Pathogen infection diagnosis marker based on DPH1-DPH7 gene mRNA and diphtheria amide modification level

Abstract

The invention discloses a pathogen infection diagnosis marker based on DPH gene family mRNA and diphtheria amide modification level, a kit and application. The marker combination comprises the mRNA expression level of the DPH1-DPH7 gene and the diphtheria amide modification level of the eukaryotic elongation factor 2. The invention reveals that the combination is remarkably related to pathogen infection for the first time, and provides a detection kit containing a specific primer, biotinylated NAD, pseudomonas exotoxin A and other reagents. The indexes are respectively detected through qPCR (quantitative polymerase chain reaction) and non-denatured immunoblotting, and efficient diagnosis can be realized through combined analysis. Experiments show that the combined marker is excellent in diagnostic performance, the area AUC under a curve of a combined logistic regression model can reach 0.917, the sensitivity is 90.9%, the specificity is 85.7%, and a novel and reliable molecular scheme is provided for early-stage differential diagnosis of infection.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical detection, and particularly relates to a novel molecular marker for diagnosing pathogen infection, a detection kit comprising the marker and application thereof. BACKGROUND

[0002] Infectious diseases are one of the main causes of morbidity and mortality worldwide, and accurate diagnosis of infectious pathogens is crucial for clinical treatment decisions, which is the key to implementing effective treatment, controlling antibiotic abuse, and reducing patient mortality.

[0003] Current main methods and limitations of clinical infection diagnosis:

[0004] 1. Marker rapid screening method

[0005] The marker rapid screening method mainly detects corresponding markers in blood for instant detection. The main markers detected include procalcitonin (PCT), C-reactive protein (CRP), and serum amyloid A (SAA), etc.

[0006] PCT significantly increases in severe systemic bacterial infection, and its response is more pathogen-specific, which is much more valuable than viruses and non-infectious inflammation in indicating bacterial infection, and is a key indicator for guiding the initiation and discontinuation of antibiotics. However, its specificity is limited, and it can also significantly increase in non-bacterial systemic inflammatory conditions such as severe trauma, major surgery, and burns. In the case of chronic inflammation, autoimmune diseases, or renal dysfunction, the PCT level may also be disturbed, limiting its differential diagnostic ability. At the same time, it may not be sensitive to local infection or early infection, which can easily cause false negatives.

[0007] CRP and SAA detection has the advantages of rapidity and convenience, and is suitable for preliminary screening. CRP and SAA belong to acute phase reactants, which are synthesized by the liver. When the body has an inflammatory response such as infection or trauma, the plasma concentration can rapidly increase within 4-6 hours. As a broad-spectrum acute phase protein, CRP and SAA can rapidly increase in various infections and non-infectious inflammation. Compared with SAA, the increase of CRP is more relevant to bacterial infection, and it is often used as a key indicator for evaluating the severity of bacterial infection and monitoring the efficacy of antibiotic therapy. SAA is more sensitive and rapid in response to viral infection, and its concentration can significantly increase in the early stage of viral infection. Therefore, the two are often used in combination for diagnosis to improve the sensitivity of infection screening. SAA helps to detect viral infection early, while CRP focuses more on indicating the risk of bacterial infection. However, the common limitation of both is the lack of specificity in pathogen diagnosis, which cannot distinguish between infection types or between infectious and non-infectious inflammation, so they are often used as "early warning" indicators to indicate the presence of inflammation, and cannot be used as a basis for diagnosis.

[0008] 2. Direct detection of pathogens

[0009] The current direct detection methods of pathogens mainly include: microbial culture, molecular detection, etc. The core advantage of direct detection technology of pathogens is its high specificity, which can directly identify and confirm pathogenic microorganisms, providing "gold standard" basis for infectious disease diagnosis. However, this method also has obvious shortcomings: the culture is time-consuming and low positive rate, the sensitivity of antigen detection is limited and depends on the specificity of the pathogen; molecular detection has the characteristics of rapidity and high sensitivity, especially suitable for difficult-to-culture pathogens and acute and severe infections, but molecular detection is fast but cannot distinguish the survival state of pathogens (DNA / RNA residues may cause false positives), and this method cannot assess the overall immune response of the host and the severity of inflammation, which limits its comprehensive judgment of the infection process and guidance of individualized treatment.

