CN108828235A - Application of the PGLYRP1 albumen as marker in diagnostic activities tuberculosis - Google Patents

Abstract

Application the invention discloses PGLYRP1 albumen as marker in diagnostic activities tuberculosis.Compared with Healthy People and tuberculosis latent infection person, the expression quantity of PGLYRP1 gene is dramatically increased in the PBMCs of active tuberculosis patient；The expression quantity of PGLYRP1 gene can be used for distinguishing active tuberculosis patient and tuberculosis latent infection person；The expression quantity of PGLYRP1 gene can be used for distinguishing active tuberculosis patient and Healthy People.Therefore, PGLYRP1 albumen and/or PGLYRP1 gene can be used as marker diagnostic activities tuberculosis.The present invention has great application value.

Description

Application of PGLYRP1 protein as a marker in the diagnosis of active tuberculosis

technical field

The invention belongs to the technical field of medical immunology diagnosis, and in particular relates to the application of PGLYRP1 protein as a marker in the diagnosis of active tuberculosis.

Background technique

Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis (MTB), which is mainly transmitted through the respiratory tract. After MTB infects the human body, three different results can occur. One is that the body’s immunity is better, and MTB is directly cleared; the other is that MTB is suppressed by the body’s immune system, but cannot be completely cleared, and develops into latent tuberculosis infection (LTBI). ; The third is that MTB proliferates rapidly in the body and develops into active tuberculosis. Tuberculosis is a major infectious disease that my country needs to focus on prevention and control.

At present, the diagnosis of tuberculosis mainly includes imaging diagnosis, tuberculosis diagnosis and immunological diagnosis, but all of them have certain shortcomings. Imaging diagnosis is difficult to distinguish tuberculosis from other lung diseases. The false negative rate of tuberculosis diagnosis was high. Immunological diagnosis mainly includes antibody detection and cellular immune detection (such as tuberculin skin test (TST) and interferon gamma release assays (IGRA)). Both TST and IGRA evaluate tuberculosis infection by detecting the body's main anti-tuberculosis immunity, that is, cellular immune response. In China, TST is now used as the main detection method. Generally, those who are strongly positive for pure protein derivatives (PPD) or change from negative to positive in a short period of time without clinical evidence of tuberculosis are judged as latent tuberculosis infections. Because TST is characterized by simple operation and low price, it has become the most commonly used and convenient method for diagnosing tuberculosis infection in clinical practice. However, PPD is an antigen mixture roughly extracted from Mycobacterium tuberculosis, which contains more than 200 proteins, many of which are common antigenic components of non-tuberculous mycobacteria and BCG. Therefore, the specificity of TST detection is poor. False-positive results are prone to occur in vaccinated populations and non-tuberculous mycobacterium-infected populations. TST can only be used as an auxiliary diagnosis according to the strength of the skin reaction, and its sensitivity is only 70-80%. In addition, TST has the disadvantages of time-consuming detection, the need for subject return visit (72h), and the subjective dependence of skin test operation and result interpretation. IGRA is to quantitatively detect Mycobacterium tuberculosis specific antigens (ESAT6, CFP10 and TB7. 7) The release reaction of IFN-γ detection is used for the diagnosis of tuberculosis infection, but it is difficult for IGRA to distinguish active tuberculosis and latent tuberculosis infection. Failure to diagnose active tuberculosis early will lead to delayed treatment, increased treatment costs and mortality on the one hand; on the other hand, failure to effectively control the source of infection will result in the spread of tuberculosis. Therefore, it is of great significance to develop specific and effective active tuberculosis diagnostic reagents for tuberculosis control.

Peptidoglycan is a pathogen-associated molecular pattern of Gram-positive bacteria, and peptidoglycan recognition protein is an important pattern recognition receptor, which plays an important role in the process of resisting pathogen invasion. Peptidoglycan recognition protein 1 (PGLYRP1), also known as PGRP-S or Tag7, can bind to the immune receptor TREM-1 on the surface of monocytes and activate secretory cytokines with antibacterial effects. Studies have found that PGLYRP1 can combine with HSP70 to form a complex, which has a direct killing effect on tumor cells and thus controls tumor growth.

