Marker for severity and complication of mycoplasma pneumoniae pneumonia infection and application thereof in preparation of diagnostic kit for mycoplasma pneumoniae infection

Abstract

The application discloses a marker for the severity and complication of mycoplasma pneumoniae pneumonia infection and application thereof in the preparation of a mycoplasma pneumoniae infection diagnosis kit, and belongs to the field of epigenetics and microbiology. In the application, the editing levels of six RNA editing sites of five genes in the severe side and the control side of MPP patients, i.e. CCRL2:chr3:46408483, CCNI:chr4:77058527, CTSS:chr1:150732881 / 150732032, IRAK3:chr12:66194142 and LYN:chr8:56010885, are obviously different, the five different genes are related to the severity and complication of MPP, and the combination of RNA editing has a very high accuracy in diagnosing the severity and complication of MPP.

Description

Technical Field

[0001] This invention relates to markers of the severity and complications of Mycoplasma pneumoniae pneumonia infection and their application in the preparation of diagnostic kits for Mycoplasma pneumoniae infection, belonging to the fields of epigenetics and microbiology. Background Technology

[0002] Mycoplasma pneumoniae pneumonia (MPP) is an important human respiratory pathogen that can cause mild to moderate community-acquired mycoplasma pneumoniae pneumonia, especially in children.

[0003] RNA editing is a branch of epigenetics. Epigenetics studies heritable changes in gene expression or cellular phenotypes caused by alterations in the nucleotide sequence of genes. It mainly includes DNA methylation, histone modification, non-coding RNA, RNA editing, and genomic imprinting. RNA editing is an important post-transcriptional epigenetic regulation that alters RNA sequences. The most common type of RNA editing is the adenosine-inosine (AI) conversion mediated by the adenosine deaminase ADAR family. RNA editing can increase transcriptomic or proteomic diversity and may alter the sequence of encoded proteins or the stability and transport of RNA. Studies have shown that RNA editing plays an important regulatory role in processes such as microbial infection and immunity. In mycoplasma pneumoniae (MPP), ADAR1 and ADAR2, as major members of the ADAR family, exert infectious and anti-infective effects, respectively, through their catalytic deaminase domains. The level of AI editing is significantly positively correlated with the infection rate and complications in MPP patients, indicating that RNA editing is of great significance in the treatment of mycoplasma pneumoniae infection.

[0004] The CCRL2 (CC motif chemokine receptor like 2) gene encodes a chemokine receptor-like protein, predicted to be a seven-transmembrane protein most closely associated with CCR1. Chemokines and their receptors mediate signal transduction, crucial for the recruitment of effector immune cells to sites of inflammation. This gene is highly expressed in primary neutrophils and primary monocytes, and further upregulated during neutrophil activation and monocyte differentiation into macrophages. The function of this gene is unknown. It is located within the chemokine receptor gene cluster, but the alterations of CCRL2 RNA editing in MPP and its clinical significance are currently unclear.

[0005] CCNI (cyclin I) The protein encoded by this gene belongs to the highly conserved cyclin family that members are characterized by their periodicity in protein abundance that is highly conserved among species. Cyclins act as regulators of CDK kinases. Different cyclins exhibit distinct expression and degradation patterns that contribute to the timing of each mitotic event. The transcript of this gene is expressed throughout the cell cycle progression with the highest similarity to cyclin G. But there is no clear report on the changes of CCNI gene and its RNA editing in MPP and its clinical significance.

[0006] CTSS (cathepsin S) The preproprotein encoded by this gene is a member of the peptidase Cl family, a lysosomal cysteine protease involved in the degradation of antigenic proteins to peptides and presentation on MHC class II molecules. The mature protein cleaves invariant chains of MHC class II molecules in the lumen of endosomes, allowing the formation of antigenic MHC class II complexes and proper display of extracellular antigenic peptides by MHC class II. The mature protein also functions as an elastase in a wide pH range. When secreted from cells, this protein can remodel components of the extracellular matrix, such as elastin, collagen, and fibronectin. The gene is associated with the pathology of many inflammatory and autoimmune diseases and plays an important role in some lung diseases due to its elastase activity. Alternative splicing variants of this gene have been found to encode different isoforms. But there is no clear report on the changes of CTSS RNA editing in MPP and its clinical significance.

