Exhaled gas enrichment mask for detecting bacteria in exhaled gas

Abstract

The utility model discloses an exhaled air enrichment mask for detecting bacteria in exhaled air, which comprises a mask body and a filter valve, the filter valve comprises a filter element, and the filter element sequentially comprises five layers of non-woven fabric, melt-blown fabric, hot air cotton, melt-blown fabric and non-woven fabric from inside to outside.

Description

Technical Field

[0001] The utility model relates to an exhaled air enrichment mask for detecting microorganisms (such as Mycobacterium tuberculosis) in exhaled air, and belongs to the field of medical detection. Background Art

[0002] Tuberculosis (TB) remains a major public health problem worldwide due to its human-to-human transmission and high morbidity and mortality rates. In 2021, there were approximately 8 million new recorded cases worldwide, in addition to approximately 4.1 million TB patients who were missed due to lack of timely diagnosis, especially in third world countries. The key to controlling the spread of TB and reducing mortality is to diagnose the source of infection in the population in a timely and accurate manner. Tuberculosis (TB) is caused by Mycobacterium tuberculosis (MTB) and is mainly transmitted through the air. Healthy people become infected by inhaling live MTB released in the form of aerosols when infected people cough, sneeze, talk or spit. The infection mode of TB leads to its high prevalence and high transmission rate, and also makes early diagnosis of TB more difficult. At present, conventional detection methods for Mycobacterium tuberculosis include culture, imaging detection, sputum smear microscopy, serological detection and molecular diagnosis. However, most of these diagnostic methods use sputum as samples, which is time-consuming, relies on large equipment, and requires professional technicians. Sputum, the main clinical sample for TB analysis, is a particularly difficult matrix to obtain and handle, and needs to be collected and analyzed in a laboratory setting. In addition, sputum samples have low diagnostic sensitivity for children, HIV-infected patients, and extrapulmonary TB patients. These issues have become a bottleneck for conducting widespread TB screening.

[0003] An ideal point-of-care (POC) test device for infectious disease screening in resource-limited areas should be rapid, economical, accurate, non-invasive, well-compliant, and easy to operate. As a commonly used non-invasive sampling method, exhaled breath samples are widely considered to be a promising strategy for diagnosing tuberculosis. Compared with the complex composition of sputum, serum, and urine samples, the components of exhaled breath are simple, mainly including small molecules, fats, proteins, and pathogens. Exhaled breath samples are mostly collected non-invasively, which is simple to operate and has good patient compliance, which is more conducive to the early diagnosis and disease monitoring of tuberculosis. Recently, the study of Benjamin Patterson et al. suggested that non-cough transmission may exist in patients with pulmonary tuberculosis, and confirmed that the exhaled breath of tuberculosis patients contains culturable MTB. It is feasible and repeatable to collect exhaled breath samples for testing.

[0004] At present, various POC platforms for disease diagnosis using exhaled breath samples have been developed. For example, Zhu Tong et al. developed a real-time monitoring system for airborne H3N1 influenza virus by integrating silicon nanowire field effect transistor (SiNW) sensor devices, microfluidics and bioaerosol air sampling technology; Sousa et al. developed an electrostatic air sampler for collecting cough samples from tuberculosis patients; Ruth McNerney et al. designed a new device RBS breathalyzer system using immunosensors and bio-optical technology to detect Mycobacterium tuberculosis antigens in human breath. Unfortunately, the above-mentioned exhaled breath detection devices all rely on complex, precise and expensive exhaled breath collection devices, and require instrument assistance to complete the detection. Therefore, POC detection of tuberculosis diagnosis using pathogenic microorganisms collected in exhaled breath is still challenging.

[0005] Since the pathogen content in exhaled breath samples is low, it is necessary to combine signal amplification strategies for detection. Nucleic acid isothermal amplification technology is increasingly widely used for pathogen POC detection due to its advantages of high sensitivity, strong specificity, rapidity (5-60 minutes), and no need for special equipment. Common isothermal amplification technologies include rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), and recombinase polymerase amplification (RPA). Among the many emerging nucleic acid isothermal amplification technologies, RPA technology has a low reaction temperature (25-42°C), strong amplification ability, simple system, and high sample tolerance. Although it appeared later, it has developed rapidly and has become a popular technology for the development of POC detection methods. In addition, lateral flow analysis (LFA) as a visual signal reading method has been widely used in pathogen nucleic acid detection. For example, Wansadaj Jaroenram et al. developed a method for visual detection of MTB in sputum by combining LAMP with lateral flow test paper. Unfortunately, this isothermal amplification relies on complex primer design, and the samples used for diagnosis are still sputum samples. The complex sample pretreatment process is not suitable for POC testing. It is precisely because the content of MTB in exhaled breath is low, the sample loss is high, and the sensitivity of the detection method is required to be high that there is no research on detecting MTB in exhaled breath through nucleic acid analysis to achieve TB diagnosis. Utility Model Content

[0006] The purpose of the utility model is to provide a detection method for detecting bacteria in exhaled breath, to solve the problems of long time consumption, complexity, high cost, poor patient compliance, etc. caused by the prior art relying on sputum detection to screen respiratory infectious diseases, and to provide a fast, economical, accurate, non-invasive, compliant, and easy-to-operate detection equipment and detection method suitable for conducting infectious disease (such as tuberculosis) screening at nursing points in resource-limited areas.

[0007] In the present utility model, we designed and assembled for the first time a mask for enriching pathogenic microorganisms in exhaled air (N95 exhaled air enrichment mask, N95-Enrichment-Respirator, N95-ER) that combines a cardiopulmonary resuscitation (CPR) first aid mask with an N95 mask, and successfully applied it to a tuberculosis diagnosis platform. N95 masks are widely used in the field of infectious disease protection due to their superior pathogenic microorganism barrier capability (PM2.5 barrier rate is greater than 95%). In addition, the CPR first aid mask used to prevent cross-infection between first aid personnel and the injured during first aid is used in the present utility model for the collection of exhaled air due to its advantages of acid resistance, alkali resistance, corrosion resistance, high temperature resistance, high temperature sterilization, and reusability. Compared with the existing exhaled air collection device, N95-ER has low cost, simple assembly, simple operation, high enrichment efficiency, and is more conducive to mass production and promotion. Therefore, the N95-ER designed by the present utility model is more suitable for enriching MTB in exhaled air to achieve TB diagnosis detection equipment.

[0008] In order to achieve the above-mentioned purpose, the utility model provides an exhaled air enrichment mask for detecting bacteria in exhaled air, which is composed of a cardiopulmonary resuscitation mask, in which the original one-way valve of the cardiopulmonary resuscitation mask is replaced with a filter valve for capturing and enriching bacteria in exhaled air, and the filter valve is composed of three parts: a valve cover, a filter element, and a valve bottom, wherein the filter element is composed of five layers from the inside to the outside, namely, non-woven fabric, melt-blown fabric, hot air cotton, melt-blown fabric, and non-woven fabric.

