Exhaled air condensate microorganism combination for predicting benign and malignant pulmonary nodules, kit and application

Abstract

The invention relates to an exhaled air condensate microorganism combination for predicting benign and malignant pulmonary nodules, a kit and application, and the exhaled air condensate microorganism combination for predicting benign and malignant pulmonary nodules comprises pneumococcus bartgooensis, twin coccus, streptococcus, Rosella, methylobacterium and marine colibacillus. Aeromonas kirschenii and yellow aureomonas sp. The invention discloses a kit for predicting benign and malignant pulmonary nodules. The kit comprises internal standard substances which are used for detecting the contents of pneumococcus bartgoeni, twin coccus, streptococcus, Rosella, methylobacterium, marine colibacillus, aeromonas kirschenii and yellow aureomonas in an exhaled gas condensate sample extraction solution. The combined marker provided by the invention has the characteristics of high sensitivity and high specificity in diagnosis of benign and malignant pulmonary nodules.

Description

Technical Field

[0001] The present invention relates to a method for predicting benign and malignant pulmonary nodules, and in particular to an exhaled breath condensate microbial combination, a kit and an application thereof for predicting benign and malignant pulmonary nodules. Background Art

[0002] Pulmonary nodules are a type of imaging manifestation characterized by focal, rounded, solid or subsolid lung shadows with a diameter of ≤3 cm. In my country, the incidence of pulmonary nodules is 35.5%, of which 0.54% are malignant pulmonary nodules diagnosed as lung cancer. The incidence and mortality of lung cancer are among the highest in the world. In China, the incidence and mortality of lung cancer have increased significantly. One of the main reasons is the lack of timely diagnosis, which results in most patients being in the middle and late stages at the time of diagnosis, reducing the effectiveness of treatment. Therefore, how to accurately distinguish between benign and malignant pulmonary nodules early is a hot topic and difficulty in current clinical research.

[0003] At present, the clinical methods for diagnosing and identifying benign and malignant pulmonary nodules include clinical manifestations, imaging examinations, pathological biopsy by thoracentesis, blood tests, etc. The most commonly used method is cytological examination by thoracentesis. If tumor cells are found in tissue samples, it has diagnostic value for malignant pulmonary nodules. However, due to the complex cellular composition of pulmonary nodules, it is sometimes difficult to distinguish tumor cells from macrophages. At the same time, it is also affected by factors such as the level of the diagnostician and sample collection. It has the disadvantages of low sensitivity and strong subjectivity, which can easily lead to missed diagnosis or misdiagnosis. In addition, some methods are highly invasive, risky, or time-consuming, and cannot meet the needs of clinical work. Therefore, the development of new and highly sensitive diagnostic technologies for benign and malignant pulmonary nodules is of great clinical significance. Summary of the invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and to provide an exhaled breath condensate microbial combination, a kit and an application for predicting benign and malignant pulmonary nodules.

[0005] The technical solution of the present invention to solve the above technical problems is as follows: The exhaled breath condensate microbiome panel for predicting benign and malignant pulmonary nodules included Trichococcus patagonicus, Geminicoccus, Streptococcus, Roseburia, Methylobacterium, Myxobacterium marineum, Aeromonas kluyveri, and Aureomonas aureus.

[0006] A kit for predicting benign or malignant pulmonary nodules, comprising reagents required for detecting the contents of Trichococcus patagonica, Gemini coccus, Streptococcus, Roseburia, Methylobacterium, Myxobacterium marineum, Aeromonas kluyveri and Aureomonas aureus in an extract solution of an exhaled breath condensate sample.

[0007] Furthermore, the detection reagent includes 16S rRNA gene V3-V4 variable region specific amplification primers.

[0008] Preferably, the nucleotide sequence of the primer is shown in SEQ ID NO: 1-2.

[0009] Application of the above-mentioned kit in predicting benign and malignant pulmonary nodules.

[0010] The beneficial effect of the present invention is that the microbial combination of Patagonian Trichococcus, Geminicoccus, Streptococcus, Roseburia, Methylobacterium, Myxobacterium marineum, Aeromonas kluyveri and Aureomonas aureus in exhaled breath condensate can be used in combination to identify patients with benign and malignant pulmonary nodules. Through the combined markers of the present invention, the diagnosis of lung cancer can have high sensitivity and high specificity.