Contents of the invention

The purpose of the present invention is to diagnose active tuberculosis.

The present invention firstly protects the application of the substance used to detect PGLYRP1 protein in the preparation of products; the function of the product can be at least one of the following a1) to a3): a1) diagnosis of active tuberculosis; a2) diagnosis of the test subject Whether they are patients with active tuberculosis; a3) Prevention and control of tuberculosis.

The present invention also protects the application of the substance used to detect PGLYRP1 protein and the carrier recorded with judgment standard A and/or judgment standard C in the preparation of products; the function of the product can be at least one of the following a1) to a3) : a1) diagnosis of active tuberculosis; a2) diagnosis of whether the test subject is a patient with active tuberculosis; a3) prevention and control of tuberculosis;

The judgment standard A can be: if the expression level of PGLYRP1 protein in the peripheral blood of the test subject is higher than the expression level of PGLYRP1 protein in the peripheral blood of the control, the test subject is or suspected to be a patient with active tuberculosis; If the expression of PGLYRP1 protein in the blood is lower than the expression of PGLYRP1 protein in the peripheral blood of the control, the subject is not or suspected to be not a patient with active tuberculosis;

The judgment standard C can be: if the concentration of PGLYRP1 protein in the peripheral blood of the subject is higher than the concentration of PGLYRP1 protein in the peripheral blood of the control, the subject is or is suspected to be a patient with active tuberculosis; if the concentration of PGLYRP1 protein in the peripheral blood of the subject is If the concentration of PGLYRP1 protein is lower than the concentration of PGLYRP1 protein in the peripheral blood of the control, the subject is not or suspected not to be a patient with active tuberculosis;

The control peripheral blood can be the peripheral blood of tuberculosis latent infection patients or healthy people.

The expression level of PGLYRP1 protein in the peripheral blood can specifically be the expression level of PGLYRP1 protein in PBMCs isolated from peripheral blood, the expression level of PGLYRP1 protein in serum or the expression level of PGLYRP1 protein in plasma.

The present invention also protects the application of the substance used to detect PGLYRP1 gene in the preparation of products; the function of the product can be at least one of the following a1) to a3): a1) diagnosis of active tuberculosis; a2) diagnosis of the test subject Whether they are patients with active tuberculosis; a3) Prevention and control of tuberculosis.

The present invention also protects the use of the substance used for detection of PGLYRP1 gene and the carrier with the judgment criterion B recorded in the preparation of products; the function of the product can be at least one of the following a1) to a3): a1) diagnostic activity tuberculosis; a2) diagnose whether the test subject is an active tuberculosis patient; a3) prevent and control tuberculosis;

The judgment standard B can be: if the expression level of PGLYRP1 gene in the peripheral blood of the test subject is higher than the expression level of the PGLYRP1 gene in the peripheral blood of the control, the test subject is or suspected to be a patient with active tuberculosis; If the expression level of PGLYRP1 gene in the blood is lower than the expression level of PGLYRP1 gene in the control peripheral blood, then the test subject is not or suspected not to be a patient with active tuberculosis; Blood.

The expression level of the PGLYRP1 gene in the peripheral blood can specifically be the expression level of the PGLYRP1 gene in PBMCs isolated from the peripheral blood.

The present invention also protects a kit, which may include substances for detecting PGLYRP1 protein and/or substances for detecting PGLYRP1 gene; said kit may have at least one function in the following a1) to a3): a1) Diagnosis active tuberculosis; a2) diagnosis whether the test subject is a patient with active tuberculosis; a3) prevention and control of tuberculosis.

Any of the above-mentioned "substances for detecting PGLYRP1 protein" can be a substance for detecting the expression level of PGLYRP1 protein (corresponding to the judgment standard A) and/or a substance for detecting the concentration of PGLYRP1 protein (corresponding to the judgment standard A) Standard C).