[0007] IRAK3 (interleukin 1 receptor associated kinase 3) The gene encodes a member of the interleukin-1 receptor associated kinase protein family. The family members are important components of the Toll / IL-R immune signal transduction pathway. The protein is mainly expressed in monocytes and macrophages and acts as a negative regulator of toll-like receptor signaling. Mutations in this gene are associated with susceptibility to asthma. But there is no clear report on the changes of IRAK3 RNA editing in MPP and its clinical significance.

[0008] LYN (LYN Proto-Oncogene, Src Family Tyrosine Kinase) The gene encodes a tyrosine-protein kinase that may be involved in the regulation of mast cell degranulation and erythroid differentiation. Diseases associated with LYN include autoinflammatory diseases, systemic diseases, congenital vasculitis, and keratodermias, autosomal dominant. Related pathways include ADORA2B-mediated production of anti-inflammatory cytokines and signaling of erythropoietin. But there is no clear report on the changes of LYN RNA editing in MPP and its clinical significance. SUMMARY

[0009] The inventors found in the research that there are significant differences in RNA editing of 6 editing sites of 5 different genes in the tumor mucosa and adjacent normal mucosa of MPP patients, and the 5 different genes are related to the occurrence and development of mycoplasma pneumoniae pneumonia infection.

[0010] In the present application, we found that there are significant differences in the editing levels of 5 different genes: CCRL2:chr3:46408483, CCNI:chr4:77058527, CTSS:chr1:150732881 / 150732032, IRAK3:chr12:66194142, and LYN:chr8:56010885 in the tumor mucosa and adjacent normal mucosa of MPP patients, and the 5 different genes are related to the occurrence and development of mycoplasma pneumoniae pneumonia, and the combination of the above 6 different RNA editing sites has a high accuracy for diagnosing the occurrence of MPP.

[0011] The present application provides a molecular marker for the severity and complications of mycoplasma pneumoniae pneumonia infection based on gene expression and RNA editing level, which takes human reference genome hg38 as a reference, and the molecular marker comprises the following RNA editing sites: CCRL2:chr3:46408483, CCNI:chr4:77058527, CTSS:chr1:150732881 / 150732032, IRAK3:chr12:66194142, and LYN:chr8:56010885.

[0012] In an embodiment of the present application, the RNA editing site: CCRL2:chr3:46408483 is located at position 46408483 on chromosome 3 with NCBI number NC_000003.12;

[0013] The RNA editing site: CCNI:chr4:77058527 is located at position 77058527 on chromosome 4 with NCBI number NC_000004.12;

[0014] The RNA editing site: CTSS:chr1:150732881 is located at position 150732881 on chromosome 1 with NCBI number NC_000001.11;

[0015] The RNA editing site: CTSS:chr1:150732032 is located at position 150732032 on chromosome 1 with NCBI number NC_000001.11;

[0016] The RNA editing site IRAK3:chr12:66194142 is at position 66194142 on chromosome 12 with NCBI number NC_000012.12;

[0017] The RNA editing site LYN:chr8:56010885 is at position 56010885 on chromosome 8 with NCBI number NC_000008.11.

[0018] The application also provides application of the molecular marker for severity and complication of Mycoplasma pneumoniae pneumonia infection in preparation of a product for detecting severity and complication of Mycoplasma pneumoniae pneumonia infection.

[0019] In one embodiment of the application, the product includes but is not limited to a biochip for predicting severity and complication of Mycoplasma pneumoniae pneumonia infection, and a kit for detecting severity and complication of Mycoplasma pneumoniae pneumonia infection.

[0020] The application also provides a biochip for predicting risk level of a colorectal cancer patient, which comprises a solid phase carrier and oligonucleotide probes orderly fixed on the solid phase carrier, and the oligonucleotide probes specifically correspond to the molecular marker for severity and complication of Mycoplasma pneumoniae pneumonia infection.

[0021] The application also provides a device for detecting or diagnosing severity and complication of Mycoplasma pneumoniae pneumonia infection, wherein the device comprises one or more devices for specifically detecting editing levels of the RNA editing sites CCRL2:chr3:46408483, CCNI:chr4:77058527, CTSS:chr1:150732881 / 150732032, IRAK3:chr12:66194142 and LYN:chr8:56010885.