[0011] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is the ROC curve of the combined marker in distinguishing benign and malignant pulmonary nodule disease groups; Figure 2 This is a graph showing the difference in microbial content in exhaled breath condensate between patients with benign and malignant pulmonary nodules. DETAILED DESCRIPTION

[0013] The principles and features of the present invention are described below. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0014] Exhaled breath condensate samples were collected under the same conditions: exhaled breath condensate samples were collected from 24 patients with benign pulmonary nodules and 24 patients with malignant pulmonary nodules to establish a screening set; Through metagenomics technology, the entire microbial community in the sample is taken as the research object, the DNA of the sample is directly extracted and screened for sequencing, and the species classification of the microorganisms in the exhaled breath condensate is studied. Figure 2As shown, it can be seen that the contents of Patagonian Trichococcus, Streptococcus, Roseburia, Methylobacterium, Marine Myxobacterium, Aeromonas Kluyveri and Aureomonas aureus in the exhaled condensate corresponding to malignant pulmonary nodules are basically 0, and their contents in the exhaled condensate corresponding to benign pulmonary nodules are significantly increased, while the content of Gemini in the exhaled condensate corresponding to malignant pulmonary nodules is significantly higher than that in the exhaled condensate corresponding to benign pulmonary nodules. Therefore, the present invention determines that there are 8 significantly different microbial genera in the exhaled condensate corresponding to benign pulmonary nodules and malignant pulmonary nodules, specifically: Patagonian Trichococcus, Gemini, Streptococcus, Roseburia, Methylobacterium, Myxobacterium marineum, Aeromonas kluyveri and Aureomonas aureus are all important microorganisms in human exhaled breath condensate.

[0015] The detection method according to the kit is as follows: (1) Exhaled breath condensate sample pretreatment method: The exhaled condensate sample was thawed at 4°C, 200 μL of it was taken, and 800 μL of methanol extract containing internal standard was added to precipitate protein: vortex for 30-90s, let stand for 15-30mins, centrifuge at 10000-14000×g for 10-15 mins at 4°C, take the supernatant and freeze-dry; add 50 uL 10-20% (v / v) methanol aqueous solution, centrifuge at 10000-14000×g for 10-15 mins at 4°C, and take the supernatant for sequencing analysis.

[0016] (2) PCR amplification and sequencing library construction to detect target microorganisms: The V3-V4 variable region of the 16S rRNA gene was amplified by PCR using the upstream primer 338F and the downstream primer 806R carrying the Barcode sequence. The PCR reaction system was: 4 μL of 5×TransStart FastPfu buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of upstream primer (5 uM), 0.8 μL of downstream primer (5 uM), 0.4 μL of TransStart FastPfu DNA polymerase, and 10 ng of template DNA, which was made up to 20 μL.

[0017] 338F: 5'-ACTCCTACGGGAGGCAGCAG-3' (SEQ ID NO: 1); 806R: 5'-GGACTACHVGGGTWTCTAAT-3' (SEQ ID NO: 2).

[0018] The amplification program was as follows: 95°C pre-denaturation for 3 min, 27 cycles (95°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 30 s), then 72°C stable extension for 10 min, and finally stored at 4°C (PCR instrument: ABI GeneAmp® 9700). Each sample was repeated 3 times. The PCR products of the same sample were mixed and recovered using 2% agarose gel and purified. The size of the band fragments was detected by 2% agarose gel electrophoresis, and the recovered products were detected and quantified using Quantus™ Fluorometer (Promega, USA).

[0019] The purified PCR products were constructed using the NEXTFLEX Rapid DNA-Seq Kit (Bioo Scientific, Austin, Texas, USA): (1) adapter ligation; (2) using magnetic beads to screen and remove adapter self-ligation fragments; (3) using PCR amplification to enrich the library template; (4) using magnetic beads to recover the PCR products to obtain the final library. Sequencing was performed using the Illumina PE300 / PE250 platform (Shanghai Meiji Biopharmaceutical Technology Co., Ltd.). The raw data were uploaded to the NCBI SRA database.

[0020] (3) Judgment model based on mass spectrometry data: A prediction model was established using the random forest model algorithm, and exhaled breath condensate samples were collected from 38 patients with malignant pulmonary nodules and 12 patients with benign pulmonary nodules to establish a training set. The prediction model was trained using the training set, and the model directly outputs the judgment conclusion of benign and malignant pulmonary nodules. The trained prediction model was obtained by minimizing the loss function.

[0021] The relevant information of the prediction model is as follows: Basic model architecture: Model type: Random Forest Classification Classification target: Binary classification model, distinguishing between "Control" and "Case" samples Number of decision trees (ntree): 500 decision trees Feature sampling number (mtry): Each node randomly selects 3 features for splitting decision Maximum number of tree nodes: 19 nodes; Model performance indicators Classification accuracy: about 82% (based on OOB estimation) Control group accuracy: about 90.5% (38 / (38+4)) Accuracy of case group: about 66.7% (8 / (8+4)) Number of samples: 50 samples in total (based on the votes matrix) Feature composition Number of features: The model uses 8 microbial classification features Feature type: bacterial genus and species level classification information An exhaled condensate sample is collected from the subject, microorganisms are extracted, and the mass fractions of Patagonian Trichococcus, Gemini Coccus, Streptococcus, Roseburia, Methylobacterium, Myxobacterium marineum, Aeromonas kluyveri and Aureomonas aureus in the exhaled condensate sample of the patient to be tested are detected, and the trained prediction model is output to obtain a conclusion on the judgment of benign and malignant pulmonary nodules.