Any of the "substances for detecting PGLYRP1 gene" described above may be substances for detecting the expression level of PGLYRP1 gene.

The expression level of any one of the above-mentioned PGLYRP1 proteins can be the relative expression level of the PGLYRP1 protein reference internal reference protein.

The expression level of any one of the PGLYRP1 genes mentioned above can be the relative expression level of the PGLYRP1 gene with reference to the internal reference gene.

The detection of the expression level of PGLYRP1 protein described above can be specifically performed by Western Blot experiment.

The detection of PGLYRP1 protein concentration in any of the above-mentioned methods can be specifically used in Elisa experiments.

Any of the above-mentioned substances for detecting the expression level of the PGLYRP1 gene or any of the above-mentioned substances for detecting the "relative expression level of the PGLYRP1 gene reference internal reference gene" includes a pair of primers composed of a specific primer pair and an internal reference primer pair combination;

The specific primer pair can be composed of primer PGLYRP1-F and primer PGLYRP1-R; the target gene of the specific primer pair contains the DNA fragment shown in position 626 to 786 from the 5' end of sequence 6 in the sequence listing;

The internal reference primer pair can be composed of primer F and primer R; the target gene of the internal reference primer pair can be all or part of the human internal reference gene.

The primer PGLYRP1-F may be a1) or a2) as follows:

a1) a single-stranded DNA molecule shown in sequence 1 of the sequence listing;

a2) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to Sequence 1 and has the same function as Sequence 1.

The primer PGLYRP1-R may be a3) or a4) as follows:

a3) a single-stranded DNA molecule shown in sequence 2 of the sequence listing;

a4) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to Sequence 2 and has the same function as Sequence 2.

The primer F can be as follows b1) or b2):

b1) a single-stranded DNA molecule shown in sequence 1 of the sequence listing;

b2) A DNA molecule having the same function as Sequence 1 after one or several nucleotide substitutions and/or deletions and/or additions to Sequence 1.

The primer R can be as follows b3) or b4):

b3) a single-stranded DNA molecule shown in sequence 2 of the sequence listing;

b4) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to Sequence 2 and has the same function as Sequence 2.

Any one of the specific primer pairs mentioned above also belongs to the protection scope of the present invention.

Using any one of the specific primer pairs described above to detect the expression level of the PGLYRP1 gene or to detect the relative expression level of the reference internal reference gene of the PGLYRP1 gene also falls within the protection scope of the present invention.

The detection of the expression level of the PGLYRP1 gene or the detection of the relative expression level of the reference internal reference gene of the PGLYRP1 gene by using any of the primer pairs described above also falls within the protection scope of the present invention.

In the above, the method for detecting the relative expression level of the PGLYRP1 gene reference internal reference gene in the cDNA of the test subject by using any of the above-mentioned primer pairs can specifically be: using the test cDNA as a template, using any of the above-mentioned specific primers Perform real-time fluorescent quantitative PCR on or any of the above-mentioned internal reference primer pairs, and then use the 2 ^-ΔCt method to calculate and obtain. The subject cDNA can be the cDNA of PBMCs isolated from the peripheral blood of the subject.

Any of the above internal reference proteins can be GAPDH protein.

Any of the above-mentioned internal reference genes can be GAPDH gene.

The invention also protects Y1) or Y2) or Y3) or Y4):

Y1) The application of PGLYRP1 protein as a marker in the development of reagents for diagnosing active tuberculosis;

Y2) The application of PGLYRP1 protein as a marker in the diagnosis of active tuberculosis;

Y3) The application of PGLYRP1 gene as a marker in the development of reagents for diagnosing active tuberculosis;

Y4) The application of PGLYRP1 gene as a marker in the diagnosis of active tuberculosis.