[0009] The utility model relates to a detection method for detecting bacteria in exhaled air, comprising the following steps: S1 enriching microorganisms in the exhaled air; S2 eluting the microorganisms; S3 extracting microorganism nucleic acids from the eluate; S4 isothermal amplification; S5 detecting microorganism nucleic acids, wherein the step S1 is to enrich the microorganisms in the exhaled air through an exhaled air enrichment mask, the step S3 is to extract microorganism nucleic acids through an electric lysis method, the step S4 is RPA isothermal amplification, and the step S5 is to detect the result through a lateral flow analysis experiment.

[0010] The utility model is an exhaled air enrichment mask for detecting bacteria in exhaled air, which is used in step S1 of the aforementioned detection method for detecting bacteria in exhaled air. The use of an enrichment mask to collect the patient's exhaled air is to replace the traditional sputum sample detection method. The sputum sample must be the deep sputum of the patient when he wakes up early in the morning, and deep coughing is required to obtain sputum that meets the requirements (for example, containing tuberculosis bacteria), and it must be collected by professionals in a medical unit with certain conditions. When collecting the patient's exhaled air, no special time or special place is required, so that the patient can wear a mask for enough time to meet the detection requirements. The enrichment efficiency of enrichment using the exhaled air enrichment mask of the utility model is much higher than that of microfluidic chip enrichment, and it can be done quickly. The error of chip enrichment is relatively low.

[0011] In step S2, the optimal elution method developed by ourselves and matched with the exhaled air enrichment mask in S1 is preferably used to obtain the optimal elution efficiency.

[0012] In step S3, the optional nucleic acid extraction methods include mechanical lysis, enzymatic lysis, chemical lysis, electric lysis, etc. Preferably, electric lysis of a direct current bacterial nucleic acid extraction microfluidic chip is used, which has the advantages of short time, miniaturization, low consumption, integration, small sample amount, and avoidance of chemical reagent contamination.

[0013] In step S4, the optional nucleic acid amplification methods include rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA) and recombinase polymerase amplification (RPA), etc., and the RPA nucleic acid amplification method is preferred, which has the advantages of low reaction temperature (25-42°C), strong amplification ability, simple system and high sample tolerance.

[0014] In step S5, the optional nucleic acid detection methods include lateral flow analysis (LFA), real-time fluorescence quantitative PCR (RT-qPCR), etc., preferably LFA, which can be detected by LFA test paper, has the advantages of rapid detection, convenient operation, visualization, etc. RT-qPCR, as a mature detection method, can verify the correctness of LFA results.

[0015] If the process of exhaled breath enrichment mask enrichment + elution + chip electrolysis + RPA + LFA is used for detection, the whole detection process will not exceed 1.5 hours, and no large-scale instrument assistance is required. Patients can complete the detection independently at home, which is more suitable for large-scale screening of tuberculosis. Moreover, it is simple to operate, highly sensitive, and low-cost, and its application can be expected to be extended to other aerosol infectious disease diagnosis and infection detection. This has great advantages for respiratory disease detection in areas with scarce medical resources, greatly reducing costs, and also reducing the burden on patients and medical workers, solving technical problems that have long been wanted to be solved but have not been solved. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The scheme of the utility model and its beneficial technical effects are described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0017] Figure 1 A workflow for a method for detecting bacteria in exhaled breath;

[0018] Figure 2 The structure diagram of the aerosol enrichment mask; the shape and size of the filter element is preferably a circle with a diameter of 2.5 cm

[0019] Figure 3To optimize the pathogen enrichment conditions, including (A) cartridge structure (from inside to outside: ① non-woven fabric; ② melt-blown non-woven fabric; ③ ES hot air cotton; ④ melt-blown non-woven fabric; ⑤ non-woven fabric); (B) eluent composition; (C) elution volume; (D) elution time; Cartridge Construciton (cartridge structure); Composition of Eluent (elution liquid composition); Elution Liquid Volume (elution liquid volume); Elution Times (elution time)

[0020] Figure 4 (A) Structure diagram and (B) Physical diagram of bacterial nucleic acid extraction chip; Lysis well; ITO electrode; PDMS lysis wells; Microfluidic chip; ITO coated glasses

[0021] Figure 5 The conditions for the direct current bacterial nucleic acid extraction chip to extract nucleic acid from Mycobacterium smegmatis were investigated; (A) Optimization of the electric field intensity of Mycobacterium smegmatis; (B) The results of Mycobacterium smegmatis plate culture after lysis; (C) The results of Mycobacterium smegmatis plate culture without treatment; Thermal lysis (thermal cycle lysis)

[0022] Figure 6 The results of condition optimization for recombinase polymerase amplification, (A) gel electrophoresis of reaction temperature, (B) lateral flow analysis of reaction temperature, (C) gel electrophoresis of reaction time, (D) lateral flow analysis of reaction time; Reaction Temperature (reaction time); Reaction Times (reaction time); Test Band (test line); Control Band (quality control line)

[0023] Figure 7 This is the gel electrophoresis of RPA amplification with palm heating, proving that palm heating can complete RPA amplification

[0024] Figure 8Results of the study on the detection performance of RPA-LFA. (A) Agarose gel electrophoresis of RPA products with different concentrations of plasmid DNA as template. (B) Lateral flow analysis of RPA products with different concentrations of plasmid DNA as template. (C) Agarose gel electrophoresis of RPA products amplified from different bacterial species (Escherichia coli, Staphylococcus epidermidis, Staphylococcus aureus, Bacillus subtilis, Salmonella typhimurium, Pseudomonas aeruginosa, Pneumococcus pneumoniae, Mycobacterium smegmatis, and Mycobacterium tuberculosis). Negative control: sterile water. (D) Lateral flow analysis of RPA products amplified from different bacterial species. The Detection Limit; Marker: E. coli; S. epidermidis; S. aureus; B. subtilis; S. typhimurium; P. aeruginosa; K. pneumoniae; M. smegmatis; M. tuberculosis; Negative; Test Band; Control Band; Figures A and B are to illustrate that the quantitative limit of RPA-LFA detection is as low as 10-18 mol L-1, and the electrophoresis diagram in Figure A is to prove the reliability of LFA detection in Figure B. Figures C and D are to illustrate the specificity of RPA-LFA detection, which is not interfered by other bacteria. Positive amplification will only occur when specific 16S rRNA is present. The electrophoresis image in Figure C is to demonstrate the reliability of the LFA detection in Figure D.

[0025] Fig. 9 (A) Linear relationship diagram and (B) corresponding fluorescence intensity-time curve of RT-qPCR detection of 16S rRNA gene of Mycobacterium; CT (cycle threshold)

[0026] Fig.10 The detection results of the detection method of the present invention on aerosol simulation samples, (A) Agarose gel electrophoresis analysis of nucleic acid of Mycobacterium smegmatis in exhaled breath after RPA amplification. (B) Lateral flow analysis of nucleic acid of Mycobacterium smegmatis in exhaled breath after RPA amplification. (C) RT-qPCR quantitative analysis of nucleic acid concentration of Mycobacterium smegmatis in exhaled breath. (D) Fluorescence intensity-time curve of nucleic acid concentration detection of Mycobacterium smegmatis in exhaled breath. Aerosol (aerosol); M. smegmatisAerosol (Mycobacterium smegmatis aerosol); FL Intensity (fluorescence intensity); Negative (negative control); Test Band (test line); Control Band (quality control line) DETAILED DESCRIPTION

[0027] In order to make the invention purpose, technical solution and beneficial technical effects of the utility model clearer, the utility model is further described in detail below in conjunction with the accompanying drawings and specific implementation methods. It should be understood that the specific implementation methods described in this specification are only for explaining the utility model, not for limiting the utility model.