[0010] 3. Antibody detection method

[0011] Antibody detection (serological detection) indirectly judges infection by detecting pathogen-specific antibodies (such as IgM, IgG) in serum. Its advantage is high pathogen specificity, which is suitable for epidemiological investigation and retrospective diagnosis of some pathogens. However, this method has obvious limitations: there is a "window period" in the early stage of infection, which is easy to cause false negatives; IgG antibodies are difficult to distinguish between current and past infections; and antibody response is delayed, which cannot meet the clinical needs of rapid differential diagnosis in the early stage of acute infection.

[0012] Therefore, constructing a multi-dimensional, multi-marker combination diagnosis model may be an ideal direction. In this context, developing more new markers that can provide specific information for differential diagnosis can effectively complement the existing rapid screening methods, and have important clinical value and practical significance.

[0013] Biological significance of DPH gene family and diphthamide modification

[0014] The DPH1-DPH7 (Diphthamide Biosynthesis Protein, DPH) gene family encodes proteins involved in the diphthamide modification process unique to eukaryotes, which occurs on eukaryotic elongation factor 2 (eEF2) and plays a key role in maintaining protein synthesis. It is known that diphtheria toxin can inhibit host protein synthesis by specifically targeting the modification site, but the following scientific problems have not been clearly defined: whether the expression of DPH genes and diphthamide modification are regulated by pathogen infection and whether their dynamic changes are pathogen-specific or related to the infection stage? Existing researches are mostly focused on the role of diphthamide modification in tumorigenesis (such as DPH gene mutations found in some cancers), while its function in infection and immune response has hardly been explored. Currently, there is no literature reporting the correlation between the mRNA expression level of DPH1-DPH7 genes and the level of diphthamide modification and pathogen infection, nor has it been developed as a diagnostic marker. SUMMARY

[0015] The present application finds that the mRNA of DPH1-DPH7 genes and the level of diphthamide modification are correlated with pathogen infection. Compared with traditional host inflammatory markers, the markers based on the diphthamide modification process are directly related to the interference mechanism of pathogens on the translation mechanism, and may have certain pathogen identification specificity. In addition, combined detection of the level of diphthamide modification and gene expression indicators can help overcome the instability of single mRNA markers, thereby constructing a more robust multi-dimensional infection diagnosis system. The present application solves the limitations of existing pathogen infection diagnosis methods in specificity, early identification and comprehensive evaluation ability, and provides a diagnostic scheme based on a new type of host molecular marker.

[0016] To achieve the above object, the technical scheme adopted by the present application is as follows:

[0017] In a first aspect of the present application, a biomarker for diagnosing pathogen infection is provided. The biomarker is selected from at least one of the following:

[0018] (a) the mRNA expression level of DPH1, DPH2, DPH3, DPH4, DPH5, DPH6 and / or DPH7 genes;

[0019] (b) the level of diphthamide modification on eukaryotic elongation factor 2 (eEF2).

[0020] Preferably, the biomarker is a combination of (a) and (b). The present application firstly relates the biological process to pathogen infection and finds that the combination can provide more stable and discriminative diagnostic information than single indicator.

[0021] In a second aspect of the present application, the above biomarker combination is provided for use in preparing a kit for diagnosing pathogen infection. The use is based on the correlation between the biomarker disclosed in the present application and the infection status.

[0022] In a third aspect of the present application, a kit for diagnosing pathogen infection is provided. The kit comprises reagents for detecting the above biomarker combination, specifically including:

[0023] A primer pair for specifically detecting mRNA of DPH1 to DPH7 genes, preferably having the sequence shown in Table 1 of the present specification.

