Exhaled Breath Condensate Microbiota Profile, Kit and Method for Predicting Lung Cancer Metastasis

Abstract

The present invention relates to an exhaled breath condensate microbial combination, a kit and a method for predicting lung cancer metastasis, wherein the exhaled breath condensate microbial combination for predicting lung cancer metastasis comprises blood-borne Gemini coccus, Clostridium and Osakabacterium. The kit for predicting lung cancer metastasis comprises an internal standard substance required for detecting the content of blood-borne Gemini coccus, Clostridium and Osakabacterium in an exhaled breath condensate sample extraction solution. Through the combined markers of the present invention, the diagnosis of lung cancer metastasis can have high sensitivity and high specificity characteristics.

Description

Technical Field

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

[0002] Lung cancer metastasis refers to the process by which cancer cells break away from the primary tumor site, migrate through the bloodstream or lymphatic system, and continue to grow in other parts of the body, forming new tumor lesions. Metastasis is a key prognostic indicator; if lung cancer has not yet spread or metastasized, the prognosis is relatively good. However, if lung cancer is left untreated and progresses to the middle or late stages, with significant spread and metastasis, the prognosis is poor, significantly impacting patient survival. Therefore, how to accurately and early identify lung cancer metastasis remains a hot topic and a challenge in current clinical research.

[0003] Current clinical methods for diagnosing and identifying lung cancer metastasis include imaging and pathological examinations. The most commonly used method is CT scanning, which can clearly demonstrate the size, shape, and location of the primary lung tumor. It can also detect metastases in other parts of the body, such as the liver and bones. However, this method is affected by the diagnostician's level and the accuracy of the instrument, and has the disadvantages of low sensitivity and high subjectivity, which can easily lead to missed or misdiagnosed cases. Therefore, the development of new, highly sensitive diagnostic technologies for lung cancer metastasis 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 lung cancer metastasis.

[0005] The technical solution of the present invention to solve the above technical problems is as follows:

[0006] Exhaled breath condensate microbial panel for predicting lung cancer metastasis, including Geminis, Clostridium, and Osakabacterium.

[0007] A kit for predicting lung cancer metastasis, comprising reagents required for detecting the levels of blood-borne Geminis, Clostridium, and Osakabacterium in an extract solution of an exhaled breath condensate sample.

[0008] Furthermore, the detection reagent includes 16S rRNA gene V3-V4 variable region specific amplification primers.

[0009] Furthermore, the nucleotide sequences of the specific amplification primers are shown in SEQ ID NO: 1 and SEQ ID NO: 2.

[0010] The method for predicting lung cancer metastasis comprises the following steps:

[0011] Step 1: extracting a solution from a sample of exhaled breath condensate from a subject, and detecting the mass fractions of Gemini cocci, Clostridium difficile, and Osakabacterium in the extract;

[0012] Step 2: Calculate the lung cancer metastasis probability Prob. If Prob>0.17, the subject is judged to have lung cancer metastasis. The calculation method of the lung cancer metastasis probability is: Prob = 1 / (1+e -X ), where X = 3.967a-217.946b-109.974c-56.418, where a, b, and c are the mass fractions of blood-borne Gemini cocci, Clostridium, and Osakabacterium obtained in step 1, respectively.

[0013] The present invention has the beneficial effect of detecting a combination of blood-borne Gemini bacilli, Clostridium, and Sakaibacter osakaiensis in exhaled breath condensate to identify patients with metastatic lung cancer. This combination of markers enables high sensitivity and specificity in the diagnosis of lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] 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 paying any creative work.

[0015] Figure 1 This is the ROC curve of the combined marker in distinguishing lung cancer metastasis disease group;

[0016] Figure 2 Schematic diagram of the changes in the levels of blood-borne Gemini cocci, Clostridium and Osakabacterium in exhaled breath condensate of patients with metastatic and non-metastatic lung cancer (mean ± standard error). DETAILED DESCRIPTION

[0017] 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.