The amino acid sequence of any one of the above-mentioned PGLYRP1 proteins (GeneID number: NP_005082.1) is shown in sequence 5 in the sequence listing. The nucleotide sequence of any one of the above-mentioned PGLYRP1 genes (Genebank number: NM_005091.2) is shown in sequence 6 in the sequence listing.

Experiments have shown that compared with healthy people and latent tuberculosis infection, the expression of PGLYRP1 gene in PBMCs of patients with active tuberculosis is significantly increased; the expression of PGLYRP1 gene can be used to distinguish active tuberculosis patients from latent tuberculosis infection; The expression level of can be used to distinguish active tuberculosis patients from healthy people. Therefore, the expression level of PGLYRP1 gene has important application value in the diagnosis of active tuberculosis.

Description of drawings

Figure 1 shows the relative expression of PGLYRP1 gene in PBMCs of patients with active tuberculosis, latent tuberculosis infection and healthy people detected by real-time fluorescent quantitative PCR.

Figure 2 is the ROC curve analysis of the relative expression of PGLYRP1 gene in PBMCs of patients with active tuberculosis and latent tuberculosis infection.

Figure 3 is a ROC curve analysis of the relative expression of PGLYRP1 gene in PBMCs of patients with active tuberculosis and healthy people.

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. Quantitative experiments in the following examples were all set up to repeat the experiments three times, and the results were averaged.

Ficoll-Paque PLUS is a product of GE Company of the United States. The 96-well plate is a product of Millipore Company. AIMV ^TM Medium serum-free medium is a product of gibco company, the product catalog number is 12055091. RPMI 1640 culture medium is the product of Gibco Company, the product catalog number is 11875-093. The IFN-γ ELISPOT detection kit is a product of Daktronics. IFN-γ monoclonal capture antibody, IFN-γ detection antibody, Mycobacterium tuberculosis-specific mixed polypeptide A, Mycobacterium tuberculosis-specific mixed polypeptide B and phytohemagglutinin are all components in the IFN-γ ELISPOT detection kit. TRIzol ^TM Reagent is a product of Invitrogen. PrimeScript ^TM RT reagent Kit with gDNA Eraser is a product of TaKaRaBiotechnology. KAPA ^™ The rapid quantitative PCR kit is a product of KapaBiosystems. 2 x Green Master Mix for KAPA ^TM Components in the Rapid Quantitative PCR Kit. Nuclease-free water is a product of Ambion, USA. Fluorescence quantitative PCR instrument is a product of Roche.

Washing solution: 0.01 M PBS buffer, pH 7.4, containing 0.05% (v/v) Tween 20.

Embodiment 1, the application of the relative expression of PGLYRP1 gene in the diagnosis of active tuberculosis

1. Obtaining of peripheral blood samples

1. Distinguish peripheral blood samples from patients with latent tuberculosis infection from those from healthy people

Latent tuberculosis infection patients and healthy people have no signs or symptoms of tuberculosis, and they can be distinguished according to the following steps:

a. Coated

(1) Take a 96-well plate, add 100 μL of IFN-γ monoclonal capture antibody to each well, and coat overnight at 4°C.

(2) After completing step (1), take the 96-well plate, discard the liquid phase, add pH 7.4, 0.01M PBS buffer solution to wash twice (1 min each time), and pat dry.

(3) After completing step (2), the 96-well plate was taken, and 200 μL of 0.01 M PBS buffer at pH 7.4 containing 2% (v/v) BSA was added to each well, and incubated at 37° C. for 1 h.

(4) After completing step (3), take the 96-well plate, discard the liquid phase, and add RPMI 1640 culture medium to rinse once.

b. Preparation of PBMCs suspension

(1) Mix 2mL of peripheral blood to be tested and 2mL of RPMI 1640 culture medium evenly; then slowly add it to a sterile centrifuge tube containing 3mL Ficoll-Paque PLUS, centrifuge at 2000rcf for 20min at room temperature, divide into three floors.

(2) After step (1), the middle layer was aspirated and transferred to a centrifuge tube containing 2 mL of RPMI 1640 culture medium.

