WO2021096466A2

WO2021096466A2 - Apparatus and usage methods for early warning and real time diagnosis of biological agents in air

Info

Publication number: WO2021096466A2
Application number: PCT/TR2020/051076
Authority: WO
Inventors: Hüseyin Avni ÖKTEM
Original assignee: Nanobi̇z Teknoloji̇ Anoni̇m Şi̇rketi̇
Priority date: 2019-11-13
Filing date: 2020-11-10
Publication date: 2021-05-20
Anticipated expiration: 2022-05-13
Also published as: WO2021096466A3

Abstract

The invention defines a system, and components and use thereof that can diagnose the biological factors within the structure of bacteria, bacteria spores, fungus, yeast, virus, algae and pollens by means of a nucleic acid based method comprising diagnosis gel including buffer, salts, detergents, micro/nano particles, enzymes, primer sets, dNTP molecules, ions, betaine, signal creating color or fluorescent molecules, buffer chemicals and gelling components, through amplification, without requiring any kind of sample preparation related to the samples taken from the air, and that can make an early warning with an SMS, e-mail and audio call by delivering the results it obtains.

Description

APPARATUS AND USAGE METHODS FOR EARLY WARNING AND REAL TIME DIAGNOSIS OF BIOLOGICAL AGENTS IN AIR

TECHNICAL FIELD OF THE INVENTION

The invention relates to the real time diagnosis of biological agents such as bacterial spore, bacteria, vims, mold, fungus, algae, pollen in the air, with an apparatus integrated into a drone and to instant transmission of an early warning to predetermined units.

KNOWN STATE OF THE ART (PRIOR ART)

Techniques that are commonly used for the detection and diagnosis of biological factors are ELISA, PCR, RT-PCR etc. These techniques are divided into two main categories as immunochemical and nucleic acid based. Immunochemical based methods are systems where antibodies (monoclonal or polyclonal) specific to different agents are used in different reaction formats. The diagnosis of the target factor is carried out with these techniques. Immunoagglutination, ELISA, horizontal flow immunographic test strips are some of the techniques that are used for this purpose. With these methods although biological agents can be diagnosed with high sensitivity, 100% verification cannot be made.

In nucleic acid based techniques, the nucleic acid (DNA or RNA) samples obtained from the agents are used; and by multiplying (i.e. amplifying) nucleic acid regions (sequences) specific to the target agent it is possible to detect and in other words to diagnose the the target factor with 100% accuracy. Techniques such as polymerase chain reaction (PCR), RT-PCR, isothermal amplification are widely used for this purpose and are still accepted as “gold standard” techniques used in the diagnosis of biological agents in several fields. Therefore, when immunochemical based methods are used in the detection of biological agents, false positivitives may occur due to the cross-affinity (potential to bind different molecules) that may arise from the antibodies used in the reactions. Since genetic information of biological agents are used in nucleic acid based techniques, and as this information is highly specific for each agent, diagnosis can be made with 100% accuracy. Due to this reason, diagnosis methods are preferred in terms of obtaining 100% accuracy in determining biological agents.

Although nucleic acid based techniques provide 100% accurate results, they have disadvantages especially in terms of non-laboratory use due to criteria such as need for trained personnel and infrastructure with equipment and relatively high costs. Another important requirement of these techniques is the step of nucleic acid isolation from the target agents.

Depending on the target agent, agent needs to be multiplied with pre-enrichment and afterwards the targeted nucleic acid (RNA or DNA) molecules need to be purified with a suitable method. In such procedures, trained personnel, laboratory infrastructure and most of the time specific isolation kits are necessary. Another important disadvantage faced when carrying out such highly sensitive methods outside the laboratory, is the risk of cross contamination created by other agents and thereby the risk of obtaining false positive results. Another important factor in not being able to perform nucleic acid based techniques outside the laboratory are the size of the devices used in the tests and the fact that they consume high amounts of energy as they create heat cycles (35 - 40 cycles between 60 to 90°C).

In recent years it is observed that techniques called isothermal amplification that can perform nucleic acid amplification at a single temperature are being used prevalently. In this method, nucleic acid samples isolated from the target agent can be amplified at a fixed temperature between 60-65°C without requiring any thermal cycling process. By means of this technique, both reaction times can be reduced (30-60 minutes) and through low energy consumption, the size and the dimension of the devices can be reduced. One of the most important problems faced in diagnosing target agents outside the laboratory using isothermal amplification based methods is the step of determining fragments created following nucleic acid purification and amplification from the target agent. For example, in the loop mediated isothermal amplification-LAMP method which is one of the most widely used isothermal amplification methods, agarose gel electrophoresis, measuring the turbidity in tubes and fluorescent methods are used in order to determine the nucleic acid particles that have been amplified as a result of reaction. These methods require different types of devices and measuring methods and the integration thereof to the technique disables the systems to be mobile, to have reduced sizes and to be used outside the laboratory.