[0018] Exhaled breath condensate samples were collected under the same conditions to establish a screening set: exhaled breath condensate samples were collected from 42 patients with invasive lung cancer and confirmed lung cancer metastasis, and 42 patients with invasive lung cancer and confirmed lung cancer non-metastasis to establish a screening set;

[0019] Through metagenomics technology, the entire microbial community in the sample is taken as the research object, the DNA of the concentrated sample is directly extracted and screened for sequencing, and the species classification of exhaled breath condensate microorganisms is studied.

[0020] The present invention determined that there are three significantly different microorganisms in the exhaled condensate of lung cancer with and without metastasis, specifically as follows: Gemini coccus, Clostridium and Sakaibacter osakaiensis, which are important microorganisms in human exhaled condensate.

[0021] The detection method according to the kit is as follows:

[0022] (1) Exhaled breath condensate sample pretreatment method:

[0023] Thaw the exhaled condensate sample at 4°C, take 200 μL of it, add 800 μL of methanol extract containing internal standard to precipitate protein: vortex for 30-90 seconds, let it stand for 15-30 minutes, centrifuge at 10,000-14,000 × g for 10-15 minutes at 4°C, and freeze-dry the supernatant; add 50 μL of 10-20% (v / v) methanol aqueous solution, centrifuge at 10,000-14,000 × g for 10-15 minutes at 4°C, and collect the supernatant for sequencing analysis.

[0024] (2) PCR amplification and sequencing library construction to detect target microorganisms:

[0025] 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 the upstream primer (5 uM), 0.8 μL of the 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.

[0026] 338F: 5'-ACTCCTACGGGAGGCAGCAG-3' (SEQ ID NO: 1);

[0027] 806R: 5'-GGACTACHVGGGTWTCTAAT-3' (SEQ ID NO: 2).

[0028] The amplification program was as follows: 95°C initial denaturation for 3 minutes, 27 cycles of (95°C denaturation for 30 seconds, 55°C annealing for 30 seconds, 72°C extension for 30 seconds), followed by a 10-minute stabilization extension at 72°C, and storage at 4°C (PCR instrument: ABI GeneAmp® 9700). Each sample was replicated three times. PCR products from the same sample were pooled and recovered on a 2% agarose gel. Purification was performed, and fragment size was determined by 2% agarose gel electrophoresis. Recovered products were quantified using a Quantus™ Fluorometer (Promega, USA).

[0029] The purified PCR products were constructed using the NEXTFLEX Rapid DNA-Seq Kit (Bioo Scientific, Austin, Texas, USA): (1) adapter ligation; (2) removal of adapter self-ligated fragments using magnetic bead screening; (3) library template enrichment using PCR amplification; and (4) magnetic bead recovery of the PCR products to obtain the final library. Sequencing was performed using the Illumina PE300 / PE250 platform (Shanghai Meiji Biopharmaceutical Technology Co., Ltd.). Raw data were uploaded to the NCBI SRA database.

[0030] (3) Judgment model based on mass spectrometry data:

[0031] The obtained data were subjected to binary logistic regression analysis using SPSS software, and the regression equation obtained from the constructed model is as follows:

[0032] X=3.967a-217.946b-109.974c-56.418.

[0033] Prob (lung cancer metastasis) = 1 / (1+e -X )

[0034] An exhaled breath condensate sample was collected from the subject, microorganisms were extracted, and the levels of blood-borne Gemini cocci, Clostridium difficile, and Osakabacterium in the exhaled breath condensate sample were detected. Prob (lung cancer metastasis) was the probability of predicting lung cancer metastasis, and the cutoff value was 0.17, i.e., when the Prob (lung cancer metastasis) value was greater than 0.17, lung cancer metastasis was predicted.

[0035] The model has good predictive ability for lung cancer metastasis, such as Figure 1 The AUC of the combined marker was 0.833, the sensitivity was 80%, and the specificity was 66.7%. Figure 2 As shown in the data, compared with the patients with non-metastatic lung cancer, the blood-borne Gemini cocci and Osaka bacteria in the exhaled breath condensate of the patients with lung cancer metastasis were significantly increased, and Clostridium was significantly decreased.

[0036] Example

[0037] Before the collection of exhaled breath condensate samples, volunteers signed an informed consent form.

[0038] Inclusion criteria for patients with invasive lung cancer: patients with clinical manifestations of invasive lung cancer: cough, blood in sputum, wheezing, weight loss, etc.; and subsequent diagnosis of invasive lung cancer.

