WO2018210128A1 - Méthode de collecte en ligne et de détection automatique en ligne de bactéries ou de virus dans l'air - Google Patents

Méthode de collecte en ligne et de détection automatique en ligne de bactéries ou de virus dans l'air Download PDF

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Publication number
WO2018210128A1
WO2018210128A1 PCT/CN2018/085293 CN2018085293W WO2018210128A1 WO 2018210128 A1 WO2018210128 A1 WO 2018210128A1 CN 2018085293 W CN2018085293 W CN 2018085293W WO 2018210128 A1 WO2018210128 A1 WO 2018210128A1
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WIPO (PCT)
Prior art keywords
sampling
air
online
viruses
bacteria
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PCT/CN2018/085293
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English (en)
Chinese (zh)
Inventor
王大平
董承智
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上海德具生物科技有限公司
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Publication of WO2018210128A1 publication Critical patent/WO2018210128A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/24—Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • the invention belongs to the technical field of microbial detection, in particular to an online collection and online automatic detection method for bacteria or viruses in the air.
  • Airborne transmission is the main route for some pathogens or viruses to spread, and it is the most difficult way to detect and control in the current environment.
  • the SARS virus is mainly found in the nose, sputum and saliva of the source of infection (human or animal) and is transmitted by air droplets and contact. When a patient coughs, sneezes, or even speaks loudly, the virus spreads to the surrounding air with saliva droplets, dust particles, etc., and spreads with the air.
  • the object of the present invention is to provide an online collection and online automatic detection method for bacteria or viruses in the air to solve the problem that the bacteria or viruses in the air cannot be early warning or monitored, so as to realize the rapid and accurate detection of bacteria or viruses in the air. On-site inspection.
  • the online collection and online automatic detecting device comprises a sample loading control device, a PCR amplification device, a detecting device and an automatic control device, and further comprises a pressing device and a negative pressure device; and the following steps are included:
  • the negative pressure device is activated, the liquid in the sampling area is sucked into the waste liquid bottle, and the particles adsorbed with DNA or RNA are trapped on the filter medium in the sampling area;
  • the detection result is output.
  • the sampling integrated sampling box is connected to the sample loading control device through a pipeline; the sample loading control device is matched with the PCR amplification device;
  • the automatic control device is electrically connected to the sample loading control device, the PCR amplification device, the detection device, the pressing device, and the negative pressure device, respectively;
  • the pressing device and the negative pressure device are both connected to the sampling integrated sampling box through a pipeline.
  • the sampling integrated sampling box includes a sampling container, an eluent storage container and a waste liquid tank; the eluent storage container is connected to the sampling container through a pipeline; the waste liquid tank and the bottom of the sampling container pass through the tube Road connection
  • sampling container and the waste liquid tank are respectively connected to the negative pressure device through a pipeline;
  • a porous air distributor and a filter are disposed in the sampling container; an inlet of the porous air distributor is connected to a pipeline connecting the sampling container and the negative pressure device, and the filter is disposed on the sampling At the bottom outlet of the container, an adsorption liquid is disposed in the sampling container, and an air outlet of the porous air distributor is lower than a liquid surface of the adsorption liquid; the adsorption liquid is composed of a microorganism lysate and adsorbed particles;
  • An eluent is disposed in the eluent storage container.
  • the volume ratio of the microorganism lysate to the adsorbed particles is 10-20:1.
  • the pressurizing device is a first peristaltic pump and the vacuum device is a vacuum pump.
  • It also includes a plug-in interface device for sampling the quick insertion of the integrated sampling box.
  • the sample loading control device includes a second peristaltic pump and a one-way valve; an outlet of the second peristaltic pump corresponds to the PCR amplification device, an inlet of the second peristaltic pump and an outlet of the one-way valve Connected by a line; the inlet of the one-way valve is connected to the bottom outlet of the sampling container through a line.
  • the PCR amplification device includes a sample plate, a PCR kit, and a PCR temperature control device.
  • the sample plate is connected to the PCR kit, and the PCR kit is connected to the PCR temperature control device.
