GB2583901A

GB2583901A - Apparatus and method for the automated detection of airborne biological particles

Info

Publication number: GB2583901A
Application number: GB1905583.9A
Authority: GB
Inventors: Wili Stuart
Original assignee: Agri Samplers Ltd
Current assignee: Agri Samplers Ltd
Priority date: 2019-04-18
Filing date: 2019-04-18
Publication date: 2020-11-18
Anticipated expiration: 2039-04-18
Also published as: GB201905583D0; GB2583901B; WO2020212708A1

Abstract

Apparatus 100 comprises means 102 for intercepting airborne biological particles. A collection device (e.g. linear actuator 110) is configured to select an empty one of a plurality of collection vessels 108 and collect the particles therein. Disruption device 112 (e.g. rotary magnetic device) mechanically disrupts the particles to release DNA and other cell contents. Means (e.g. liquid and/or dried reagents) for amplifying targeted DNA of a specific type of particle and a detector (e.g. fluorimeter 130) for detecting DNA are provided. In an aspect, a predetermined amount of liquid and two magnetic objects are placed into the collection vessel. A magnet rotatable around the vessel actuates the magnetic objects to mechanically disrupt spores. A predetermined volume of liquid reagent is added, together with the disrupted spores, to a test vial containing dried reagents. The vial is heated and illuminated, and emitted light is measured with a fluorimeter.

Description

APPARATUS AND METHOD FOR THE AUTOMATED DETECTION OF AIRBORNE

BIOLOGICAL PARTICLES

The present invention relates to an apparatus and method for the automated detection of airborne biological particles. In particular, the apparatus and method of the present invention find application for the detection and quantification of airborne pathogens which could affect agricultural crops with a view to providing near real-time information about their presence in a given area.

Many crop diseases are caused by airborne pathogens. Air dispersal is one of the major mechanisms by which a disease can spread to reach new susceptible plants. Some pathogens -such as fungal spores, plant spores, pollens, and bacteria -remain viable and capable of causing outbreaks of the disease even after travelling very far from their source. This can affect crops and have a serious impact on the economic output of farms.

To counter this, farmers and growers make use of pesticides and fungicides to protect their crops while they are growing. However, it is difficult to predict whether a disease is likely to affect a particular crop, and how severe it is likely to become. Forecasts based solely on weather-related information, in particular, may lead to pesticides and fungicides being applied inappropriately, at the wrong time, or when they are not required at all. Further, if too much fungicide or pesticide is applied, then resistance may occur as the pathogens may become insensitive to a particular treatment. As such, it is very important that the correct dose of the pesticide or fungicide is applied to a crop at the right time.

For this reason, it is desirable to be able to access accurate information relating to the presence of certain pathogens in a given area. Air samplers adapted to detect the presence of airborne pathogens such as fungal spores can be used within the framework of a disease forecasting protocol, which may effectively enable early warning of disease risk and facilitate the control of epidemics. In addition, one such disease forecasting protocol can help growers to better manage diseases and the fungicides that are used to control them, since application of a treatment to a crop may thus be more closely timed and tailored to the need estimated by forecasting models.

It would be particularly desirable to be able to provide farmers and growers with information about the presence of airborne spores in the fields that is as up-to-date as possible, preferably alongside accurate weather data, since this could help inform their decisions on if, when, and where to spray fungicides on a preventative basis.

A number of air samplers have been developed over the past decades for sampling airborne biological particles, starting with the Hirst trap and successive modifications thereof, in which airborne biological particles like pollen and spores are collected by suction and impacted onto a sticky surface, such as an adhesive tape or greased microscope slide. The impaction surface is moved slowly past the orifice to enable time-discriminate analysis, a number of different methods being available for analysing the concentration and type of the trapped biological particles. Traditionally, airborne biological particles have been analysed by performing plate counts or direct counts using biological stains to discriminate between biological and non-biological particles. However, such methods have been found to fail to detect a significant fraction of the total microbial diversity in a given environment. Accordingly, the need has been felt to apply more accurate analytical methods for the identification of airborne biological particles, including DNA-based diagnostic methods.

More recently, a multi-vial cyclone has been provided that is adapted to capture and sample airborne biological particles directly into tubes suitable for carrying out immunological or DNA-based diagnostic methods. In an embodiment, the multi-vial cyclone comprises a cyclone sampler mounted on a carousel of eight collection tubes, which can be programmed to rotate a new collection tube into place for collection at a desired time. Another, further improved air sampling device is known from UK patent application GB 2510501, wherein a virtual impactor and a plurality of vessels for collecting biological particles that pass through the virtual impactor are provided for use in combination with means for detecting biological particles within the same collection vessels. Devices of this type facilitate and improve the efficiency of the air sampling process. As such, they may help improve the overall detection sensitivity of an analytical protocol by providing samples that give clearer readings when tested.

