A microbial detection system for exhaled breath condensate samples

Abstract

The embodiment of the specification provides a kind of microbial detection system of exhaled breath condensate, it is related to medical instrument and in-vitro diagnosis technical field.The system includes the sequential communication of closed flow path structure in turn exhaled breath collection module, condensation and quantification module, microbial enrichment and pretreatment module and microbial detection module, exhaled breath collection module, for the non-invasive collection of exhaled breath of subject;Condensation and quantification module, for the condensation of exhaled breath into condensate, and the volume of obtained condensate is quantitatively monitored;When the volume of condensate reaches preset threshold, control flows into downstream module;Microbial enrichment and pretreatment module, for receiving and automatically enriching and in situ lysing microorganism in condensate, obtain the condensate of improved microbial target concentration;Microbial detection module, with the output end of microbial enrichment and pretreatment module is communicated, for detecting the condensate of improved microbial target concentration.

Description

Technical Field

[0001] This invention relates to the field of medical devices and in vitro diagnostics, and more specifically, to a microbial detection system for exhaled breath condensate samples. Background Technology

[0002] Exhaled breath condensate (EBC), as a completely non-invasive respiratory sample collection method, can reflect the microbial composition and aerosol information of the airway. It is particularly suitable for high-risk populations with interstitial lung disease, rheumatic immune diseases, and other conditions requiring long-term immunosuppressant therapy, as well as clinical scenarios requiring frequent sampling to monitor dynamic changes. However, despite the significant clinical translational value of EBC, its entire process from collection to detection still faces multiple technical bottlenecks in practical applications, resulting in low success rates, unreliable results, and difficulty in integrating it into standardized clinical diagnostic pathways. Existing EBC collection devices (such as the RTube handheld EBC collector)... Figure 2 A) EcoScreen (a commonly used condensation platform for research / clinical studies) Figure 2 (B) Domestic portable "exhaled breath condensate collector / BioscreenII" type products ( Figure 2 The C) etc. method mainly solves the problems of condensation and collection, but has the following significant drawbacks: 1. The problem of fragmented collection and detection processes: In existing technologies, the EBC collection device and the microbial detection system are independent of each other. After collection, samples must undergo open operation steps such as manual opening, pipetting, transferring, and sample addition before entering the detection stage. For EBC, a natural ultra-dilute matrix, its microbial load is much lower than that of sputum or lavage fluid. Open manual operation may introduce exogenous contamination (leading to false positives), or cause significant loss of the already scarce sample due to adsorption, volatilization, and residue on the tube wall (leading to false negatives). This loss has a non-linear amplification effect under low biomass conditions, directly determining the detection results.

[0003] 2. Most existing collection devices cannot accurately quantify exhaled air volume, condensate volume, and collection efficiency. Condensation efficiency is easily affected by fluctuations in ambient temperature, humidity, and the subject's expiratory air flow, resulting in a lack of quantitative comparability between different subjects, and even between samples from the same subject at different time points, which cannot meet clinical monitoring needs.

[0004] 3. Mismatch between condensation and detection processes. EBC matrix lacks cell sedimentation carriers and has low viscosity, making it impossible to use traditional centrifugation sedimentation methods for microbial enrichment. Existing microbial detection platforms (including various POCT devices) are mostly designed for high biomass samples (such as swabs and sputum), and their sample loading volume, purification process, and amplification conditions are seriously mismatched with the characteristics of EBC: "small volume, high dilution, and weak signal". Summary of the Invention

[0005] This invention aims to solve at least one of the technical problems existing in the prior art. Addressing the issues of the separation between existing EBC collection and detection processes and the susceptibility of low biomass samples to contamination and loss, this invention provides a microbial detection system for exhaled breath condensate samples. Taking into account the special physicochemical properties of EBC, this system integrates collection, quantification, enrichment, processing, and detection into a closed-loop integrated flow path to eliminate contamination and loss caused by manual transfer and to achieve quantitative control and enrichment processing of low biomass samples.