[0024] A reagent for specifically detecting diphthamide modification of eEF2, comprising biotinylated nicotinamide adenine dinucleotide (biotinylated NAD) and Pseudomonas exotoxin A.

[0025] In a fourth aspect of the present application, a method for diagnosing pathogen infection is provided. The method comprises the following steps:

[0026] (1) obtaining a biological sample from a subject, preferably peripheral blood lymphocytes;

[0027] (2) detecting the mRNA expression level of DPH1 to DPH7 genes and the diphthamide modification level of eEF2 in the sample. The mRNA level is preferably detected by real-time fluorescent quantitative PCR (qPCR), and the diphthamide modification level is preferably detected by non-denaturing immunoblotting analysis using biotinylated NAD and Pseudomonas exotoxin A;

[0028] (3) comparing the detection results of step (2) with healthy controls. When the mRNA expression level and / or the diphthamide modification level is significantly reduced, it indicates that the subject has pathogen infection.

[0029] Preferably, the method further employs a logistic regression model to jointly analyze the detection results of the mRNA expression level of DPH1 to DPH7 genes and the diphthamide modification level of eEF2, so as to achieve more accurate diagnosis. As shown in the results of the examples, the joint analysis method exhibits excellent diagnostic performance, with an area under the receiver operating characteristic curve (AUC) greater than 0.9 (e.g. 0.917), high sensitivity (e.g. 90.9%) and specificity (e.g. 85.7%).

[0030] The present application has the following beneficial effects:

[0031] 1. The present application first discloses the direct correlation between DPH1-DPH7 gene mRNA level and diphthamide modification level and pathogen infection, and provides a new specific marker for molecular diagnosis of pathogen infection.

[0032] 2. The present application remedies some limitations of existing pathogen infection diagnosis methods. For example, traditional pathogen culture method takes a long time, and has low sensitivity to viruses, anaerobic bacteria and other difficult-to-culture pathogens; immunological detection has a detection window period, and antibody level is easily affected by individual immune state difference, resulting in false negative or false positive results, etc.

[0033] 3. The present application fills the application gap of the biological process in infection diagnosis, and has good transformation prospect. The prior art has never involved the dynamic changes of DPH gene family and diphthamide modification in the infection process and their diagnostic value. The present application not only first clarifies the correlation between the biological process and infection, but also further develops the corresponding detection method and kit. The detection system has the characteristics of simple operation, short detection period, strong pertinence, etc., and is easy to carry out in clinical laboratory, and can be used as an important supplement to the existing rapid screening and pathogen direct detection method, and improves the comprehensive diagnosis ability of infection. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 : Clinical case grouping for detecting DPH1-DPH7 gene mRNA level;

[0035] Figure 2 : Clinical case CRP / SAA level for detecting DPH1-DPH7 gene mRNA level;

[0036] Figure 3 : Comparison of DPH1-DPH7 gene mRNA level in peripheral blood lymphocytes of each group of cases;

[0037] Figure 4 : ROC curve of control group and High CRP / SAA group to verify the sensitivity and specificity of DPH1-DPH7 as an infection diagnostic marker;

[0038] Figure 5 ROC curve of control group and High SAA group to verify the sensitivity and specificity of DPH1-DPH7 as an infection diagnostic marker;

[0039] Figure 6 : Clinical case grouping for detecting diphthamide modification level;

[0040] Figure 7 : Blood CRP / SAA level of clinical cases for detecting diphthamide modification level;

[0041] Figure 8 : The level of diphthamide modification in peripheral blood lymphocytes of each group of cases;

[0042] Figure 9 : ROC curve of the control group and the infection group to verify the sensitivity and specificity of diphthamide modification level as an infection diagnostic marker;

[0043] Figure 10 : Joint ROC curve analysis of DPH1-DPH7 gene mRNA and diphthamide modification level. DETAILED DESCRIPTION

[0044] The embodiments of the present application will be further described below through several specific examples. However, it should be made clear that the material ratio, process conditions, and experimental results described herein are only used to illustrate the principles and effects of the present application, and do not constitute a limitation on the scope of protection of the present application. Any equivalent modifications or adjustments made without deviating from the basic idea and spirit of the present application should be included in the scope of protection of the present application.