(3) After completing step (2), add 8 mL of RPMI 1640 culture solution preheated to 37° C. into the centrifuge tube, gently blow and mix with a dropper, and centrifuge at room temperature at 1400 rpm for 7 minutes.

(4) After completing step (3), take the centrifuge tube, discard the supernatant, add 6 mL of RPMI 1640 culture medium preheated to 37° C. to resuspend, and centrifuge at room temperature at 1400 rpm for 7 minutes.

(5) After completing step (4), take the centrifuge tube, discard the supernatant, and resuspend in AIM V ^TM Medium serum-free medium preheated to 37°C to obtain PBMCs with a concentration of 2.5×10 ⁶ cells/mL Suspension.

c. Immunospot detection

Refer to the instruction manual of the IFN-γ ELISPOT detection kit, and use the kit for immunospot detection. The dosage of reagents was carried out according to the instructions. Specific steps are as follows:

(1) Take the 96-well plate that completed step a, and add 100 μL of the PBMCs suspension prepared in step b (about 2.5×10 ⁵ PBMCs) to each well.

(2) After completing step (1), add Mycobacterium tuberculosis-specific mixed polypeptide A or Mycobacterium tuberculosis-specific mixed polypeptide B to each detection well; add serum-free medium to each negative control well; add phytohemagglutinin to each positive control well .

(3) After completing step (2), the 96-well plate was placed in an incubator, and cultured at 37° C. and 5% CO ₂ for 20 h.

(4) After completing step (3), take the 96-well plate, discard the supernatant, add 200 μL of pre-cooled ice water, and place it at 4° C. for 10 minutes (the purpose is to lyse the cells).

(5) After completing step (4), take the 96-well plate, discard the supernatant, wash with washing solution 5 times (add 200 μL of washing solution for 1 min each time), and pat dry.

(6) After completing step (5), take the 96-well plate and add 100 μL of IFN-γ detection antibody (avidin-labeled) dilution (99 parts by volume of PBS buffer solution of pH 7.4 and 0.01M and 1 volume part of IFN-γ detection antibody mixture), incubated at 37°C for 1h.

(7) After completing step (6), take the 96-well plate, discard the supernatant, wash with washing solution 5 times (add 200 μL of washing solution for 1 min each time), and pat dry.

(8) After completing step (7), take the 96-well plate and add 100 μL of HRP-labeled streptomycin dilution (consisting of 99 parts by volume of PBS buffer solution of pH 7.4 and 0.01M and 1 part by volume of HRP-labeled Streptomycin mixture), incubated at 37°C for 1 h.

(9) After completing step (8), take the 96-well plate, discard the supernatant, wash 5 times with washing solution (add 200 μL of washing solution for 1 min each time), and pat dry.

(10) After completing step (9), take the 96-well plate, add an enzyme substrate to each well, and develop color for 15-45 minutes at room temperature with light blocked.

(11) After completing step (10), take the 96-well plate and wash it three times with distilled water (for the purpose of stopping the reaction), then leave the 96-well plate at room temperature and let it dry naturally.

(12) After completing step (11), take the 96-well plate, use an immunospot counter (CellularTechnology Ltd, USA) to perform image and spot counting, and then make the following judgment: when the number of spots in the negative control well is less than 6, if If the number of spots in the detection well minus the number of spots in the negative control well is more than 6, the test well is positive; if the number of spots in the test well minus the number of spots in the negative control well is less than 6, the test well is negative; When the number of spots is more than 6, if the number of spots in the detection well is more than 2 times the number of spots in the negative control well, the detection well is positive; if the number of spots in the detection well is less than 2 times that of the negative control well, Then the detection hole is negative. If the detection hole is positive, the peripheral blood to be tested is provided by a person with latent tuberculosis infection (that is, the peripheral blood sample of a person with latent tuberculosis infection); if the detection hole is negative, the peripheral blood to be tested is provided by a healthy person (that is, the peripheral blood sample of a healthy person ).