[0006] This application discloses a microbial detection system for exhaled breath condensate samples, comprising an exhaled breath collection module, a condensation and quantification module, a microbial enrichment and pretreatment module, and a microbial detection module, which are sequentially connected through a closed flow path structure. The exhaled breath collection module is used for non-invasive collection of the subject's exhaled breath; The condensation and quantification module is sealed and connected to the exhaled air acquisition module. It is used to condense the exhaled air into condensate and quantitatively monitor the volume of the condensate. When the volume of the condensate reaches a preset threshold, it controls the condensate to flow into the downstream module. The microbial enrichment and pretreatment module is sealed and connected to the condensation and quantification module. It is used to receive and automatically enrich and lyse the microorganisms in the condensate to obtain a condensate with increased microbial target concentration. The microbial detection module is connected to the output of the microbial enrichment and pretreatment module and is used to detect the condensate with increased microbial target concentration.

[0007] By constructing a closed-loop, integrated sample transfer architecture and specifically introducing microbial enrichment and pretreatment modules, this application has the following beneficial effects: 1. This application utilizes a closed-loop flow path structure to achieve completely manual transfer without opening the cap, from breath sample collection, condensation quantification, microbial enrichment, in-situ lysis to final detection. This design fundamentally blocks the introduction of exogenous contamination and avoids the loss of low-biomass samples due to adsorption and volatilization on the tube walls during pipetting and transfer. Addressing the characteristic of EBC samples where microbial signals are close to the detection limit, this system maximizes the retention of effective sample volume, transforming potential false negatives due to trace losses into detectable true positives, significantly improving the detection success rate. It ensures sample integrity from the source, blocks contamination and loss, and guarantees the accuracy of the detection.

[0008] 2. This application achieves repeatable and comparable standardized detection through quantitative condensation. The system disclosed in this application integrates a liquid volume sensing structure and an automatic control system in the condensation module, which can monitor the volume of condensate in real time and automatically start the quantitative transfer program when the preset threshold is reached. This mechanism ensures that the condensation volume, sample loading amount and detection conditions are highly consistent each time, eliminating the uncertainty of dilution effect caused by environmental fluctuations or differences in human operation. This makes the test results of different time points and different subjects have strict quantitative comparability, meeting the data consistency requirements of clinical longitudinal monitoring and multi-center application.

[0009] 3. Addressing the extremely low microbial abundance in EBC samples, this application incorporates a dedicated microbial enrichment and pretreatment module within a closed flow path. Utilizing microfiltration structures and surface modification technologies (charged coatings and bioaffinity layers), it efficiently traps and enriches target microorganisms during sample flow, raising their concentration to a level identifiable by conventional detection methods. Subsequently, the enriched microorganisms are lysed in situ via an integrated thermal or chemical lysis unit, releasing the nucleic acid template without transfer. This design, employing a dual enrichment mechanism of size trapping and affinity adsorption, not only solves the problem of traditional centrifugation methods being unsuitable for EBCs but also achieves efficient in-situ enrichment of ultra-diluted samples, raising the microbial concentration to a level identifiable by conventional detection methods and overcoming the bottleneck in detecting low-abundance samples.

[0010] 4. The system adopts a 1–100 μL micro-scale detection chamber, equipped with optimized nucleic acid detection, optical detection or electrochemical detection units, which are adapted to the small volume and weak signal matrix characteristics of EBC, and solve the problem of incompatibility between existing detection platforms and EBC samples.

[0011] 5. This system retains the advantages of EBC collection being non-invasive and painless, while reducing the operational threshold through automated integration. It is particularly suitable for high-risk groups such as the elderly, those with impaired lung function, and those with immunosuppression who are not suitable for invasive sampling (such as bronchoalveolar lavage). It provides a reliable tool for early screening of respiratory infections, pathogen spectrum assessment, and monitoring of treatment response. Attached Figure Description

