Mycoplasma pneumoniae infection severity and complication marker and application thereof in preparation of mycoplasma pneumoniae infection diagnostic kit

Abstract

The invention discloses a mycoplasma pneumonia infection severity and complication marker and application thereof in preparation of a mycoplasma pneumonia infection diagnostic kit, and belongs to the field of epigenetics and microbiology. In the invention, it is studied that the editing levels of six RNA editing sites of five genes in the severe side and the control side of an MPP patient are obviously different, namely CCRL2: chr3: 46408483, CCNI: chr4: 77058527, CTSS: chr1: 150732881 / 150732032, IRAK3: chr12: 66194142 and LYN: chr8: 56010885, the five different genes are associated with the severe degree and complications of MPP, and the RNA editing combination has very high accuracy in diagnosing the severity and complications of MPP.

Description

Technical Field

[0001] The invention relates to Mycoplasma pneumoniae pneumonia infection severity and complication markers and application thereof in preparing a Mycoplasma pneumoniae infection diagnosis kit, and belongs to the field of epigenetics and microbiology. Background Art

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

[0003] RNA editing is a type of epigenetics. Epigenetics is a branch of genetics that studies the changes in gene expression or cell phenotype that can be inherited without changing the nucleotide sequence. 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 changes RNA sequences. The most common type of RNA editing is adenosine-inosine (AI) conversion mediated by the adenosine deaminase ADAR family. RNA editing can increase the diversity of the transcriptome or proteome and may change the stability and transport of the encoded protein sequence or RNA. Studies have shown that RNA editing plays an important regulatory role in processes such as microbial infection or immunity. In MPP, ADAR1 and ADAR2, as the main members of the ADAR family, play an infection and anti-infection role through their catalytic deaminase domains, respectively. The level of AI editing is significantly positively correlated with the infection and complications of MPP patients, indicating that RNA editing is of great significance for the treatment of mycoplasma pneumonia infection.

[0004] The CCRL2 (CC motif chemokine receptor like 2) gene encodes a chemokine receptor-like protein that is predicted to be a seven-transmembrane protein that is most closely related to CCR1. Signal transduction mediated by chemokines and their receptors is essential for the recruitment of effector immune cells to sites of inflammation. This gene is highly expressed in primary neutrophils and primary monocytes and is further upregulated during neutrophil activation and monocyte differentiation into macrophages. The function of this gene is unknown. The gene is mapped to the region where the chemokine receptor gene cluster is located, but there is currently no clear report on the changes in CCRL2 RNA editing in MPP and its clinical significance.

[0005] The protein encoded by CCNI (cyclin I) belongs to the highly conserved cyclin family, whose members are characterized by a significant periodicity in protein abundance during the cell cycle. Cyclins are regulators of CDK kinases. Different cyclins show different expression and degradation patterns, which contribute to the temporal coordination of each mitotic event. The transcript of this gene is continuously expressed during the cell cycle progression and has the highest similarity to cyclin G. However, there is no clear report on the changes in the CCNI gene and its RNA editing in MPP and its clinical significance.

[0006] CTSS (cathepsin S) is a member of the peptidase C1 family and a lysosomal cysteine ​​protease that is involved in the degradation of antigenic proteins into peptides and presents them on MHC-II molecules. The mature protein cleaves the invariant chain of MHC-II molecules in the endolysosomal compartment, allowing the formation of antigen-MHC-II complexes and the proper display of extracellular antigenic peptides by MHC-II. The mature protein also acts as an elastase over a wide pH range. When secreted from cells, this protein can remodel components of the extracellular matrix, such as elastin, collagen, and fibronectin. This 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 transcript variants of this gene have been found to encode different isoforms. However, there is no clear report on the changes in CTSS RNA editing in MPP and its clinical significance.

[0007] The IRAK3 (interleukin 1 receptor associated kinase 3) gene encodes a member of the interleukin-1 receptor-associated kinase protein family. Members of this family are important components of the Toll / IL-R immune signaling pathway. This 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 asthma susceptibility. However, there are currently no clear reports on the changes in IRAK3 RNA editing in MPP and their clinical significance.

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

[0009] In the study, the inventors found that there were significant differences in RNA editing at six editing sites of five different genes in the tumor mucosa and adjacent normal mucosa of MPP patients, and these five different genes were associated with the occurrence and development of Mycoplasma pneumonia infection.