2. Obtaining peripheral blood samples

(1) Active tuberculosis group: 25 peripheral blood samples.

25 peripheral blood samples: Take 2-3mL of peripheral blood from 25 patients with clinically diagnosed active pulmonary tuberculosis (all patients gave informed consent), put them in EDTA anticoagulant blood collection tubes, and turn them upside down 5-6 times (The purpose is to mix the anticoagulant and peripheral blood), and 25 peripheral blood samples were obtained.

(2) Tuberculosis latent infection group: 30 peripheral blood samples.

30 peripheral blood samples: Take 2-3mL of peripheral blood from 30 cases of latent tuberculosis infection who have been clinically diagnosed as tuberculosis latent infection (all with informed consent), put them in EDTA anticoagulant blood collection tubes, and turn them upside down 5-6 times (The purpose is to mix the anticoagulant and peripheral blood), and obtain 30 peripheral blood samples.

(3) Healthy control group: 40 peripheral blood samples.

40 peripheral blood samples: take 2-3mL peripheral blood from 40 healthy people (with informed consent), put them in anticoagulant blood collection tubes containing EDTA, turn them upside down 5-6 times (the purpose is to mix the anticoagulant and peripheral blood) ), and 40 peripheral blood samples were obtained.

95 peripheral blood samples need to be kept at room temperature (do not freeze or refrigerate), and the storage time should be less than 6 hours.

2. The application of the relative expression of PGLYRP1 gene in the diagnosis of active tuberculosis

The amino acid sequence of PGLYRP1 protein (GeneID number: NP_005082.1) is shown as sequence 5 in the sequence listing. The nucleotide sequence of the gene encoding PGLYRP1 protein (PGLYRP1 gene for short, Genebank number: NM_005091.2) is shown in sequence 6 in the sequence listing.

1. Acquisition of cDNA from 95 peripheral blood samples

(1) Preparation of PBMCs suspension

Replace the peripheral blood to be tested in Step 1, 1b, with the 95 peripheral blood samples in Step 1, 2, and keep the other steps unchanged to obtain the PBMCs suspension of 95 peripheral blood samples.

(2) RNA extraction

RNA was extracted from the PBMCs suspension of 95 peripheral blood samples. Specific steps are as follows:

① Take a 96-well plate and add the PBMCs suspension of 95 peripheral blood samples respectively. 150 μL per well.

2. After completing step 1., take the 96-well plate, and add 50 μL of the dilution of Mycobacterium tuberculosis H37Rv lysate to each hole (obtained by diluting the Mycobacterium tuberculosis H37Rv lysate with AIM V ^TM Medium serum-free medium; the protein concentration is 10μg/mL), mix well.

The preparation method of the lysate of Mycobacterium tuberculosis H37Rv: take the cells of Mycobacterium tuberculosis H37Rv, first inactivate them by cobalt 60 irradiation, and then resuspend them with PBS buffer to obtain a resuspension; Shred the bacterial cells with a crusher, centrifuge at 12,000 rcf for 10 min at 4°C, and collect the supernatant; take the supernatant, and use the BCA method to determine the protein concentration.

③After completing step ②, take the 96-well plate and culture it at 37° C., 5% CO ₂ for 16 hours, and then use TRIzol ^™ Reagent to extract RNA.

(3) Synthesis of cDNA

The RNA of the PBMCs suspension of 95 peripheral blood samples was collected, and the PrimeScript ^TM RT reagent Kit with gDNA Eraser was used for reverse transcription to obtain the cDNA of 95 peripheral blood samples.

2. Preparation of primer pair combinations

According to the nucleotide sequence of the PGLYRP1 gene, the specific primer pairs shown in Table 1 were designed and synthesized.

According to the nucleotide sequence of the GAPDH gene, the internal reference primer pairs shown in Table 1 were designed and synthesized.

The primer pair combination consists of a specific primer pair and an internal reference primer pair.