However, it would be desirable to provide a novel and improved apparatus and method for the automated detection of airborne biological particles capable of allowing quicker and more accurate quantifications of airborne species. In particular, it would be desirable that such a novel and improved apparatus could allow samples to be analysed reliably in-situ and in a short period of time, such that results relating to the presence of predetermined pathogens could be made available to farmers and growers within hours or a day at most by automatically and wirelessly transmitting the results of in-situ analysis.

According to an aspect of the present invention, there is provided an apparatus comprising means for intercepting airborne biological particles; a plurality of collection vessels; and a collection device configured to select an empty one of the plurality of collection vessels and coiled the intercepted biological particles therein. The apparatus further comprises means for preparing the collected biological particles for analysis which comprises a disruption device for generating a sample by mechanically disrupting the collected biological particles to release DNA and other cell contents and means for amplifying targeted DNA of a specific type of biological particle. The apparatus further comprises a detector for detecting the presence of the amplified targeted DNA of the specific type of biological particle within the sample.

The apparatus of the invention allows for providing near-real-time information on the risk of disease contamination in agricultural crops. Because the samples are analysed in-situ and results transmitted wirelessly and automatically, the apparatus allows for the results of analysis to be made available to the user within hours.

The term "airborne biological particles" is used herein to denote particles of biological origin such as bacteria, viruses, pollen, spores, nucleic acids, proteins, and toxins.

Means for intercepting and associated collection devices can be selected from a range of filters or inertial, gravitational, centrifugal, electrostatic, magnetic, or thermal techniques. Preferably, the collection device is a high-volume cyclone. Advantageously, using a high-volume cyclone allows for airborne biological particles to be intercepted with high efficiency at a high throughput.

Preferably, the means for intercepting airborne biological particles can intercept up to 300 litres of air per minute. Advantageously, intercepting such a high flow of air allows for airborne biological particles to be intercepted at a higher rate, allowing for more precise detection, detection of lower concentration of biological particles, and/or for the device to be used in a more secure location, further away from the crops of interest.

In preferred embodiments, the disruption device for mechanically disrupting the collected biological particles to release DNA or other cell contents is a rotary disruptor. More preferably, the disruption device for mechanically disrupting the collected biological particles to release DNA or other cell contents is a magnetic rotary disruptor. Preferably, the magnetic rotary disruptor comprises at least one magnet rotatable about the longitudinal axis of the selected collection vessel and around said vessel.

Preferably, the apparatus comprises means for depositing at least two magnetic objects into the selected collection vessel. Preferably, each one of the at least two magnetic objects comprise a protective coating. Said protective coating could be a coatings of chromium, stainless steel, or any other suitable coating material. Optionally, the magnetic objects are added to the vessel containing intercepted particles for analysis along with a predetermined volume of water.

Preferably, each of the at least two magnetic objects is an elongate cylinder. Advantageously, using a magnetic rotary disruptor allows for a high proportion of disruption (over 95 °/0) for different types of biological particles in a short period of time. Preferably, each of the magnetic objects are round needle rollers, such as those used in needle roller bearings, which have been chrome-plated before use.

In preferred embodiments, the mechanical disruption of the collected biological particles is carried out for between about 1 and about 10 minutes, preferably for between about 2 minutes and about 5 minutes, and in one particularly preferable embodiment about 3 minutes. Advantageously, it has been found that carrying out the mechanical disruption for only 3 minutes saves time and allows for quicker operation, while maintaining a high proportion of disruption.

Preferably, the apparatus comprises a plurality of sets of test vials, each set of test vials comprising a plurality of test vials and means for transferring a predetermined amount of disrupted biological particles into a test vial for analysis. Advantageously, this allows to only use part of the disrupted biological particles for analysis.

Preferably, the test vials belonging to each of the plurality of sets of test vials comprise means for amplifying the targeted DNA of a different one of a plurality of types of biological particles. Preferably, the transferring means is operable to transfer subsamples of the sample of disrupted biological particles into at least two separate test vials. Preferably, the at least two separate test vials belong to different sets of test vials. Advantageously, this allows for the same sample to be used for the detection of multiple specific types of biological particle. This can allow for more thorough detection of airborne biological particles by allowing for the detection of different targeted types of biological particles using the same sample.

