GB2612780A - Method and system for sampling of airborne microbial activity - Google Patents

Abstract

An airborne microbial detection system comprises a housing comprising an air sampler 105 configured to collect and process ambient air to create a liquid sample, the air sampler comprising an air inlet (305, fig 3) and a liquid outlet (330, fig 3). The housing also comprises a removable cassette 130 that has a plurality of vials (210, fig 2a) for receiving a volume of liquid. A first vial is configured for receiving the liquid sample from the air sampler. At least one other vial of the plurality of vials comprises a reagent for a microbial assay and the system is configured to effect an introduction of at least a portion of the liquid sample into the at least one other vial to create a sample under test. A lysing station 120 is also provided so that the sample under test can be lysed, and a monitoring station is provided for determining optical changes in the sample under test and to provide an output indicative of monitored optical changes. The system is configured to effect a movement of the cassette within the housing for selective presentation of the cassette to each of the air sampler, lysing station and monitoring station 150. Preferably, the assay is a loop mediated isothermal amplification (LAMP) assay.

Description

Method and system for sampling of airborne microbial activity

Field

The present invention relates to a system and method for sampling of airborne microbial activity. More specifically, the invention relates to an automated air-sampling system which is configured to output an indication of the presence of specific microbes within any sample under test. In exemplary arrangements the system may utilise nucleic acid (NA) amplification test regimes designed for quasi-real time detection of specific airborne pathogen targets.

Background

In food, healthcare and agricultural industries, airborne pathogens can result in serious disease outbreaks and mass food spoilage ultimately leading to increased mortality, illnesses and costs.

Airborne pathogens generically can be defined as bacterium, virus or other microorganisms. Within this generic definition aspergillus, legionella, Methicillinresistant Staphylococcus aureus (MRSA), klebsiella, c. difficile, "viruses". All of these exist in different ways in the environment and their transport in the air is complex and most likely different in each case.

Microbial contaminants may be spread by various means e.g. direct transmission from contaminated surfaces or individuals by touch or through contaminated water supplies. However, in the aforementioned industries even with strict decontamination methods for surfaces, individuals and water supplies in place, microbial contamination still remains commonplace. An aspect of microbial contamination which is far more difficult to control is that of airborne contamination. Microbes may persist on hard to reach surfaces for up to several months or more. When disturbed, these microbes become airborne, enabling transmission to other areas of the particular facility in question. Furthermore, poor compliance with cleaning procedures and high footfall in other areas of a facility may result in regions of high levels contamination. Again, as this area is disturbed by the movement of individuals or machinery, microbial contamination from a low risk' (e.g. warehouse) to a 'high risk' (e.g. food production line) area, through airborne contaminants, is likely.

Current methods for assessing the levels of microbial contamination involve the use of (a) sampling and plating, (b) biochemical laboratory analysis and (c) optical methods.

Assessments using methods (a) and (b) usually involve relatively long timescales (several days), dedicated highly trained staff or investment in costly instrumentation and chemicals, or both. For example, samples must be prepared by an individual skilled in the field of microbiology before growing on nutrient media and enumeration or, in the case of polymerase chain reaction (PCR), samples must be isolated and one or a series of chemical preparations performed before the microbes are identified. Therefore, such methods are usually invoked in very specific laboratory analyses.

Prior art proposes the use of optical techniques for the rapid identification of airborne microbes. For example, US patent publication no. 2003/0098422 Al proposes the use of a UV laser light source to induce auto-fluorescence of compounds in airborne biological matter. However, coherent sources of UV radiation are rather costly and the capacity of these sources to discern differences in signals em itted from bacteria and moulds requires one or more of more complicated equipment, signal processing techniques, or both. It is appreciated that a less costly approach, using widely available technologies and with increased selectivity, represents an attractive alternative to such methods.

Existing optical methods for determining the presence of microbes in optically transparent solids or fluids use the properties of light scattering by microbial species present in the media. For example, US patent publication no. US 6107082A discloses an apparatus and process for automated detection of bacteria in a fluid through measurement of the changes in optical density or turbidity of the medium. However, the process requires several preparatory steps which are most suited to supervised laboratory analyses.