[0022] The model has good predictive ability for benign and malignant pulmonary nodules (see Figure 1 ), the AUC of the combined marker was 0.813, the sensitivity was 81.3%, and the specificity was 80%. Example

[0023] 1. Sample Preparation Before the collection of exhaled breath condensate samples, the volunteers signed an informed consent.

[0024] Inclusion criteria for benign and malignant pulmonary nodules: patients with clinical manifestations of benign and malignant pulmonary nodules: cough, sputum, chest tightness and shortness of breath, etc.; and subsequent imaging examinations diagnosed them as patients with benign and malignant pulmonary nodules.

[0025] Exhaled breath condensate samples were used under the same conditions: exhaled breath condensate samples from 21 patients with pulmonary nodules; 50 ml of exhaled breath condensate was collected from the patients before any treatment after admission, and the exhaled breath condensate was separated within half an hour and stored in a -80°C refrigerator for inspection.

[0026] 2. Analytical methods 2.1 Pretreatment of exhaled condensate samples The exhaled condensate sample was thawed at 4°C, 200 μL of it was taken, and 800 μL of methanol extract containing internal standard was added to precipitate protein: vortex for 30-90s, let stand for 15-30mins, centrifuge at 10000-14000×g for 10-15 mins at 4°C, take the supernatant and freeze-dry; add 50 uL of 10-20% (v / v) methanol aqueous solution, centrifuge at 12000×g for 15mins at 4°C, and take the supernatant for subsequent analysis.

[0027] 2.2 PCR amplification and sequencing library construction The DNA extracted above was used as a template, and the upstream primer 338F and the downstream primer 806R carrying the Barcode sequence were used to perform PCR amplification of the variable region of the 16S rRNA gene V3-V4. The PCR reaction system was: 4 μL of 5×TransStartFastPfu buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of upstream primer (5 uM), 0.8 μL of downstream primer (5 uM), 0.4 μL of TransStart FastPfu DNA polymerase, 10 ng of template DNA, and the total was 20 μL. The amplification program was as follows: 95°C pre-denaturation for 3 min, 27 cycles (95°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 30 s), then 72°C stable extension for 10 min, and finally stored at 4°C (PCR instrument: ABI GeneAmp® 9700). Each sample was repeated 3 times. The PCR products of the same sample were mixed and recovered using 2% agarose gel and purified. The size of the band fragments was detected by 2% agarose gel electrophoresis, and the recovered products were detected and quantified using Quantus™ Fluorometer (Promega, USA).

[0028] The purified PCR products were constructed using the NEXTFLEX Rapid DNA-Seq Kit (Bioo Scientific, Austin, Texas, USA): (1) adapter ligation; (2) using magnetic beads to screen and remove adapter self-ligation fragments; (3) using PCR amplification to enrich the library template; (4) using magnetic beads to recover the PCR products to obtain the final library. Sequencing was performed using the Illumina PE300 / PE250 platform (Shanghai Meiji Biopharmaceutical Technology Co., Ltd.). The raw data were uploaded to the NCBI SRA database.

[0029] Exhaled condensate samples were collected from 21 subjects, and microorganisms were extracted. The mass fractions of Patagonian Trichococcus, Gemini, Streptococcus, Roseburia, Methylobacterium, Myxobacterium marineum, Aeromonas kluyveri and Aureomonas aureus in the exhaled condensate samples of the tested patients were detected. The trained prediction model was input to obtain the conclusion on the judgment of benign and malignant pulmonary nodules.

[0030] Through this method, 14 patients were finally diagnosed as having malignant pulmonary nodules, 13 of the 16 patients diagnosed as having malignant pulmonary nodules by pathological biopsy were diagnosed as having malignant pulmonary nodules, and 4 of the 5 patients diagnosed as having benign pulmonary nodules by pathological biopsy were diagnosed as having benign pulmonary nodules. The AUC of the combined marker was 0.813, the sensitivity was 81.3%, and the specificity was 80%, indicating that the evaluation accuracy of the present invention is relatively high.

Claims

1. An exhaled breath condensate microbial combination for predicting benign and malignant pulmonary nodules, characterized in that: These include Trichococcus patagonicus, Gemini coccus, Streptococcus, Roseburia, Methylobacterium, Myxobacterium marineum, Aeromonas kluyveri and Aureomonas xanthophylla.

2. A kit for predicting benign and malignant pulmonary nodules, characterized in that: Contains reagents necessary for the detection of Trichococcus patagonicus, Geminicoccus, Streptococcus, Roseburia, Methylobacterium, Myxobacterium marineum, Aeromonas kluyveri, and Aureomonas aureus in an extract solution of an exhaled breath condensate sample.

3. The kit for predicting benign and malignant pulmonary nodules according to claim 2, characterized in that: The detection reagent includes 16S rRNA gene V3-V4 variable region specific amplification primers.

4. The kit for predicting benign and malignant pulmonary nodules according to claim 3, characterized in that: The nucleotide sequences of the primers are shown in SEQ ID NO: 1-2.

5. Use of the kit according to any one of claims 2 to 4 in predicting benign and malignant pulmonary nodules.

Images