WO2018210128A1 - 一种空气中细菌或病毒在线采集及在线自动检测方法 - Google Patents

一种空气中细菌或病毒在线采集及在线自动检测方法 Download PDF

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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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sampling
air
online
viruses
bacteria
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PCT/CN2018/085293
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English (en)
French (fr)
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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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    • 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]

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  • 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

本发明涉及一种空气中细菌或病毒在线采集及在线自动检测方法，结合生物芯片或荧光定量PCR技术，可快速、灵敏地检测出空气中可能存在的致病微生物。该装置主要由负压吸气装置、空气流量计、吸附液及吸附粒子组成。利用抽气装置，通过负压吸附空气，以每分钟恒定气流量，经过液体采集区，在溶液和溶液内的吸附粒子的作用下，使空气中的病毒粒子收集在小体积的液体中，同时裂解收集的微生物，释放微生物DNA(RNA)，吸附在吸附粒子上。然后通过在线过滤，收集液体中的粒子，用市售的DNA(RNA)洗脱液洗脱DNA(RNA)，然后采用PCR技术检测微生物量，测定的拷贝数除以吸附的空气体积，即检测所得的单位空气中的微生物数量。

Description

一种空气中细菌或病毒在线采集及在线自动检测方法 技术领域
本发明属于微生物检测技术领域，特别是指一种空气中细菌或病毒在线采集及在线自动检测方法。
背景技术
空气传播是一些致病菌或病毒传播的主要途径，也是目前环境中最难以检测和控制的途径。
从2003年给全球带来巨大冲击的SARS疫情，到2009年影响国内的H1N1和2013年爆发的禽流感疫情，都是可通过空气传播的病毒性疾病。以SARS病毒为例：病毒主要存在于传染源(人或动物)的鼻涕、痰和唾液中，通过空气飞沫和接触传播。患者通过咳嗽、打喷嚏、甚至大声说话时都会将病毒随唾液飞沫、尘埃粒子等扩散到周围的空气中，随空气流动传播。
SARS初期之所以造成大量人员感染特别是医护人员带来巨大牺牲，除防护措施的原因外，很大程度上是由于空气中的病毒难以监测和防范造成的。
SARS疫情虽然过去十几年了，但它给社会生活带来的巨大影响和社会的恐慌情绪让我们记忆犹新。时至今日，对于空气中有害微生物的预警与监测仍然缺乏有效的工具和方法。现有检测仪器及检测方法程序复杂，耗时较长，难以及时提供疫情现场准确的数据信息以便迅速采取防范措施。
随着社会的发展，不同国家、地区人们的交往越来越频繁，因此公共交通工具、交通站点、学校、医院、商场、写字楼等人流密集的封闭区域已经成为空气中传播疾病的主要场所。特别是在医院中，院内交叉感染一直是令医院头疼的问题。