In preferred embodiments, the means for amplifying the targeted DNA comprises at least one dried reagent and/or at least one liquid reagent. Advantageously, dried reagents typically allow for long shelf-life, easier transport to the end-user site, ease of storage, and ease of handling. Preferably, drying occurs by first freezing the reagents and then drying preferably under a vacuum. For example, options for freezing include snap-freezing in liquid nitrogen or more slow freezing in a conventional freezer. Preferably, the dried reagents are freeze-dried. Advantageously, liquid reagents typically allow for high sensitivity and specificity to DNA of a specific kind. Advantageously, using both dried and liquid reagents allows for the advantages of dried and liquid reagents to be combined.

Preferably, the transferring means is further operable to transfer a predetermined volume of at least one liquid reagent into one of the test vials. Preferably, the apparatus comprises at least one actuator to move the transferring means. Preferably, the apparatus comprises at least one actuator to move the collection vessel. In preferred embodiments, the actuators are linear actuators. Advantageously, multiple actuators to move different components of the apparatus allow for faster and more variable operation. Preferably, the apparatus comprises three linear actuators to move the transferring means. More preferably, the three linear actuators are configured to move the transferring means along three orthogonal axes.

Preferably, the apparatus further comprises a carousel configured to hold the plurality of test vials, indexable about the detector.

In preferred embodiments, the means for amplifying the targeted DNA further comprises a heating device. Advantageously, this can allow for faster and better amplification. Heating can be used to improve the specificity of a primer and reduce background amplification, depending on the targeted DNA.

The assays can be Loop Mediated Isothermal Amplification (LAMP) assays or Recombinase Polymerase Amplification (RPA) assays. LAMP assays are carried out at a constant temperature and use two or three sets of primers as well as a polymerase. Preferably, the temperature for LAMP assays is about 60 to about 72 "C depending on the targeted DNA and the assay. RPA assays are carried out using three enzymes, including a recombinase, a DNA-binding protein and a polymerase. Preferably, the temperature for RPA assays is about 37 to about 42 °C depending on the targeted DNA and the assay.

Preferably, the means for amplifying the targeted DNA comprises means for preparing the disrupted biological particles for optical analysis. More preferably, said means for preparing the disrupted biological particles for visual analysis comprises a dye, preferably a fluorescent dye.

Advantageously, dye can be used to both detect and quantify the amount of DNA of a specific kind. DNA assays require carefully designed primers to bind to the target DNA and replicate a piece of DNA many times so that the amount of DNA in the test vial increases greatly. Preferably, a fluorescent dye that binds to the targeted DNA can be used. If light of a first wavelength is directed at the test vial, it causes emission of light at a different wavelength (fluorescence), which can be measured and quantified.

More preferably still, dried and liquid reagents, dye, and heating are used in combination, depending on the requirements of the targeted DNA, to optimise the trade-off between sensitivity, specificity, shelf-life, ease of storage, and ease of handling. Possible combinations include a wet master mix and dye with dried primers, wet master mix with dried primers and dye, wet dye with dried master mix and primers, wet primers with dried master mix and dye, or wet primers and dye with dried master mix.

The term "primer' is used herein to denote short single strand RNA or DNA which serve as starting points for DNA synthesis. The term "master mix" is used herein to denote solutions of DNA polymerase and all other components required for polymerase chain reaction (PCR) except for primers. Such master mixes are commercially available, for example Thermo ScientificTm PCR Master Mix or LAVALAMPTm DNA Master Mix. The term "dye" is used herein to denote substances capable of visualising and staining DNA. For example, ethidium bromide can be used to adhere to DNA strands and when radiated with UV light, it fluoresces orange. Other potential dyes include propidium iodide, crystal violet, DAPI, 7-AAD, Hoechst 33258, YOYO-1, TOTO-1, DiTOTm-1, or EvaGreen®.

Preferably, the efficacy of the means for amplifying the DNA, which can be PCR, is temperature dependent. Advantageously, temperature dependence can allow for improved sensitivity and specificity.

In preferred embodiments, the apparatus comprises means for controlling the temperature at which the means for amplifying the targeted DNA is stored within the apparatus. Advantageously, this allows for improved shelf-life of the means for amplifying the targeted DNA. Liquid reagents (such as master mixes), especially, become unstable quickly at temperature above room temperature and therefore require cooling to ensure long shelf-life, in particular if direct sunlight and other adverse weather conditions are expected.

Preferably, the detector is an optical detector. More preferably, the optical detector is a fluorimeter. Advantageously, optical detectors and especially fluorimeters are cheap, low-maintenance, and offer consistent performance. Measurements with fluorimeters are quick, reliable, and produce limited amounts of data. Preferably, fluorescence readings are recorded every 5 seconds while assays are being carried out.