[0012] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0013] Figure 1 This is a schematic diagram of a microbial detection system for exhaled breath condensate samples provided in an embodiment of the present invention; Figure 2 This is a schematic diagram of an existing EBC acquisition device provided in an embodiment of the present invention, wherein, Figure 2 In this context, A represents the RTube handheld EBC data collector. Figure 2 B in the text stands for EcoScreen (a condensation platform commonly used in research / clinical studies). Figure 2 The "C" in the text refers to domestic portable "exhaled breath condensate collector / BioscreenII" products. Figure 3 This is a schematic diagram of the exhaled breath collection module provided in an embodiment of the present invention; Figure 4 This is a schematic diagram of the condensation and metering module structure provided in an embodiment of the present invention; Figure 5 This is a schematic diagram of the structure of the microbial enrichment and pretreatment module provided in an embodiment of the present invention; Figure 6 This is a schematic diagram of the microbial detection module structure provided in an embodiment of the present invention; Figure 7 This is a schematic diagram of the result output module structure provided in an embodiment of the present invention; 1. Disposable mouthpiece; 2. One-way valve assembly; 3. Saliva backflow prevention chamber; 4. Quick-connect interface; 5. Condensation chamber; 6. Condensation element; 7. Liquid collection port; 8. Liquid volume sensing structure; 9. Microvalve / pump; 10. Control system; 11. Inlet; 12. Microfluidic enrichment chamber; 13. Microfiltration structure; 14. Electrostatic coating; 15. Bioaffinity layer; 16. Drain port; 17. Pyrolysis unit; 18. Detection chamber; 19. Detection element; 20. Signal output interface; 21. Data interface; 22. Display screen; 23. Indicator light. Detailed Implementation

[0014] To enable those skilled in the art to better understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0015] In some of the processes described in the specification, claims, and accompanying drawings of this invention, multiple operations appearing in a specific order are included. However, it should be clearly understood that these operations may not be executed in the order they appear herein, or may be executed in parallel. The operation numbers, such as 101, 102, etc., are merely used to distinguish different operations and do not represent any execution order. Furthermore, these processes may include more or fewer operations, and these operations may be executed sequentially or in parallel. It should be noted that the descriptions such as "first," "second," etc., in this document are used to distinguish different messages, devices, modules, etc., and do not represent a sequential order, nor do they limit "first" and "second" to different types.

[0016] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0017] Figure 1 This is a schematic diagram of a microbial detection system for exhaled breath condensate samples provided in an embodiment of the present invention. Specifically, the system includes an exhaled breath collection module, a condensation and quantification module, a microbial enrichment and pretreatment module, and a microbial detection module, which are sequentially connected through a closed flow path structure. It also includes a control system 10 for receiving liquid volume sensing signals, controlling the operation of microvalves / micropumps, and recording volume information. After the exhaled breath enters the system, the sample remains in a closed flow during condensation, transfer, enrichment, lysis, and detection processes to reduce contamination, loss, and operational errors caused by open processing.

[0018] The exhaled air collection module is used to non-invasively collect the exhaled air of the subject and introduce the gas into the condensation and quantification module; In some embodiments, the terms “subject” or “test subject” or “sample” as used herein refer to any animal (e.g., a mammal), including but not limited to humans, non-human primates, rodents, etc., which will become the recipient of a particular treatment. Generally, the terms “subject” and “patient” are used interchangeably herein when referring to human subjects. Preferably, the subject is a human.

[0019] In some embodiments, the exhaled air collection module includes a disposable mouthpiece 1, a one-way valve, and a saliva backflow prevention chamber 3 connected in sequence; the cross-sectional area of ​​the saliva backflow prevention chamber 3 is enlarged and a saliva collection section is provided to reduce the airflow speed and allow saliva droplets to settle.

[0020] In a more specific embodiment, as an example, a detailed structural description is given of the exhaled air collection module used to guide the exhaled air of a subject into the system. However, those skilled in the art will understand that this structure is not unique, and it can also be other reasonably deducible structures, such as... Figure 3 As shown, in this embodiment, the exhaled air collection module includes a disposable mouthpiece 1, a one-way valve assembly 2, a saliva backflow prevention chamber 3, and a quick-connect interface 4 (detachable) connected in sequence. The disposable mouthpiece 1 is detachably connected to the module body and is used by the subject to exhale through their mouth. The one-way valve assembly 2 is located downstream of the disposable mouthpiece 1, allowing airflow from the oral cavity to the interior of the system and restricting reverse flow. The saliva backflow prevention chamber 3 is located downstream of the one-way valve assembly 2, and its cross-section is larger than that of the adjacent gas flow channel. It has a saliva collection groove at the bottom to trap saliva carried in the exhaled air. The quick-connect interface 4 is located downstream of the saliva backflow prevention chamber 3 and is used for a detachable sealed connection between the exhaled air module and the condensation and metering module. The shape of the saliva collection groove, the shape of the disposable mouthpiece 1, and other reasonably deformable structures are not specifically limited in this embodiment.