[0028] The research and development process of the utility model is described in detail below.

[0029] 1. Experimental part

[0030] 1.1 Materials and reagents

[0031] The RPA amplification kit was purchased from Hangzhou Zhongce Biotechnology Co., Ltd. (Hangzhou, China). The single-target HybriDetect lateral flow test strips were purchased from Nanjing Wobo Biotechnology Co., Ltd. (Nanjing, China). The DNA sequences used in the experiments were synthesized and purified by Sangon Biotechnology (Shanghai) Co., Ltd. (Shanghai, China).

[0032] Polydimethylsiloxane (PDMS) was purchased from Dow Corning, USA; kanamycin, glycerol, and PBS buffer (pH 7.4) were purchased from Shenggong Biotechnology (Shanghai) Co., Ltd.; mycobacterium culture medium was purchased from Jingnuo Biotechnology; Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, Salmonella typhimurium, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Mycobacterium smegmatis were purchased from Guangdong Microbiological Culture Collection Center (Guangzhou, China); real-time fluorescence quantitative PCR amplification kit BeyoFast SYBR Green qPCR Mix (2X) was purchased from Shanghai Biyuntian Biotechnology Co., Ltd.; nutrient agar medium, Luria-Bertani (LB) broth medium, and Roche slant medium were purchased from Guangdong Huankai Microbiological Technology Co., Ltd.; bacterial lysis buffer was purchased from Beibo Biotechnology (Shanghai, China); Tween 80, hydrogen peroxide solution, and hydrochloric acid were purchased from Sinopharm Chemical Reagent Co., Ltd.; other reagents used in the experiment were of analytical grade, and the experimental water was ultrapure water (18.2 MΩcm). The DNA sequences used in the experiment were synthesized and purified by Sangon Biotechnology (Shanghai) Co., Ltd.

[0033] Table 1 Related nucleic acid sequences

[0034]

[0035]

[0036] 1.2 Instruments and Equipment

[0037] Bacteria were revived and cultured using a constant temperature shaking shaker (THZ-100, Shanghai Yetuo Technology Co., Ltd.) and a biochemical incubator (LRH-150, Shanghai Yiheng Scientific Instrument Co., Ltd.); bacterial morphology was observed using an inverted microscope (EclipseTs2, Nikon, Japan); an oxygen plasma generator (PDC-32G-2, Harrick Plasma, USA) was used to bond the electrolysis chip lysis pool to the ITO glass; an autoclave (LDZM-40L, Shanghai Shen'an Medical Device Factory) was used for sterilization; a real-time fluorescence quantitative PCR instrument (LightCycler480Ⅱ, Roche, USA) was used to record the real-time fluorescence quantitative PCR test results; agarose gel electrophoresis imaging results were obtained in the Azure Biosystems c300 automatic gel imaging system (USA); a medical ultrasonic nebulizer (402AI, Shanghai Yuyue Company, China) was used to simulate the generation of bioaerosols. The direct current required for the bacterial nucleic acid extraction electrolysis chip was generated by a laboratory-made high-voltage direct current generator. The Langer syringe pump (LSP02-1B, USA) was used to adjust the aerosol flow rate and the microfluidic chip channel flushing flow rate. The multifunctional microplate reader (Synergy H1, Biotek, USA) was used to measure the optical density of the bacterial solution so as to calculate the bacterial concentration by turbidimetry. The centrifugal precipitation of the bacterial solution was completed on a desktop high-speed refrigerated centrifuge (H1850R, Hunan Xiangyi Laboratory Instrument Development Co., Ltd.).

[0038] 1.3 Bacterial culture and clinical samples

[0039] Considering the safety of the experiment, we still used Mycobacterium smegmatis, which has the same genetic characteristics as Mycobacterium tuberculosis, can secrete mucus but is not pathogenic, as the bioaerosol model bacteria.

[0040] After the Mycobacterium smegmatis strains were restored to room temperature, they were inoculated into Roche slant medium and placed horizontally in a biochemical incubator at 37°C for 20 hours. After the strains were revived, a single colony was picked and inoculated into 5 mL of mycobacterium culture medium, tilted 45° and placed upright in a constant temperature shaking incubator at 200 rpm min. -1 , shaken at 37°C for 20 hours, and the concentration was about 5×10 8 CFU mL -1 The confirmation of Mycobacterium smegmatis in the bacterial solution was carried out by observing the bacterial morphology under a microscope and amplifying the Mycobacterium smegmatis mc after bacterial DNA extraction. 2The 358bp fragment downstream of the 155 strain-specific MSMEG_6935 gene (Genbank: NC_008596.1) was completed together. The real-time fluorescence quantitative PCR amplification program was: 95℃ heat denaturation for 2 minutes, 95℃ for 30 seconds, 54℃ for 30 seconds, 72℃ for 30 seconds, 40 cycles, and 72℃ for 5 minutes. After the RT-qPCR was completed, the cycle threshold (Ct value) was used to determine whether the target fragment was successfully amplified. The primers used are shown in Table 2. The dilution plate method was used to count Mycobacterium smegmatis.

[0041] The 7 clinical bacteria used for specificity verification in the utility model, including Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, Salmonella typhimurium, Pseudomonas aeruginosa and Klebsiella pneumoniae, were inoculated in a nutrient agar medium at 37°C and revived for 20 hours. After revivification, a single colony was picked and placed in 5 mL LB medium at 200 rpm min. -1 , cultured at 37°C with shaking for 20 hours.

[0042] The morphology of the cultured bacteria was observed under a microscope and nucleic acid analysis was performed to confirm that the revived Mycobacterium smegmatis was the target strain. Nucleic acid analysis was performed using real-time fluorescence quantitative PCR, and the target gene was Mycobacterium smegmatis mc 2 155 strain-specific gene MSMEG_6935 (Genbank: NC_008596.1), the amplified fragment was a 358 bp gene fragment downstream of MSMEG_6935, and the DNA sequence of this nucleotide region is shown in Table 1.

[0043] To calculate the bacterial concentration of the bacterial solution used, the dilution spread plate count method and the turbidimetric count method were used.

[0044] Guangzhou Chest Hospital collected clinical sputum samples from three patients with pulmonary tuberculosis, and hospital staff used Lab-Acid824 Mycobacterium tuberculosis nucleic acid extraction Maxi reagent (Xiamen Zhishan Biotechnology Co., Ltd.) to extract genomic DNA from the sputum samples. We directly used this nucleic acid extract for subsequent specificity verification experiments.