[0010] In the present invention, we studied the editing levels of 5 different genes in the tumor mucosa and adjacent normal mucosa of MPP patients: CCRL2:chr3:46408483, CCNI:chr4:77058527, CTSS:chr1:150732881 / 150732032, IRAK3:chr12:66194142, LYN:chr8:56010885. There were significant differences. These 5 different genes were associated with the occurrence and development of Mycoplasma pneumonia, and the combination of the above 6 differential RNA editing sites was used to diagnose the occurrence of MPP with a high accuracy.

[0011] The present invention provides a molecular marker for severity and complications of Mycoplasma pneumonia infection based on gene expression and RNA editing levels. Taking the human reference genome hg38 as a benchmark, the molecular marker includes the following RNA editing sites: CCRL2: chr3:46408483, CCNI: chr4:77058527, CTSS: chr1:150732881 / 150732032, IRAK3: chr12:66194142, LYN: chr8:56010885.

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

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

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

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

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

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

[0018] The present invention also provides the use of the above-mentioned Mycoplasma pneumoniae pneumonia infection severity and complication molecular markers in the preparation of products for detecting the severity and complications of Mycoplasma pneumoniae pneumonia infection.

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

[0020] The present invention also provides a biochip for predicting the risk level of colorectal cancer patients, wherein the biochip comprises a solid phase carrier and oligonucleotide probes fixed in order on the solid phase carrier, wherein the oligonucleotide probes specifically correspond to the above-mentioned Mycoplasma pneumonia infection severity and complication molecular markers.

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

[0022] The present invention also provides a kit, which contains the above-mentioned Mycoplasma pneumonia infection severity and complication molecular markers or the above-mentioned device, or the above-mentioned biochip.

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

[0024] In one embodiment of the present invention, in order to verify the diagnostic effectiveness of the present invention, the inventors conducted verification by the following method:

[0025] (1) Collect bronchoalveolar lavage fluid and tissue samples from the critically ill and contralateral sides of patients with Mycoplasma pneumoniae pneumonia and clinical data.

[0026] (2) Verification of RNA editing differences in bronchoalveolar lavage fluid of patients with Mycoplasma pneumoniae pneumonia: Through transcriptome sequencing and post-sequencing data analysis, we obtained data on tissue RNA editing and verified the differences in RNA editing between the acute side and the control side of MPP.

[0027] In one embodiment of the present invention, the inventors collected tissue samples that met the standards according to the standard operating procedure (SOP), systematically collected complete clinical data, etc., and used high-throughput sequencing methods for verification. 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 bronchoalveolar lavage fluid samples from patients with MPP;

[0031] (3) Sample collection from the critically ill side and the control side.

[0032] 2. Database data analysis

[0033] (1) MPP transcriptome sequencing data;

[0034] (2) RNA editing analysis;

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

[0036] 3. Transcriptome sequencing

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

[0038] (2) RNA editing analysis;

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

[0040] In one embodiment of the present invention, by performing a logistic regression analysis on the level differences of the six RNA editing sites in the severe side and the control side, with complications and without complications, the formula is obtained:

[0041] (1) Diagnosis of the critically ill side and the control side:

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

[0043] Cutoff value: P1 = 0.788, a value greater than this value is diagnosed as severe;

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

[0045] (2) Diagnosis with and without complications:

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

[0047] Cutoff value: P1 = 0.973, a value greater than this value is diagnosed as having complications;

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

[0049] The present invention also provides a use of an MPP molecular marker in preparing a product for predicting Mycoplasma pneumonia and guiding medication selection or therapy selection. Taking the human reference genome hg38 as a benchmark, the molecular marker includes the following A>IRNA editing sites: CCRL2: chr3: 46408483, CCNI: chr4: 77058527, CTSS: chr1: 150732881 / 150732032, IRAK3: chr12: 66194142, LYN: chr8: 56010885.

[0050] The present invention also provides a reagent for detecting the presence or level of molecular markers of the severity and complications of Mycoplasma pneumonia in the preparation of a product for predicting the severity and complications of Mycoplasma pneumonia and guiding medication selection or therapy selection. Taking the human reference genome hg38 as a benchmark, the molecular markers include the following A>I RNA editing sites: CCRL2: chr3: 46408483, CCNI: chr4: 77058527, CTSS: chr1: 150732881 / 150732032, IRAK3: chr12: 66194142, LYN: chr8: 56010885.

