Exhaled air condensate microorganism combination, kit and method for predicting invasive lung adenocarcinoma

Abstract

The invention relates to an exhaled air condensate microorganism combination, a kit and a method for predicting infiltrating lung adenocarcinoma. The exhaled air condensate microorganism combination for predicting the infiltrating lung adenocarcinoma comprises purple bacillus delavayi, microbacterium aerobacter and pseudomonas rubra. The kit for predicting the invasive lung adenocarcinoma comprises an internal standard substance required for detecting the content of purple blue bacillus, microbacterium aerobacter and pseudomonas rubra in an exhaled gas condensate sample extraction solution, wherein the internal standard substance is used for detecting the content of the purple blue bacillus, the microbacterium aerobacter and the pseudomonas rubra in the exhaled gas condensate sample extraction solution. The combined marker provided by the invention has the characteristics of high sensitivity and high specificity on the diagnosis of the invasive lung adenocarcinoma.

Description

Technical Field

[0001] The present invention relates to a method for predicting invasive lung adenocarcinoma, and in particular to an exhaled breath condensate microbial combination, a kit and a method for predicting invasive lung adenocarcinoma. Background Art

[0002] Invasive lung adenocarcinoma refers to a malignant tumor in which cancer cells invade and infiltrate the surrounding lung tissues. The tumor shows unclear boundaries, rough edges, lobes, and burrs. The staging is an important indicator for judging the prognosis. If the invasive lung adenocarcinoma is staged earlier and has not yet spread or metastasized, the corresponding prognosis is better. However, if it is not treated in time, the invasive lung adenocarcinoma will develop to the middle and late stages, and there will be obvious spread and metastasis. The prognosis is poor, which has a greater impact on the patient's survival time. Therefore, how to identify invasive lung adenocarcinoma early and accurately is a hot spot and difficulty in current clinical research.

[0003] At present, the clinical diagnosis and identification methods of invasive lung adenocarcinoma include clinical manifestations, imaging examinations, surgical biopsy, etc. The most commonly used method is surgical biopsy. After surgical removal of the tumor, the tumor sample is subjected to pathological histological examination to determine the pathological subtype, degree of differentiation and pathological TNM stage of lung adenocarcinoma. However, this method is 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. It is also traumatic, risky, or takes a long time, which cannot meet the needs of clinical work. Therefore, the development of new and highly sensitive diagnostic technology for invasive lung adenocarcinoma 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 a method for predicting invasive lung adenocarcinoma.

[0005] The technical solution of the present invention to solve the above technical problems is as follows: Exhaled breath condensate microbiome panel for predicting invasive lung adenocarcinoma, including Pseudomonas aeruginosa, Microbacterium aeruginosa, and Pseudomonas erythrophilum.

[0006] A kit for predicting invasive lung adenocarcinoma comprises reagents required for detecting the content of Bacillus cyanobacterium, Microbacterium aerogenes and Pseudomonas erythrocytes in an extraction solution of an exhaled breath condensate sample.

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

[0008] Furthermore, the nucleotide sequences of the primers are shown in SEQ ID NO: 1 and SEQ ID NO: 2.

[0009] A method for predicting invasive lung adenocarcinoma, comprising the following steps: Step 1, extracting a solution of a sample of exhaled breath condensate from a subject, and detecting the mass fractions of Cercobacterium cyanobacterium, Microbacterium aerogenes, and Pseudomonas erythrorhizium in the extract; Step 2: Calculate the probability of invasive lung adenocarcinoma Prob. If Prob>0.38, the subject is judged to have invasive lung adenocarcinoma. The calculation method of the probability of invasive lung adenocarcinoma is: Prob = 1 / (1+e -X ), where X = -2.356a-3.006b-0.332c+2.995, where a, b and c are the mass fractions of Cercobacterium cyanobacterium, Microbacterium aerogenes and Pseudomonas erythrophilum obtained in step 1, respectively.

[0010] The beneficial effects of the present invention are as follows: the microbial combination of Bacillus cyanobacterium, Microbacterium aerogenes and Pseudomonas erythrophilum in exhaled breath condensate can be used in combination to identify patients with invasive lung adenocarcinoma. The combined markers of the present invention can provide high sensitivity and high specificity for the diagnosis of lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] 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.

[0013] Figure 1 The ROC curve of the combined marker in distinguishing the invasive lung adenocarcinoma disease group; Figure 2 The figure is a schematic diagram of the changes in the contents of Cercobacterium cyanobacterium, Aerobacterium oxybacterium and Pseudomonas erythrocytes in the exhaled breath condensate of patients with invasive and non-invasive lung adenocarcinoma (mean ± standard error). Figure 3 This is a pathological picture of in situ lung adenocarcinoma; Figure 4 This is a pathological picture of invasive lung adenocarcinoma. DETAILED DESCRIPTION

[0014] 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.