[0045] 1.4 Primer design

[0046] The complete genome sequence of Mycobacterium tuberculosis H37Rv strain (Genbank: AL123456.3) and Mycobacterium smegmatis mc 2The 16S rRNA gene of mycobacteria shared by the whole genome sequence of 155 strains (Genbank: CP000480.1) was used as the detection target. The total length of the nucleotide sequence of the 16S rRNA gene (GenBank: X52922.1) is 1454 bp, and nucleotide regions of 214 bp and 202 bp in length were selected for RPA primer design and RT-qPCR primer design. By quantitatively determining the DNA fragment, the content of mycobacteria in the aerosol can be determined. The DNA sequence of the nucleotide region used is shown in Table 1.

[0047] The primer design and modification for RPA detection were designed according to the instructions of the RPA amplification kit and lateral flow test strips. The primers were based on a 214 bp fragment of the 16S rRNA gene of Mycobacterium smegmatis (GenBank: X52922.1). Several initial primer combinations were screened by BLAST and NCBI nucleotide databases, and then evaluated by actual tests to determine the best primer combination. To verify the accuracy of our detection method, we used RT-qPCR to perform quantitative analysis of aerosol samples.

[0048] The primers used in this invention are listed in Table 1.

[0049] 1.5 Design of N95 enrichment mask

[0050] On the basis of the cardiopulmonary resuscitation mask, the original one-way valve of the mask is replaced with a filter valve for capturing and enriching bacteria in exhaled air. The design of the filter valve is similar to that of the condensing breathing valve. It consists of three parts: a valve cover, a filter element, and a valve bottom. The filter element part plays an enrichment role. The filter element has five layers, from inside to outside, non-woven fabric, meltblown non-woven fabric, ES hot air cotton, meltblown non-woven fabric, and non-woven fabric. Among them, ES hot air cotton is prepared by heat treatment of ES (ethylene-propylene side by side) fiber. ES fiber is a two-component (polypropylene / polyethylene) low-melting-point skin-core hot-melt fiber. After heat treatment, the fibers are bonded to each other to form a non-woven fabric molding without adhesive. After the filter valve is assembled, use the breathing tube extension tube to connect the resuscitation mask and the filter valve together, and finally use a sealing film to seal it to reduce the leakage of exhaled air during the collection process. See the design schematic of the enrichment mask. Figure 2 .

[0051] 1.6 Simulation and enrichment of aerosol samples

[0052] The purchased Mycobacterium smegmatis strain was inoculated into the mycobacterial culture medium at 200 rpm min. -1, 37℃ shaking culture for 20 hours. Centrifuge the bacterial solution at 6000rpm for 5 minutes, discard the supernatant, and redisperse in PBS solution. Slightly shake and mix, do not use vortex mixing to prevent bacterial rupture. The redispersed bacterial solution is used to generate bioaerosols of different concentrations.

[0053] A medical ultrasonic nebulizer was used to simulate the generation of bioaerosol. The bioaerosol generated by the ultrasonic nebulizer was dispersed in the nebulizer and introduced into the enrichment mask through a catheter. The interface was sealed with plastic wrap and sealing film. The enrichment filter valve and the injection pump were connected together with a syringe. The extraction flow rate of the injection pump was set to 1 mL min -1 , the extraction time is 60 minutes. Under the action of the syringe pump, the inside of the enrichment mask is in a negative pressure state, so that the bioaerosol in the nebulizer will slowly flow through the enrichment filter valve to achieve the enrichment simulation of the bioaerosol in the exhaled air. After the bioaerosol enrichment is completed, the filter valve of the enrichment mask is removed, and the filter element is immersed in 200μL of PBS buffer containing 1% Tween 80 and placed at room temperature for 15 minutes. The obtained filter element eluate can be used for the subsequent nucleic acid extraction process. In order to calculate the number of bacteria enriched by the filter element, the filter element eluate is directly inoculated on the nutrient agar medium, and the total number of enriched bacteria is calculated by the dilution plate coating method. In order to further calculate the enrichment efficiency of the mask, the bioaerosol generated by the nebulizer is directly collected in a 1.5mL EP tube through a catheter as a control group, and the tube is pre-filled with 200μL of PBS buffer containing 1% Tween 80. The calculation of bacterial concentration in the control group is consistent with the counting method of the filter element eluate.

[0054] 1.7 Nucleic acid extraction

[0055] When bacteria are exposed to a strong electric field, a transmembrane potential is formed inside and outside the cell membrane. At 0.2V-1V, reversible holes will be created in the cell membrane. When the voltage is greater than 1V, it will cause irreversible changes in the permeability of the cell membrane. Under strong electric fields, the bacterial cell membrane will be electroporated, and an osmotic pressure imbalance will occur between the bacterial cytoplasm and the extracellular medium, eventually leading to bacterial rupture. The value of is given by:

[0056]

[0057] where F is determined by the bacterial cell shape:

[0058] F=l / (l-0.67a)

[0059] Where l is the length of the cell and a is the radius. For spherical cells, F = 1.5, l = 2a; for rod-shaped cells, F = 1, l >> 2a. E is the electric field strength, approximately E = U / d, where U is the applied voltage and d is the distance between the electrodes. The angle α between the cell's dipole moment and the direction of the electric field can be 0° or 180°, so cosα = 1 (α = 0°) or -1 (α = 180°).

[0060] Based on the above concept, the utility model designs a microfluidic bacterial nucleic acid extraction chip, which extracts DNA by lysing bacteria through high-voltage direct current. Figure 4 A, see the actual picture Figure 4 B. The nucleic acid extraction chip is composed of a double-layer microfluidic chip. The upper lysis pool is made of PDMS, and there is a group of cylinders with a diameter of 4.5mm and a height of 3mm on the copper mold for PDMS injection molding. PDMS is mixed with a monomer (C2H6OSi) and a curing agent in a ratio of 10:1 (w / w), and degassed at a pressure of 40Torr (1Torr is 1mmHg) to remove residual bubbles. The PDMS is then poured into the copper mold with a thickness of 3mm, thermally cured at 65°C for 2 hours, and removed from the copper mold after cooling to room temperature (25°C) to obtain a lysis layer with holes (diameter 4.5mm; height 3mm). The bottom layer is made of optically transparent ITO-coated glass. The thickness of the ITO layer is about 1200nm, and the square resistance (the resistance between the edges of the square conductive film) value is 1~2Ω (cm 2 ) -1 . By laser etching, 10 sets of parallel electrode pairs were formed on the ITO-coated glass, with an electrode width of 0.8 mm and an electrode spacing of 0.5 mm. The upper layer (PDMS) and the bottom layer (ITO-coated glass) were treated with an oxygen plasma generator and bonded at 80°C for 3 hours to form an irreversibly bonded microfluidic chip. The holes in the PDMS layer and the electrodes in the ITO-coated glass constitute the electrolysis unit. The bacteria are placed in the lysis pool, and a DC power supply is connected to a set of parallel electrode pairs to generate a strong electric field between the two electrodes. This state is maintained for several seconds. The bacterial cell membrane is broken down by the electric field, which in turn causes an osmotic pressure imbalance between the cytoplasm and the medium outside the cell membrane, ultimately causing the bacteria to rupture. The cell lysate (including DNA and protein) is collected in the lysis pool. After the lysis is completed, the extraction efficiency of the nucleic acid can be calculated using RT-qPCR without further purification of the DNA.

