this method was 94.87%, the specificity was 100%, and the discrimination accuracy was 97.06% (Fig. 2D).

In this example, TRIM35, TRIM47, TRIM65 and TRIM68 have significant differential expression between active tuberculosis patients and latent infection patients. The TRIM gene is used as a template to design specific primers for qRT-PCR detection, which can be used for the rapid detection of peripheral blood of patients with active tuberculosis. Early diagnosis rate.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made. It should be regarded as the protection scope of the present invention.

SEQUENCE LISTING

<110> Beijing Chest Hospital Affiliated to Capital Medical University

<120> Use of products for detection of TRIM gene and/or protein levels

<130> None

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<170> PatentIn version 3.5

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Claims (1)

1. Use of a product for detecting the level of the TRIM gene in the manufacture of a product for the identification, diagnosis or screening of active tuberculosis, wherein the gene is TRIM 47;

when the relative expression multiple of TRIM47 is used, if the relative expression multiple of TRIM47 is less than 2.059, the detection result is positive, and the active tuberculosis is judged; if the detection result is negative when the detection result is more than or equal to 2.059, the tuberculosis latent infection is judged;

the products for detecting the TRIM gene level are shown as SEQ ID No.3 and SEQ ID No. 4.

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