[0045] The embodiments of the present application will be further described below through several specific examples. However, it should be made clear that the material ratio, process conditions, and experimental results described herein are only used to illustrate the principles and effects of the present application, and do not constitute a limitation on the scope of protection of the present application. Any equivalent modifications or adjustments made without deviating from the basic idea and spirit of the present application should be included in the scope of protection of the present application.

[0046] Example 1

[0047] One of the technical solutions of the present application is to provide a pathogen infection diagnostic method based on DPH1 to DPH7 gene mRNA level detection. The method first separates lymphocytes from the peripheral blood of the subject to be tested and extracts total RNA, and then reversely transcribes the RNA into cDNA. Using specifically designed primers, the mRNA expression levels of DPH1, DPH2, DPH3, DPH4, DPH5, DPH6, and DPH7 are quantitatively detected by real-time fluorescent quantitative PCR technology. By setting up a reference gene for standardization, the relative expression amount of the target gene is calculated and compared with the expression level of the healthy control group for analysis.

[0048] The specific steps are as follows:

[0049] 1. Lymphocyte total RNA extraction

[0050] (1) Take the patient's anticoagulant peripheral whole blood (4-5ml), prevent coagulation by adding EDTA to the blood. Add the blood sample to a centrifuge tube, and add an equal volume of PBS buffer, mix well to obtain a mixed blood sample;

[0051] (2) Add an equal volume of lymphocyte separation medium (commercial product) to another centrifuge tube. Gently add the mixed blood sample, then centrifuge at 600g / min for 15 minutes;

[0052] (3) After centrifugation, the blood sample is layered, and the lymphocyte layer is removed and added to 5ml PBS buffer, mixed and centrifuged at 1500r / min for 10 minutes. Repeat the process and again suck the lymphocyte layer to obtain the sample to be tested.

[0053] (4) Add TRIzol reagent to each sample to be tested (about 1 mL per 10 6 cells), and mix well by blowing;

[0054] (5) Add chloroform (1 / 5 of the volume of TRIzol), shake vigorously for 15-30 seconds, and stand at room temperature for 2-3 minutes. Centrifuge at 4°C (12,000 x g, 15 minutes), and separate into three layers: upper layer (water phase containing RNA), middle layer (DNA), and lower layer (organic phase)

[0055] (6) Take the upper water phase to a new tube, avoiding the middle layer, add an equal volume of isopropanol, mix gently by inverting, stand at room temperature for 10 minutes, and centrifuge at 4°C (12,000 x g, 10 minutes). A white RNA precipitate can be seen at the bottom of the tube;

[0056] (7) Discard the supernatant, add 75% ethanol (prepared with DEPC water), gently shake the precipitate, centrifuge at 4°C (7,500 x g, 5 minutes), discard the ethanol, and dry the precipitate at room temperature for 5-10 minutes (do not completely dry), and dissolve the precipitate with 20-50 μl of RNase-free DEPC water;

[0057] (8) Take 1 μl of the sample, and measure the total RNA concentration on a nucleic acid quantifier, to ensure that the total RNA concentration is above 100 ng / μl, to provide a suitable template for subsequent mRNA reverse transcription into cDNA.

[0058] 2. cDNA reverse transcription

[0059] Select TaKaRa's PrimeScript™ RT Master Mix reverse transcription kit, and perform the following operations:

[0060] (1) Reaction system

[0061] 2X RT Master Mix 10 μL

[0062] Total RNA template 1 μg

[0063] RNase-free water to a total volume of 20 μL

[0064] (2) Reaction conditions

[0065] Run in the PCR instrument according to the following program: 25°C for 5 minutes → 42°C for 60 minutes → 85°C for 5 minutes → 4°C for keeping. The cDNA can be used immediately or stored at -20°C.