[0061] In short, the lysis pool of the chip is prepared using PDMS, and the lysis layer is bonded to a conductive coated glass with an indium tin oxide (ITO) electrode pair using an oxygen plasma cleaner. According to the results of the previous optimization, the width of the ITO electrode used in the DC bacterial nucleic acid extraction chip is 0.8mm, and the electrode square resistance is 1-2Ω (cm 2 )-1 , the electrode spacing between the electrode pairs is 0.5 mm, the optimal lysis time is 3 seconds, and the sample volume required for lysis is 10 μL. The optimal electric field strength for lysing bacteria on this chip needs to be appropriately adjusted according to the difference in bacterial species. For the Mycobacterium smegmatis used in this study, the optimal electric field strength of the chip is 3.0 kV mm -1 Therefore, 10 μL of the enriched filter eluate was placed in the lysis pool of the chip at 3.0 kV mm -1 The electric field strength is 1000 μg / cm2, and the lysis is continued for 3 seconds. The obtained nucleic acid extract can be directly used for subsequent nucleic acid analysis. The nucleic acid extraction of the strains used in the utility model is completed using the above-mentioned bacterial nucleic acid extraction microfluidic chip.

[0062] 1.8 Recombinase polymerase amplification coupled with lateral flow assay (RPA-LF)

[0063] The RPA amplification reaction was completed using a purchased kit. A pair of RPA amplification primers were designed for the target gene. To better cooperate with the subsequent lateral flow analysis, the 5' end of the forward primer was modified with a biotin group, and the 5' end of the reverse primer was modified with a FAM group. The size of the RPA amplification product was 214 bp. The RPA amplification sequences of the primers and target DNA are shown in Table 1. The RPA reaction mixture (50 μL) included freeze-dried enzyme particles, 25 μL rehydration buffer, 2 μL forward primer (2 μmol L -1 ), 2 μL reverse primer (2 μmol L -1 ), 12.9 μL sterile water, 2.5 μL magnesium acetate and 5 μL synthetic DNA template. The negative control (NC) used 5 μL sterile water as the DNA template. Subsequently, the mixture was incubated at 36°C or handheld conditions for 8 minutes. To verify the RPA amplification results, agarose gel electrophoresis was used to verify the target 214bp band (Note: agarose gel electrophoresis is a type of electrophoresis that requires gel preparation and running, which is cumbersome and time-consuming. Its main feature is that different bands can be displayed on the gel according to the length of the DNA fragment. It cannot distinguish the DNA category, and can only separate DNA fragments of different lengths according to the difference in fragment length. It is generally used to verify nucleic acid amplification products and primer lengths). The electrophoresis conditions are 100V, 40min, and the concentration of low electroosmotic agarose used is 2.5%.

[0064] The obtained RPA amplification product is detected using a lateral flow test strip (Note: RPA amplification products, also called amplicons, are generally determined by agarose gel electrophoresis. Because our primers are designed with special biotin and FAM modifications, our amplicons can be detected with test strips. If the primers are modified with special fluorescent groups or special probes, fluorescence detection can also be achieved). Dilute the amplification product with PBS buffer (pH 7.4) at a volume ratio of 1:5. Add 20μL of amplification product to a 200μL EP tube pre-filled with 100μL PBS buffer and vortex to mix. Place the test strip vertically into the EP tube in the indicated direction, and read the test results after standing at room temperature for 10 minutes. When the test (Test) T line and the control (Control) C line are clearly visible at the same time, it is judged as a positive result. If only the control C line is visible, it is considered a negative result.

[0065] To evaluate the sensitivity of RPA-LF, synthetic plasmid DNA of the complete gene of 16S rRNA of Mycobacterium smegmatis was isolated from 10 -20 mol L -1 to 10 -8 mol L -1 The amplified products were diluted stepwise in TE buffer. The amplified products were analyzed by agarose gel electrophoresis and lateral flow strips to verify the accuracy of lateral flow analysis in detecting amplicons.

[0066] 1.9 Real-time fluorescence quantitative PCR

[0067] The results of real-time fluorescence quantitative PCR (RT-qPCR) were used as controls. RT-qPCR was performed in a 20 μL reaction system, including 10 μL BeyoFast SYBR Green qPCR mixture (2X), 1 μL 10 mmol L -1 Forward primer, 1 μL 10 mmol L -1 Reverse primer, 6 μL sterile water and 2 μL synthetic plasmid were used as DNA templates. Negative control used 2 μL sterile water as DNA template. The PCR amplification program was as follows: heat denaturation at 95°C for 2 minutes, followed by 40 cycles at 95°C for 30 seconds, 54°C for 30 seconds and 72°C for 30 seconds. The concentration of the template DNA to be tested was calculated based on the cycle threshold (Ct value) measured by the RT-qPCR instrument.

[0068] 1.10 Detection of aerosol samples

[0069] Prepare gradient concentrations of (10 9 , 10 8 , 10 7 , 10 6 , 10 5 , 10 4, 10 3 , 10 2 , 10 1 CFU mL -1 ) of Mycobacterium smegmatis bioaerosol, collected by N95-ER for subsequent testing. After enrichment, the filter was soaked in 200 μL 1% Tween80PBS buffer for 15 minutes. 10 μL of the eluate was placed in the lysis pool of the DC nucleic acid extraction chip at 3 kV mm -1 , 3 seconds. Take 5μL of lysate in a 50μL RPA amplification system, place the RPA reaction tube in the palm of your hand, and hold it tightly for 8 minutes. Add 20μL of amplification product to a 200μL EP tube containing 100μL PBS and mix thoroughly. Place the lateral flow test strip in the EP tube in the indicated direction. After standing for 10 minutes, read the test results. In addition, RT-qPCR quantitatively detects the amount of target nucleic acid in the lysate, and agarose gel electrophoresis and lateral flow analysis simultaneously detect amplicons (Note: electrophoresis is currently a widely recognized amplicon detection method). All tests were performed under the same conditions and in triplicate.

[0070] 2. Results and Discussion

[0071] 2.1 Principles of diagnosing tuberculosis through visualization of exhaled breath

[0072] Since the content of Mycobacterium tuberculosis in exhaled gas bioaerosol is relatively low, each step of its detection requires high efficiency. Based on this demand, the utility model provides a detection method for detecting bacteria in exhaled gas, which can be used to detect Mycobacterium tuberculosis in exhaled gas. The workflow of the method is as follows Figure 1 As shown. Based on this diagnostic platform, simulation experiments were conducted on the enrichment of bioaerosols and nucleic acid analysis in exhaled breath. Considering safety, the experiment used Mycobacterium smegmatis as the model bacteria. Mycobacterium smegmatis bioaerosols with different concentration gradients were prepared, and an ultrasonic nebulizer was used to simulate exhaled breath. The team's homemade enrichment mask and direct current nucleic acid extraction microfluidic chip were used to collect pathogens in exhaled gas and quickly extract nucleic acids. The extracted nucleic acids were amplified isothermally by RPA, and the visual reading of the test results was performed with the help of lateral flow analysis experiments. At the same time, agarose gel electrophoresis and RT-qPCR were used to jointly verify the test results.