US patent no. US7465560B2 discloses a rapid bacterial detection method based on light scattering by bacterial colonies. The samples are prepared on growth medium and placed in the optical path of a light source. Detectors measure the pattern and intensity of the forward scattered light, and the bacterial species are identified by analysis of the unique "fingerprint" of the forward scattered light patterns. However, there is no method proposed for rapid identification of other microbes, for example moulds, or for automatically sampling from the environment.

Our previous US Patent, US 10 456 785, describes an assembly and method for capturing and proliferating airborne microbes and to remotely determine their presence by means of optical scattering and absorption of a collimated light source. The technology is based on capturing using an growth media substrate which evidently restricts the nature of the analysis that can be done.

Despite the advantages that can be derived from such a device that provides remote sampling and monitoring of microbial activity suitable for use in ambient monitoring applications, there continues to be a need for a system and method that facilitates more complex analysis of airborne pathogens

Summary

Accordingly the present teaching provides an apparatus for remote sampling and sensing of the presence of air borne microbes as detailed in claim 1. Advantageous embodiments are provided in the dependent claims.

Brief Description of the Drawings

The present application will now be described with reference to the accompanying drawings in which: Figure 1 is a system in accordance with the present teaching; Figures 2a and 2b show components of the system during a selective presentation of an individual vial into a heating block; Figure 3 is a section through an air sampler in accordance with the present teaching; Figure 4 shows a filling of a vial.

Figure 5 is a perspective view of a sampling cassette comprising a plurality of vials; Figure 6 is a plan view of another sampling cassette.

Figure 7 is schematic showing a view of an exemplary housing in accordance with the present teaching.

Detailed Description of the Drawings

The present teachings relate to a system for the capture and detection of airborne microbes. While exemplary implementations and aspects of the present teaching will be described with reference to the accompanying drawings, it will be appreciated that the scope and spirit of the present teaching should not be construed as being limited in any fashion to the specifics now described which are illustrative of benefits associated with the present teaching and not intended to limit the present teaching to these specifics. Furthermore, where one or more elements are described with reference to one or more figures these could be replaced or used with one or more elements described with reference to other figures. For the purposes of the features that are described with reference to the accompanying figures the following reference numerals are used: An exemplary aspect of a system 100 for detecting airborne microbes in accordance with the present teaching is detailed in Figure 1. Provided within the same housing, which typically has dimensions of the order of 300mm x 300 mm is provided an air sampler 105 within which air can be collected and processed to create a liquid sample for testing purposes.

A syringe 110 for inter well liquid transfer is also provided.

The system includes a platform configured to receive and support a disposable disc 130, also referred to as a cassette or cartridge herein -which will be discussed in more detail below. The disc includes a plurality of individual chambers containing liquids and reagents as required for, in this example, a completed DNA amplification process.

A heating block 120 within which one or more wells may be defined may also be provided. In this exemplary arrangement, a single well is provided. As detailed in each of Figures 2A and 2B, the well 200 is dimensioned to receive individual ones of the chambers 210 to effect a localised heating of the content of that chamber 210. Each of the chambers may contain different reagents or be used for different samples. Two or more of the chambers may be used for the same reagent and/or sample to provide a multiplexing functionality.

The system may also include a further heating block 140 which can be used for other sample analysis technique-for example this exemplary arrangement shows 4 wells (141) are provided. This configuration is, in this configuration, to facilitate 3 sample-based LAMP reactions and 1 for negative control.

Figure 3 shows detail of the air sampler 105 which operates similar in principle to that described in US 5902385. The sampler comprises a housing 300 having an inlet 305 through which environmental air may be introduced into an internal volume 306 defined by the housing. Air can be extracted out of the internal volume 306 by coupling a vacuum pump (not shown) to an outlet 310.

Clean water (which may be filtered and deionised as appropriate) is introduced through a fluid inlet 315 and collects as a liquid reservoir 320 in a lower region of the housing. The air that is introduced in from the inlet 305 passes downwardly within the housing through channels 325 that are defined within the inner volume 306.