当禽流感病毒流行时，因为没有快速检测装置，政府往往在没有直接证据情况下对家禽养殖场进行盲目地扑杀，给政府和养殖场都造成巨大损失。
发明内容
本发明的目的是提供一种空气中细菌或病毒在线采集及在线自动检测方 法，以解决不能对空气中细菌或病毒进行预警或监测的问题，以实现对空气中的细菌或病毒进行迅速准确的现场检测。
本发明是通过以下技术方案实现的：
一种空气中细菌或病毒在线采集及在线自动检测方法，
使用在线采集及在线自动检测装置，包括加样控制装置、PCR扩增装置、检测装置及自动控制装置，还包括加压装置及负压装置；包括以下步骤：
1)开启在线采集及在线自动检测装置，根据待检测的对象及对应的PCR试剂盒要求，预设置PCR扩增程序，然后插入一体化采样盒；
2)设置取样体积，然后开启所述负压装置，自动采集样品至设置取样体积；
3)静置设置时间，所负压装置启动，将采样区的液体吸入废液瓶，吸附了DNA或RNA的颗粒被截留在所述采样区的过滤介质上；
4)开启所述加压装置，在所述采样区加入DNA或RNA洗脱液，将所述颗粒上的所述DNA或所述RNA洗脱下；
5)通过所述加样控制装置，将洗脱下所述DNA或所述RNA的洗脱液加入到所述PCR扩增装置通过所述预设置PCR扩增程序进行PCR扩增；
6)将扩增后的样品通过检测装置进行检测后，输出检测结果。
所述采样一体化采样盒与所述加样控制装置通过管路连接；所述加样控制装置与所述PCR扩增装置配合；
所述自动控制装置分别与所述加样控制装置、所述PCR扩增装置、所述检测装置、所述加压装置及所述负压装置电连接；
所述加压装置与所述负压装置均与所述采样一体化采样盒通过管路连接。
所述采样一体化采样盒包括采样容器、洗脱液存储容器及废液缸；洗脱液存储容器与所述采样容器通过管路连接；所述废液缸与所述采样容器的底部通过管路连接；
在所述采样容器与所述洗脱液存储容器之间的管路上设置有所述加压装置；
所述采样容器与所述废液缸分别通过管路与所述负压装置连接；
在所述采样容器内设置有多孔空气分布器及过滤器；所述多孔空气分布器 的入口与连接所述采样容器与所述负压装置的管路连接，所述过滤器设置于所述采样容器的底部出口处，在所述采样容器内设置有吸附液，所述多孔空气分布器的出气口低于所述吸附液的液面；所述吸附液由微生物裂解液及吸附粒子组成；
在所述洗脱液存储容器内设置有洗脱液。
在所述吸附液中，所述微生物裂解液与所述吸附粒子的体积比为10-20：1。
所述加压装置为第一蠕动泵，所述负压装置为真空泵。
还包括插拨式接口装置，用于采样一体化采样盒的快速插拨。
所述加样控制装置包括第二蠕动泵及单向阀；所述第二蠕动泵的出口与所述PCR扩增装置相对应，所述第二蠕动泵的入口与所述单向阀的出口通过管路连接；所述单向阀的入口通过管路与所述采样容器的底部出口连接。
所述PCR扩增装置包括加样板、PCR试剂盒及PCR温度控制装置，所述加样板与所述PCR试剂盒连通，所述PCR试剂盒与所述PCR温度控制装置连接。
所述检测装置包括激发光发射器、荧光接收器及人机对话系统；所述激发光发射器与所述荧光接收器相对设置于所述PCR扩增装置的检测区，所述激发光发射器与所述荧光接收器均与所述人机对话系统电连接，所述人机对话系统与所述自动控制装置电连接。
所述自动检测装置包括中央控制器，所述中央控制器为程序控制器、单片机或工控机中的一种；
所述自动检测装置还包括蓝牙接口、无线WIFI接口、以太网接口、USB接口、RS232串口或RS485串口中的一种或多种组合。
本发明的有益效果是：
本技术方案的细菌或病毒在线采集及在线自动检测方法，包括负压抽气、微生物在线采集、空气微生物吸附、DNA或RNA在线提取、DNA在线扩增及检测等部件，通过采集空气中的微生物粒子，在液体中进行实时裂解、提取DNA(或RNA)，通过PCR扩增后，进行在线实时检测。
本技术方案通过采样、提取及检测集合于一体，能将采集的样品分别分析，并适合测定单位体积空气中不同种类微生物数量。