[0021] The condensation and quantification module is sealed and connected to the exhaled air acquisition module. It is used to condense the exhaled air into condensate and quantitatively monitor the volume of the condensate. When the volume of the condensate reaches a preset threshold, it controls the condensate to flow into the downstream module. In some embodiments, the condensation and metering module includes a condensation chamber 5, a liquid volume sensing structure 8, an automatic transfer channel, and a control system 10; The liquid volume sensing structure 8 is disposed at the sample collection point of the condensation chamber 5. The automatic transfer channel connects the condensation chamber 5 and the inlet of the microbial enrichment and pretreatment module, and is equipped with a controlled microvalve and micropump. The control system 10 is connected to the liquid volume sensing structure 8, the microvalve, and the micropump. Based on the feedback signal from the liquid volume sensing structure 8, when the condensate volume reaches the preset threshold, it automatically opens the microvalve and starts the micropump to deliver a fixed amount of condensate into the downstream module, thereby ensuring consistent sample loading volumes across different collections. Of course, to guarantee quantitative measurement, this embodiment includes a liquid level sensor, such as a flow-through sheath tank.

[0022] In a more specific embodiment, as an example, a condensation and quantification module for condensing water vapor and aerosols in exhaled breath to form a liquid sample and controlling the collection process is described in detail. However, those skilled in the art will understand that this structure is not unique, and it can also be other structures that can be reasonably derived, such as... Figure 4As shown, in this embodiment, the top of the condensing chamber 5 is provided with a quick-connect interface 4 for sealing connection with the exhaled air collection module. The condensing chamber 5 is provided with a condensing element 6 inside or on the outer wall for condensing water vapor and aerosol in the exhaled air into a liquid sample. The bottom of the condensing chamber 5 is provided with a liquid collection port 7. The liquid volume sensing structure 8 is set at or near the liquid collection port 7 for real-time monitoring of the volume of condensate or the number of droplets. The automatic transfer channel connects the liquid collection port 7 to the inlet of the downstream microbial enrichment and pretreatment module. The microvalve / pump 9 is set on the automatic transfer channel between the liquid collection port 7 and the inlet 11. The control system 10 is electrically connected to the liquid volume sensing structure 8, the microvalve, and the micropump. When the liquid volume sensing structure 8 detects that the volume of condensate reaches a preset threshold, the control system 10 controls the microvalve to open and starts the micropump to quantitatively transfer the condensate to the microbial enrichment and pretreatment module. Microvalves and micropumps are the two core control and actuation components in a lab-on-a-chip microfluidic chip, working together to achieve precise control of minute amounts of liquid (microliters / picolittles). In this embodiment, the micropump provides the power for liquid flow, while the microvalves provide precise control over the path and timing of the flow; existing brands can be used directly.

[0023] The microbial enrichment and pretreatment module is sealed and connected to the condensation and quantification module. It is used to receive and automatically enrich and lyse the microorganisms in the condensate to obtain a condensate with increased microbial target concentration. In some embodiments, the microbial enrichment and pretreatment module includes an integrated microfluidic chip, wherein a microfluidic enrichment cavity 12 and a lysis unit 17 are integrated within the microfluidic chip; The microfluidic enrichment chamber 12 is equipped with a microfiltration structure 13 for physically retaining microorganisms from the flowing condensate. The surface of the microfiltration structure 13 is coated with a coating to enhance microorganism adsorption. The lysis unit 17 is located in the microfluidic enrichment chamber 12 and is used to perform in-situ lysis of the microorganisms retained on the microfiltration structure 13.

[0024] In some embodiments, the pore size of the microfiltration structure 13 is 0.22-0.45 μm.

[0025] In some embodiments, the surface of the microfiltration structure 13 is sequentially modified with a charged coating 14 and a bioaffinity layer 15; the charged coating 14 is used to non-specifically capture microorganisms by electrostatic adsorption; the bioaffinity layer 15 is immobilized with specific antibodies, lectins or aptamers for specific capture of target microorganisms.

[0026] In some embodiments, the pyrolysis unit 17 includes a thermal pyrolysis component and / or a chemical pyrolysis component. The thermal pyrolysis component includes a micro heater and a temperature sensor integrated into the region of the microfluidic enrichment cavity 12; the chemical pyrolysis component includes a lyophilized pyrolysis reagent chamber pre-embedded in the chip flow path.