Preferably, the apparatus comprises a communication device for sending data to a server. Advantageously, this allows for the data to be processed, combined with data from other stations, and used to predict risk of infection in near-real-time.

In preferred embodiments, the apparatus comprises means for collecting weather data. Weather conditions significantly influence the likelihood of infection of crops. Advantageously, collecting weather data allows to improve the assessment of risk of infection.

Preferably, the apparatus comprises a processor for processing data. Advantageously, processing the analytical and/or weather data allows for the data to be used in the analysis of risk of infection. This can help to prevent pesticides and fungicides being applied inappropriately, at the wrong time, or when they are not required at all. This can further help to prevent resistance which may occur if too much fungicide or pesticide is applied.

Preferably, the data sent by the communication device comprises information such as the amount and identity of biological particles and/or weather data and/or predictive information.

Advantageously, detector and/or weather data can be sent to a server so that data processing and predictive modelling may be carried out. If a plurality of apparatuses are employed, sending the data to a server may allow for the data of the plurality of apparatuses to be combined to improve analysis of the results and predictive models. Alternatively, the data could be shared between nearby apparatuses such that processing of data from multiple apparatuses can occur on site.

The detector and/or weather data can be used to alert users of imminent risk of infections via a web-based system. Further information may be sent, such as error messages or messages indicating the status of the apparatus, such as "sampling", "processing sample", "operator on site", or "consumables required".

A variety of different prediction models may be used to produce predictive information. Qualitative and quantitative models based on published research may be used, or may be developed depending on the species of the targeted biological particles. Beyond the presence of potentially infectious biological particles, temperature, relative humidity, and presence of recent rainfall may contribute to the risk of infection determined by predictive models.

The communication device may be connected via a Personal Area Network (PAN; standards such as Bluetooth, ZigBee, or Wireless USB), a Wireless Local Area Network (WLAN), or Wireless Wide-Area Network (WAN; standards such as GSM, EDGE, GPRS, LTE, 4G, 5G). In preferred embodiments, the communication device is fitted with a SIM card. Advantageously, this allows for the data to be sent over a GSM, LTE, 4G, or 5G network.

Preferably, the SIM card is an AnyNet SecureTM SIM card. Advantageously, this allows for the data to be sent via the best available network in the area the apparatus is located in, rather than being tied to any one network. This is particularly beneficial in rural locations, which are the most likely location for the application of an apparatus according to the present invention. According to another aspect of the present invention, there is provided a method comprising the use of an apparatus according to the previous summary. According to another aspect of the present invention, there is provided a method comprising the steps of: collecting intercepted airborne biological particles into a collection vessel; adding a predetermined volume of liquid into the collection vessel containing the collected biological particles; dropping at least two magnetic objects into the collection vessel; mechanically disrupting the spores for a predetermined amount of time using a magnet rotatable around the collection vessel and configured to actuate the at least two magnetic objects; adding a predetermined volume of liquid reagent to a test vial containing dried reagents; collecting a set volume of disrupted spore sample from the collection vessel and adding it into the test vial; heating the test vial; illuminating the test vial; and measuring the emitted light with a fluorimeter.

Any feature in one aspect of the invention may be applied to other aspects of the invention, in any appropriate combination. In particular, method aspects may be applied to apparatus aspects, and vice versa. Furthermore, any, some and/or all features in one aspect can be applied to any, some and/or all features in any other aspect, in any appropriate combination.

It should also be appreciated that particular combinations of the various features described and defined in any aspects of the invention can be implemented and/or supplied and/or used independently.

The invention will now be further described with reference to the figures in which: Figure 1 shows a perspective view of an apparatus for the automated detection of airborne biological particles in accordance with the present invention; Figure 2 shows a schematic side view of the apparatus of Figure 1; Figure 3 shows another schematic side view of the apparatus of Figure 1 as viewed from the opposite side to Figure 2; Figure 4 shows a plan top view of the apparatus of Figures 1; Figure 5 shows a perspective view of the apparatus components responsible for the collection of airborne biological particles, the translation of collection vessels; and the disruption of the particles; Figure 6 shows a perspective view of the apparatus components responsible for the disruption of the particles, the amplification of DNA, and the detecting of specific DNA; Figure 7 shows a cross section of the components of the apparatus of Figure 1 responsible for the disruption of the particles; Figure 8 shows a cross section of the components of the apparatus of Figure 1 responsible for the amplification of DNA and the detecting of specific DNA; Figure 9 shows a perspective view of the components of the apparatus of Figure 1 responsible for the transferring of liquids; and Figures 10 shows a block diagram showing the handling of the data created by the apparatus of Figure 1.

