WO2024047567A1

WO2024047567A1 - Method, device and system for detecting microbial or viral infection

Info

Publication number: WO2024047567A1
Application number: PCT/IB2023/058597
Authority: WO
Inventors: Željko PERDIJA; Tadej TOFANT; Tomaz BÜDEFELD
Original assignee: VD-ING d.o.o.
Priority date: 2022-08-31
Filing date: 2023-08-31
Publication date: 2024-03-07

Abstract

A method of detecting a microbial or viral infection in a putative host. The method comprises reverse transcribing RNA determinative of the microbial or viral infection into cDNA and amplifying the reverse transcribed cDNA in an assay solution comprising a buffer, deoxynucleotides, primers, reverse transcriptase, DNA polymerase and a biological sample extracted from the putative host to produce high molecular weight amplicons. The method also comprises measuring with a spectrophotometer the amplified assay solution to detect the high molecular weight amplicons; and comparing the measurement with a reference measurement, optionally a calibration curve for the high molecular weight amplicons, to provide a result of the measurement to thereby detect the microbial or viral infection. A device and system for detecting a microbial or viral infection in a putative host are also described. The comparison may be conducted locally on a personal computing device such as, a smart phone.

Description

METHOD, DEVICE AND SYSTEM FOR DETECTING MICROBIAL OR VIRAL INFECTION

FIELD OF THE INVENTION

[0001] The present invention relates to a method, device and system for detecting a microbial or viral infection. More particularly the invention relates to a method, device and system for detecting a microbial or viral invention wherein the detection provides a measurement with a reference measurement, which is optionally a calibration curve for the high molecular weight amplicons associated with microbial or viral infection, and a system for collecting, storing and managing measurement results

BACKGROUND TO THE INVENTION

[0002] Pathogenic infection is detrimental to both humans and animal life. Accurate detection of a pathogen is important for treatment and minimisation of further infection. [0003] US7122799 describes a PCR assay which is performed by absorption detection. The system includes a multi-well plate which is adapted to retain a collection of sample wells. This system includes a thermal cycler for the multi-well plate. The system additionally includes a collection of photodetectors, and a corresponding number of light sources. The light sources are positioned such that light emitted from each of the respective light sources passes through a corresponding well retained in the multi-well plate and to a corresponding photodetector. The system also includes a processor or other means for analyzing the output signals from the photodetectors. In certain versions of the system, ultra-violet light is used.

[0004] WO201518929 is directed to method for operating a real-time polymerase chain reaction (rtPCR) system and a device for operating the method. It is the object of the invention to provide a new method, which allows the manufacture of a small and portable rtPCR system. The object is met by the method for operating a rtPCR system comprising a control system for temperature regulation of a sample holder, whereby this regulation is of the closed-loop type, for at least two temperature set values, whereby at a first time the closed-loop temperature regulation is started and at a second time, which is after the given first time, the measurement of the fluorescence intensity and/or luminescence decay time starts, whereby at the second time the components of the control system required for the closed-loop temperature regulation are at least partly used for the fluorescence intensity and/or luminescence decay time measurements, while at the second time the closed-loop temperature regulation is interrupted for the measuring period.

[0005] WO2015095929 teaches a method and kit for detecting a target nucleic acid in which an amplified nucleic acid is combined with a particle such as a paramagnetic bead form a flocculated complex which can be detected by visual inspection. The volume of nucleic acid sample that can be detected is as low as a few microlitres. The method can be applied to in-the-field or point-of-care diagnosis for a rapid determination of the presence or absence of the target nucleic acid. The methylation status of a target nucleic acid can also be determined. The method and lit may have general applicability to detecting diseases in plants and animals, environmental testing and testing for contamination of foods and other edible products.

[0006] There remains a need for other methods, devices and systems to detect microbial and viral infections.

SUMMARY OF THE INVENTION

[0007] The present invention is broadly directed to a method, device and system for detecting a microbial or viral infection and, optionally, a system for collecting, storing and managing measurement results.

[0008] In a broad form, the invention relates to a method, device and system for detecting a microbial or viral infection wherein the detection provides a measurement with a reference measurement, which is optionally a calibration curve for the microbial or viral infection.

[0009] In one aspect, although it need not be the only or indeed the broadest form, the invention provides a method of detecting a microbial or viral infection in a putative host, the method comprising: reverse transcribing RNA determinative of the microbial or viral infection into cDNA and amplifying the reverse transcribed cDNA in an assay solution comprising a buffer, deoxynucleotides, primers, reverse transcriptase, DNA polymerase and a biological sample extracted from the putative host to produce high molecular weight amplicons; measuring with a spectrophotometer the amplified assay solution to detect the high molecular weight amplicons; and comparing the measurement with a reference measurement, optionally a calibration curve for the high molecular weight amplicons, to provide a result of the measurement to thereby detect the microbial or viral infection.

[0010] In a second aspect the invention provides a device for detecting a microbial or viral infection in a putative host, the method comprising: a reaction chamber for holding a reaction vessel in which RNA determinative of the microbial or viral infection is reverse transcribed into cDNA and amplified in an assay solution comprising a buffer, deoxynucleotides, primers, reverse transcriptase, DNA polymerase and a biological sample extracted from the putative host to produce high molecular weight amplicons; a spectrophotometer to measure the amplified assay solution to detect the high molecular weight amplicons; and a processor for comparing the measurement with a reference measurement, optionally a calibration curve for the high molecular weight amplicons, to provide a result of the measurement to thereby detect the microbial or viral infection.