[0022] The application also provides a kit, which contains the molecular marker for severity and complication of Mycoplasma pneumoniae pneumonia infection, or contains the device, or contains the biochip.

[0023] In one embodiment of the application, the detection method used in the diagnostic kit is transcriptome sequencing, and the inventors find that the combination of editing levels of the differential genes CCRL2 and CCNI, and the multiple differential RNA editing sites of the genes CTSS, IRAK3 and LYN in the transcriptome sequencing results has good sensitivity and specificity for diagnosing severity and complication of Mycoplasma pneumoniae pneumonia infection, and has a very high accuracy for diagnosing severity and complication of Mycoplasma pneumoniae pneumonia infection.

[0024] In an embodiment of the present application, in order to verify the diagnostic effectiveness of the present application, the inventors verified by the following method:

[0025] (1) Collecting alveolar lavage fluid samples from the severe side and the control side of patients infected with mycoplasma pneumoniae pneumonia and clinical data.

[0026] (2) Verification of RNA editing differences in alveolar lavage fluid of patients infected with mycoplasma pneumoniae pneumonia: Through data analysis after transcriptome sequencing, obtain tissue RNA editing data, and verify the differences of RNA editing in the acute side and the control side of MPP.

[0027] In an embodiment of the present application, the inventors collected standard tissue samples according to standard operating procedures (SOP), systematically collected complete clinical data, etc., and verified using high-throughput sequencing methods. Specifically, the experimental methods of the study mainly include the following parts:

[0028] 1. Selection of research samples:

[0029] (1) MPP cases diagnosed by pathology;

[0030] (2) Collection of MPP alveolar lavage fluid samples from patients;

[0031] (3) Collection of samples from the severe side and the control side.

[0032] 2. Database data analysis

[0033] (1) MPP transcriptome sequencing data;

[0034] (2) RNA editing analysis;

[0035] (3) Screening of differentially expressed genes and differentially edited RNA sites in MPP alveolar lavage fluid samples from the severe side and the control side;

[0036] 3. Transcriptome sequencing

[0037] (1) RNA from MPP alveolar lavage fluid samples from the severe side and the control side;

[0038] (2) RNA editing analysis;

[0039] (3) Comparison of RNA editing levels in CCRL2, CCNI, ACSL1, CTSS, and IRAK3 genes in MPP alveolar lavage fluid samples from the severe side and the control side.

[0040] In an embodiment of the present application, by performing logistics regression analysis on the level differences of the 6 RNA editing sites in the severe side and the control side, with and without complications, the formula is obtained:

[0041] (1) Severe side and control side diagnosis:

[0042] n = -1.477 + 0.056x1 + 0.0575x2;

[0043] Cut-off value: P1 = 0.788, greater than the value is diagnosed as severe;

[0044] x1-x2 are the RNA editing levels of CCRL2:chr3:46408483 and CCNI:chr4:77058527, respectively.

[0045] (2) Complications and no complications diagnosis:

[0046] n = -4.316 + 1.530x1 - 0.174x2 + 0.725x3 - 0.199x4;

[0047] Cut-off value: P1 = 0.973, greater than the value is diagnosed as having complications;

[0048] x1-x4 are the RNA editing levels of CTSS:chr1:150732881 / 150732032, IRAK3:chr12:66194142, LYN:chr8:56010885, respectively.

[0049] The application also provides a use of an MPP molecular marker in the preparation of a product for predicting Mycoplasma pneumoniae pneumonia and guiding drug selection or therapy selection, wherein the molecular marker comprises the following A>I RNA editing sites: CCRL2:chr3:46408483, CCNI:chr4:77058527, CTSS:chr1:150732881 / 150732032, IRAK3:chr12:66194142, and LYN:chr8:56010885, based on the human reference genome hg38.

[0050] The application also provides a use of a reagent for detecting the presence or level of a molecular marker for predicting the severity of Mycoplasma pneumoniae pneumonia and complications in the preparation of a product for predicting the severity of Mycoplasma pneumoniae pneumonia and complications and guiding drug selection or therapy selection, wherein the molecular marker comprises the following A>I RNA editing sites: CCRL2:chr3:46408483, CCNI:chr4:77058527, CTSS:chr1:150732881 / 150732032, IRAK3:chr12:66194142, and LYN:chr8:56010885, based on the human reference genome hg38.