本技术方案开发出的新型的空气微生物采样检测仪器，结合生物芯片或荧光定量PCR技术，可快速、灵敏地检测出空气中可能存在的致病微生物。该装置主要由负压吸气装置(手持式或真空泵)、空气流量计、吸附液及吸附粒子组成。利用抽气装置，通过负压吸附空气，以每分钟恒定气流量，经过液体采集区，在溶液和溶液内的吸附粒子的作用下，使空气中的病毒粒子收集在小体积的液体中，同时裂解收集的微生物，释放微生物DNA(RNA)，吸附在吸附粒子上。然后通过在线过滤，收集液体中的粒子，用市售的DNA(RNA)洗脱液洗脱DNA(RNA)，然后采用PCR技术检测微生物量，测定的拷贝数除以吸附的空气体积，即检测所得的单位空气中的微生物数量。
附图说明
图1为本发明装置的结构示意图；
图2为本发明采样一体化采样盒工作示意图。
附图标记说明
1真空泵，2第一电磁阀，3采样容器，4第一蠕动泵，5洗脱液存储容器，6洗脱液，7吸附液，8第二蠕动泵，9单向阀，10第二电磁阀，11废液缸，12激发光发射器，13荧光接收器，14人机对话系统，15加样板，16PCR温度控制装置，17过滤器。
具体实施方式
以下通过实施例来详细说明本发明的技术方案，以下的实施例仅是示例性的，仅能用来解释和说明本发明的技术方案，而不能解释为是对本发明技术方案的限制。
本发明开发出新型的空气微生物采样检测仪器，结合生物芯片或荧光定量PCR技术，可快速、灵敏地检测出空气中可能存在的致病微生物。该装置主要由负压吸气装置(手持式或真空泵)、空气流量计、吸附液及吸附粒子组成。利用抽气装置，通过负压吸附空气，以每分钟恒定气流量，经过液体采集区，在溶液和溶液内的吸附粒子的作用下，使空气中的病毒粒子收集在小体积的液体中，同时裂解收集的微生物，释放微生物DNA(RNA)，吸附在吸附粒子上。 然后通过在线过滤，收集液体中的粒子，用市售的DNA(RNA)洗脱液洗脱DNA(RNA)，然后采用PCR技术检测微生物量，测定的拷贝数除以吸附的空气体积，即检测所得的单位空气中的微生物数量。
目前的市场上该类采样器只适用于高浓度的空气微生物采样，对于较低密度的病毒，由于空气在较小体积的液体中的停留时间等因素的限制，无法捕集微小病毒粒子，因此灵敏度不高，同时也不能在线测定。本发明通过采样、提取及检测集合于一体，能将采集的样品分别分析，并适合测定单位体积空气中不同种类微生物数量。
本申请提供一种空气中细菌或病毒在线采集及在线自动检测装置，如图1和图2所示，包括采样一体化采样盒、加样控制装置、PCR扩增装置、检测装置及自动控制装置，还包括与采样一体化采样盒相配合的加压装置及负压装置。
采样一体化采样盒与加样控制装置通过管路连接；加样控制装置与PCR扩增装置配合；本申请的采样一体化采样盒通过插拨式接口装置，用于采样一体化采样盒的快速插拨。
自动控制装置分别与加样控制装置、PCR扩增装置、检测装置、加压装置及负压装置电连接；自动检测装置包括中央控制器，中央控制器为程序控制器、单片机或工控机中的一种，通过编程实现从空气采样到检测出微生物种类及浓度全自动进行。
自动检测装置还包括蓝牙接口、无线WIFI接口、以太网接口、USB接口、RS232串口或RS485串口中的一种或多种组合，通过有线或无线方式与上位机或移动终端通讯，实现对本装置的远程监控，检测人员无需深入疫区便可得到检测结果，避免了检测人员被感染的风险。同时，这些接口可以与其它设备如上位机、手机等进行通讯，使检测结果可被其它设备应用，自动检测装置还可以被其它设备所控制，也可实现对检测数据的远程传输或云端管理以及对自动检测装置的远程控制。
加压装置与负压装置均与采样一体化采样盒通过管路连接。
加压装置为第一蠕动泵4；在本申请中负压装置为真空泵1。
采样一体化采样盒包括采样容器3、洗脱液存储容器5及废液缸11；洗脱液存储容器与采样容器通过管路连接；废液缸与采样容器的底部通过管路连接。 在采样一体化采样盒中，液体的流动通过气压控制，管路上通过单向阀控制气体流动方向，从而控制采样容器、洗脱液存储容器或废液缸中的压力，实现液体(吸附液或洗脱液)的定向流动。