  • the detecting device comprises an excitation light emitter, a fluorescence receiver and a human-machine dialogue system; the excitation light emitter is disposed opposite to the fluorescence receiver in a detection area of the PCR amplification device, and the excitation light emitter
  • the fluorescent receiver is electrically connected to the human-machine dialog system, and the human-machine dialog system is electrically connected to the automatic control device.
  • the automatic detecting device includes a central controller, and the central controller is one of a program controller, a single chip microcomputer, or an industrial computer;
  • the automatic detecting device further comprises one or more combinations of a Bluetooth interface, a wireless WIFI interface, an Ethernet interface, a USB interface, an RS232 serial port or an RS485 serial port.
  • the invention adopts the online collection and online automatic detection method of bacteria or virus, including negative pressure pumping, on-line microbial collection, air microbial adsorption, DNA or RNA online extraction, DNA online amplification and detection, etc., by collecting microorganisms in the air.
  • the particles are lysed in real time in a liquid, extracted DNA (or RNA), amplified by PCR, and subjected to online real-time detection.
  • the technical scheme can analyze the collected samples separately by sampling, extracting and detecting, and is suitable for determining the number of different kinds of microorganisms in a unit volume of air.
  • the new air microbial sampling and detecting instrument developed by the technical solution, combined with biochip or real-time PCR technology, can quickly and sensitively detect pathogenic microorganisms that may exist in the air.
  • the device is mainly composed of a negative pressure suction device (handheld or vacuum pump), an air flow meter, an adsorption liquid and adsorbed particles.
  • a suction device adsorbing air through a vacuum, at a constant gas flow rate per minute, through the liquid collection zone, under the action of adsorbed particles in the solution and the solution, the virions in the air are collected in a small volume of liquid, while The collected microorganisms are lysed to release microbial DNA (RNA) and adsorbed on the adsorbed particles.
  • RNA microbial DNA
  • RNA DNA
  • RNA DNA
  • RNA DNA
  • Figure 1 is a schematic view showing the structure of the device of the present invention.
  • FIG. 2 is a schematic view showing the operation of the sampling integrated sampling box of the present invention.
  • the invention develops a novel air microbial sampling and detecting instrument, and combines biochip or real-time PCR technology to quickly and sensitively detect pathogenic microorganisms which may exist in the air.
  • the device is mainly composed of a negative pressure suction device (handheld or vacuum pump), an air flow meter, an adsorption liquid and adsorbed particles.
  • a suction device adsorbing air through a vacuum, at a constant gas flow rate per minute, through the liquid collection zone, under the action of adsorbed particles in the solution and the solution, the virions in the air are collected in a small volume of liquid, while The collected microorganisms are lysed to release microbial DNA (RNA) and adsorbed on the adsorbed particles.
  • RNA microbial DNA
  • RNA DNA
  • RNA DNA
  • RNA DNA
  • the invention integrates sampling, extraction and detection into one body, can separately analyze the collected samples, and is suitable for determining the number of different kinds of microorganisms in a unit volume of air.
  • the present application provides an online collection and online automatic detection device for bacteria or viruses in the air, as shown in FIG. 1 and FIG. 2, including a sampling integrated sampling box, a sample loading control device, a PCR amplification device, a detection device and an automatic control device. It also includes a pressurizing device and a vacuum device matched with the sampling integrated sampling box.
  • sampling integrated sampling box and the sample loading control device are connected by a pipeline; the sample loading control device is matched with the PCR amplification device; the sampling integrated sampling box of the present application is used for sampling the integrated sampling box by the plug-in interface device. Plug in.
  • the automatic control device is respectively electrically connected with the sample control device, the PCR amplification device, the detection device, the pressing device and the negative pressure device;
  • the automatic detection device comprises a central controller, and the central controller is a program controller, a single chip microcomputer or an industrial computer One is to fully automate the process from sampling air to detecting the type and concentration of microorganisms.
  • the automatic detecting device further comprises one or a combination of a Bluetooth interface, a wireless WIFI interface, an Ethernet interface, a USB interface, an RS232 serial port or an RS485 serial port, and communicates with the host computer or the mobile terminal through a wired or wireless manner to implement the device.
  • Remote monitoring allows the tester to obtain test results without going deep into the affected area, thus avoiding the risk of the tester being infected.