[0051] The application also provides a reagent or kit for detecting MPP, wherein the reagent or kit contains a reagent capable of detecting the expression amount of a molecular marker, and the molecular marker comprises the following A>I RNA editing sites: CCRL2:chr3:46408483, CCNI:chr4:77058527, CTSS:chr1:150732881 / 150732032, IRAK3:chr12:66194142, and LYN:chr8:56010885.

[0052] The application provides application of a reagent for detecting RNA editing level of a molecular marker of severity and complication of Mycoplasma pneumoniae pneumonia infection in preparation of a product for diagnosing, predicting, evaluating and / or identifying severity and complication of Mycoplasma pneumoniae pneumonia infection and guiding drug selection or therapy selection, wherein the molecular marker comprises the following adenosine- inosine (A-I) RNA editing sites: CCRL2:chr3:46408483, CCNI:chr4:77058527, CTSS:chr1:150732881 / 150732032, IRAK3:chr12:66194142, and LYN:chr8:56010885; and the product diagnoses severity and complication of Mycoplasma pneumoniae pneumonia infection by detecting the RNA editing level of the molecular marker of severity and complication of Mycoplasma pneumoniae pneumonia infection.

[0053] In an embodiment of the application, the application further comprises evaluating or diagnosing the sample by using the following formula:

[0054] n=-1.477+0.056x1+0.0575x2;

[0055] Cut-off value: P1=0.788, greater than which is diagnosed as severe;

[0056] x1-x2 are respectively RNA editing levels of CCRL2:chr3:46408483 and CCNI:chr4:77058527;

[0057] n=-4.316+1.530x1-0.174x2+0.725x3-0.199x4;

[0058] Cut-off value: P1=0.973, greater than which is diagnosed as having complication;

[0059] x1-x4 are RNA editing levels of CTSS:chr1:150732881 / 150732032, IRAK3:chr12:66194142, LYN:chr8:56010885, respectively.

[0060] In an embodiment of the present application, the product comprises a kit for detecting severity and complications of Mycoplasma pneumoniae pneumonia infection, a device for diagnosing severity and complications of Mycoplasma pneumoniae pneumonia infection and / or predicting severity and complications of Mycoplasma pneumoniae pneumonia infection, a biochip.

[0061] In an embodiment of the present application, the biochip comprises a solid support and oligonucleotide probes immobilized in order on the solid support, wherein the oligonucleotide probes specifically correspond to molecular markers of severity and complications of Mycoplasma pneumoniae pneumonia infection.

[0062] In an embodiment of the present application, the device comprises one or more devices for specifically detecting RNA editing levels of the RNA editing sites: CCRL2:chr3:46408483, CCNI:chr4:77058527, CTSS:chr1:150732881 / 150732032, IRAK3:chr12:66194142, LYN:chr8:56010885.

[0063] In an embodiment of the present application, the kit comprises reagents for detecting expression levels of the molecular markers of severity and complications of Mycoplasma pneumoniae pneumonia infection.

[0064] The present application also provides use of the molecular markers of severity and complications of Mycoplasma pneumoniae pneumonia infection based on RNA editing levels in the preparation of a product for diagnosing severity and complications of Mycoplasma pneumoniae pneumonia infection and / or predicting, evaluating or identifying severity and complications of Mycoplasma pneumoniae pneumonia infection, wherein the molecular markers are based on human reference genome hg38, and the molecular markers comprise the following adenosine-inosine (A-I) RNA editing sites: CCRL2:chr3:46408483, CCNI:chr4:77058527, CTSS:chr1:150732881 / 150732032, IRAK3:chr12:66194142, LYN:chr8:56010885; and the product diagnoses severity and complications of Mycoplasma pneumoniae pneumonia infection by detecting RNA editing levels of the molecular markers of severity and complications of Mycoplasma pneumoniae pneumonia infection.