Reference numeral 100 in Figure 1 identifies an apparatus for the automated detection of airborne biological particles in accordance with the present invention.

The apparatus 100 is adapted to collect spores and other airborne particles for analysis and identification. To this purpose, the apparatus 100 comprises means for intercepting airborne biological particles 102, which includes a cyclone fluidly connected to a pump 106, and is configured to deliver the collected airborne particles into a collection vessel 108. Means for intercepting airborne biological particles 102 further comprises a vane 109 which allows for the opening of the means for intercepting airborne biological particles 102 to be aligned with the wind direction to improve collection efficiency. Collection vessel 108 may be made of any suitable material and may be of any suitable shape. Preferably, collection vessel 108 is made from a polymer, such as nylon. The apparatus 100 further comprises means for sequentially advancing empty sample collection vessel 108a, 108b, 108c to a collection location adjacent an outlet of the cyclone. By way of example, the advancing means may comprise a piston driven by a dedicated motor.

Further, apparatus 100 comprises means for preparing a sample for a subsequent detection step. A linear actuator 110 is configured to move collection vessel 108 from the collection location first to a filling location and then to a disruption location along a linear track 111. At the disruption location, apparatus 100 comprises a disruption device 112, which is configured to mechanically disrupt the spores; in this example, it has been found that mechanical disruption for three minutes is effective.

The apparatus comprises a pipette 114 adapted to be moved along three orthogonal axes.

Pipette 114 is configured to pick up a pipette tip 116 from a rack of pipette tips 118 and to be moved to collect a set volume of liquid reagent (master mix) from a vial 119 in a rack 120 of foil-sealed vials 119. Said rack 120 is positioned above a cool-plate 121. Cool-plate 121 is configured to cool the sealed vials 119 in rack 120 because master mix may become unviable if exposed to temperatures above about 30 °C for extended periods of time.

Pipette 114 is further configured to add the liquid reagent to a test vial 122 located in a carousel 124 of test vials 126. The test vials 126 contain dried reagents, comprising primers and dye, and are covered with a foil seal which can be pierced by the pipette 114 using pipette tip 116. Pipette 114 is further configured to collect a set volume (in this example, 18 pL) of the disrupted spore sample from collection vessel 108 and transfer it to test vial 122. Test vial 122 in carousel 124 is aligned with heater 128 and fluorimeter 130.

Heater 128 comprises a heater block 129 and a cartridge heater. At a first end, heater block 129 comprises a connection point positioned on the bottom side of heater block 129. The connection point is configured to receive a pin to hingedly connect a solenoid to said first end of the heater block 129. Heater block 129 comprises a protrusion on the bottom side of the middle of heater block 129 configured to receive a fulcrum pin. Said fulcrum pin and protrusion are configured to act as a pivot, such that when the solenoid hingedly connected to the first end of heater block 129 moves upwards, the second end of heater block 129 moves downwards.

Heater block 129 is made of metal characterised by relatively high heat conductivity such as iron or steel. Heater block 129 is configured to conduct heat to a metal sleeve provided at the second end of the heater block 129. The metal sleeve is made of the same metal as heater block 129. The metal sleeve is configured to receive test vial 122, to radially heat the content of test vial 122.

Fluorimeter 130 is mounted below the metal sleeve and configured to illuminate test vial 122 from beneath and measure the light emitted by the contents of test vial 122. If test vial 122 is illuminated by light of a first wavelength and the content of test vial 122 contains a fluorescent dye, fluorescence causes emission of light at a different wavelength which can be measured and quantified.

Apparatus 100 further comprises a switch 132 for turning on and off as well as multiple buttons 134a, 134b allowing for a user to provide input.

Apparatus 100 further comprises a thermometer 136 to measure the temperature inside apparatus 100, which is connected to rack 119 and cool-plate 121 to ensure that the master mix in foil-sealed vials 120 is not exposed to excessive temperature.

The process for obtaining the data pertinent to the presence and quantity of airborne biological particles involves the following steps: A collection vessel 108 (nylon pot) is selected and placed beneath an outlet 510 of a high-volume cyclone 204. Collection vessel 108 is configured to collect spores and other airborne particles for a predetermined sampling period.

After the sampling period, collection vessel 108 containing the collected particles is moved by a linear actuator 110. A predetermined volume of liquid On this example, about 300 pL of water) is added to the collected particles.