[0027] In a more specific embodiment, as an example, a microbial enrichment and pretreatment module for enriching, retaining, stabilizing, and / or lysing microorganisms in condensed samples is described in detail. However, those skilled in the art will understand that this structure is not unique, and it can also be other structures that can be reasonably derived, such as... Figure 5 As shown, in this embodiment, the microbial enrichment and pretreatment module specifically includes an integrated microfluidic chip. The chip is equipped with an inlet 11, a microfluidic enrichment cavity 12, a microfiltration structure 13, a charged coating 14, a bioaffinity layer 15, a drain 16, and a lysis unit 17. The inlet 11 receives condensate flowing in through the automatic transfer channel. The microfluidic enrichment cavity 12 has pores with a diameter of 0.22–0.45 mm. The chip features a microfiltration structure 13 (μm in diameter), with a charged coating 14 and a bioaffinity layer 15 sequentially coated on its surface. The charged coating 14 can capture negatively charged microbial cell walls through electrostatic adsorption. The bioaffinity layer 15 is coated with specific antibodies, lectins, or aptamers to specifically capture target microorganisms through bioaffinity. The chip also integrates a lysis unit 17, which can be configured for both thermal and chemical lysis. Thermal lysis utilizes a micro-heating membrane (platinum resistance thermometer or indium tin oxide) attached to the bottom of the enrichment chamber, coupled with a temperature sensor, to achieve precise temperature control from 50–95°C. Chemical lysis can be pre-positioned with a lysis reagent chamber (containing proteinase K and lyophilized spheres of surfactants). The lysis unit 17, corresponding to the microfluidic enrichment chamber 12, is used for in-situ lysis of microorganisms enriched on the microfiltration structure 13 and / or the inner wall of the flow channels. A drain port 16 is used to transfer the condensate from microbial lysis to the next module.

[0028] The microbial detection module is connected to the output of the microbial enrichment and pretreatment module and is used to detect the condensate with increased microbial target concentration.

[0029] In some embodiments, the microbial detection module includes a detection chamber 18 with a volume of 1–100 μL, and at least one of a nucleic acid detection unit, an optical detection unit, or an electrochemical detection unit; the detection chamber 18 is connected to the drain port 16 of the microbial enrichment and pretreatment module through a zero dead volume interface.

[0030] In a more specific embodiment, as an example, a specific structural description is given of a microbial detection module for detecting microorganisms or their derived signals. However, those skilled in the art will understand that this structure is not unique, and it can also be other structures that can be reasonably derived, such as... Figure 6 As shown, in this embodiment, the microbial detection module specifically includes a detection chamber 18, a detection element, and a signal output interface 20. The detection chamber 18 is directly connected to the drain port 16 of the microbial enrichment and pretreatment module, and the two can be integrally formed or connected through a zero-dead-volume interface. The internal volume of the detection chamber 18 is 1-100 μL. The detection element 19 can be a nucleic acid amplification detection unit, an optical detection unit, or an electrochemical detection unit. The signal output interface 20 is electrically connected to the detection unit and is used to convert the detection signal into a standardized digital signal for output to an external terminal.

[0031] In some embodiments, the system further includes a result output module electrically connected to the microbial detection module, for receiving, displaying, storing or transmitting detection results and sample volume information for this detection.

[0032] In a more specific embodiment, as an example, a specific structural description is given of a result output module used to display, store, or output detection results. However, those skilled in the art will understand that this structure is not unique, and it can also be other structures that can be reasonably derived, such as... Figure 7 As shown, in this embodiment, the result output module specifically includes a display screen 22, an indicator light 23, and a data interface 21. The display screen 22 is used to display test results, quality control status, and sample traceability information. The display screen 22 can be a touch screen or a non-touch screen. The indicator light 23 is a multi-color LED light group, which indicates the instrument's operating status and test results through the combination of light color, on / off state, and flashing frequency. The data interface 21 includes a wired interface and a wireless communication module, which is used to output the test results to an external terminal in a standardized format. The result output module also has a non-volatile memory for local storage of test records.

[0033] Furthermore, the working principle of this system during use is as follows: Subjects exhale through the exhaled breath collection module. The exhaled breath enters the condensation and quantification module and is condensed to form an exhaled breath condensate sample. Once the condensate sample meets the preset conditions, it automatically enters the microbial enrichment and pretreatment module, where microbial enrichment and necessary pretreatment are completed.