A large air sample, typically several hundred litres is introduced over time. Within the housing a swirling vortex of liquid is created as a result of the air flow in and out of the housing similarly to that described as the operation of US 5902385. This vortex motion brings the liquid from the reservoir into contact with incoming air. The contact of the liquid with the air causes a transfer of the originally airborne pathogens into the liquid sample. It will be appreciated that this mechanism of sampling ambient air is one mechanism that can be utilised within the context of the present teaching and that other methods for extracting particulates and aerosols from the air into the sample liquid can also be utilised within the context of the present teaching. After a sufficient period of time, a small volume of liquid is extracted out from the outlet 330.

The sample liquid is introduced into one or more vials 210 that are located within the disc or cartridge 130. The cartridge is moveable within the system to effect an individual presentation of individual ones of the vials 210 to a cannula or needle 115 that is moveable within the system so as to provide a fluid connection to the outlet 330 of the air sampler. Individual ones of the vials can be preloaded with different reagents and primers 410 as required for different sampling techniques. As shown in Figure 5, the vials may differ in their dimensions to facilitate receipt of different volumes of liquid as appropriate for different reactions. By preloading the vials with different reagents and primers, it is possible to introduce liquid from the same sample into different vials so as to provide a cartridge 130 that can facilitate multiple different tests on the same liquid sample.

A cartridge, such as that shown in Figure 5, preferably comes preloaded with specific reagents and primers. In this way a user can select different cartridges for different tests. For example a first cartridge may include reagents and primers that are particularly appropriate for testing for the presence of a first set of microbes and a second cartridge may include reagents and primers that are particularly appropriate for testing for the presence of different microbes. Each of the two cartridges will adopt the same physical form factor such that a user can simply select an appropriate cartridge for an appropriate test and insert that cartridge into the system for the processing of a sample. The cartridges may include a machine readable identifier that facilitates the automatic changing of the processing steps that are conducted by the machine, to those steps that are consistent with desired analysis for that cartridge type. For example, a first test sequence may include the use of 4 reagents and primers that are provided in vials 1-4, which requires the selective deposition of samples into vials 1-4. A second test, in contrast may require the use of reagents and primers in vials 1-3, and 5-8. Evidently this will require a presentation of different vials to the outlet 230 of the air sampler to facilitate the deposition of the sample from the air sampler.

As shown in Figure 6, The cartridge may also be configured to include reagents and primers for repeating the same test over time. For example, a specific test may require the use of three vials (610A-610C). By configuring the cassette with 9 vials (block A, block B, block C), the same test can be conducted at 3 separate occasions over time, by simply changing the vials that are used at any one test occasion. This advantageously facilitates the provision of a temporal test regime without having to replace the individual cartridge at each test occasion.

In this regard, it will be understood that the cartridge can be a disposable and replaceable disk shaped component that contains all the necessary reagents and primers pre-loaded into the reaction chambers in order to facilitate multiple NA (LAMP) reactions over a number of weeks before it needs replacing.

The cartridge may include one or more physical identifiers -to facilitate alignment of the cartridge when presented into the system. For example the cartridge may include serrations or perforations at the outer perimeter which will only allow the location of the cartridge at one predetermined position on its supporting substrate. It will be appreciated that accurate alignment of the cassette within the system is important as the selective use of individual ones of the vials requires a knowledge of which vial is which.

As each of the individual cassettes facilitate a standalone test regime, it is important that the integrity of the test reagents and primers is maintained up to use. To facilitate the effective storage, transportation, and use of the cassettes, it is preferred that the cassettes are preloaded with reagents that are then individually sealed within their respective vials. Figure 5 shows an example of such a cassette which includes a seal layer 500 on the top of each vial. For those vials that are loaded with a reagent and primer, the seal layer prevents the spillage of the contents before actual use of same. For those vials that are empty until required in the processing of a sample, the vials can be sealed to ensure that they are not contaminated by dirt of other contaminants in the period before use.