[0066] 3. Primer design

[0067] The upstream and downstream primers for amplifying the DPH1-DPH7 genes and the primers for the internal reference gene GAPDH were designed using the Primer 5.0 software, and the primer sequences were synthesized by Shanghai Sangon Biological Engineering Co., Ltd. The nucleotide sequences of the upstream and downstream amplification primers of the DPH1-DPH7 genes are shown in Table 1.

[0068]

[0069] 4. Realtime-PCR

[0070] The cDNA levels of the DPH1 to DPH7 genes in the samples were detected by the SYBR Green method, and the single-well reaction system was: cDNA 0.4 μL, forward and reverse primers each 0.4 μL, 2X Master Mix 10 μL, enzyme-free water 8.8 μL, total volume 20 μL. Amplification was performed in a fluorescence quantitative PCR instrument, and the cycle amplification program was: first pre-denaturation, 95°C, 2 minutes; then denaturation, 95°C, 10-15 seconds, annealing / extension, 60°C, 30 seconds, and amplification repeated for 40 cycles.

[0071] Example 2

[0072] The second technical solution of the present application is to provide a pathogen infection diagnosis method based on diphthamide modification level detection. The method extracts total protein from the peripheral blood lymphocytes of the subject to be tested, and uses biotinylated NAD and Pseudomonas exotoxin A to specifically label the diphthamide modification in the sample. Then, the labeled diphthamide modification level is detected by immunoblotting under non-denaturing conditions, and the total amount of translation elongation factor eEF2 is used as an internal reference for standardization to calculate the relative level of diphthamide modification, which is then compared and analyzed with the modification level of the healthy control group.

[0073] The specific steps are as follows:

[0074] 1. Cell total protein supernatant acquisition

[0075] (1) Take the patient's anticoagulant peripheral whole blood (4-5ml), prevent coagulation by adding EDTA to the blood. Add the blood sample to a centrifuge tube, and add an equal volume of PBS buffer, mix to obtain a mixed blood sample;

[0076] (2) Add an equal volume of lymphocyte separation medium (commercial product) to another centrifuge tube. Gently add the mixed blood sample, then centrifuge horizontally at 600g / min for 15 minutes;

[0077] (3) After centrifugation, the blood sample is layered, and the lymphocyte layer is removed and added to 5ml PBS buffer, mixed and centrifuged again at 1500r / min for 10 minutes. Repeat the process and again suck the lymphocyte layer.

[0078] (4) Add 100ul of cell lysis solution RIPA (Radio Immunoprecipitation Assay) with protease inhibitors (commercially available) to the lymphocyte pellet obtained in step (3) and mix well, place on ice for 20min, then ultrasonic treatment at 20kHz ultrasonic frequency for 20s, stand on ice for 20min, 4℃, 14000r / min speed centrifugation for 15min, collect the obtained cell protein supernatant.

[0079] (5) Take 5ul of cell protein supernatant to determine the total protein concentration of the sample with BCA protein concentration determination kit (commercially available), and dilute the concentration of the control cell and the test cell protein supernatant to 3ug / ul.

[0080] (6) Add biotinylated NAD (commercially available) and Pseudomonas exotoxin A (commercially available) to the cell protein supernatant (the amount of control and test cell protein supernatant is the same total protein amount), the final concentration of biotinylated NAD is 10uM / ul, and the final concentration of Pseudomonas exotoxin A is 20ng / ul, mix well, and react at room temperature for 60min. Add immunoblotting test (WB) loading buffer (commercially available) and mix well, and directly proceed, all samples cannot be denatured.

[0081] (7) After the WB experiment is finished, put the transfer film into 0.01% Streptavidin-HRP (commercially available) at 4℃ overnight, the next day after washing, use enhanced chemiluminescence reagent ECL (commercially available) to develop color.