[0034] The processed sample then enters the microbial detection module, where microbial signals are acquired through appropriate detection methods. The results are ultimately displayed or output by the results output module. Throughout the entire process, the sample remains within a closed system to prevent external interference.

[0035] In some embodiments, the threshold in the above scheme can be determined based on experience or obtained by training with training set samples. It can be a specific threshold or an interval range. The specific form is not specifically limited in this embodiment.

[0036] In some embodiments, to verify the advantages of the present invention, the following experiments were designed to simulate "artificial transfer loss" and "enrichment module synergy": Experiment 1: Simulating "Artificial Transfer Loss" 1. Experimental Materials The materials used in this experiment are as follows: (1) Sample source Exhaled breath condensate (EBC) sample, Source: Data collected from the exhaled breath of healthy subjects. Single collection volume: approximately 1500 μL. Dispensing method: Divided into 2 portions, each 750 μL, for different processing methods.

[0037] (2) DNA extraction kit Name: QIAamp PowerFecal Pro DNA Kit, Manufacturer: QIAGEN (Germany) Item No.: 51804 Function: Used for microbial DNA extraction from low biomass samples.

[0038] (3) Nucleic acid precipitation reagent (enrichment treatment) Sodium acetate: 3 M concentration, pH 5.2.

[0039] Anhydrous ethanol (Ethanol): Concentration is 95%.

[0040] 70% ethanol (for washing); TE buffer: Composed of Tris-HCl + EDTA, used to dissolve DNA precipitates.

[0041] (4) PCR and qPCR reagents SYBR Green qPCR reaction system; primer sequences.

[0042] (5) Electrophoresis-related reagents Agarose, DNA Marker (100 bp–2000 bp), Electrophoresis Buffer (TAE), Nucleic Acid Dyes.

[0043] (6) Experimental results One transfer → Effective nucleic acid / DNA recovery decreases by approximately 40%. After 10 transfers, the effective nucleic acid / DNA recovery rate decreases by approximately 60%. Adding an open exposure step results in an approximately 75% decrease in effective nucleic acid / DNA recovery. The above results suggest that manual opening of the cap, pipetting, transfer, and open exposure can reduce the effective nucleic acid recovery in EBC samples; for samples with low biomass and close to the detection limit, this loss may lead to a decrease in detection rate or fluctuation in results.

[0044] Experiment 2: The synergistic effect of the simulated enrichment module on detection 1. Experimental objective: To verify the effect of enrichment treatment on improving the detection capability of microbial DNA in EBC samples.

[0045] 2. Group design:

[0046] 3. Experimental Procedure Method 1 (Enrichment): 1) Add 1 / 10 of the original sample volume of 3M sodium acetate to 1 mL of EBC sample. 2) Add 2.5–3 times the volume of 95% ethanol. 3) Incubate overnight at room temperature. 4) Centrifuge at 4℃ and 10000 rpm for 30 min. 5) Abandon Shangqing, 6) Wash with 70% ethanol. 7) Centrifuge for another 15 minutes. 8) Resuspend in 300 μL TE buffer. 9) Store at -80℃, 10) DNA extraction will be performed subsequently.

[0047] Method 2 (Non-enrichment) 1) EBC samples were directly frozen (-80℃). 2) Extract DNA directly according to the kit instructions.

[0048] 4. Detection Method DNA quantification (ng / μL) qPCR testing Agarose gel electrophoresis was used to verify the amplified products.

[0049] 5. Experimental Results Under the conditions of this batch of experiments, after enrichment treatment, the DNA quantification value was increased by approximately 68% compared to the unenriched direct detection group. The purity is basically the same (indicating that it is not a false signal).

[0050] Conclusion: Enrichment processing helps improve the detection signal of low biomass EBC samples.

[0051] The results of the above experiments show that: 1) Manual opening, pipetting, and tube transfer may cause loss of effective nucleic acids in EBC samples; 2) Open exposure may increase sample instability and the risk of exogenous contamination; 3) Enrichment treatment can improve the recovery and detection signal of microbial nucleic acids in low biomass samples; 4) At low concentrations and close to the detection limit, differences in transfer loss, open exposure, and enrichment efficiency can affect the stability of detection results.

[0052] Therefore, this application solves the problem of unstable detection of exhaled condensate samples from a system-level perspective by using a closed sample transfer and enrichment processing structure.