在采样容器与洗脱液存储容器之间的管路上设置有加压装置；
采样容器3与废液缸分别通过管路与负压装置连接；在本申请中，负压装置为真空泵1，在真空泵与采样容器之间设置有第一电磁阀2，在真空泵与废液缸之间设置有第二电磁阀10；第一电磁阀与第二电磁阀均与自动控制装置电连接，通过自动控制装置控制第一电磁阀和第二电磁阀的开启或关闭。
在采样容器内设置有多孔空气分布器及过滤器17；多孔空气分布器的入口与连接采样容器与负压装置的管路连接，过滤器设置于采样容器的底部出口处，在采样容器内设置有吸附液7，多孔空气分布器的出气口低于吸附液的液面；多孔空气分布器的出气口通过多个细孔方式将抽入的带菌空气分布到吸附液中，为了防止气流太大，直接短路冲出液体，多个细孔将带菌空气分布成小气泡，增加带菌空气与吸附液的接触面积，提高吸附空气中微生物的效率。
吸附液由微生物裂解液及吸附粒子组成。
在洗脱液存储容器5内设置有洗脱液6，在本申请中，洗脱液为DNA(RNA)洗脱液，均为市售的DNA(RNA)洗脱液。
在吸附液中，微生物裂解液与吸附粒子的体积比为10-20：1。
吸附粒子为DNA吸附粒子或RNA吸附粒子。
吸附液在采样容器中的高径比为6：1-3：1。
在本申请中，微生物裂解液采用磷酸盐缓冲液PBS，配置方法为，将NaCl、KCl、Na 2HPO 4和KH 2PO 4,溶于蒸馏水中，用HCl调节溶液的pH值至7.4，最后加蒸馏水定容即可，并在121℃高压下蒸气灭菌20分钟，保存存于室温或4℃冰箱中。
例如，8g NaCl、0.2g KCl、1.44g Na 2HPO 4和0.24g KH 2PO 4,溶于800ml蒸馏水中，用HCl调节溶液的pH值至7.4，最后加蒸馏水定容至1L即可。121℃高压下蒸气灭菌20分钟，保存存于室温或4℃冰箱中。
在本申请中，广谱高效吸附液组成：1ml PBS溶液中添加粒径分布为10-30毫克直径为10-200nm的壳聚糖纳米粒子。
在本申请中，如采用特异性吸附液组成，1ml PBS溶液中添加粒径分布为10-30.毫克直径为10-200nm的表面带有流感病毒抗体修饰的纳米粒子。
在本申请中，DNA吸附粒子或RNA吸附粒子均可以为市售的吸附树脂，如上海生工或北京全式金等公司生产的DNA(RNA)吸附树脂。
加样控制装置包括第二蠕动泵8及单向阀9；第二蠕动泵的出口与PCR扩增装置相对应，第二蠕动泵的入口与单向阀的出口通过管路连接；单向阀的入口通过管路与采样容器的底部出口连接。在本申请的加样板的加样区，采用第二蠕动泵进行加样，采用计滴器进行加样量的计量。
PCR扩增装置包括加样板15、PCR试剂盒及PCR温度控制装置16，加样板与PCR试剂盒连通，PCR试剂盒与PCR温度控制装置连接。在PCR试剂盒中添加液体石蜡对反应液进行密封。本申请的PCR试剂盒为市售的PCR试剂盒。
检测装置包括激发光发射器12、荧光接收器13及人机对话系统14；激发光发射器与荧光接收器相对设置于PCR扩增装置的检测区，荧光发射器与荧光接收器均与人机对话系统电连接，人机对话系统与自动控制装置电连接。同时人机对话系统还可以设定相关检测参数及设备维护管理。
具体的工作原理是：
根据检测对象及PCR试剂盒要求，设置PCR扩增程序，插入采样一体化采样盒；设置好采样体积，然后开启装置，自动控制装置会控制真空泵启动，实现自动采样至设定体积；通过静置2分钟后，自动控制装置控制真空泵再次启动，使废液缸中为负压，将采样容器中的液体吸入到废液缸，吸附了DNA(RNA)的颗粒截止在采样容器中的过滤介质上；自动控制装置控制第一蠕动泵启动，为洗脱液存储容器加压，将DNA(RNA)洗脱液压入到采样容器中，将明附粒子上吸附的DNA(RNA)洗脱下，通过单向阀及第二蠕动泵将洗脱下的DNA(RNA)的洗脱液加入到预先加有市售DNA(RNA)检测样品PCR试剂盒中，并根据PCR试剂盒的要求预设的程序进行PCR扩增；扩增后的样品通过检测区的检测装置检测后，输出结果给自动控制装置，获得微生物的种类信浓度，并将检测结果通过有线或无线通讯传递给上位机或移动终端。