Collection vessel 108 is again moved by linear actuator 110 to be accessible by a disruption device 112. Two magnetic objects are dropped into collection vessel 108 containing the collected particles and the predetermined volume of liquid Disruption device 112 mechanically disrupts the spores for three minutes.

Collection vessel 108 is again moved by linear actuator 110.

A pipette 114, mounted on multiple linear actuators 900, 902, 904 is configured to collect a pipette tip 116 from a rack of pipette tips 118. Further, the pipette 116 is configured to collect a set volume of liquid reagent (master mix) from a foil-sealed vial 119 which is located on a rack of foil-sealed vials 120.

The liquid reagent (master mix) is added by the pipette to a test vial 122 which is located in a carousel 124 attest vials 126 Said test vials 126 contain dried reagents (e.g. primers, fluorescent dye).

A set amount of disrupted sample is collected from collection vessel 108 by pipette 114 and is transferred to the same test vial 122 that the liquid reagent was added to.

Test vial 122 is then heated via a metal sleeve 800 connected to a heater 128.

Test vial 122 is illuminated from beneath (e.g. at a wavelength of 475 nm) and the emitted light (e.g. at a wavelength of 540 nm) is measured, S1000, by a fluorimeter 130.

The fluorescence results are used to determine if DNA associated with a specific type of biological particles is present.

The measured fluorescence data and measured weather data are relayed, 51002, by a 4G router to a server.

A web-based application displays the fluorescence data and weather data. Predictive models are used to combine the fluorescence data and weather data to give, 51004, an indication of risk of infection. An alert may be sent, S1006, automatically to the user if a high risk of infection is detected.

The collection device is shown in more detail in Figures 2, 3, 5, and 6. Apparatus 100 comprises an air inlet 200 connected to pipes 202 which act as means for intercepting airborne biological particles 102. Vane 109 is configured to align air inlet 200 with the wind direction. Pipes 202 are fluidly connected to cyclone 204. Multiple collection vessels are stored in tube 300 which is connected to linear actuator 302 configured to release the next of collection vessels 108a, 108b, 108c in tube 300. After a used collection vessel 108a, 108b, 108c has been disposed of automatically, a new collection vessel 108a, 108b, 108c is configured to be selected for a new collection and detection process. Collection vessels 108a, 108b, 108c have a tubular midsection 500 with an upper annular flange 502 and a lower annular flange 504 at two opposite ends of tubular midsection 500. Collection vessel 108 is secured to linear actuator 110 and linear track 111 by slotting lower annular flange 504 into a slot 506 of holding assembly 508. Slot 506 is configured to receive lower annular flange 504. For collection, collection vessel 108 is placed beneath outlet 510 of cyclone 204. Outlet 510 is formed to conform with upper annular flange 502 of collection vessel 108.

The linear actuators configured to move collection vessel 108 and pipette 114 are shown in more detail in Figures 2 to 6 and 9. Linear actuator 110 is configured to move collection vessel 108 from the collection location underneath outlet 510 of cyclone 204 to a filling location underneath a liquid dispenser 512. Liquid dispenser 512 is configured to add a predetermined amount of liquid to selected collection vessel 108, which contains collected biological particles.

Linear actuator 110 is configured to move collection vessel 108 into a disruption location in which collection vessel 108 is accessible to disruption device 112 and then to move collection vessel 108 further to a location at which collection vessel 108 is accessible to pipette 114. Pipette 114 is configured to collect the disrupted sample from collection vessel 108. Linear tracks 900, 902, 904 are configured to allow pipette 114 to move along three orthogonal axes. After the disrupted sample in collection vessel 108 has been analysed (possibly more than once, for multiple analyses), collection vessel 108 is disposed into waste pot 613. If part of the same disrupted sample is used for multiple analyses, collection vessel 108 may be moved back into the disruption location by linear actuator 110 to enable the disrupted spore to be mixed by the disruption device 112 for a few seconds. This is to ensure that the disrupted sample collected by pipette 114 is well dispersed.

Carousel 124 containing test vials 126 is shown in more detail in Figures 4 and 8. Test vials 126 located in carousel 124 are organised into sets of test vials 400, 402, 404, 406. These sets of test vials 400, 402, 404, 406 can comprise means for amplifying targeted DNA of a different one of a plurality of types of biological particles. The carousel is indexable about the detector, allowing for the selection of a test vial for a specific targeted type of DNA for each test. This may allow for the testing of targeted DNA of different biological particles using the same disrupted sample. It will be appreciated that the test vials 126 of each set of test vials 400, 402, 404, 406 do not need to be placed adjacent one another and that all test vials 126 may belong to the same set. When test vial 122 has been prepared for analysis, content 818 of test vial 122 comprises dried reagents 810 (also present in each of the other test vials 126), liquid reagent (master mix), and a subsample of the disrupted sample of collected biological particles from collection vessel 108. Carousel 124 is connected to a perpendicular rod 814 in its centre which allows for rotary movement of carousel 124 via a rotary actuator 816.