[0073] 2.2 Optimization of aerosol enrichment conditions

[0074] In order to obtain the best bioaerosol enrichment performance, the filter composition of the enrichment mask, the composition of the filter eluent, the eluent volume, the elution time and other parameters were optimized. RT-qPCR was used to quantitatively evaluate the results of condition optimization, and the amount of nucleic acid was used as the evaluation standard for enrichment efficiency.

[0075] The enrichment performance of the enrichment mask depends on the composition of the filter element. Therefore, the structure of the filter element was optimized first. N95 protective masks generally have 3-5 layers, mostly composed of non-woven fabrics, meltblown fabrics, and hot air cotton, among which meltblown fabrics play the main protective role. The composition of the enrichment mask with 5 layers of filter membrane is as follows Figure 2 As shown in the figure, the filter element is composed of non-woven fabric, melt-blown fabric, hot air cotton, melt-blown fabric, and non-woven fabric from the inside to the outside. For the convenience of marking, they are numbered 1-5. Here, the enrichment efficiency of filter elements with different structures is investigated, and the results are as follows Figure 3 As shown in A. When the filter element is a single layer, among the three different materials of non-woven fabric, meltblown fabric and hot air cotton, meltblown fabric shows the best barrier efficiency. When the single-layer meltblown fabric is increased to two layers, the barrier effect of the double-layer meltblown fabric is significantly stronger than that of the single layer. On the basis of two layers of meltblown fabric, adding hot air cotton in the middle can significantly improve the enrichment efficiency. When the filter membrane is superimposed to 5 layers, it shows excellent performance that exceeds the sum of all single layers. Continue to superimpose the number of meltblown fabric layers. At this time, the total number of filter membranes is 7 layers, of which 4 layers are meltblown fabrics. The enrichment efficiency has not increased significantly, and is even slightly lower than before. When the total number of filter membrane layers is superimposed to 10 layers, the enrichment efficiency does not increase but decreases. It is speculated that the reason for this phenomenon may be that there are too many filter membrane layers. Although it has a higher barrier capacity, it also makes elution difficult and the amount of bacterial nucleic acid extraction is low. Therefore, a combination of 5 layers is selected as the filter element composition of the enrichment mask, which is consistent with the composition of the common N95 medical surgical protective masks on the market.

[0076] Secondly, the elution efficiency will affect the concentration of pathogens in the eluent and interfere with the nucleic acid detection results. Therefore, the filter elution conditions were further studied. Considering the subsequent nucleic acid analysis, we selected water and PBS buffer, which have no effect on the RPA system, as eluents. However, experiments have shown that the elution ability of the above solutions is poor. In order to improve the elution performance, after consulting the literature, it is considered to add surfactants to the eluent to increase the wettability of the filter material. As a non-ionic surfactant, Tween 80 has long-chain oxyethylene groups in its molecular structure, which makes Tween 80 have good surface activity and emulsification ability. In addition, the octanoic acid group contained in the Tween 80 molecule makes it have good solubility and wettability. Compared with other reagents in the Tween series, it has only a weak hemolytic effect and high safety. The above advantages make Tween 80 often used in biological experiments. Researchers often add a suitable concentration of Tween 80 to the eluent to improve the elution performance. Therefore, we chose to add a small amount of Tween 80 to the elution solution in order to improve the elution efficiency. At the same time, in order not to affect the subsequent bacterial count, the concentration of Tween 80 should not be too high. Our experimental results show that the number of pathogens eluted by elution solutions with different compositions is significantly different, such as Figure 3As shown in B in the figure. When the eluent contained only water or PBS buffer, only a trace amount of pathogens were eluted. When a surfactant Tween 80 with a concentration of 1% was added to the water, the pathogen elution ability was significantly improved, and Tween 80 performed better in PBS buffer than in water. In addition, it was investigated whether increasing the concentration of Tween 80 could further improve the elution performance. The results showed that a Tween 80 content of 1% was better than 5%. Finally, PBS buffer containing 1% Tween 80 was selected as the eluent. In addition, the effect of the volume of the eluent on the elution was also investigated, as shown in Figure 3 As shown in C. Because the enrichment filter element is composed of 5 layers of filter membranes, when the volume of the elution solution is only 100μL, it can be observed during the experiment that the filter element cannot be completely wetted, resulting in the inability to completely release the pathogens in the filter element. On the contrary, when the volume of the elution solution is as high as 500μL, there is a large amount of elution solution in the EP tube, which will cause the concentration of pathogens in the solution to decrease. Therefore, based on the results of the optimization experiment, an elution volume of 200μL was finally selected. However, elution does not occur instantaneously, so the elution time of the filter element will also affect the elution effect. In order to find the time required for the pathogens enriched by the filter element to be completely eluted, we examined the elution time spanning 5-1440 minutes. The results are shown below. Figure 3 As shown in D. It can be seen that when the elution time is extended from 5 minutes to 15 minutes, the elution effect improves with time. However, when the time is further extended to 30 minutes, or even up to 1440 minutes (24 hours), the elution effect is not significantly improved. Based on this result, it can be considered that the elution of pathogens in the filter element can be completed in 15 minutes.

[0077] 2.3 Comparison of enrichment performance between N95 enrichment mask and microfluidic chip

[0078] In previous studies by our team, microfluidic chips were used to enrich bioaerosols in exhaled breath. The microfluidic chip achieves bioaerosol enrichment due to its special fishbone structure. However, due to the miniaturization of the microfluidic chip, its disadvantage of low gas flow is particularly prominent. This shortcoming makes it difficult to improve the aerosol enrichment efficiency of the microfluidic chip. The enrichment mask designed and used in this study can circumvent this shortcoming and significantly improve the enrichment efficiency. The comparison of the enrichment efficiency of the two different pathogen enrichment methods in aerosols is shown in Tables 2 and 3. According to the data in the two tables, when the aerosol concentration of Mycobacterium smegmatis is in the range of 10 2 -10 9 CFU mL -1 When the concentration of bioaerosol is as low as 10 2 CFU mL -1The number of pathogens collected was too low to be counted, so we believe that the minimum concentration of the chip is 10 3 CFU mL -1 The low concentration of pathogens in exhaled breath is one of the difficulties in using exhaled breath to diagnose tuberculosis. The enrichment mask we designed can detect pathogens in exhaled breath at lower concentrations by increasing the total amount of exhaled breath to be detected. The total amount of pathogens in bioaerosols is 4.97×10 6 When comparing the efficiency of the two enrichment methods, the enrichment efficiency of the microfluidic chip was only 23.2%, while the enrichment efficiency of the mask using the same control group was as high as 61.5%, which was 2.65 times that of the chip. In addition, the repeatability of the two enrichment methods was good. When n=3, the error of the chip enrichment efficiency was only 0.33%, and the enrichment mask was slightly inferior, with a minimum error of 1.40%. The precise design of the internal channel and fishbone structure of the chip ensures its good repeatability, so that it has similar performance in the enrichment process of different aerosol bacterial concentrations. From this, we can see that the maximum error of the chip enrichment efficiency is only 2.40% (Table 2), which is much lower than the 6.83% of the mask (Table 3). Obviously, the two pathogen enrichment methods have their own advantages, but given that our purpose is to improve the enrichment efficiency and to ensure the smooth progress of subsequent nucleic acid analysis, the mask is more suitable for this diagnostic platform because of its higher enrichment efficiency. In summary, the enrichment mask designed in the utility model can very effectively collect Mycobacterium smegmatis from aerosols of different concentrations, has a wide range of applicable concentrations, and has better enrichment efficiency than the microfluidic chip enrichment method. Further research believes that the reason is that the gas flow of the enrichment mask is high-throughput, which collects a larger amount of samples relative to the microfluidic chip. The microfluidic chip is subject to its micro-nano structure, and the flux of collecting aerosol samples is very low, so the pathogens collected in the same time are much lower than those of the mask. For example, the channel that plays an enrichment role inside the microfluidic chip used to compare the microfluidic chip in this study is only 150 microns wide, which is a huge difference compared to the size of the enrichment filter element (diameter 2.5 cm = 25,000 microns).