Once inserted into the system, the seal layer can be selectively removed as required. This can be effected, for example, by a piercing of the surface layer by the cannula or needle 115 of the air sampler. In another arrangement a single layer can be provided across the entirety of the top surface of the cassette and this can be removed in a single action to effect an opening of a plurality of vials in a single action.

To facilitate the movement of the cartridge 130 within the housing it is desirable that the cartridge is mountable on a support that can effect a movement or rotation of the cartridge to present individual ones of the vials separately to the outlet 330 of the air sampler. Once sufficient sample has been loaded into the individual vials 210, then the cartridge itself can be moved -in one or more of an X, Y, and X direction-within the system to facilitate the subsequent presentation of the individual vials 210 to other components of the system.

As referenced above, different test regimes will require different reagents and primers. Many tests require the use of clean water. This can be facilitated by either having a source of water within the system and then selectively accessing that water supply by presentation of the cassette to the water supply as required.

In another configuration, a cassette can be preloaded with water in identifiable ones of the plurality of vials.

For any test regime that requires the selective use of a plurality of different vials, it is necessary for the sample in a first vial to be transferrable to a second vial.

This can be achieved using a syringe arrangement which selectively provides a mechanism by which predetermined volumes of a sample can be moved from one vial to another. Given that it is important that a sample is not contaminated by the apparatus that is used to transport the sample, the cassette may include another set of vials or wells dedicated to the storage of a sterilizing agent such as 70% alcohol. The tips of the syringes will need cleaning between phases and after/beginning to use a disc. By having a well or wells on the disc containing a sterilizing agent, the syringes can also clean themselves between samples, further removing the need for the syringe systems to be removed and making sure they do not become contaminated.

Figure 6 shows an exemplary layout configuration of a disc 130 that provides for three separate Loop Mediated Isothermal Amplification, LAMP, tests-each associated with a separate block: Block A, Block B, Block C. It will be appreciated that a LAMP test is one of the WHO's recommended nucleic acid amplification tests (NAAT) to detect the presence of infectious molecules (either DNA or RNA) from a sample. As the person of ordinary skill will appreciate, a LAMP test uses isothermal technology to detect the presence of infectious molecules (either DNA or RNA) from a patient sample. By using a number of primer sequences unique to the molecule being detected and a special polymerase enzyme, the amplification reaction can take place at a single temperature. The system of the present teaching is particularly suited for such LAMP test regimes.

On commencement, a first syringe action fills with water from the Clean Water 30 Supply which is provided in a common vial 600. The system then moves on to Block A and the disc 130 is presented to the air sampler -as discussed above. By locating vial 605 relative to the outlet of the air sampler, a first liquid sample, Sample A, is loaded into what is now termed the Sample Well 605, using the decided air sampling method (typically 1.5-2m1 created). Sample A is then distributed among the 3 LAMP Reaction Tubes, 610A, 6103, 610C (shown in orange) using a second syringing action.

Clean water can be introduced into the LAMP Negative Control tube 615. Once the LAMP process is completed, a sterilizing agent can be used to effect a cleaning of the used syringes by pumping in and out of the sterilizing agent well 620 to clean out their needle tips. This process repeats for Block B and Block C. So in this iteration, we would get 3 complete tests out of a cartridge/disc.

By sacrificing real-estate/wells on the disc for these extra fluids, the cassette itself becomes the source of all sample fluids, cleaning fluids and reagents thereby avoiding a need to locate sources of these fluids elsewhere within the system. In this way it is possible to not only keep the syringe needles from interacting with anything outside of the disc area, but also the need for manual intervention to refresh some of the finite liquids required for any one test is obviated. The tradeoff is system usability vis-a-vis the number of samples that can be run on a single disc.

Having loaded a sample into the individual vials of a cassette, the cassette is then moved to other processing stations within the system. In the context of a LAMP test regime, it is necessary for the samples to be exposed to a constant temperature for a prescribed period. As was discussed above with reference to Figure 1, the system includes a lysing heating block, or temperature control module 120. The heating block 120 is configured to provide a constant temperature for the amplification process (65C) and additionally a cooling station (5C) for post lysing. It will be appreciated that the specific of the heating and cooling are with reference to the exemplary loop-mediated isothermal amplification process (LAMP) that requires a constant temperature provided to the reaction chambers.