[0051] The present invention 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 based on the human reference genome hg38, the molecular marker includes the following A>IRNA editing sites: CCRL2: chr3:46408483, CCNI: chr4:77058527, CTSS: chr1:150732881 / 150732032, IRAK3: chr12:66194142, LYN: chr8:56010885.

[0052] The present invention provides a reagent for detecting RNA editing levels of molecular markers for the severity and complications of Mycoplasma pneumoniae pneumonia infection in the preparation of products for diagnosing, predicting, evaluating and / or identifying the severity and complications of Mycoplasma pneumoniae pneumonia infection, and guiding medication selection or therapy selection. The molecular markers are based on the human reference genome hg38, and the molecular markers include the following adenosine-inosine (AI) RNA editing sites: CCRL2: chr3: 46408483, CCNI: chr4: 77058527, CTSS: chr1: 150732881 / 150732032, IRAK3: chr12: 66194142, LYN: chr8: 56010885. The product diagnoses the severity and complications of Mycoplasma pneumoniae pneumonia infection by detecting RNA editing levels of molecular markers for the severity and complications of Mycoplasma pneumoniae pneumonia infection.

[0053] In one embodiment of the present invention, the use further comprises evaluating or diagnosing the sample using the following formula:

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

[0055] Cutoff value: P1 = 0.788, a value greater than this value is diagnosed as severe;

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

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

[0058] Cutoff value: P1 = 0.973, a value greater than this value is diagnosed as having complications;

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

[0060] In one embodiment of the present invention, the product includes a kit for detecting the severity and complications of Mycoplasma pneumonia infection, a device for diagnosing the severity and complications of Mycoplasma pneumonia infection and / or predicting the severity and complications of Mycoplasma pneumonia infection, and a biochip.

[0061] In one embodiment of the present invention, the biochip comprises a solid phase carrier and oligonucleotide probes fixed in order on the solid phase carrier, wherein the oligonucleotide probes specifically correspond to molecular markers of severity and complications of Mycoplasma pneumoniae pneumonia infection.

[0062] In one embodiment of the present invention, 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.

[0063] In one embodiment of the present invention, the kit contains reagents for detecting the severity of Mycoplasma pneumonia infection and the expression levels of molecular markers for complications.

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

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

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

[0067] Cutoff value: P1 = 0.788, a value greater than this value 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] Cutoff value: P1 = 0.973, a value greater than this value is diagnosed as having complications;

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

[0072] In one embodiment of the present invention, the product includes a kit and a biochip for detecting the severity and complications of Mycoplasma pneumoniae pneumonia infection, and 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 one embodiment of the present invention, the biochip comprises a solid phase carrier and oligonucleotide probes fixed in order on the solid phase carrier, wherein the oligonucleotide probes specifically correspond to molecular markers of severity and complications of Mycoplasma pneumoniae pneumonia infection;

[0074] In one embodiment of the present invention, 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 one embodiment of the present invention, the kit contains reagents for detecting the severity of Mycoplasma pneumonia infection and the expression levels of molecular markers for complications.

[0076] Beneficial Effects

[0077] (1) The present invention combines the differences in editing levels of six RNA editing sites to determine the severity of MPP and the occurrence of its complications with an accuracy of more than 80%, and this effect has been verified in clinical samples;

[0078] (2) The classifier generated by combining the differences in editing levels of the six RNA editing sites in the present invention is more effective in determining the occurrence of Mycoplasma pneumonia than the classifier generated by combining the differences in editing levels of the six sites alone;

[0079] (3) This method is simpler in terms of operation technology, has lower detection costs, and the results are less likely to be affected by control samples and are more stable. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1 : There were significant differences in the two RNA editing sites between the severe side and the control side of patients with Mycoplasma pneumoniae pneumonia infection (p<0.05).

[0081] Figure 2 : There were significant differences in the four RNA editing sites between patients with severe Mycoplasma pneumonia infection and those without complications (p<0.05).

[0082] Figure 3 : ROC curve analysis showed the diagnostic performance of the combination of two RNA editing sites in the severe side of Mycoplasma pneumoniae pneumonia infection in the severe side tissue and the control side tissue.