在本申请中，所有的开启动作均由自动控制装置控制各装置自动完成，运动部分采用步进电机由自动控制装置控制完成。
本申请提供一种空气中细菌或病毒在线采集及在线自动检测方法，
使用在线采集及在线自动检测装置，包括以下步骤：
1)开启在线采集及在线自动检测装置，进行开机预热，根据待检测的对象及对应的PCR试剂盒要求，预设置PCR扩增程序，然后插入一体化采样盒，在本申请中，所有耗材均需要无菌处理。
2)设置取样体积，然后开启负压装置，在本实施例中，负压装置为真空泵，自动采集样品至设置取样体积。
3)静置设置时间，在本实施例中选用静置2分钟，所负压装置启动，将采样区的液体吸入废液瓶，吸附了DNA或RNA的颗粒被截留在采样区的过滤介质上。
4)开启加压装置，在本实施例中，加压装置为第一蠕动泵，在采样区加入DNA或RNA洗脱液，将颗粒上的DNA或RNA洗脱下。
5)通过加样控制装置，将洗脱下DNA或RNA的洗脱液加入到PCR扩增装置通过预设置PCR扩增程序进行PCR扩增。
6)将扩增后的样品通过检测装置进行检测后，输出检测结果，获得微生物的种类及浓度。
实施例1
液体采集区的液体添加1ml市售DNA(RNA)吸附粒子，分别用0.01mol/L的NaOH和HCl调节溶液的pH至6-8，进行空气中流感病毒的采集实验。将鼠肺适应株甲型流感病毒经鸡胚复壮后，用0.1mol/L的磷酸盐溶液配置成100000个病毒/ml溶液，然后向体积约50m 3的密闭空间中，用空气分布器在空气中喷洒病毒，具体的病毒粒子数根据喷雾器中液体的减少量计算(空气中的病毒粒子约为1000个/m 3)。液体采集区的液体体积15ml，高径比为3:1，采用不同方式采集0.1m 3的流感病毒，分两次进行采样检测实验，每次三个批次，实验结果(表1)。PCR试剂盒采用市售的H1N1定量试剂盒检测。
表1 空气中病毒粒子的采集结果
Figure PCTCN2018085293-appb-000001
Figure PCTCN2018085293-appb-000002
注：采样方式，喷雾后，立刻用线采集及在线自动检测装置分别在房间的四个角落和中间位置离地1.6m处同时取样，然后计算平均值，在表1中1个病毒为1个copy。
实施例2
液体采集区的液体添加1ml市售DNA(RNA)吸附粒子，分别用0.01mol/L的NaOH和HCl调节溶液的pH至6-8，进行空气中大肠杆菌的采集实验。将大肠杆菌DH5α采用常规方法培养至浓度为5×10 6后，用0.1mol/L的磷酸盐溶液配置成100000个细胞/ml溶液，然后向体积约50m 3的密闭空间中，用空气分布器在空气中喷洒病毒，具体的微生物粒子数根据喷雾器中液体的减少量计算(空气中的微生物细胞浓度约为1000个/m 3)。液体采集区的液体体积15ml，高径比为3:1，采用不同方式采集0.1m 3的大肠杆菌，分两次进行采样检测实验，每次三个批次，实验结果(表2)。采用市售的大肠杆菌PCR定量检测试剂盒检测。
表2 空气中大肠杆菌的采集结果
Figure PCTCN2018085293-appb-000003
注：注:采样方式，喷雾后，立刻用采样器分别在房间的四个角落和中间位置离地1.6m处同时取样，然后计算平均值。在表2中1个大肠菌群为1个pfu。
以上仅是本发明的优选实施方式的描述，应当指出，由于文字表达的有限性，而在客观上存在无限的具体结构，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (10)

  1. 一种空气中细菌或病毒在线采集及在线自动检测方法，其特征在于：
    使用在线采集及在线自动检测装置，包括加样控制装置、PCR扩增装置、检测装置及自动控制装置，还包括加压装置及负压装置；包括以下步骤：