Having exposed the sample within its specific reagent vial to the necessary heating regime, it is anticipated per conventional LAMP tests, that a change in colour of the sample within the vial is indicative of a positive or negative test. The system includes a process detection module, PDM, 150 which is configured to effect a determination of any changes resultant from the LAMP chemistry which includes a visible pH indicator. Based on the production of protons and subsequent drop in pH that occurs from the extensive DNA polymerase activity in a LAMP reaction, a change in solution color from pink to yellow is produced in our reaction. The PDM includes a light source and a camera and is configured to utilise a machine vision technique to monitor process/colour changes. The PDM is configured to examine the colour of the individual test vials within a cassette block and then -based on pre-programmed calibration routines to effect a determination of the presence of otherwise of the microbe being tested for.

Equally other optical changes such as turbidity or fluorescence can be detected, depending on the specific chemistry of the assay.

It is appreciated that by means of suitable electronic circuitry the signals produced by the PDM can be electrically transmitted from a transmitter to a remote receiver and stored for analysis by for example, a computing device. To facilitate this, the system will typically include a wireless transmitter-such as those provided using ZigBee technology or the like.

Figure 7 is an external representation of an exemplary system in accordance with the present teaching. The air sampler 105 is desirable located in an upper region of the housing and has an air intake 305 open to the ambient air to allow an introduction of air into the housing. Control of the system is provided via user interface 700, in this exemplary configuration one including a touch panel screen 710. The removable cartridge can be presented into the housing through a door 720 provided on a side of the housing, through which a user can selectively introduce and remove cassettes as appropriate. The functionality of a processor and the wireless transmitters 730are also provided within the housing It will be appreciated that the present teaching has been described with reference to preferred aspects and implementations but changes and modifications can be made without departing from the spirit or scope of the present teaching which should be limited only insofar as is deemed necessary in the light of the appended claims.

The words comprises/comprising when used in this specification are to specify the presence of stated features, integers, steps or components but does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

Claims (8)

1. Claims 1. An airborne microbial detection system for detecting airborne microbes, the system comprising a housing comprising: an air sampler configured to collect and process ambient air to create a liquid sample, the air sampler comprising an air inlet and a liquid outlet; a removable cassette comprising a plurality of vials, each vial dimensioned for receiving a volume of liquid, a first vial of the plurality of vials being configured for receiving the liquid sample from the air sampler, at least one other vial of the plurality of vials comprising a reagent for a microbial assay, wherein the system is configured to effect an introduction of at least a portion of the liquid sample into the at least one other vial to create a sample under test; a lysing station for effecting a-lysis of the sample under test; a monitoring station for determining optical changes in the sample under test and to provide an output indicative of monitored optical changes; and wherein the system is configured to effect a movement of the cassette within the housing for selective presentation of the cassette to each of the air sampler, lysing station and monitoring station.
2. 2. The system of claim 1 further comprising a processor configured to receive the output indicative of the monitored colorimetric changes and to generate a result indicative of the presence of airborne microbes based on the received output
3. 3. The system of claim 1 or 2 wherein the assay is a loop mediated isothermal amplification, LAMP, assay.
4. 4. The system of any preceding claim wherein the assay is a DNA or RNA assay.
5. 5. The system of any preceding claim comprising a support substrate for receiving the removable cassette, the support substrate being moveable or rotatable to effect a selective presentation of individual ones of the vials.
6. 6. The system of claim 5 wherein the support substrate is moveable within the housing to effect the selective presentation of the cassette to each of the air sampler, lysing station and monitoring station.
7. 7. The system of any preceding claim comprising a sampling syringe configured to effect a transfer of liquid between individual ones of the vials so as to effect the introduction of at least a portion of the liquid sample into the at least one other vial to create a sample under test.
8. 8. The system of any preceding claims wherein individual ones of the vials are initially sealed, the system being configured to effect a selective breaking of the seals to allow at least one of the introduction, or removal, of a liquid from at least one vial.