Each primer was synthesized by Shanghai Sangon Biotechnology Co., Ltd. (HPLC purification).

Table 1

3. Real-time fluorescent quantitative PCR detection of relative expression of PGLYRP1 gene

Using the cDNA of 95 peripheral blood samples as templates, the specific primer pairs or internal reference primer pairs prepared in step 2 were used to perform real-time quantitative PCR, and then the relative expression of PGLYRP1 gene in each template was obtained. Specific steps are as follows:

(1) Prepare reaction system 1 and reaction system 2

Reaction system 1 is 20 μL, consisting of 10 μL 2×Green Master Mix, 0.4 μL PGLYRP1-F aqueous solution (concentration is 10 μM), 0.4 μL PGLYRP1-R aqueous solution (concentration is 10 μM), 2 μL template (5-20ng) and 7.2 μL nuclease-free water composition.

Reaction system 2 is 20 μL, consisting of 10 μL 2×Green Master Mix, 0.4 μL GAPDH-F aqueous solution (concentration is 10 μM), 0.4 μL GAPDH-R aqueous solution (concentration is 10 μM), 2 μL template (5-20ng) and 7.2 μL nuclease-free water composition.

(2) Real-time quantitative PCR detection

Each reaction system of step (1) preparation is in Real-time quantitative PCR detection was performed on a fluorescent quantitative PCR instrument. The 2 ^-ΔCt method was used to calculate the relative expression of PGLYRP1 gene in each template.

Reaction conditions: pre-denaturation at 95°C for 3min; 40 cycles at 95°C for 5s and 60°C for 30sec, and fluorescence signals were collected during the extension stage.

The experimental results are shown in Figure 1 (TB is the active tuberculosis group, LI is the tuberculosis latent infection group, and Nor is the healthy control group). The results showed that the relative expression of PGLYRP1 gene in PBMCs of the active tuberculosis group was significantly increased compared with the healthy control group and the tuberculosis latent infection group.

(3) Statistical analysis

The results of step (2) were statistically analyzed using GraphPad Prism 5.

According to the relative expression of PGLYRP1 gene in PBMCs of active tuberculosis group and latent tuberculosis infection group, receiver operating characteristic curve analysis was performed using GraphPad Prism 5. The results are shown in Figure 2. The results showed that the relative expression of PGLYRP1 gene can be used to distinguish active tuberculosis patients from latent tuberculosis infection patients.

According to the relative expression of PGLYRP1 gene in the PBMCs of the active tuberculosis group and the healthy control group, the receiver operating characteristic curve was analyzed using GraphPad Prism 5. The results are shown in Figure 3. The results showed that the relative expression of PGLYRP1 gene can be used to distinguish active tuberculosis patients from healthy people.

The above results show that the relative expression of PGLYRP1 gene has important application value in the diagnosis of active tuberculosis.

<110> The 309th Hospital of the Chinese People's Liberation Army

<120> Application of PGLYRP1 protein as a marker in the diagnosis of active tuberculosis

<160> 6

<170> PatentIn version 3.5

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Claims (10)