Disruption device 112 is shown in more detail in Figures 5,6, and 7. Figure 7 shows a cross section of disruptor device 112 along cross section AA-AA'. Disruption device 112 is configured to mechanically disrupt the collected biological particles for, in this example, three minutes. Liquid dispenser 512 is configured to add a predetermined amount of liquid to collection vessel 108 before disruption occurs. Disruption device 112 comprises magnet 514 located in one of two prongs 516 of a rotating disc 518. Magnet 514 is configured to rotate about the longitudinal axis of collection vessel 108 and around collection vessel 108 when collection vessel 108 is in the disruption location. Rotating disc 518 is connected to rotary actuator 600. Prongs 516 are spaced apart such that collection vessel 108 can be moved into the disruption location in between prongs 516. Before disruption, liquid dispenser 512 is configured to dispense a predetermined volume On this example, about 300 pL) of liquid (water, but buffer solution is also possible) into collection vessel 108. One of prongs 516 of rotating disc 518 comprises a circular recess 700 for receiving button magnet 514.

Heater 128, fluorimeter 130, and carousel 124 are shown in more detail in Figures 6 and 8.

Figure 8 shows a cross section of heater 128 and carousel 124 along cross section AA-AA'. Metal sleeve 800 is connected to a second end of heater block 129 and is configured to heat test vial 122. Solenoid 602 is hingedly connected to a first end of heater block 129 via pin 604 which is connected to two hinge mounts 606 protruding from the bottom of heater block 129. Solenoid 602 is configured to push a first end of heater block 129 upwards. In the middle of the bottom of heater block 129, heater block 129 further comprises protrusion 608 containing bore 610, which is configured to receive pin 612 configured to form fulcrum 614. Because heater block 129 is connected to fulcrum 614, when solenoid 602 pushes up the first end of heater block 129, the second end of heater block 129 and metal sleeve 800 connected to said second end of heater block 129 are pushed downwardly. By moving the second end of the heater block 129, comprising the sleeve for receiving a vial, down, the carousel may be indexed to position a vial above the sleeve. A connector 616 connected to fluorimeter 130 forces fluorimeter 130 downwards simultaneously. Connector 616 is connected to fluorimeter 130 via a sleeve 802 and a spring 804 to ensure that fluorimeter 130 returns to its initial position when metal sleeve 800 is moved back up. Heater block 128 comprises a cylindrical cavity 806 for receiving a cartridge heater via opening 808.

A weather station is provided as part of apparatus 100. The weather station may comprise instruments such as thermometers, barometers, hygrometers, and anemometers. Other sensors, for example a solar panel to record solar intensity, may be provided. The solar panel may also provide power to the system.

Weather data and detector results are transmitted automatically and wirelessly via a 4G router using an AnyNet SecureTM SIM; however, as will be appreciated, any suitable wireless 20 communication system may be used. The data is sent to the AWS Cloud and analysed and reported on a web-portal.

Weather data is sent every 10 minutes, although this may be varied depending in circumstances. Fluorescence data is sent every 5 seconds while assays are being carried out. Both fluorescence data and weather data is sent via text message. Text messages may further contain information such as apparatus ID and location. Further information may be sent, such as error messages or messages indicating the status of the apparatus, such as "sampling", "processing sample", "operator on site", or "consumables required". The fluorescence data is analysed to indicated no detection, a low number of spores, or a high number of spores.

The data from the weather station and the detector can be combined in risk models to give an indication of the risk of infection. Both qualitative and quantitative models can be used, depending on the species of biological particle(s) of interest. The detection of the presence and quantity of biological particles can be combined with information such as temperature, relative humidity, UV intensity, and presence of recent rainfall to predict imminent risks. If there is a risk of infection, the user can be alerted by the server or the web-based system.

As weather data and fluorescence data is collected, S1000, it is sent, S1002 to a server using a 4G router. The data is analysed, S1004, to be displayed in a web-based application and further to use predictive models to determine risk of infections. If a high risk of infection is detected, S1006, an alert may be sent to the user by the server.