[0083] Figure 4 : ROC curve analysis showed the diagnostic performance of the combination of four RNA editing sites in patients with complications and those without complications in the severe side of Mycoplasma pneumoniae pneumonia infection. DETAILED DESCRIPTION

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

[0085] Table 1: Information of instruments used

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

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

[0088] Table 2: Information of reagents used

[0089]

[0090]

[0091] Example 1: Sample collection and processing

[0092] Patients with Mycoplasma pneumoniae pneumonia infection were collected clinically. Alveolar lavage fluid from the severe side and the control side (mild side) of the patients with Mycoplasma pneumoniae pneumonia infection were obtained respectively. After obtaining the alveolar lavage fluid, RNA was extracted from a part of the samples immediately, and the other part of the samples were placed in cryovials and frozen in liquid nitrogen. The tissues frozen in liquid nitrogen were transferred to a -80℃ refrigerator for long-term storage. The clinical data of the patients were systematically collected. The statistics of MPP patients are shown in Table 3.

[0093] Table 3: Clinical statistics of MPP patients

[0094]

[0095] Example 2: RNA extraction and transcriptome sequencing

[0096] 1. Collect bronchoalveolar lavage fluid from the critical side and the control side of patients with Mycoplasma pneumonia, extract RNA to obtain transcriptome samples to be sequenced, and obtain the RNA editing level of each sample by transcriptome sequencing analysis. The specific steps are as follows:

[0097] RNA extraction from bronchoalveolar lavage fluid:

[0098] The bronchoalveolar lavage fluid from the critical side and the control side of patients with Mycoplasma pneumonia frozen in a -80℃ refrigerator were taken out, placed in a biosafety cabinet, and thawed on ice. After the sample was dissolved, 250uL of lavage fluid was measured using a pipette tip that was soaked in DEPC water and sterilized by high pressure and placed in an RNase-free EP tube. 750uL Trizol was added and stored on ice.

[0099] (1) Invert the sample tube to mix the sample with Trizol thoroughly. After lysing on ice for 5 min, perform RNA extraction.

[0100] (2) Centrifuge at 12,000 g and 4°C for 15 min and remove 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 half the volume of Trizol to the aspirated supernatant. Invert the EP tube to mix thoroughly. Let stand at room temperature for 10 min. Centrifuge at 12,000 g, 4°C for 10 min.

[0102] (4) After discarding the supernatant, add the same volume of 75% ethanol as that of Trizol, wash the tube wall by inverting it, centrifuge at 7500g, 4°C for 5 min, and carefully discard the supernatant;

[0103] (5) Open the EP tube cap in a clean bench and dry it at room temperature. After the alcohol is completely evaporated, add an appropriate amount of DEPC water to dissolve the RNA precipitate to obtain RNA from the bronchoalveolar lavage fluid on the severe side of Mycoplasma pneumoniae pneumonia and the bronchoalveolar lavage fluid on the control side.

[0104] 2. Transcriptome sequencing

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

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

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

[0108] The original transcriptome sequencing data obtained above were quality controlled and analyzed using FASTQC software. The sequencing reads that passed the quality control were aligned with the human hg38 genome sequence using RNA STAR (version 2.7.0e) software, and their RNA splicing junctions were analyzed, mapped to the genome, and the alignment result file in BAM format was generated. The reads in the BAM file were deduplicated using samtools (version 1.9) software, and only reads with a single location were retained. The BAM file was recalibrated for base quality score using GATK (version 4.1.3) software;

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

[0110] VarScan (version 2.4) software was used to identify RNA single nucleotide variants (SNVs) in the calibrated BAM files. The identification criteria were set as base quality ≥ 25, sequencing depth ≥ 10, U base (converted to T base in RNA sequencing) depth ≥ 2 and editing frequency ≥ 1%, and false positive variants were filtered using the fpfilter command of VarScan and default parameters. SNV annotation was performed using Ensembl Variant Effect Predictor (VEP, https: / / www.ensembl.org / vep);

[0111] (4) Statistical analysis:

[0112] The comparison of RNA editing level or gene expression level between groups was performed using paired or paired Kruskal-Wallis (KW) nonparametric test, with P < 0.05 as the screening standard for significant difference.

[0113] The results showed that the RNA editing levels of CCRL2:chr3:46408483 and CCNI:chr4:77058527 sites were significantly different between the severe side and the control side (mild side). Figure 1 ), among which, the RNA editing levels of CCRL2:chr3:46408483 and CCNI:chr4:77058527 sites were significantly increased on the severe side.