[0082] (8) Another protein total amount of the same and not treated with biotinylated NAD and Pseudomonas exotoxin A concentration of 3 ug / ul cell protein supernatant sample, add WB loading buffer (commercial, can be purchased) mixed, 95℃ temperature treatment 5 min, WB, membrane transfer blocking, eEF2 antibody (commercial, can be purchased), the final concentration of eEF2 antibody is 0.1 ug / ml; 4℃ overnight, the next day horseradish peroxidase secondary antibody room temperature incubation 1h, after washing with enhanced chemiluminescence reagent ECL luminescence color.

[0083] Result analysis

[0084] (I) DPH1 to DPH7 gene mRNA level result analysis

[0085] First step: experimental quality control

[0086] Negative control check, its amplification curve should be a flat straight line, Ct value shows "Undetermined" or more than 35-40.

[0087] Positive control check, known concentration of positive template, its Ct value should be within the expected range.

[0088] Technical repeatability, the Ct value of 3 repeated holes of the same sample should be very close (usually standard deviation < 0.5).

[0089] Second step: evaluate the amplification curve

[0090] Observe the amplification curve of the target sample, all samples should have normal amplification curve shape.

[0091] Third step: melting curve analysis

[0092] The melting curve of each gene (including internal reference gene) shows a single, sharp peak

[0093] Fourth step: quantitative analysis and result determination

[0094] Compare the relative difference of target gene expression between different groups, determine the method (use 2^(-ΔΔCt) method). First calculate ΔCt = Ct (target gene) - Ct (internal reference gene), then calculate ΔΔCt = ΔCt (treatment group) - ΔCt (control group), finally calculate the expression change fold = 2^(-ΔΔCt).

[0095] (II) Diphthamide level result analysis

[0096] WB results were color-developed and photographed on a fully automatic chemiluminescence analyzer. The photographed results were measured for gray value of WB color-developed bands on Quantity One software. The gray ratio of the control (C = control diphthamide modification / control eEF2) and the gray ratio of the subject to be detected (P = subject to be detected diphthamide modification / subject to be detected eEF2) were calculated respectively, and then the ratio C / P of the gray ratio C and the gray ratio P was calculated.

[0097] (Three) ROC curve analysis of the sensitivity and specificity of clinical diagnosis

[0098] The receiver operating characteristic curve (i.e. ROC curve) between different groups was drawn. The diagnostic performance evaluation curve was used to comprehensively evaluate the discrimination ability of a diagnostic method or model. According to international standards, the area under the ROC curve (i.e. AUC value) was analyzed to analyze the sensitivity and specificity of the clinical diagnosis of DPH1-DPH7 gene mRNA level and diphthamide modification level.

[0099] General standard AUC is between 0.5 and 1.0: the closer the value is to 1, the better the discrimination ability of the model.

[0100] 0.5 < AUC ≤ 0.7: poor or general discrimination ability.

[0101] 0.7 < AUC ≤ 0.8: acceptable discrimination ability.

[0102] 0.8 < AUC ≤ 0.9: excellent discrimination ability.

[0103] AUC > 0.9: very good discrimination ability.

[0104] At the same time, the detection results of the above DPH gene mRNA level and diphthamide modification level were combined and analyzed. Through the ROC curve, the diagnostic performance of single or combined markers for distinguishing the infection group from the healthy control group was evaluated, including the area under the curve, sensitivity and specificity, etc. so as to determine the clinical application value of the markers as pathogen infection diagnostic markers.

[0105] Results verification

[0106] (One) Diagnostic verification of DPH1 to DPH7 gene mRNA level

[0107] The repeatability verification of DPH1 to DPH7 gene mRNA level was carried out according to the above steps, and the clinical cases included (see Figure 1): 28 cases of healthy children group (CRP <0.8 mg / L, SAA <10 mg / L; Normal group); 44 cases of CRP / SAA abnormal children group (CRP >0.8 mg / L, SAA >10 mg / L; High CRP / SAA group) and 25 cases of SAA abnormal children group (CRP <0.8 mg / L, SAA >10 mg / L; High SAA group).