  • these interfaces can communicate with other devices such as the top computer, mobile phone, etc., so that the detection result can be applied by other devices, the automatic detection device can also be controlled by other devices, and can also realize remote transmission or cloud management of detection data and Remote control of the automatic detection device.
  • Both the pressurizing device and the negative pressure device are connected to the sampling integrated sampling box through a pipeline.
  • the pressurizing device is the first peristaltic pump 4; in the present application, the vacuum device is the vacuum pump 1.
  • the sampling integrated sampling box comprises a sampling container 3, an eluent storage container 5 and a waste liquid tank 11; the eluent storage container and the sampling container are connected by a pipeline; the waste liquid tank and the bottom of the sampling container are connected by a pipeline.
  • the flow of the liquid is controlled by the air pressure, and the flow direction of the gas is controlled by the check valve on the pipeline, thereby controlling the pressure in the sampling container, the eluent storage container or the waste liquid tank to realize the liquid (adsorbed liquid or Directional flow of the eluent).
  • a pressurizing device is disposed on the pipeline between the sampling container and the eluent storage container;
  • the sampling container 3 and the waste liquid tank are respectively connected to the vacuum device through a pipeline; in the present application, the vacuum device is a vacuum pump 1, and a first electromagnetic valve 2 is disposed between the vacuum pump and the sampling container, in the vacuum pump and the waste liquid cylinder A second solenoid valve 10 is disposed therebetween; both the first solenoid valve and the second solenoid valve are electrically connected to the automatic control device, and the first solenoid valve and the second solenoid valve are controlled to be turned on or off by the automatic control device.
  • a porous air distributor and a filter 17 are disposed in the sampling container; the inlet of the porous air distributor is connected to a pipeline connecting the sampling container and the negative pressure device, and the filter is disposed at the bottom outlet of the sampling container, and is disposed in the sampling container There is an adsorption liquid 7, the outlet port of the porous air distributor is lower than the liquid level of the adsorption liquid; the air outlet of the porous air distributor distributes the drawn air into the adsorption liquid through a plurality of pores, in order to prevent the airflow from being too large The direct short circuit rushes out the liquid, and the plurality of pores distribute the carrier air into small bubbles, increasing the contact area between the carrier air and the adsorption liquid, and improving the efficiency of adsorbing microorganisms in the air.
  • the adsorbent consists of a microbial lysate and adsorbed particles.
  • the eluent 6 is provided in the eluent storage container 5.
  • the eluate is a DNA (RNA) eluate, and both are commercially available DNA (RNA) eluents.
  • the volume ratio of the microorganism lysate to the adsorbed particles is 10-20:1.
  • the adsorbed particles are DNA adsorbing particles or RNA adsorbing particles.
  • the height-to-diameter ratio of the adsorbent in the sampling container is 6:1-3:1.
  • the microbial lysate is phosphate buffered PBS by dissolving NaCl, KCl, Na 2 HPO 4 and KH 2 PO 4 in distilled water, and adjusting the pH of the solution to 7.4 with HCl. Add distilled water to make up to volume, and steam sterilize at 121 ° C for 20 minutes under high pressure, and store in room temperature or 4 ° C refrigerator.
  • a broad-spectrum high-efficiency adsorption liquid composition is added to a 1 ml PBS solution to which a chitosan nanoparticle having a particle size distribution of 10 to 30 mg and a diameter of 10 to 200 nm is added.
  • a particle size distribution of 10-30. mg of a diameter of 10-200 nm is added to a 1 ml PBS solution with influenza virus antibody-modified nanoparticles.
  • the DNA adsorption particles or the RNA adsorption particles may each be a commercially available adsorption resin such as a DNA (RNA) adsorption resin produced by a company such as Shanghai Shenggong or Beijing Quanjin.
  • a DNA (RNA) adsorption resin produced by a company such as Shanghai Shenggong or Beijing Quanjin.
  • the sample loading control device comprises a second peristaltic pump 8 and a check valve 9; the outlet of the second peristaltic pump corresponds to the PCR amplification device, and the inlet of the second peristaltic pump and the outlet of the check valve are connected by a pipeline; The inlet is connected to the bottom outlet of the sampling vessel via a line.