[0079] Table 2 Efficiency of microfluidic chip in enriching Mycobacterium smegmatis bioaerosol (n=3)

[0080]

[0081] Table 3 Efficiency of N95-ER in enriching Mycobacterium smegmatis bioaerosol (n=3)

[0082]

[0083] 2.4 Performance of nucleic acid extraction chip

[0084] We conducted a detailed study on the design and performance of a microfluidic chip for bacterial nucleic acid extraction. By combining an ITO electrode with a PDMS substrate, we constructed a microfluidic chip that can achieve lysis of multiple bacteria and nucleic acid extraction in a DC electric field. The chip can be applied to both Gram-positive and Gram-negative bacteria, including cocci and bacilli. We also explored in detail the effects of electric field strength, electrode spacing, lysis time, indium tin oxide electrode square resistance, and electrode width on bacterial nucleic acid extraction. Our results show that at 3.0 kV mm -1 Under an electric field strength of , the chip can lyse a variety of common bacteria (including Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, Klebsiella pneumoniae, Salmonella typhimurium and Pseudomonas aeruginosa) within 3 seconds and release nucleic acids. The extracted nucleic acids do not need to be purified and can be directly used for subsequent nucleic acid amplification detection. The chip needs to adjust the optimal lysis conditions appropriately according to different bacterial types. Compared with traditional nucleic acid extraction methods, nucleic acid extraction chips are particularly suitable for small volume samples because they are chemically free and the lysis solution does not need to be purified and can be used for amplification. The performance of the chip in bacterial lysis and nucleic acid extraction was verified by quantitative PCR experiments, which can be better combined with subsequent detection processes. This study provides a simple, fast, efficient and chemical reagent-free nucleic acid extraction method for pathogen analysis and medical diagnosis of bacterial infections.

[0085] Here we continue to use the above-mentioned direct current bacterial nucleic acid extraction chip to complete the extraction of bacterial nucleic acids in the eluate. The chip used needs to optimize the nucleic acid extraction conditions according to different bacteria. By optimizing the effects of different electric field strengths E values ​​on nucleic acid extraction under the same lysis conditions, the optimal electric field strength (E) is selected for nucleic acid extraction experiments. In short, 20 thermal cycles (90°C for 30 seconds, 30°C for 30 seconds as one cycle) were used as a control for nucleic acid extraction for complete bacterial lysis. The results showed that the chip can be used at E of 3.0 kV mm -1 The highest extraction efficiency of Mycobacterium smegmatis nucleic acid was obtained when Figure 5 A), so 3.0kV mm -1 The optimal E value for nucleic acid extraction of Mycobacterium smegmatis. In order to more intuitively observe the performance of the chip in lysing Mycobacterium smegmatis and extracting its nucleic acid, the lysed and unlysed Mycobacterium smegmatis were inoculated on agar plates and cultured at 37°C for 20 hours. The growth of the colonies was observed. Figure 5 B and C. It can be seen that only a few colonies of Mycobacterium smegmatis grew after electrolysis, and the number of colonies decreased significantly compared with the unlysed bacterial solution, proving that its complete cell structure has been destroyed.

[0086] 2.5 Optimization of RPA-LFA conditions

[0087] In order to achieve rapid visual detection at the point of care, the RPA amplification temperature and the reaction time of the RPA-LF analysis were optimized. Our preliminary experiments and previous literature have demonstrated that a 10-minute incubation time is indeed sufficient to display the test and control bands and is applicable to all tests. Combined with our final choice of 10 minutes as the final incubation time for the test strip (Note: After the test strip is placed in the test sample, the antibodies and probes fixed on the test strip itself need to bind to the probes and modifications on the sample before they are intercepted and colored when the sample solution flows through the test strip. The process of combining the modifications in the sample with the modifications on the test strip is called incubation). To explore the optimal temperature for amplification, LF-RPA tests were performed at different temperatures for 30 minutes. At 100 a mol L -1 The 16S rRNA gene of Mycobacterium tuberculosis and Mycobacterium smegmatis was used as the target DNA template to synthesize the plasmid. The core region of the plasmid fragment is the nucleotide region of the 16S rRNA gene sequence, totaling 1454bp. The temperature range investigated was 35℃-41℃. The amplification product was detected by lateral flow test strips, and the amplicon was detected by agarose gel electrophoresis to verify the test results of the test strips. The optimization results of RPA amplification temperature are shown in Figure 6 As shown in A and B. When the amplification temperature is 35℃-41℃, after 30 minutes, the positive samples can be observed with obvious red test bands and blue control bands. The corresponding negative samples have no obvious bands in the test band area, and only clear blue quality control bands are displayed, indicating that amplification only occurs in positive samples containing 16SrRNA genes, and no false positives occur in negative samples. Because the length of the target DNA is 214bp, Figure 6 From the agarose gel electrophoresis results of A, we can see that in the 200-300bp region, closer to the 200bp position, the positive samples all have bright bands, while the negative samples only have faint bands near 30bp and 60bp, which should be the bands formed by primers and primer dimers. The detection results of gel electrophoresis are consistent with the detection results of lateral flow test strips. And according to the results of gel electrophoresis, we can further know that compared with other temperature points, the positive samples have brighter bands at 36°C, 37°C and 39°C. Considering the needs of point-of-care testing, we chose a lower temperature of 36°C as the RPA amplification temperature. In addition, at the selected amplification temperature, the effect of amplification time on RPA-LF analysis was further investigated, such as Figure 6 As shown in C and D. At a reaction temperature of 36°C, the amplification lasted for 5-35 minutes. Bright bands were amplified in the positive samples. The test bands on the test strips were obvious, and the negative samples were not disturbed. Figure 6It can be seen from the C that although the amplification was successful at 5 minutes, the electrophoresis bands were obviously brighter than 5 minutes when the amplification time was extended to 8 minutes or longer. In order to make the visual results clearer, the RPA amplification time was selected as 8 minutes. In summary, by consulting the literature and experimental optimization results, 36°C amplification for 8 minutes and lateral flow test strip incubation for 10 minutes were finally selected as the reaction conditions for RPA-LF analysis.