Another problem encountered during the diagnosis of biological agents with nucleic acid based techniques is the time that passes between the sampling of the biological agent and its diagnosis. This time is the pre-enrichment process when the sampled biological agent is not sufficiently dense (this is a frequently faced condition during sampling from the field). This process requires the factor to be taken into a suitable medium and incubated at a certain temperature for 3-18 hours. Following this nucleic acid purification and amplification reaction can be carried out. Therefore, a time of 5-20 hours can pass between the sampling of the biological agent and its diagnosis. During this time, the biological agent in the air can spread significantly and can be contagious and this increases the risk of an epidemic.

Due to these reasons, the biological agents particularly in the air need to be determined (diagnosed) in real time with high accuracy and in order to take rapid precautions against said biological agents, it is crucial to be able to notify the related people and units immediately.

Most of the conditions mentioned above can be met with the prior art, however it not possible with this invention to diagnose the biological agents in the air in a very short period of time and to submit this information to the related people and units immediately.

BRIEF DESCRIPTION OF THE INVENTION

The invention defines a system, and components and usage are thereof that can diagnose the biological agents in the form of bacteria, bacteria spores, fungus, yeast, virus, algae and pollens by means of a nucleic acid based method (loop mediated isothermal amplification method), without requiring any kind of sample preparation of the samples taken from the air, and that can deliver the results it obtains to the control unit to make an early warning to the control unit via SMS, e-mail and audio call.

By means of the method, apparatus and applications defined in the invention that overcomes the technical problems that cannot be solved by the prior art, it is possible to diagnose the biological factors in the air in under an hour and to notify this information to the related persons and units.

The invention that can carry out biological agent diagnosis and early warning comprises several components therein. One of the components that constitute the invention is the diagnosis unit and it sucks air automatically from the environment, and it comprises all of the components that can carry out the diagnosis of biological agents in the air using a method based on nucleic acid amplification.

The air that is sucked is applied on a reactive (diagnosis gel) gel. The reactive gel comprises all components that are required for isothermal application such as primers, enzymes, buffers, ions, chemicals and dyes. Additionally, it comprises agents such as chemicals, detergents and enzymes that promote the emerging of nucleic acids from biological agents.

The gel is located on a heating unit. Following the suction of sufficient air, the heating plate increases up to the required temperature and reaction in the gel is carried out. Following the reaction, in the case that there is a biological agent on the gel, nucleic acid amplification takes place and this changes the colour of the gel.

The change in gel color as a result of the reaction is sensed by a unit located in the unit and processed in the relevant electronic units and the results can be transferred wirelessly to another unit by means of the communication unit located in the unit. The unit to which the information is transferred can make an audio call and an audio alert and send an SMS, if necessary, to the phone numbers previously registered in the system, depending on the content of the incoming data, by means of the software included. Moreover, information can be submitted via e-mail to the registered addresses.

By means of this feature the system enables to send an early warning message to the related people, organizations and units in order to take the necessary precautions and to make a diagnosis before any kind of damage and contamination occurs from the biological agent without encountering a risk, and without necessitating human labor regarding any kind of biological threats that may occur in a remote location.

The system has the potential to be used for different purposes in different sectors such as the remote diagnosis of biological warfare agents in aerosol format, especially in the defense industry and the concept of homeland security, and in the pharmaceutical, environment, agriculture, animal husbandry sectors; but it not limited the examples given here and below.

For example, by means of the invention rapid diagnosis of biological agents (such as mold, bacteria, fungus, virus) that may cause a disease can be carried out on samples taken from the air, by placing the said invention in greenhouses and fields and a large-scale disease and thus product loss that may occur can be prevented by taking the necessary precautions.

Similarly, it can provide the diagnosis of pathogenic biological agents that may be found in the samples collected from the air in poultry houses and bams where animals are collectively sheltered and it can ensure that precaution is taken.

Another field that can employ the system is monitoring and determining biological attacks 7/24 that may be carried out in aerosol form by placing the said system at borders and creating an early warning by taking the necessary precautions.

In another application, the polens in the air that cause allergies can be diagnosed and an early warning can be sent to allergic patients to ensure such people move away from areas where such pollens are abundant.