[0015] Exhaled breath condensate samples were collected under the same conditions to establish a screening set: Exhaled breath condensate samples were collected from 34 patients with invasive lung adenocarcinoma and 34 patients with non-invasive in situ lung adenocarcinoma to establish a screening set. Figure 3 and Figure 4These are the pathological pictures of in situ lung adenocarcinoma and invasive lung adenocarcinoma respectively; Through metagenomics technology, the entire microbial community in the sample is taken as the research object, and the sample DNA is directly extracted and screened for sequencing to study the species classification of exhaled breath condensate microorganisms. The present invention determined that there are three significantly different microorganisms in the exhaled condensate of invasive lung adenocarcinoma and non-invasive lung adenocarcinoma, specifically as follows: Cercobacterium cyanobacterium, Aerobacterium and Pseudomonas erythrocytes, which are important microorganisms in human exhaled breath condensate.

[0016] 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.

[0017] (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.

[0018] 338F: 5'-ACTCCTACGGGAGGCAGCAG-3' (SEQ ID NO: 1); 806R: 5'-GGACTACHVGGGTWTCTAAT-3' (SEQ ID NO: 2).

[0019] 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).

[0020] 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.

[0021] (3) Judgment model based on mass spectrometry data: The obtained data were analyzed by binary logistic regression using SPSS software, and the regression equation obtained by the constructed model is as follows: X=-2.356a-3.006b-0.332c+2.995 Prob (invasive lung adenocarcinoma) = 1 / (1+e -X ) An exhaled condensate sample is collected from the subject, microorganisms are extracted, and the contents of Cercobacterium cyanobacterium, Aerobacterium oxytoca and Pseudomonas erythrocytes in the exhaled condensate sample are detected. Prob (invasive lung adenocarcinoma) is the probability of predicting invasive lung adenocarcinoma, and Cutoff=0.38, that is, when the Prob (invasive lung adenocarcinoma) value is greater than 0.38, invasive lung adenocarcinoma is predicted.

[0022] The model has good predictive ability for invasive lung adenocarcinoma (see Figure 1 ), the AUC of the combined marker was 0.968, the sensitivity was 100%, and the specificity was 85.7%. Figure 2 As shown, compared with the patients with non-invasive lung cancer group, the dark blue-purple bacilli, aerobic microbacteria and red pseudomonas in the exhaled breath condensate of the patients with invasive lung cancer group were significantly increased.

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

[0024] Inclusion criteria for lung adenocarcinoma: patients with clinical manifestations of lung adenocarcinoma: cough, blood in sputum, wheezing, weight loss, etc.; and subsequently diagnosed as lung adenocarcinoma patients.

[0025] Exhaled breath condensate samples were collected under the same conditions: Exhaled breath condensate samples were collected from 34 patients with lung adenocarcinoma (without invasive and non-invasive screening) to establish a validation set for verification; the collection method was as follows: 50 ml of exhaled breath condensate was collected from the patient after admission and before any treatment, 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] 2.3 Exhaled breath condensate test results and auxiliary diagnostic methods The relative content of the flora was brought into the regression equation to calculate the probability. The cutoff value used was 0.38, that is, when the probability of the combined marker was greater than 0.38, it was considered to be invasive lung adenocarcinoma. The AUC of the combined marker was 0.968, and the sensitivity and specificity were also relatively high, at 100% and 85.7%, respectively. Figure 1 Through this method, 28 out of 34 patients with lung adenocarcinoma were finally diagnosed as having invasive lung cancer, 27 out of 27 patients diagnosed as having invasive lung cancer through pathological biopsy were diagnosed as having invasive lung cancer, and 6 out of 7 patients diagnosed as having non-invasive lung cancer through pathological biopsy were diagnosed as having non-invasive lung cancer, indicating that the method of the present invention has a high prediction success rate.

Claims

1. An exhaled breath condensate microbial panel for predicting invasive lung adenocarcinoma, characterized in that: Including dark blue-violet bacteria, aerobic bacteria and red pseudomonas.

2. A kit for predicting invasive lung adenocarcinoma, characterized in that: Contains reagents necessary for detecting the presence of Bacillus cyanobacterium, Microbacterium aerogenes, and Pseudomonas erythrophilum in an extract solution of an exhaled breath condensate sample.

3. The kit for predicting invasive lung adenocarcinoma 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 invasive lung adenocarcinoma according to claim 3, characterized in that: The nucleotide sequences of the primers are shown in SEQ ID NO:1 and SEQ ID NO:

2.

5. Use of the kit according to any one of claims 2 to 4 in predicting invasive lung adenocarcinoma.

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