[0053] The exemplary embodiments of this disclosure described in detail above are merely illustrative and not restrictive. Those skilled in the art will understand that various modifications and combinations can be made to these embodiments or their features without departing from the principles and spirit of this disclosure, and such modifications should fall within the scope of this disclosure.

Claims

1. A microbial detection system for exhaled breath condensate samples, characterized in that, It includes an exhaled breath collection module, a condensation and quantification module, a microbial enrichment and pretreatment module, and a microbial detection module, which are sequentially connected through a closed flow path structure. The exhaled breath collection module is used for non-invasive collection of the subject's exhaled breath; The condensation and quantification module is sealed and connected to the exhaled air acquisition module. It is used to condense the exhaled air into condensate and quantitatively monitor the volume of the condensate. When the volume of the condensate reaches a preset threshold, it controls the condensate to flow into the downstream module. The microbial enrichment and pretreatment module is sealed and connected to the condensation and quantification module. It is used to receive and automatically enrich and lyse the microorganisms in the condensate to obtain a condensate with increased microbial target concentration. The microbial detection module is connected to the output of the microbial enrichment and pretreatment module and is used to detect the condensate with increased microbial target concentration.

2. The microbial detection system for exhaled breath condensate samples according to claim 1, characterized in that, The condensation and metering module includes a condensation chamber, a liquid volume sensing structure, an automatic transfer channel, and a control system. The liquid volume sensing structure is located at the sample collection point of the condensation chamber; The automatic transfer channel connects the condensation chamber and the inlet of the microbial enrichment and pretreatment module, and is equipped with a controlled microvalve and micropump. The control system is connected to the liquid volume sensing structure, micro valve, and micro pump signal, and is used to automatically open the micro valve and start the micro pump when the condensate volume reaches the preset threshold based on the feedback signal from the liquid volume sensing structure, so as to flow a certain amount of condensate into the downstream module.

3. The microbial detection system for exhaled breath condensate samples according to claim 1, characterized in that, The microbial enrichment and pretreatment module includes an integrated microfluidic chip, which integrates a microfluidic enrichment cavity and a lysis unit. The microfluidic enrichment chamber is equipped with a microfiltration structure for physically retaining microorganisms from the flowing condensate, and the surface of the microfiltration structure is coated to enhance microbial adsorption. The lysis unit corresponds to the position of the microfluidic enrichment cavity and is used to perform in-situ lysis of microorganisms trapped on the microfiltration structure.

4. The microbial detection system for exhaled breath condensate samples according to claim 3, characterized in that, The pore size of the microfiltration structure is 0.22-0.45 μm.

5. The microbial detection system for exhaled breath condensate samples according to claim 3, characterized in that, The surface of the microfiltration structure is sequentially modified with a charged coating and a bioaffinity layer; the charged coating is used to non-specifically capture microorganisms by electrostatic adsorption; the bioaffinity layer is immobilized with specific antibodies, lectins or aptamers for specific capture of target microorganisms.

6. The microbial detection system for exhaled breath condensate samples according to claim 3, characterized in that, The pyrolysis unit includes a thermal pyrolysis component and / or a chemical pyrolysis component.

7. The microbial detection system for exhaled breath condensate samples according to claim 6, characterized in that, The thermal pyrolysis assembly includes a micro heater and a temperature sensor integrated into the microfluidic enrichment cavity region; the chemical pyrolysis assembly includes a freeze-dried pyrolysis reagent chamber pre-embedded in the chip flow path.

8. The microbial detection system for exhaled breath condensate samples according to claim 1, characterized in that, The exhaled air collection module includes a disposable mouthpiece, a one-way valve, and a saliva backflow prevention chamber connected in sequence; the cross-section of the flow channel of the saliva backflow prevention chamber is enlarged and a saliva collection part is provided to reduce the airflow speed and cause saliva droplets to settle.

9. The microbial detection system for exhaled breath condensate samples according to claim 1, characterized in that, The microbial detection module includes a detection chamber with a volume of 1–100 μL, and at least one of a nucleic acid detection unit, an optical detection unit, or an electrochemical detection unit; the detection chamber is connected to the drain port of the microbial enrichment and pretreatment module through a zero dead volume interface.

10. The microbial detection system for exhaled breath condensate samples according to claim 1, characterized in that, The system also includes a result output module electrically connected to the microbial detection module, used to receive, display, store or transmit the detection results and the sample volume information of this test.

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