DEFINITION OF THE FIGURES DESCRIBING THE INVENTION The figures and the related descriptions necessary for further understanding of the subject of the invention are as follows.

Figure 1. Diagnosis unit and parts forming the unit that can diagnose the biological agents by taking samples from the air.

Figure 2. Diagnosis gel tray (carrier) A) Side section view of the tray B) Top view of the tray. P: Positivie control section, N: Negative control section, T: Test section. C) Top view of the tray that has been designed to simultaneously determine multiple biological agents. T1-T6 test sections represent the sections that detect different biological agents. P and N; are as described in article B.

Figure 3. The operation of the diagnosis unit as being integrated to the drone or being independent from the drone, and its communication with the ground unit.

Figure 4. Test result carried out in gel format.

Figure 5. Electronic processor and control unit, heating unit, sensor unit constituting the components

DEFINITIONS OF THE ASPECTS AND PARTS FORMING THE INVENTION

1. Diagnosis unit

2. Air suction unit

3. Diagnosis gel

4. Heating unit

5. Communication unit

6. Electronic processor and control unit

7. Battery unit

8. Sensing unit 9. Connection units

10. Air flow

11. Diagnosis gel tray /carrier

12. Drone unit

13. Ground unit

14. Drone control unit

P: Positive control section

N: Negative control section T: Test section

T1-T6: Test sections determining the different biological agents

DETAILED DESCRIPTION OF THE INVENTION

The invention defines a system, and components and usage are thereof that can diagnose the biological agents in the form of bacteria, bacteria spores, fungus, yeast, virus, algae and polens by means of a nucleic acid based method (loop mediated isothermal amplification method), without requiring any kind of sample preparation of the samples taken from the air, and that can deliver the results it obtains to the control unit to make an early warning to the control unit via SMS, e-mail and audio call. The parts, sections in the figures that are used in order to further describe the components of the applications developed with this invention have each been numbered and the references of each number shown on the prepared figures, have been provided below.

The components of the system forming the invention have been illustrated in Figure 1., Figure 2. and Figure 3.

Figure 1. Shows the diagnosis unit (1) and the components within this unit. The diagnosis unit (1) carries out the suction of the biological agents in the ambient air (10) into the said diagnosis unit (1) by means of the air suction unit (2). The sucked air is applied on the diagnosis gel (3) and air penetrates the gel. The air suction unit (2) comprises the fans, filters and electronic components that are required for carrying out air suction and the controlling of the unit is performed by the electronic processor and the control module (6).

After air suction and penetration of the biological factors into the diagnosis gel (3) from the sucked air (10) is carried out for the desired period of time, diagnosis processes are initiated. The diagnosis gel (3) has a gel form and is made of gelling agents such as agar, agarose and alginate and has different functional features. The gel has three basic functions: a) To establish a suitable medium so that the nucleic acid (DNA, RNA) can be released out of the biological agents that are absorbed with components such as buffer, salts, detergents, enzymes, micro/nano particles located therein, b) To accommodate enzymes, primer sets, dNTP molecules, betaine, ions, buffer solution chemicals that are required for the amplification of specific nucleic acid sequences of target biological agents with the nucleic acid amplification method. c) To create a signal following amplification of nucleic acid fragments by accommodating dye or fluorescent molecules that can enable the generation of a detectable signal (color change or fluorescent) in case of nucleic acid amplification from the specific DNA/RNA sequences of the target biological agent takes place.

The diagnosis gel (3) has been placed on a tray/carrier (11) located on the heating unit (4) (Figure 2). The diagnosis gel tray (11) is produced from a material that is heat conductive (such as aluminum), and is formed of three sections being positive (P), negative (N) and test (T) (Figure 2).

The diagnosis gel in the positive section (Figure 3-P), comprises a nucleic acid whose sequence is known, primer sets that will amplify all or part of this nucleic acid, dNTP molecules, betaine, ions, buffer solution, chemicals and dye or fluorescent molecules that provide the generation of signals in case of amplification. The function of this section is to generate signals following the heating process carried out by the heating unit (6) without taking into consideration if there is a biological factor in the air or not. As a result, it is verified that the system is operating in a reliable manner.

The gel at the negative section (Figure 3-N), comprises all the other components such as the primer sets besides enzymes that provide nucleic acid amplification, dNTP molecules, betaine, ions, buffer solution chemicals, and dye and fluorescent molecules that generate signals. The function of this section is to determine a background signal that can be generated. Under normal conditions signal generation is not expected in this section.