[0065] In an embodiment of the present application, the use further comprises evaluating or diagnosing the sample using the following formula:

[0066] n = -1.477 + 0.056x1 + 0.0575x2;

[0067] Cut-off value: P1 = 0.788, greater than which is diagnosed as severe;

[0068] x1-x2 are the RNA editing levels of CCRL2:chr3:46408483 and CCNI:chr4:77058527, respectively;

[0069] n = -4.316 + 1.530x1 - 0.174x2 + 0.725x3 - 0.199x4;

[0070] Cut-off value: P1 = 0.973, greater than which is diagnosed as having complications;

[0071] x1-x4 are the RNA editing levels of CTSS:chr1:150732881 / 150732032, IRAK3:chr12:66194142, LYN:chr8:56010885, respectively.

[0072] In an embodiment of the present application, the product comprises a kit and a biochip for detecting the severity and complications of Mycoplasma pneumoniae pneumonia infection, a device for diagnosing the severity and complications of Mycoplasma pneumoniae pneumonia infection and / or predicting the severity and complications of Mycoplasma pneumoniae pneumonia infection;

[0073] In an embodiment of the present application, the biochip comprises a solid support and oligonucleotide probes orderly fixed on the solid support, wherein the oligonucleotide probes specifically correspond to the molecular markers of the severity and complications of Mycoplasma pneumoniae pneumonia infection.

[0074] In an embodiment of the present application, the device comprises one or more devices for specifically detecting the RNA editing levels of the RNA editing sites: CCRL2:chr3:46408483, CCNI:chr4:77058527, CTSS:chr1:150732881 / 150732032, IRAK3:chr12:66194142, LYN:chr8:56010885.

[0075] In an embodiment of the present application, the kit comprises reagents for detecting the expression levels of the molecular markers of the severity and complications of Mycoplasma pneumoniae pneumonia infection.

[0076] Advantages

[0077] (1) The application judges the severity of MPP and the occurrence of complications by combining the editing level differences of 6 RNA editing sites, with a true rate of more than 80%, and this effect is verified in clinical samples;

[0078] (2) The classifier generated by combining the editing level differences of 6 RNA editing sites has a better effect on judging the occurrence of mycoplasma pneumoniae pneumonia than the classifier generated by combining the editing level differences of the 6 sites alone;

[0079] (3) The method is simpler in operation and lower in detection cost, and the results are not easily affected by the control sample, and the results are more stable. BRIEF DESCRIPTION OF DRAWINGS

[0080] Figure 1 : 2 RNA editing sites have significant differences (p<0.05) between the severe side and the control side of mycoplasma pneumoniae pneumonia infection in patients.

[0081] Figure 2 : 4 RNA editing sites have significant differences (p<0.05) between individuals with complications and individuals without complications in the severe side of mycoplasma pneumoniae pneumonia infection in patients.

[0082] Figure 3 : ROC curve analysis shows the performance of the combination of 2 RNA editing sites in diagnosing the severe side tissue and the control side tissue in the severe side of mycoplasma pneumoniae pneumonia infection.

[0083] Figure 4 : ROC curve analysis shows the performance of the combination of 4 RNA editing sites in diagnosing the patients with complications and the patients without complications in the severe side of mycoplasma pneumoniae pneumonia infection. DETAILED DESCRIPTION

[0084] The instruments used in the application are shown in Table 1 below:

[0085] Table 1: Information of instruments used

[0086] Instrument name Manufacturer Vertical automatic pressure steam sterilizer (G54DWS) Zhiwei (Xiamen) Instrument Co., Ltd. Freeze high-speed centrifuge (Legend Micro 21R) USA, Thermo Fisher Ice maker (IMS-20) Changshu Xueke Electrical Appliance Co., Ltd. Medical refrigeration and freezing refrigerator (HYCD-205) Qingdao Haier Special Electrical Appliance Co., Ltd. Pipette (1000 / 200 / 100 / 10 / 2.5 μL) Germany, Eppendorf -80 ℃ refrigerator (902) USA, Thermo Fisher -20 ℃ refrigerator (DW-25L262) USA, Thermo Fisher NANODROP ONE USA, Thermo Fisher

[0087] The experimental reagents and consumables used in the application are shown in Table 2 below:

[0088] Table 2: Information of reagents used

[0089]

[0090]

[0091] Example 1: Sample collection and processing

[0092] Clinical collection of mycoplasma pneumoniae pneumonia infection patients, respectively, take mycoplasma pneumoniae pneumonia infection patients severe side alveolar lavage fluid and control side alveolar lavage fluid (mild side), after obtaining the alveolar lavage fluid, a part of the sample is immediately extracted for RNA, another part of the sample is loaded into the frozen tube and placed in liquid nitrogen for freezing, the tissue stored in liquid nitrogen is transferred to-80℃ refrigerator for long-term storage, the clinical data of patients are collected, and the statistical conditions of MPP patients are shown in Table 3.