1. The application of the substance used to detect PGLYRP1 protein in the preparation of products; the function of the product is at least one of the following a1) to a3): a1) diagnosis of active tuberculosis; a2) diagnosis of whether the subject is active tuberculosis patients; a3) prevention and control of tuberculosis. 2. The application of the substance used to detect PGLYRP1 protein and the carrier recorded with judgment standard A and/or judgment standard C in the preparation of products; the function of the product is at least one of the following a1) to a3): a1) Diagnose active tuberculosis; a2) diagnose whether the test subject is a patient with active tuberculosis; a3) prevent and control tuberculosis; The judgment standard A is: if the expression level of PGLYRP1 protein in the peripheral blood of the test subject is higher than the expression level of PGLYRP1 protein in the peripheral blood of the control, the test subject is or suspected to be a patient with active tuberculosis; If the expression level of PGLYRP1 protein in blood is lower than the expression level of PGLYRP1 protein in peripheral blood of the control, the test subject is not or suspected not to be a patient with active tuberculosis; The judgment criterion C is: if the concentration of PGLYRP1 protein in the peripheral blood of the subject is higher than the concentration of PGLYRP1 protein in the peripheral blood of the control, the subject is or suspected to be a patient with active tuberculosis; if the concentration of PGLYRP1 in the peripheral blood of the subject is If the concentration of the protein is lower than the concentration of PGLYRP1 protein in the peripheral blood of the control, the subject is not or suspected not to be a patient with active tuberculosis; The control peripheral blood is the peripheral blood of tuberculosis latent infection patients or healthy people. 3. The application of the substance used to detect the PGLYRP1 gene in the preparation of products; the function of the product is at least one of the following a1) to a3): a1) diagnosis of active tuberculosis; a2) diagnosis of whether the subject is active tuberculosis patients; a3) prevention and control of tuberculosis. 4. The application of the substance used to detect the PGLYRP1 gene and the carrier with the judgment standard B recorded in the preparation of the product; the function of the product is at least one of the following a1) to a3): a1) diagnosis of active tuberculosis; a2 ) Diagnose whether the test subject is an active tuberculosis patient; a3) prevent and control tuberculosis; The judgment standard B is: if the expression of PGLYRP1 gene in the peripheral blood of the test subject is higher than the expression of PGLYRP1 gene in the peripheral blood of the control, the test subject is or suspected to be a patient with active tuberculosis; if the test subject's peripheral blood If the expression level of PGLYRP1 gene in the blood is lower than the expression level of the PGLYRP1 gene in the control peripheral blood, the test subject is not or suspected not to be an active tuberculosis patient; the control peripheral blood is the peripheral blood of tuberculosis latent infection patients or healthy people. 5. A kit comprising a substance for detecting PGLYRP1 protein and/or a substance for detecting PGLYRP1 gene; said kit has at least one function in the following a1) to a3): a1) diagnosis of active tuberculosis; a2) Diagnose whether the test subject is a patient with active tuberculosis; a3) Prevent and control tuberculosis. 6. The application according to any one of claims 1 to 4 or the kit according to claim 5, characterized in that: The "substance for detecting PGLYRP1 protein" is a substance for detecting the expression level of PGLYRP1 protein and/or a substance for detecting the concentration of PGLYRP1 protein; The "substance for detecting PGLYRP1 gene" is a substance for detecting the expression level of PGLYRP1 gene. 7. application or test kit as claimed in claim 6, is characterized in that: The expression level of the PGLYRP1 protein is the relative expression level of the reference internal reference protein of the PGLYRP1 protein; The expression level of the PGLYRP1 gene is the relative expression level of the reference internal reference gene of the PGLYRP1 gene. 8. The application or test kit as claimed in claim 6 or 7, characterized in that: The substance used to detect the expression level of PGLYRP1 gene or the substance used to detect the "relative expression level of PGLYRP1 gene reference internal reference gene" includes a primer pair combination composed of a specific primer pair and an internal reference primer pair; The specific primer pair is composed of primer PGLYRP1-F and primer PGLYRP1-R; the target gene of the specific primer pair contains the DNA fragment shown in the 626th to 786th position from the 5' end of sequence 6 in the sequence listing; The internal reference primer pair is composed of primer F and primer R; the target gene of the internal reference primer pair is all or part of the human internal reference gene. 9. The pair of specific primers as claimed in claim 8. 10. Y1) or Y2) or Y3) or Y4): Y1) The application of PGLYRP1 protein as a marker in the development of reagents for diagnosing active tuberculosis; Y2) The application of PGLYRP1 protein as a marker in the diagnosis of active tuberculosis; Y3) The application of PGLYRP1 gene as a marker in the development of reagents for diagnosing active tuberculosis; Y4) The application of PGLYRP1 gene as a marker in the diagnosis of active tuberculosis.