The test section (Figure 3-T) comprises all of the components that can generate signals by amplification of a specific sequence of target agents in the sucked air, such as enzymes, primer sets dNTP molecules, ions buffer solution chemicals, and dye or fluorescent molecules that enable the generation of signals, and it generates signals when target agents are available in the air.

The diagnosis gel tray (11) may comprise a multitude of test sections (T1-T6) in order to be able to detect a multitude of factors in the air. In Figure 3, six test sections are shown as examples and the number of these test sections can be increased or decreased optionally. Each test section may comprise different diagnosis gel contents that are to be used in the diagnosis of a different biological factor. By this means the diagnosis of plurality of and different quality biological factors is possible.

The heating unit (3) is controlled by the electronic processor and control unit. The main function of the heating unit is to reach different temperatures according to the commands received from the electronic processor and control unit and to establish the desired temperatures within the diagnosis gel (3). Different temperatures in different sections can be established in the diagnosis gel (3) by being able to constitute the heating unit (3) from different parts, if desired. As a result, it is possible to simultaneously carry out reactions in the diagnosis gel (3) at different temperatures.

The electronic processor and control unit (6) is the unit that is responsible of controlling and operating different units that are accommodated in the diagnosis unit (1).

The communication unit (5) is the unit responsible for the wireless communication between the diagnosis unit (1) and the ground unit (13).

The battery unit (7) provides the power required for the independent operation of the diagnosis unit (1). The diagnosis unit (1) does not only operate by means of the battery unit (7) located therein, but also with the mains power, if necessary.

The diagnosis unit (8) is responsible for capturing the signals that are generated on the diagnosis gel (3). It may vary according to the characteristic of the signal (colour, fluorescent, magnetic etc.) generated.

The connection units (9) are units that enable the diagnosis unit (1) to be able to be mounted to unmanned air vehicles such as drones (12) if desired, and they may have different designs according to the unmanned unit that the diagnosis unit will be mounted to.

The ground unit is the unit that can communicate with the drone (12) and the diagnosis unit (1) by means of different unit and functions therein, such as batteries, computer, internet connection, radio frequency (RF), software etc. The drone (12) can be controlled via the ground unit (13) or the drone control unit (14).

Figure 3, shows the general operation principle of the system. The diagnosis unit (1) mounted to the drone (12) takes samples from the air during motion (flight). The diagnosis unit (1) can also be operated by being mounted to other land and sea platforms. Moreover, it can carry out independent activity by being left in any location desired (such as the hot zone). The sampled air (10) penetrates into the diagnosis gel (3) located in the diagnosis unit (1). Following this, the biological agents(s) in the air interact with the components in the gel. At this moment, the heating unit (4) steps in and it initiates the nucleic acid amplification process in the diagnosis gel. The diagnosis gel (3) generates a signal if the target biological factor is present following amplification. This signal that is generated is recorded by the sensing unit (8).

The data is processed by the electronic processor and the control unit (6) and transferred to the ground unit (13) via the communication unit (5) wirelessly or via internet. The ground unit (13) processes the information received from the diagnosis unit (1) that is mounted on the drone (12), from the drone (12) itself or from the units that operate independently, and it can send the information such as the type of biological agent, the GPS data of the location of detected agent, photos of the location, live video streaming, temperature, wind speed/direction, etc., wirelessly or via the internet, to the addresses and numbers recorded via e-mail, SMS message or an audio call, using the software available in the ground unit.

In order to prove that the concept works, some examples have been given from studies that were conducted.

EXAMPLE 1: Determining Bacillus subtilis spores with the LAMP method without DNA isolation

T Using spores obtained from Bacillus subtilis , which is considered as Bacillus anthracis (Anthrax) simulant, which is one of the most important biological warfare agents, the amplification of gene regions specific to B. subtilis, by loop mediated isothermal amplification (LAMP) method is carried out in the invention. In the invention, besides LAMP, amplification methods such as PCR, conventional PCR, real time -PCR, can also be used. Accordingly, the LAMP reaction mixture given in Table 1, is firstly dired via lyophilization, following this, B. subtilis spores, are added directly into the reaction tubes, without carrying out any kind of DNA purification. After a reaction of 60 minutes at 65°C, the reaction tubes containing B. subtilis change colour, the tubes that do not contain B. subtilis do not change colour.

With the results of this test, it is shown that diagnosis can be carried out by following the change of colour after reaction without carrying out any kind of DNA isolation on biological agents applied into the reaction tubes that are prepared by drying all of the components that are required for a reaction, using the LAMP method. These tests, show that the specified amplification reaction can be carried out in the diagnosis gel (3) and that a signal can be received when a biological factor is present.