  • the second peristaltic pump is used for the sample loading, and the metering device is used for the measurement of the sample amount.
  • the PCR amplification device comprises a sample plate 15, a PCR kit and a PCR temperature control device 16.
  • the sample plate is connected to the PCR kit, and the PCR kit is connected to the PCR temperature control device.
  • the reaction solution was sealed by adding liquid paraffin to the PCR kit.
  • the PCR kit of the present application is a commercially available PCR kit.
  • the detecting device comprises an excitation light emitter 12, a fluorescence receiver 13 and a human-machine dialogue system 14; the excitation light emitter and the fluorescence receiver are disposed opposite to the detection area of the PCR amplification device, and the fluorescence emitter and the fluorescence receiver are both connected to the human-machine
  • the dialogue system is electrically connected, and the human-machine dialogue system is electrically connected to the automatic control device.
  • the human-machine dialogue system can also set relevant detection parameters and equipment maintenance management.
  • the automatic control device will control the vacuum pump to start, realize automatic sampling to the set volume; After 2 minutes, the automatic control device controls the vacuum pump to start again, so that the waste liquid cylinder is under negative pressure, and the liquid in the sampling container is sucked into the waste liquid tank, and the particles adsorbed with DNA (RNA) are cut off on the filter medium in the sampling container.
  • the automatic control device controls the first peristaltic pump to start, pressurizes the eluent storage container, and hydraulically elutes the DNA (RNA) into the sampling container, and elutes the adsorbed DNA (RNA) on the exposed particles to pass through
  • the check valve and the second peristaltic pump add the eluted DNA (RNA) eluate to a PCR kit pre-loaded with a commercially available DNA (RNA) detection sample, and preset according to the requirements of the PCR kit. Perform PCR amplification; after the amplified sample is detected by the detection device of the detection area, the output is sent to the automatic control device to obtain the type density of the microorganism, and the detection result is transmitted through wired or wireless communication.
  • PC or a mobile terminal PC or a mobile terminal.
  • all the opening actions are automatically completed by the automatic control device, and the moving part is controlled by the automatic control device using the stepping motor.
  • the present application provides an online collection and online automatic detection method for bacteria or viruses in the air.
  • the vacuum device is a vacuum pump, and the sample is automatically collected to set the sampling volume.
  • the settling time is set.
  • the static setting device is selected for 2 minutes, the negative pressure device is activated, and the liquid in the sampling area is sucked into the waste liquid bottle, and the particles adsorbed with DNA or RNA are trapped on the filter medium in the sampling area. .
  • the pressurizing device is a first peristaltic pump, and a DNA or RNA eluate is added to the sampling area to elute the DNA or RNA on the particles.
  • the elution solution of the eluted DNA or RNA is added to the PCR amplification device by a sample control device to perform PCR amplification by a preset PCR amplification program.
  • the detection result is output to obtain the type and concentration of the microorganism.
  • the liquid in the liquid collection area was added with 1 ml of commercially available DNA (RNA) adsorption particles, and the pH of the solution was adjusted to 6-8 with 0.01 mol/L NaOH and HCl, respectively, and an influenza virus collection experiment in the air was carried out.
  • the rat lung adaptor strain influenza A virus was reconstituted by chicken embryos, and then configured into a 100,000 virus/ml solution with a 0.1 mol/L phosphate solution, and then air-distributed to the air in a closed space of about 50 m 3 . The virus is sprayed, and the specific number of virions is calculated based on the amount of liquid reduction in the nebulizer (viral particles in the air is about 1000/m 3 ).
  • the liquid volume of the liquid collection area is 15 ml, the height-to-diameter ratio is 3:1, and 0.1 m 3 of influenza virus is collected in different ways, and the sampling test is performed twice, three batches each time, and the experimental results (Table 1).
  • the PCR kit was tested using a commercially available H1N1 quantification kit.
  • the liquid in the liquid collection area was added with 1 ml of commercially available DNA (RNA) adsorption particles, and the pH of the solution was adjusted to 6-8 with 0.01 mol/L NaOH and HCl, respectively, and an E. coli collection experiment in the air was carried out.