[0039] Exhaled breath condensate samples were collected under the same conditions: exhaled breath condensate samples were collected from 42 patients with invasive lung cancer who were not sure whether they had lung cancer metastasis 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 testing.

[0040] 2. Analytical methods

[0041] 2.1 Pretreatment of exhaled condensate samples

[0042] Thaw the exhaled breath condensate sample at 4°C, take 200 μL of it, add 800 μL of methanol extract containing internal standard to precipitate protein: vortex for 30-90 seconds, let it stand for 15-30 minutes, centrifuge at 10,000-14,000 × g for 10-15 minutes at 4°C, and freeze-dry the supernatant; add 50 μL of 10-20% (v / v) methanol aqueous solution, centrifuge at 12,000 × g for 15 minutes at 4°C, and collect the supernatant for subsequent analysis.

[0043] 2.2 PCR amplification and sequencing library construction

[0044] Using the extracted DNA as template, PCR amplification of the V3-V4 variable region of the 16S rRNA gene was performed using upstream primer 338F and downstream primer 806R carrying a barcode sequence. The PCR reaction system consisted of 4 μL of 5× TransStart Fast Pfu buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of the upstream primer (5 μM), 0.8 μL of the downstream primer (5 μM), 0.4 μL of TransStart Fast Pfu DNA polymerase, and 10 ng of template DNA, made up to 20 μL. The amplification program was as follows: initial denaturation at 95°C for 3 min, 27 cycles of (95°C denaturation for 30 s, 55°C annealing for 30 s, and 72°C extension for 30 s), followed by a 10-min stabilization extension at 72°C, and storage at 4°C (PCR instrument: ABI GeneAmp® 9700). Three replicates were performed for each sample. PCR products from the same sample were mixed and recovered using 2% agarose gel. The PCR products were purified and the sizes of the bands were detected by 2% agarose gel electrophoresis. The recovered products were quantified using a Quantus™ Fluorometer (Promega, USA).

[0045] The purified PCR products were constructed using the NEXTFLEX Rapid DNA-Seq Kit (Bioo Scientific, Austin, Texas, USA): (1) adapter ligation; (2) removal of adapter self-ligated fragments using magnetic bead screening; (3) library template enrichment using PCR amplification; and (4) magnetic bead recovery of the PCR products to obtain the final library. Sequencing was performed using the Illumina PE300 / PE250 platform (Shanghai Meiji Biopharmaceutical Technology Co., Ltd.). Raw data were uploaded to the NCBI SRA database.

[0046] 2.3 Exhaled Breath Condensate Test Results and Auxiliary Diagnostic Methods

[0047] The relative content of the bacterial flora was brought into the regression equation to calculate the probability. The cutoff value used was 0.17, that is, if the probability of the combined marker was greater than 0.17, it was considered to be lung cancer metastasis. The AUC of the combined marker was 0.833, and the sensitivity and specificity were also relatively high, at 80% and 66.7%, respectively. Figure 1 ; Using this method, 21 out of 42 patients with invasive lung cancer were diagnosed as having lung cancer metastasis, 12 out of 15 patients diagnosed as having lung cancer metastasis by pathological biopsy were diagnosed as having lung cancer metastasis, and 18 out of 27 patients diagnosed as having non-lung cancer metastasis by pathological biopsy were diagnosed as having non-lung cancer metastasis, indicating that the prediction success rate of this method is high.

Claims

1. An exhaled breath condensate microbial composition for predicting lung cancer metastasis, characterized in that: Including blood-borne Gemini, Clostridium and Osaka bacteria.

2. Use of the microbial combination according to claim 1 in preparing a kit for predicting lung cancer metastasis, characterized in that: The kit comprises the detection reagents required for detecting the contents of blood-borne Gemini cocci, Clostridium difficile and Osakabacterium in the exhaled breath condensate sample extraction solution.

3. The use according to claim 2, characterized in that: The detection reagent includes 16S rRNA gene V3-V4 variable region specific amplification primers.

4. The use according to claim 3, characterized in that: The nucleotide sequences of the specific amplification primers are shown in SEQ ID NO: 1 and SEQ ID NO: 2.

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