[0114] The RNA editing levels at CTSS:chr1:150732881 / 150732032, IRAK3:chr12:66194142, and LYN:chr8:56010885 were significantly different between patients with complications and those without complications ( Figure 2 ). Among them, the RNA editing levels of CTSS:chr1:150732881 and IRAK3:chr12:66194142 sites were significantly increased in the complication group, and the RNA editing levels of CTSS:chr1:150732032 and LYN:chr8:56010885 sites were significantly decreased in the complication group.

[0115] Example 3: Generating a classifier for diagnosing the severity of MPP and its complications using RNA differentially edited sites combined with differentially edited gene compositions

[0116] Take 21 pairs of bronchoalveolar lavage fluid from the severe side of Mycoplasma pneumonia patients and the control side collected in Example 1, immediately put a portion of them into a freezing tube and place them in liquid nitrogen for freezing, transfer the frozen tissue in liquid nitrogen to a -80°C refrigerator for long-term storage until taken out, and obtain transcriptome samples to be sequenced by RNA extraction for transcriptome sequencing.

[0117] The RNA editing level in the sequencing results was used to establish the ROC curve. In the transcriptome sequencing data, we first used SPSS software to perform logistics regression on the editing levels of six 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 set the state variable to 0 and 1. In the analysis of severe and non-severe tissues, the severe group was set to 1, and the ROC curve was drawn using SPSS software to obtain Figure 3 , as shown in the figure, the area under the ROC curve AUC is 0.788.

[0118] In the analysis of complications and non-complications, the complication group was set to 1, and the ROC curve was drawn using SPSS software. 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, that is, the maximum value of the Youden index, is selected as the cutoff value. The cutoff value is the judgment standard and is the boundary value for determining whether the test is positive or negative. Figure 3 The sensitivity and specificity at the middle cutoff value were 76.2% and 72.2%, respectively. Figure 4 The sensitivity at the middle cutoff value was 100% and the specificity was 81.8%.

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

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

[0121]

[0122]

[0123] Table 5: The performance of the four RNA editing sites as independent indicators and combinations in the diagnosis of bronchoalveolar lavage fluid with and without complications in patients with Mycoplasma pneumoniae pneumonia (AUC: area under the ROC curve)

[0124]

[0125] The results showed that in Tables 4 and 5, the classifiers generated by the differences in RNA editing levels of 2 sites and 4 sites combined had a veracity of 78.8% and 97.3% for judging the occurrence of Mycoplasma pneumonia, respectively.

[0126] Example 4: Diagnosis of severity and complications of MPP infection using a combination of RNA differential editing sites

[0127] 1. Logistic regression analysis was performed on the level differences of the six RNA editing sites in the severe side and the control side, as well as in complications and non-complications, to obtain the formula:

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

[0129]

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

[0131] Cutoff value: P1 = 0.788, a value greater than this value is diagnosed as severe;

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

[0133] (2) Diagnosis of complications and non-complications:

[0134]

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

[0136] Cutoff value: P1 = 0.973, a value greater than this value 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. Use the formula obtained above to determine whether the tissue contains severe factors of Mycoplasma pneumoniae pneumonia, or determine whether the individual is a complication individual.

[0139] According to the above method, multiple subjects were tested respectively, and the RNA editing levels were substituted into the above formula to diagnose the severity of MPP and complications. The results showed that the results obtained by the method of the present application were consistent with the pathological diagnosis results.

[0140] Example 1: Tissue samples from patient 1 were selected to extract RNA from the bronchoalveolar lavage fluid of the severe side of Mycoplasma pneumoniae MPP, and the RNA editing level was detected. The RNA was substituted into the formula to obtain P1=0.963, which was greater than the cutoff value of 0.788 and was judged to be the severe side, which was consistent with the actual situation.

[0141] Example 2: Tissue samples from patient 2 were selected to extract RNA from the bronchoalveolar lavage fluid of the Mycoplasma pneumonia MPP control side, and the RNA editing level was detected. The RNA was substituted into the formula to obtain P1=0.426, which is less than the cutoff value of 0.788, and was judged to be the mild side, which is consistent with the actual situation.

[0142] Example 3: Tissue samples of patient 3 were selected to extract RNA from the bronchoalveolar lavage fluid of the severe side of Mycoplasma pneumoniae MPP, and the RNA editing level was detected. The result was substituted into the formula to obtain P1=0.992, which was greater than the cutoff value of 0.973. The patient was judged to have complications, which was consistent with the actual situation.