[0088] According to the literature, the temperature of a normal person's palm is generally 36-37°C. It is speculated that our amplification method can be amplified by placing the reaction EP tube in the palm of the hand and holding it for 8 minutes. To verify this idea, we added different concentrations of target DNA to the reaction tube at the same time, ranging from 10 -7 L -1 -10 -20 L -1 After the amplification is completed, the amplicon is detected by gel electrophoresis. Figure 7 .from Figure 7 It can be seen that the hand-held heating method can achieve RPA amplification, and the detection concentration is as low as 10 -19 L -1 , to meet the detection needs, the final amplification method is to hold the RPA reaction tube tightly in the palm of your hand and heat it for 8 minutes.

[0089] 2.6 Detection Performance of RPA-LFA

[0090] Under the optimized experimental conditions, the detection performance of RPA-LFA was further investigated. First, we evaluated the detection limit of RPA-LFA. The synthesized plasmid was serially diluted (5.0×10 -8 L -1 -5.0×10 -20 L -1 , corresponding to a copy number of 1.8×10 11 copiesμL -1 -10 -1 copiesμL -1 ) as a template. The amplified product was identified by gel electrophoresis. Figure 8 As shown in A, when the DNA template (plasmid) concentration is diluted to 5.0×10 -20 L -1 (0.18 copies μL -1 ), no band of 214 bp was observed. -19 L -1 (1.8 copies μL -1), only a faint band was observed, and a faint pink test line was visible on the chromatographic test strip at the corresponding concentration. According to the instructions, this result should be judged as weakly positive. However, since the pathogens were collected from exhaled breath and the nucleic acid was extracted through electrolysis, multiple detection processes were involved. In order to improve the accuracy of the final test results, the researchers of this article believe that the test line should be clearly visible so that different users can judge it as a consistent result. Therefore, the researchers of this article believe that our method cannot accurately detect the target DNA at this concentration. When the concentration was increased to 5.0×10 -18 L -1 (18 copies μL -1 ) The lateral flow test strip test results were consistent with the gel electrophoresis, and clear target detection bands could be observed. Figure 8 From the detection results of A and B, we can know that in a 50μL reaction system, the detection limit of RPA-LFA is as low as 5.0×10 -18 L -1 (18 copies μL -1 ). In addition, to verify the specificity of RPA-LFA detection, we tested common bacteria in bioaerosols, including Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, Salmonella typhimurium, Pseudomonas aeruginosa, and Klebsiella pneumoniae. The nucleic acid was extracted using a direct current bacterial nucleic acid extraction chip, and after RPA amplification, the test results of the test strip were read, and the specificity results were verified by gel electrophoresis, see Figure 8 From the gel electrophoresis diagram, it can be seen that only the Mycobacterium tuberculosis samples and the Mycobacterium smegmatis samples can be observed with bright DNA bands. The test results of the test strips also prove the specificity of RPA amplification. Only pathogens containing 16S rRNA specific genes will undergo RPA amplification and be detected positive. The above test results prove that RPA-LFA has good specificity and a low detection limit.

[0091] 2.7 RT-qPCR detection linear range

[0092] The utility model uses RT-qPCR to quantitatively evaluate the test results. The plasmid containing 1454bp 16S rRNA gene fragment is used as the template DNA, and the plasmid DNA template is diluted step by step to obtain different concentrations. The concentration range of the RT-qPCR investigated is 10 -9 -10 -19 L -1 The highest concentration investigated was only 10 -9 L -1 This is because when the concentration is higher, the number of cycles of real-time fluorescence quantitative PCR is less than 5, which exceeds the measurement range of the instrument and cannot be quantitatively detected. The fluorescence-time curve and linear relationship are shown in Fig. 9 As shown. Fig. 9 It can be seen from A that when the template concentration is as low as 10 -19 L -1 , or up to 10 -9 L -1 When the concentration is 0.04, it is no longer linear with other concentration points, so the linear range of the detection method is 10 -10 -10 -18 L -1 The quantitative method has a good linear relationship. The linear correlation equation is Y = -3.0156Log CDNA-19.242, the linear correlation coefficient R is 0.9985, and the minimum detection concentration is 1.0×10 -18 molL -1 (3.6 copies μL -1 ).

[0093] 2.8 Test results of aerosol simulation samples

[0094] To verify the ability of the TB diagnostic platform to diagnose pathogens in exhaled breath, nine different concentration gradients (10 9 , 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 CFU mL -1 ) of Mycobacterium smegmatis liquid, ultrasonically atomized into bioaerosols of corresponding concentrations, collected by enrichment masks and eluted, and the nucleic acid in the eluate was extracted by electrolysis chip, and finally RPA-LFA was used to detect samples of different concentrations. Fig.10 As shown in B, when the concentration of bioaerosol is 10 2 -10 9 CFU mL -1 Within this range, this diagnostic platform can achieve visual detection of target pathogens through only a 1-hour enrichment process. In order to prove the reliability of direct reading of the results by the chromatographic test strips, agarose gel electrophoresis was further used to identify the amplified products. Fig.10 As shown in A, the concentration is 10 3 -10 10 CFU mL -1 The aerosol samples all had bright and clear amplification bands, and the concentration was 10 2 CFUmL -1The sample only had a weak positive band, which was consistent with the test strip. To better demonstrate the collection capacity of our enriched masks and the nucleic acid extraction capacity of the chip, the nucleic acid extract obtained by chip electrolysis was directly measured by RT-qPCR to calculate its nucleic acid content, such as Fig.10 As shown in C, it can be seen that the amount of nucleic acid we extracted is proportional to the concentration of pathogens in the aerosol, but due to the instability of ultrasonic atomization and enrichment process, the amount of nucleic acid is not linearly related to the concentration of bioaerosol. Even so, we can also see that if our enrichment mask and nucleic acid extraction chip are combined with RT-qPCR, although the detection and analysis time is prolonged (Note: According to experiments, the RPA-LFA time is about 18 minutes, and the RT-qPCR time is about 100 minutes), the detection of bioaerosols with lower concentrations, as low as 10 1 CFU mL -1 . Fig.10 D is the fluorescence-time curve of RT-qPCR detection. From the detection results of bioaerosol simulation samples, it can be seen that the diagnostic platform can detect the presence of bioaerosols at a concentration of 10 2 -10 9 CFU mL -1 The minimum detection concentration of pathogens in exhaled air by the two detection methods is compared. Fig.10 C, RT-qPCR (10 1 CFU mL -1 ) was lower than that of RPA-LFA (10 2 CFU mL -1 ). However, RPA-LFA is fast, visual, and instrument-free, making it more suitable for point-of-care testing, especially in resource-limited areas. The visual diagnostic platform constructed by the utility model has the advantages of being non-invasive, good compliance, short analysis time, simple operation, and no instrument required, and has shown great potential in the rapid screening and diagnosis of tuberculosis at the point of care.

Claims

1. An exhaled air enrichment mask, characterized in that: It contains a cover body and a filter valve. The filter valve contains a filter element, wherein the filter element is composed of five layers from inside to outside, namely, non-woven fabric, melt-blown fabric, hot air cotton, melt-blown fabric and non-woven fabric.

2. An exhaled air enrichment mask according to claim 1, characterized in that: Used to detect bacteria in exhaled air, the filter valve consists of a valve cover, a filter element, and a valve bottom. The filter element is in the shape of a circle with a diameter of 2.5 cm.

Images