    1)开启在线采集及在线自动检测装置，根据待检测的对象及对应的PCR试剂盒要求，预设置PCR扩增程序，然后插入一体化采样盒；
    2)设置取样体积，然后开启所述负压装置，自动采集样品至设置取样体积；
    3)静置设置时间，所负压装置启动，将采样区的液体吸入废液瓶，吸附了DNA或RNA的颗粒被截留在所述采样区的过滤介质上；
    4)开启所述加压装置，在所述采样区加入DNA或RNA洗脱液，将所述颗粒上的所述DNA或所述RNA洗脱下；
    5)通过所述加样控制装置，将洗脱下所述DNA或所述RNA的洗脱液加入到所述PCR扩增装置通过所述预设置PCR扩增程序进行PCR扩增；
    6)将扩增后的样品通过检测装置进行检测后，输出检测结果。
  2. 根据权利要求1所述的空气中细菌或病毒在线采集及在线自动检测方法，其特征在于：所述采样一体化采样盒与所述加样控制装置通过管路连接；所述加样控制装置与所述PCR扩增装置配合；
    所述自动控制装置分别与所述加样控制装置、所述PCR扩增装置、所述检测装置、所述加压装置及所述负压装置电连接；
    所述加压装置与所述负压装置均与所述采样一体化采样盒通过管路连接。
  3. 根据权利要求1或2所述的空气中细菌或病毒在线采集及在线自动检测方法，其特征在于：所述采样一体化采样盒包括采样容器、洗脱液存储容器及废液缸；洗脱液存储容器与所述采样容器通过管路连接；所述废液缸与所述采样容器的底部通过管路连接；
    在所述采样容器与所述洗脱液存储容器之间的管路上设置有所述加压装置；
    所述采样容器与所述废液缸分别通过管路与所述负压装置连接；
    在所述采样容器内设置有多孔空气分布器及过滤器；所述多孔空气分布器 的入口与连接所述采样容器与所述负压装置的管路连接，所述过滤器设置于所述采样容器的底部出口处，在所述采样容器内设置有吸附液，所述多孔空气分布器的出气口低于所述吸附液的液面；所述吸附液由微生物裂解液及吸附粒子组成；
    在所述洗脱液存储容器内设置有洗脱液。
  4. 根据权利要求3所述的空气中细菌或病毒在线采集及在线自动检测方法，其特征在于：在所述吸附液中，所述微生物裂解液与所述吸附粒子的体积比为10-20：1。
  5. 根据权利要求1所述的空气中细菌或病毒在线采集及在线自动检测方法，其特征在于：所述加压装置为第一蠕动泵，所述负压装置为真空泵。
  6. 根据权利要求3所述的空气中细菌或病毒在线采集及在线自动检测方法，其特征在于：还包括插拨式接口装置，用于采样一体化采样盒的快速插拨。
  7. 根据权利要求3所述的空气中细菌或病毒在线采集及在线自动检测方法，其特征在于：所述加样控制装置包括第二蠕动泵及单向阀；所述第二蠕动泵的出口与所述PCR扩增装置相对应，所述第二蠕动泵的入口与所述单向阀的出口通过管路连接；所述单向阀的入口通过管路与所述采样容器的底部出口连接。
  8. 根据权利要求3所述的空气中细菌或病毒在线采集及在线自动检测方法，其特征在于：所述PCR扩增装置包括加样板、PCR试剂盒及PCR温度控制装置，所述加样板与所述PCR试剂盒连通，所述PCR试剂盒与所述PCR温度控制装置连接。
  9. 根据权利要求1所述的空气中细菌或病毒在线采集及在线自动检测方法，其特征在于：所述检测装置包括激发光发射器、荧光接收器及人机对话系统；所述激发光发射器与所述荧光接收器相对设置于所述PCR扩增装置的检测区，所述激发光发射器与所述荧光接收器均与所述人机对话系统电连接，所述人机对话系统与所述自动控制装置电连接。
  10. 根据权利要求1所述的空气中细菌或病毒在线采集及在线自动检测方法，其特征在于：所述自动检测装置包括中央控制器，所述中央控制器为程序控制器、单片机或工控机中的一种；
    所述自动检测装置还包括蓝牙接口、无线WIFI接口、以太网接口、USB接口、RS232串口或RS485串口中的一种或多种组合。
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