[0143] Example 4: Tissue samples of patient 4 were selected to extract RNA from the bronchoalveolar lavage fluid of the severe side of Mycoplasma pneumoniae MPP, and the RNA editing level was detected. The RNA was substituted into the formula to obtain P1=0.273, which is less than the cutoff value of 0.973. It was judged that the patient had no complications, which was consistent with the actual situation.

[0144] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A reagent for detecting the level of RNA editing of molecular markers for the severity of Mycoplasma pneumonia infection and complications in the preparation of products for diagnosing, predicting, evaluating and / or identifying the severity of Mycoplasma pneumonia infection and complications, and guiding the selection of medication or therapy, characterized in that: The molecular markers are based on the human reference genome hg38, and include the following adenosine-inosine (AI) RNA editing sites: CCRL2: chr3: 46408483, CCNI: chr4: 77058527, CTSS: chr1: 150732881 / 150732032, IRAK3: chr12: 66194142, LYN: chr8: 56010885; the product diagnoses the severity and complications of Mycoplasma pneumoniae pneumonia infection by detecting the RNA editing level of molecular markers of the severity and complications of Mycoplasma pneumoniae pneumonia infection.

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

3. The use according to claim 2, characterized in that: The biochip comprises a solid phase carrier and oligonucleotide probes fixed in order on the solid phase carrier, wherein the oligonucleotide probes specifically correspond to molecular markers of severity and complications of Mycoplasma pneumoniae pneumonia infection.

4. The use according to claim 2, characterized in that 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.

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

6. Use of a molecular marker for severity and complications of Mycoplasma pneumonia infection based on RNA editing level in the preparation of a product for diagnosing severity and complications of Mycoplasma pneumonia infection and / or predicting, evaluating or identifying severity and complications of Mycoplasma pneumonia infection, characterized in that: The molecular markers are based on the human reference genome hg38, and include the following adenosine-inosine (AI) RNA editing sites: CCRL2: chr3: 46408483, CCNI: chr4: 77058527, CTSS: chr1: 150732881 / 150732032, IRAK3: chr12: 66194142, LYN: chr8: 56010885; the product diagnoses the severity and complications of Mycoplasma pneumoniae pneumonia infection by detecting the RNA editing level of molecular markers of the severity and complications of Mycoplasma pneumoniae pneumonia infection.

7. The use according to claim 6, 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 and / or predicting the severity and complications of Mycoplasma pneumoniae pneumonia infection; Preferably, the biochip comprises a solid phase carrier and oligonucleotide probes fixed in order on the solid phase carrier, wherein the oligonucleotide probes specifically correspond to molecular markers of severity and complications of Mycoplasma pneumoniae pneumonia infection; Preferably, 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; Preferably, the kit contains reagents for detecting the severity of Mycoplasma pneumonia infection and the expression levels of molecular markers for complications.

8. A molecular marker for severity and complications of Mycoplasma pneumonia infection based on RNA editing level, characterized in that: Based on the human reference genome hg38, the molecular markers include the following adenosine-inosine (AI) RNA editing sites: CCRL2: chr3:46408483, CCNI: chr4:77058527, CTSS: chr1:150732881 / 150732032, IRAK3: chr12:66194142, LYN: chr8:56010885.

9. A biochip, device or kit for detecting the severity and complications of Mycoplasma pneumonia infection, characterized in that: The biochip comprises a solid phase carrier and oligonucleotide probes fixed in order on the solid phase carrier, wherein the oligonucleotide probes specifically correspond to the molecular markers of severity and complications of Mycoplasma pneumonia infection based on RNA editing level according to claim 8; The device comprises one or more devices for specifically detecting the RNA editing levels of the RNA editing sites of claim 8: CCRL2:chr3:46408483, CCNI:chr4:77058527, CTSS:chr1:150732881 / 150732032, IRAK3:chr12:66194142, LYN:chr8:56010885; The kit contains the molecular markers for severity and complications of Mycoplasma pneumonia infection based on RNA editing level as described in claim 8 or the device, or the biochip.

10. The biochip, device or kit according to claim 9, characterized in that: The method further comprises a reagent capable of detecting the expression level of the molecular marker according to claim 8.

Images