[0108] The concentration of CRP / SAA in blood of each group of cases was detected (Fig. 1) Figure 2 ), and the average concentration was calculated, in which the CRP of Normal group was 0.4 mg / L, the SAA was 5 mg / L; the CRP of High SAA group was 0.4 mg / L, the SAA was 58.3 mg / L; the CRP of High CRP / SAA group was 13.7 mg / L, the SAA was 58.3 mg / L.

[0109] The mRNA level of DPH1-DPH7 gene in blood of each group of patients was detected (Fig. 2) Figure 3 ), the mRNA level of DPH1 / 2 / 4 / 5 / 6 / 7 gene was significantly reduced in High CRP / SAA group and High SAA group, the mRNA level of DPH3 gene was significantly reduced only in High SAA group, and all had statistical difference.

[0110] The ROC curve of DPH1-DPH7 gene of healthy control group and High CRP / SAA group was drawn, the AUC value was analyzed, and the sensitivity and specificity of infection diagnosis were determined (Fig. 3) Figure 4 ), in which DPH1 (AUC = 0.7719), DPH2 (AUC = 0.7459), DPH3 (AUC = 0.5946), DPH4 (AUC = 0.8117), DPH5 (AUC = 0.7804), DPH6 (AUC = 0.8539), DPH7 (AUC = 0.8401), except DPH3 showed no significant distinguishing ability, other indicators showed that the mRNA level of DPH1 / 2 / 4 / 5 / 6 / 7 gene changed significantly in systemic inflammatory state, which could be used as a new blood biomarker of infection-related inflammation.

[0111] Meanwhile, the ROC curve of DPH1-DPH7 gene of healthy control group and High SAA group was drawn, the AUC value was analyzed, and the sensitivity and specificity of infection diagnosis were determined (Fig. 4) Figure 5), DPH2 (AUC = 0.7200), DPH3 (AUC = 0.7286), DPH4 (AUC = 0.7879), DPH5 (AUC = 0.7836), DPH6 (AUC = 0.827), DPH7 (AUC = 0.860), the mRNA levels of DPH1-DPH7 genes also changed significantly in the systemic inflammatory state, which can be used as a new blood biomarker for infection-related inflammation.

[0112] (II) Diagnostic verification of diphthamide modification level detection

[0113] The repeatability verification of diphthamide modification level detection was carried out according to the above steps, and the clinical cases included (see Figure 6 ): 76 cases of healthy children group (CRP <0.8 mg / L, SAA <10 mg / L; Normal group) and 51 cases of CRP / SAA abnormal children group (CRP >0.8 mg / L, SAA >10 mg / L; High CRP / SAA group),

[0114] The concentrations of CRP / SAA in the blood of each group of cases were detected (see Figure 7 ), and the average concentrations were calculated, in which the CRP of the Normal group was 0.4 mg / L, and the SAA was 5 mg / L; the CRP of the High CRP / SAA group was 13.53 mg / L, and the SAA was 63.55 mg / L.

[0115] The diphthamide modification level in the blood of each group of patients was detected at the same time, the C / P of the detection sample was analyzed, and the repeatability verification of the diphthamide modification level was carried out (see Figure 8 ), and the results showed that the diphthamide modification level of the High CRP / SAA group, i.e. the infection case group, was significantly lower than that of the Normal group, with a statistically significant difference.

[0116] ROC curves of diphthamide modification levels of healthy control group and infection group were drawn, AUC values were analyzed, and the sensitivity and specificity of infection diagnosis were determined, the AUC value was 0.624, indicating that it had certain diagnostic sensitivity and specificity (see Figure 9 ), and had the possibility of being a new blood biomarker for infection diagnosis.