[0093] Table 3: Statistical table of clinical MPP patient conditions

[0094]

[0095] Example 2: RNA extraction and transcriptome sequencing

[0096] 1. Take the alveolar lavage fluid of mycoplasma pneumoniae pneumonia patients on the severe side and the control side, obtain the transcriptome sample to be sequenced by RNA extraction, and obtain the RNA editing level of each sample by transcriptome sequencing analysis. The specific steps are as follows:

[0097] Alveolar lavage fluid RNA extraction:

[0098] The alveolar lavage fluid of mycoplasma pneumoniae pneumonia patients on the severe side and the control side frozen in-80℃ refrigerator is taken out and placed in a biological safety cabinet and thawed on ice. After the sample is dissolved, 250uL of lavage fluid is taken with a DEPC water soaked high pressure sterilized gun head and placed in an RNase free EP tube. 750uL of Trizol is added and stored on ice.

[0099] (1) Invert the sample tube up and down, mix the sample and Trizol thoroughly, lyse for 5min on ice, and then extract RNA;

[0100] (2) Centrifuge at 12000g, 4℃ for 15min, and take out the EP tube from the centrifuge;

[0101] (3) Then aspirate the supernatant and transfer it to a new EP tube (do not aspirate the white middle layer), add isopropanol with half the volume of Trizol to the aspirated supernatant, mix the EP tube thoroughly up and down, and then stand at room temperature for 10min, centrifuge at 12000g, 4℃ for 10min;

[0102] (4) After discarding the supernatant, add 75% ethanol with the same volume as Trizol, invert the centrifuge tube wall, centrifuge at 7500g, 4℃ for 5min, and carefully discard the supernatant;

[0103] (5) In the super-clean workbench, open the EP tube cover, dry at room temperature, and after the alcohol is completely volatilized, add an appropriate amount of DEPC water to dissolve the RNA precipitate, and obtain the RNA of the severe side of the alveolar lavage fluid and the control side of the alveolar lavage fluid of Mycoplasma pneumoniae pneumonia.

[0104] 2. Transcriptome sequencing

[0105] (1) Acquisition of RNA sequencing data:

[0106] A total of 21 pairs of MPP patients (Table 3) were collected in the severe side of the alveolar lavage fluid and the control side of the alveolar lavage fluid. Among them, 10 patients in the severe side had complications, and 11 had no complications.

[0107] (2) Alignment of RNA sequencing data:

[0108] The transcriptome raw sequencing data obtained above was analyzed by quality control using FASTQC software. The qualified sequencing reads were aligned with the human hg38 genome sequence using RNA STAR (version 2.7.0e) software, the RNA splice junction was analyzed, and the reads were mapped to the genome to generate a BAM format alignment result file. The reads in the BAM file were deduplicated using samtools (version 1.9) software, only the uniquely mapped reads were retained, and GATK (version 4.1.3) software was used to recalibrate the base quality score of the BAM file;

[0109] (3) Identification and annotation of RNA editing sites:

[0110] The RNA single nucleotide variant (SNV) of the BAM file after calibration was identified using VarScan (version 2.4) software, with a base quality of ≥25, a sequencing depth of ≥10, a U base (converted to T base in RNA sequencing) depth of ≥2 and an editing frequency of ≥1%, and the false positive variants were filtered using the fpfilter command of VarScan and the default parameters. The annotation of SNV was performed using Ensembl Variant Effect Predictor (VEP, https: / / www.ensembl.org / vep);

[0111] (4) Statistical analysis:

[0112] The comparison of RNA editing levels or gene expression levels between groups was performed using paired or paired Kruskal-Wallis (KW) non-parametric test. P<0.05 was used as the significant difference screening standard.