  • Escherichia coli DH5 ⁇ was cultured to a concentration of 5 ⁇ 10 6 by a conventional method, and then configured into a 100,000 cells/ml solution with a 0.1 mol/L phosphate solution, and then used in an air distributor in a closed space having a volume of about 50 m 3 .
  • the virus is sprayed in the air, and the specific number of microbial particles is calculated based on the amount of liquid reduction in the nebulizer (the concentration of microbial cells in the air is about 1000/m 3 ).
  • the liquid volume of the liquid collection area is 15 ml, and the aspect ratio is 3:1.
  • 0.1 m 3 of E. coli is collected in different ways, and the sampling test is performed twice, three batches each time, and the experimental results (Table 2). It was detected using a commercially available Escherichia coli PCR quantitative detection kit.

Abstract

La présente invention concerne une méthode de collecte en ligne et de détection automatique en ligne de bactéries ou de virus dans l'air, destinée à être utilisée dans la détection rapide et sensible de micro-organismes pathogènes éventuellement présents dans l'air en combinaison avec une puce biologique ou une technologie de PCR quantitative fluorescente. L'appareil est principalement constitué d'un dispositif d'aspiration d'air à pression négative, d'un débitmètre d'air, d'une solution d'adsorption et de particules d'adsorption. Un dispositif d'extraction d'air est utilisé pour adsorber l'air au moyen d'une pression négative, de telle sorte que l'air circule à travers une zone de collecte de liquide à un débit d'air constant par minute. Sous l'action de la solution et des particules d'adsorption dans la solution, des particules de virus dans l'air sont collectées dans un faible volume de liquide, et pendant ce temps, des microorganismes collectés sont lysés, de telle sorte que l'ADN (ARN) des microorganismes est libéré et adsorbé sur les particules d'adsorption. Ensuite, l'ADN (ARN) est élué avec un éluant d'ADN (ARN) disponible dans le commerce au moyen d'une filtration en ligne, le nombre de microorganismes est ensuite evalué à l'aide de la technologie de PCR, et le nombre evalué de microorganismes dans l'air unitaire est obtenu en divisant un nombre de copies déterminé par le volume d'air adsorbé.
PCT/CN2018/085293 2017-05-16 2018-05-02 Méthode de collecte en ligne et de détection automatique en ligne de bactéries ou de virus dans l'air WO2018210128A1 (fr)

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WO2022024035A1 (fr) * 2020-07-29 2022-02-03 Istituto Nazionale Di Astrofisica Appareil et procédé d'échantillonnage et de détection d'un agent pathogène dans l'air
TWI756746B (zh) * 2020-07-21 2022-03-01 威盛電子股份有限公司 病毒收集總成、病毒儲存裝置及病毒收集方法
WO2022254044A1 (fr) 2021-06-04 2022-12-08 Compan Valerie Dispositif mobile et autonome et procédé de détection d'analytes dans l'air
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TWI756746B (zh) * 2020-07-21 2022-03-01 威盛電子股份有限公司 病毒收集總成、病毒儲存裝置及病毒收集方法
WO2022024035A1 (fr) * 2020-07-29 2022-02-03 Istituto Nazionale Di Astrofisica Appareil et procédé d'échantillonnage et de détection d'un agent pathogène dans l'air
CN112557364A (zh) * 2020-12-11 2021-03-26 天津市职业大学 一种智能室内空气质量病毒检测系统及检测方法
CN112557364B (zh) * 2020-12-11 2023-03-07 天津市职业大学 一种智能室内空气质量病毒检测系统及检测方法
WO2022254044A1 (fr) 2021-06-04 2022-12-08 Compan Valerie Dispositif mobile et autonome et procédé de détection d'analytes dans l'air
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EP4290210A1 (fr) 2022-06-08 2023-12-13 Fundació Institut de Ciències Fotòniques Dispositif, procédé et programme informatique pour la détection des particules en suspension dans les aérosols
CN115902088A (zh) * 2023-03-09 2023-04-04 合肥泷睿机器人有限责任公司 纳米二氧化钛光催化筛网病毒消杀性能测试装置

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