[0117] (III) Combined ROC curve analysis of DPH1-DPH7 gene mRNA and diphthamide modification level

[0118] The ROC curves of DPH1-DPH7 gene mRNA and diphthamide modification level were combined (see Figure 10), i.e. simultaneous detection of DPH1-DPH7 gene mRNA and diphthamide modification level in the sample, we found that this combination method based on logistic regression achieved excellent clinical diagnostic performance of infectious cases (AUC = 0.917, sensitivity = 90.9%, specificity = 85.7%), which improved the clinical diagnostic ability compared with single biomarker.

[0119] Through the above experiments, we found that in the clinical cases with abnormal increase of CRP / SAA index, i.e. in the infectious cases, the mRNA levels of DPH1 to DPH7 genes in the blood lymphocytes were significantly reduced, and the diphthamide modification level was also significantly reduced. By comprehensive analysis, it is proved that the combination of DPH gene mRNA level (DPH1-7) and diphthamide modification level can create a powerful pathogen infection detection and diagnosis panel. The combination method based on logistic regression achieves excellent diagnostic performance (AUC = 0.917, sensitivity = 90.9%, specificity = 85.7%), and this multi-biomarker method provides better clinical diagnostic sensitivity and specificity, which is suitable for infection screening and confirmatory testing, and is expected to improve early infection detection and reduce diagnostic errors. The results support that DPH1-DPH7 gene mRNA and diphthamide modification level can be used as a new marker for infection diagnosis to realize the clinical application of infection diagnosis, so it can be used for diagnosing pathogen infection or reagent kit.

[0120] The embodiments of the present application are intended to illustrate its principles and effects, and do not limit the scope of the present application. Those skilled in the art of related technology can make appropriate modifications or changes without departing from the spirit of the present application. Therefore, all equivalent changes meeting the technical idea of the present application shall be included in the protection scope of the present application.

Claims

1. A biomarker for diagnosing pathogen infection, characterized in that, The biomarker is selected from at least one of the following: (a) mRNA expression levels of the DPH1, DPH2, DPH3, DPH4, DPH5, DPH6 and / or DPH7 genes; (b) Level of diphtheria amide modification on eukaryotic elongation factor 2.

2. The biomarker according to claim 1, characterized in that, The biomarker is a combination of (a) and (b).

3. The use of the biomarker according to claim 1 or 2 in the preparation of a kit for diagnosing pathogen infection.

4. A reagent kit for diagnosing pathogen infection, characterized in that, The kit comprises reagents for detecting the combination of biomarkers of claim 1 or 2, the reagents comprising: Primer pairs for specific detection of DPH1 to DPH7 gene mRNA; and A reagent for the specific detection of eEF2 diphtheria amide modification contains biotinylated nicotinamide adenine dinucleotide and Pseudomonas exotoxin A.

5. The reagent kit according to claim 3, characterized in that, The sequences of the primer pairs used for specific detection of DPH1 to DPH7 gene mRNA are shown in SEQ NO.1-16.

6. A method for diagnosing pathogen infection, characterized in that, The method includes the following steps: (1) Obtain biological samples from the subjects; (2) Detect the mRNA expression levels of DPH1 to DPH7 genes and the diphtheria amide modification level of eEF2 in the samples; (3) Compare the detection results of step (2) with those of a healthy control, wherein the mRNA expression level and / or diphtheria amide modification level are significantly reduced, indicating that the subject has a pathogen infection.

7. The method according to claim 5, characterized in that, In step (2): The mRNA expression levels of DPH1 to DPH7 genes were detected by real-time quantitative PCR; and / or the diphtheria amide modification level of eEF2 was detected by non-denaturing immunoblotting analysis using biotinylated NAD and Pseudomonas exotoxin A.

8. The method according to claim 5 or 6, characterized in that, The biological sample was peripheral blood lymphocytes.

9. The method according to claim 5, characterized in that, The method employs a logistic regression model to jointly analyze the detection results of the mRNA expression levels of DPH1 to DPH7 genes and the diphtheria amide modification level of eEF2, in order to achieve diagnosis.

10. The method according to claim 8, characterized in that, The combined analysis showed an area under the curve (AUC) greater than 0.9 for diagnosing pathogen infection.

Images