[0113] The results show that the RNA editing levels of CCRL2:chr3:46408483 and CCNI:chr4:77058527 sites have significant differences between the severe side and the control side group (mild side) Figure 1 ), wherein the RNA editing levels of CCRL2:chr3:46408483 and CCNI:chr4:77058527 sites are significantly increased in the severe side.

[0114] The RNA editing levels of CTSS:chr1:150732881 / 150732032, IRAK3:chr12:66194142, LYN:chr8:56010885 sites have significant differences between the patients with complications and the patients without complications Figure 2 ), wherein the RNA editing levels of CTSS:chr1:150732881 and IRAK3:chr12:66194142 sites are significantly increased in the complication group, and the RNA editing levels of CTSS:chr1:150732032 and LYN:chr8:56010885 sites are significantly decreased in the complication group.

[0115] Example 3: Generating a classifier for the diagnosis of the severity of MPP and its complications based on the combination of RNA differential editing sites

[0116] Take the 21 pairs of alveolar lavage fluid from the severe side and the control side of the patients with mycoplasma pneumonia collected in Example 1, immediately put a part into a frozen tube and store in liquid nitrogen for freezing, transfer the tissue stored in liquid nitrogen to a-80℃ refrigerator for long-term storage until it is taken out, obtain the transcriptome sample to be sequenced by RNA extraction, and use it for transcriptome sequencing.

[0117] The RNA editing level in the sequencing result is used for the establishment of the ROC curve. In the transcriptome sequencing data, we first use the SPSS software to perform logistics regression on the editing level of 6 RNA editing sites (CCRL2:chr3:46408483, CCNI:chr4:77058527, CTSS:chr1:150732881 / 150732032, IRAK3:chr12:66194142, LYN:chr8:56010885) to obtain the predicted value. Set the predicted value as the test variable, and the state variable as 0 and 1. In the analysis of severe and non-severe tissues, set the severe group as 1. Use the SPSS software to make the ROC curve, and obtain Figure 3 As shown in the figure, the area AUC under the ROC curve is 0.788.

[0118] In the complication and non-complication tissue analysis, the complication group was set to 1, and the ROC curve was made using SPSS software to obtain Figure 4 As shown in the figure, the area under the ROC curve AUC is 0.973. The point closest to the upper left corner, i.e., the maximum Youden index, is selected as the cutoff value. The cutoff value, i.e., the judgment standard, is the boundary value for determining the positivity and negativity of the test. In the complication group, the sensitivity at the cutoff value is 76.2%, and the specificity is 72.2%; in the non-complication group, the sensitivity at the cutoff value is 100%, and the specificity is 81.8%. Figure 3 Figure 4

[0119] In addition, as shown in Tables 4 and 5, we generated classifiers using the RNA editing levels of the 2 sites and 4 sites, respectively.

[0120] Table 4: Performance of 2 RNA editing sites as independent indicators and their combinations in the diagnosis of Mycoplasma pneumoniae pneumonia in the severe side and control side of the bronchoalveolar lavage fluid of Mycoplasma pneumoniae pneumonia (AUC: area under the ROC curve)

[0121]

[0122]

[0123] Table 5: Performance of 4 RNA editing sites as independent indicators and their combinations in the diagnosis of Mycoplasma pneumoniae pneumonia in the severe side of the bronchoalveolar lavage fluid of Mycoplasma pneumoniae pneumonia with complications and without complications (AUC: area under the ROC curve)

[0124]

[0125] The results show that in Tables 4 and 5, the classifiers generated by the differences in RNA editing levels of 2 sites and 4 sites combinations, respectively, have a true positive rate of 78.8% and 97.3% for Mycoplasma pneumoniae pneumonia.

[0126] Example 4: Diagnosis of the severity and complications of MPP infection by RNA differential editing site composition

[0127] 1. By performing logistic regression analysis on the levels of 6 RNA editing sites in the severe side and control side and in the complication and non-complication, the formula is obtained:

[0128] (1) Diagnosis of the severe side and control side (mild side):

[0129]

[0130] n = -1.477 + 0.056x1 + 0.0575x2​​

[0131] Cut-off value: P1=0.788, greater than which is diagnosed as severe;

[0132] x1-x2 are the RNA editing levels of CCRL2:chr3:46408483 and CCNI:chr4:77058527, respectively.