Table -1

EXAMPLE 2: Diagnosis of B.subtilis spores in a matrix in gel format. In this application the reaction mixture shown in Table 1, is converted to gel format using 1% agarose. B.subtilis spores are added on this gel, and then the gel is incubated at 65°C for 60 minutes. Following reaction, the presence of B.subtilis is determined with the shifting of colour of the gel from purple to blue (Figure 4). This example verifies that the diagnosis gel

(3); a) can release the nucleic acid samples from target biological agents, b) that specific nucleic acid regions can be amplified, c) that a detectable signal can be generated following amplification.

EXAMPLE 3: Diagnosis of Salmonella, transfer of data wirelessly, and establishing an early warning. In this application, to the electronic units, heating block, sensing unit shown in Figure 5, and ready-to-use tubes containing the components in Table 2 mixture, Salmonella cells are added directly without any pre-preparation and/or DNA purification. Following this, the electronic unit raises the temperature of the heating unit up to 65 °C. At the end of the reaction, amplification and colour change occurs in the Salmonella added tubes. In the tubes where Salmonella were not added, colour change is not observed.

The change of colour of the tubes is determined by the sensing unit and the change is processed and Salmonella has been diagnosed information is produced. By means of the Nanotakip software, this result is submitted to the telephone number that is recorded in the system, via e-mail and SMS. This example shows that the signal created at the end of the reaction (colour change) is detected by the sensing unit and is processed with the electronic processor and control unit and that the results are displayed and the information is submitted to the pre-recorded people via SMS and E-mail, through the software.

Table -2

As it is illustrated in the above examples, it is possible to carry out real time diagnosis of biological agents in the air and to establish an early warning by means of the invention.

Claims

1. Biological agent diagnosis gel (3) characterized in that it comprises buffers, salts, detergent, micro/nano particles, enzymes, primer sets, dNTP molecules, ions, betaine, colored or fluorescent molecules that form signals, buffer chemicals and gelling components.

2. A biological agent diagnosis gel (3) according to claim 1, characterized in that the gelling component is selected as at least one, from the group comprising agarose, agar and alginate.

3. A method for diagnosing a biological agent real time, characterized in that the gene regions specific to the target agent of the biological agent diagnosis gel of claim 1 or 2, is amplified by means of loop mediated isothermal amplification, polymerase chain reaction (PCR), conventional PCR or by the real time PCR method.

4. Real time biological agent diagnosis unit (1) characterized by comprising, i. An air suction unit (2) that sucks the biological agents into the diagnosis unit (1), ii. A biological agent diagnosis gel (3) according to claim 1 or 2, iii. A heating unit (4) located below the diagnosis gel (3) that enables the gel to be heated, iv. A sensing unit (8) comprising sensors and electronic components that can sense colour or fluorescent signals, v. A diagnosis gel carrier (11) that comprises the diagnosis gel (3) and the heating unit

(4).

5. A diagnosis unit (1) according to claim 4, characterized by comprising, i. A communication unit (5), ii. An electronic processor and control unit (6), iii. A battery unit (7) iv. Connection units (9).

6. A diagnosis unit (1) according to claim 4, characterized in that said diagnosis gel carrier (11) is heat conductive.

7. A diagnosis unit (1) according to claim 4, characterized in that, the gel carrier (11) that comprises said gel (3) comprises at least a positive (P), at least a negative (N) and at least a test (T) section.

8. A diagnosis unit according to claim 4 or 5, characterized in that, said diagnosis unit (1) has a structure such that it can be connected to air, ground, sea, unmanned platforms.

9. A real time biological agent diagnosis system characterized by comprising a diagnosis unit (1) according to claim 4 or 5.

10. A diagnosis system according to claim 9, characterized in that said diagnosis unit (1) comprises a drone unit (12) that provides the motion of the diagnosis unit (1) it is connected to, and a diagnosis unit (1) and a ground unit (13) that communicates with the drone (12).

11. A diagnosis system according to claim 9, characterized in that said diagnosis unit (1) comprises a battery unit (7) so that it can operate independently.

12. A diagnosis system according to claim 9, characterized in that said diagnosis unit (1) has a structure such that it can deliver the data to the ground unit (13), via wireless radio frequency (RF) or by internet connection.

13. A diagnosis system according to claim 10, characterized in that, said ground unit (13) comprises a battery unit, a computer, an internet connection, electronic units, software and communication units.