Claims

CLAIMS 2. 3. 5. 6.An apparatus for the automated detection of airborne biological particles, the apparatus comprising: means for intercepting airborne biological particles; a plurality of collection vessels; a collection device configured to select an empty one of the plurality of collection vessels and collect the intercepted biological particles therein; means for preparing the collected biological particles for analysis, the preparing means comprising: a disruption device for generating a sample by mechanically disrupting the collected biological particles to release DNA and other cell contents; and means for amplifying targeted DNA of a specific type of biological particle; and a detector for detecting the presence of the amplified targeted DNA of the specific type of biological particle within the sample.
An apparatus according to claim 1, wherein the disruption device is a rotary disruptor.
An apparatus according to claim 2, wherein the rotary disruptor is a magnetic rotary disruptor.
An apparatus according to claim 3, wherein the magnetic rotary disruptor comprises at least one magnet rotatable about the longitudinal axis of the selected collection vessel and around said vessel.
An apparatus according to any of the preceding claims, wherein the apparatus comprises means for depositing at least two magnetic objects into the selected collection vessel.
An apparatus according to claim 5, wherein each one of the at least two magnetic objects comprises a protective coating and/or is an elongate cylinder.
An apparatus according to any of the preceding claim, further comprising: a plurality of sets of test vials, each set of test vials comprising a plurality of test vials; and means for transferring a predetermined amount of the sample into one of the test vials.
8. An apparatus according to claim 7, wherein the test vials belonging to each of the plurality of sets of test vials comprise means for amplifying the targeted DNA of a different one of a plurality of types of biological particles and/or wherein the transferring means is operable to transfer subsamples of the disrupted biological particles into at least two separate test vials.
9. An apparatus according to claim 8, wherein the at least two separate test vials belong to different sets of test vials.
10. An apparatus according to any of the preceding claims, wherein the means for amplifying the targeted DNA comprises at least one dried reagent and/or at least one liquid reagent.
11. An apparatus according to any of claims 7 to 10, wherein the transferring means are further operable to transfer a predetermined volume of at least one liquid reagent into one of the test vials.
12. An apparatus according to any of claims 7 to 11, wherein the apparatus comprises at least one actuator to move the transferring means.
13. An apparatus according to any of the preceding claims, wherein the apparatus comprises at least one actuator to move the collection vessel.
14. An apparatus according to any of the preceding claims, wherein the apparatus further comprises a carousel configured to hold the plurality of test vials, indexable about the detector.
15. An apparatus according to any preceding claim, wherein the means for amplifying the targeted DNA further comprises a heating device.
16. An apparatus according to any preceding claim, wherein the means for amplifying the targeted DNA comprises means for preparing the disrupted biological particles for optical analysis.
17. An apparatus according to claim 16, wherein the means for preparing the disrupted biological particles for visual analysis comprises a dye, preferably a fluorescent dye.
18. An apparatus according to any of the preceding claims, wherein the apparatus comprises means for controlling the temperature at which the means for amplifying the targeted DNA are stored within the apparatus.
19. An apparatus according to any of the preceding claims, wherein the detector is an optical detector.
20. An apparatus according to claim 19, wherein the optical detector is a fluorimeter. 5
21. An apparatus according to any of the preceding claims, wherein the apparatus comprises a communication device for sending data to a server.
22. An apparatus according to any of the preceding claims, wherein the apparatus comprises means for collecting weather data.
23. An apparatus according to any of the preceding claims, wherein the apparatus comprises a processor for processing data.
24 A method of automatically detecting airborne biological particles, carried out by the apparatus according to any of claims 1-23, said method comprising: Intercepting and collecting airborne biological particles into a collection vessel; adding a predetermined volume of liquid into the collection vessel; dropping at least two magnetic objects into the collection vessel; mechanically disrupting the collected biological particles for a predetermined amount of time using a magnet rotatable around the collection vessel and configured to actuate the at least two magnetic objects; adding a predetermined volume of liquid reagent to a test vial containing dried reagents; collecting a set volume of the disrupted biological particle sample from the collection vessel and adding it into the test vial; and amplifying and analysing the targeted DNA in the test vial.
25. A method of automatically detecting airborne biological particles, wherein the method comprises the steps of: collecting intercepted airborne biological particles into a collection vessel; adding a predetermined volume of liquid into the collection vessel containing the collected biological particles; dropping at least two magnetic objects into the collection vessel; mechanically disrupting the spores for a predetermined amount of time using a magnet rotatable around the collection vessel and configured to actuate the at least two magnetic objects; adding a predetermined volume of liquid reagent to a test vial containing dried reagents; collecting a set volume of disrupted spore sample from the collection vessel and adding it into the test vial; heating the test vial; illuminating the test vial; and measuring the emitted light with a fluorimeter.