[0133] (2) Complication and non-complication diagnosis:

[0134]

[0135] n=-4.316+1.530x1-0.174x2+0.725x3-0.199x4

[0136] Cut-off value: P1=0.973, greater than which is diagnosed as having complications;

[0137] x1-x4 are the RNA editing levels of CTSS:chr1:150732881 / 150732032, IRAK3:chr12:66194142, and LYN:chr8:56010885, respectively.

[0138] 2. Using the above obtained formula, it is determined whether the tissue contains a severe factor of mycoplasma pneumoniae pneumonia or whether the individual is a complication individual.

[0139] According to the above method, a plurality of subjects are detected, and each RNA editing level is substituted into the above formula to diagnose the severity of MPP and complications. The results show that the results obtained by the method of the present application are consistent with the pathological diagnosis results.

[0140] Example 1: Selecting the tissue sample of patient 1, extracting the RNA of the mycoplasma pneumoniae pneumonia MPP severe side alveolar lavage fluid, detecting the RNA editing level, and substituting it into the formula to obtain P1=0.963, which is greater than the cut-off value 0.788, indicating that it is a severe side, which is consistent with the actual situation.

[0141] Example 2: Selecting the tissue sample of patient 2, extracting the RNA of the mycoplasma pneumoniae pneumonia MPP control side alveolar lavage fluid, detecting the RNA editing level, and substituting it into the formula to obtain P1=0.426, which is less than the cut-off value 0.788, indicating that it is a mild side, which is consistent with the actual situation.

[0142] Example 3: Selecting the tissue sample of patient 3, extracting the RNA of the mycoplasma pneumoniae pneumonia MPP severe side alveolar lavage fluid, detecting the RNA editing level, and substituting it into the formula to obtain P1=0.992, which is greater than the cut-off value 0.973, indicating that it is a patient with complications, which is consistent with the actual situation.

[0143] Example 4: The tissue sample of patient 4 is selected, the RNA of mycoplasma pneumoniae pneumonia MPP severe side alveolar lavage fluid is extracted, the RNA editing level is detected, and the formula is brought into, P1=0.273 is obtained, which is less than the cutoff value 0.973, and it is judged that there is no complication patient, which is consistent with the actual situation.

[0144] Although the present application has been disclosed in the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, and therefore the protection scope of the present application should be defined by the claims.

Claims

1. The application of a reagent for detecting the RNA editing level, a molecular marker for the severity and complications of Mycoplasma pneumoniae pneumonia infection, in the preparation of products for diagnosing severe Mycoplasma pneumoniae pneumonia infection and its complications, characterized in that... The molecular markers, based on the human reference genome hg38, are the following adenosine-inosine (AI) RNA editing sites: CCRL2:chr3:46408483, CCNI:chr4:77058527, CTSS:chr1:150732881, CTSS:chr1:150732032, IRAK3:chr12:66194142, and LYN:chr8:56010885. The product diagnoses the severity and complications of Mycoplasma pneumoniae pneumonia infection by detecting the RNA editing levels of these molecular markers.

2. The application according to claim 1, characterized in that, The products include a kit for detecting the severity and complications of Mycoplasma pneumoniae pneumonia infection, a biochip, and a device for diagnosing the severity and complications of Mycoplasma pneumoniae pneumonia infection.

3. The application according to claim 2, characterized in that, The biochip includes a solid support and oligonucleotide probes ordered immobilized on the solid support. The oligonucleotide probes specifically correspond to molecular markers of the severity and complications of Mycoplasma pneumoniae pneumonia infection.

4. The application according to claim 2, characterized in that, The device includes one or more means for specifically detecting the RNA editing level of the RNA editing sites: CCRL2:chr3:46408483, CCNI:chr4:77058527, CTSS:chr1:150732881, CTSS:chr1:150732032, IRAK3:chr12:66194142, and LYN:chr8:56010885.

5. The application according to claim 2, characterized in that, The kit contains reagents for detecting the expression levels of molecular markers for the severity and complications of Mycoplasma pneumoniae pneumonia infection.

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