[0011] In a third aspect the invention provides a system for detecting a microbial or viral infection in a putative host, the method comprising: a reaction chamber for holding a reaction vessel in which RNA determinative of the microbial or viral infection is reverse transcribe into cDNA and amplified in an assay solution comprising a buffer, deoxynucleotides, primers, reverse transcriptase, DNA polymerase and a biological sample extracted from the putative host to produce high molecular weight amplicons; a spectrophotometer for measuring the amplified assay solution to detect the high molecular weight amplicons; a processor for comparing the measurement with a reference measurement, optionally a calibration curve for the high molecular weight amplicons, to provide a result of the measurement to thereby detect the microbial or viral infection.

[0012] According to any one of the above aspects, the comparison may be conducted locally on a personal computing device such as, a smart phone. The comparison may be conducted by a server operatively connected to a database. The personal computing device may be operatively connected through a network to the server and/or database. The personal computing device may be wirelessly connected to the spectrophotometer such as, through Bluetooth.

[0013] According to any one of the above aspects, the calibration curve may be prepared with (i) one or more measurements of one or more known concentrations of high molecular weight amplicons associated with the microbial or viral infectious agent. The calibration may further comprise (ii) one or more measurement of one or more unknown samples and/or (iii) one or more measurement of one or more reference samples of known concentration. The calibration may be prepared by comparing one or more of (i); (ii) and (iii).

[0014] According to any one of the above aspects, the database may comprise measurement comparison parameters which may comprise one or more of calibration values of the spectrophotometer; one or more baseline factory parameters; and one or more specific characteristics of sensors comprised on the spectrophotometer. The database may comprise a corpus of previous measurements.

[0015] According to any one of the above aspects, the comparison may comprise application of a model algorithm. The model algorithm may calculate the result of the detection. The model algorithm may change and/or adjust the comparison using one or more of the measurement comparison parameters. The model algorithm may qualify the measurement. The algorithm may be applied by the server and/or personal computing device. The model algorithm may be specific to the particular microbe or virus.

[0016] In one embodiment of any one of the above aspects, the model algorithm and/or calibration curve may be updated. Additional model algorithms and/or calibration curves for particular microbes or viruses may be added.

[0017] Advantageously, this provides troubleshooting during measurement and consequently improves the accuracy of the final results.

[0018] In another particular embodiment of any one of the above aspects, a plurality of calibration curves may be stored and/or received, each calibration curve associated with a particular microbe or virus. The database may store specific characteristics for a plurality of spectrophotometers.

[0019] In yet another embodiment of any one of the above aspects, the measurement may be provided to the database. The database may provide the calibration curve or a plurality of calibration curves to the personal computing device.

[0020] In still another embodiment of any one of the above aspects, the measurement step may also comprise heating and/or incubating.

[0021] According to any one of the above aspects, the measurement may be of fluorescence.

[0022] In another embodiment of any one of the above aspects, the sample collection, lysing in commercial buffer and extraction may use a commercial kit and/or may use conventional technology. The enzyme assay buffer may be a commercially available amplification buffer. The primers may be a universal primer set suitable for detecting SARS-CoV-2 by RT LAMP. A positive control may detect any human RNA such as, POP7. A negative control may use DNAse-free water.

[0023] The reverse transcription, amplification, measurement, and sample collection according to any one of the above aspects may be in a standard sample tube. The sample tube may be an Eppendorf tube.

[0024] According to any one of the above aspects, the measurement may be in a chamber shielded from ambient light.

[0025] Where the terms “comprise”, comprises”, “comprising”, “include”, “includes”, “included” or “including” are used in this specification, they are to be interpreted as specifying the presence of the stated features, integers, steps or components referred to, but not to preclude the presence or addition of one or more other feature, integer, step, component or group thereof.

[0026] Further, any prior art reference or statement provided in the specification is not to be taken as an admission that such art constitutes, or is to be understood as constituting, part of the common general knowledge.

BRIEF DESCRIPTION OF THE FIGURES

[0027] In order that the present invention may be readily understood and put into practical effect, reference will now be made to the accompanying illustrations, wherein like reference numerals refer to like features and wherein:

[0028] Figure 1 : is a schematic diagram showing one embodiment of the invention.

[0029] Figure 2: is a schematic diagram showing one embodiment of data flow according to the invention.

[0030] Figure 3: is a logic diagram showing one embodiment of the invention.

[0031] Figures 4 A and 4B: are schematic diagrams illustrating one embodiment of a computer system according to the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0032] The following description refers to specific embodiments of the present invention and is in no way intended to limit the scope of the present invention to those specific embodiments.

[0033] Advantageously, the invention represents an interdisciplinary connection of optoelectronics, electronics, analytical chemistry, data science, biology, and pharmaceutical technologies. The invention concerns the field of optoelectronics, more concrete spectrophotometry. It is the development of an advanced portable spectrophotometer that is powered by a battery and connected to a smartphone. The spectrophotometer is designed to insert a standard single-use sample-tube such as, an Eppendorf. The virus or bacterium that we want to measure may be re-entered into the database or there may be an appropriate calibration curve for it. It must also be dissuaded at the appropriate level. Advantageously, such system design allows us to capture samples and identify the virus or bacterium onsite, directly on the ground, without the need for expensive laboratory equipment.

[0034] The present invention has wide ranging application, including in in the medicine, pharmaceutical, food and chemical industries.

[0035] Although the invention will be described with reference to SARS-CoV-2, it is not so limited and has general application to any viral or bacterial infection.

[0036] As shown in Figure 1, device 100 contains three or more different sensors 120 consisting of photodiodes 122 and optic filters 124. Each sensor 120 may contains six photodiodes 122 and six optic filters 124. Each of the sensors 120 also belongs to the selected transmitting diode 124, which has an oral wavelength to which the sensor 120 is sensitive (for example a sensor from 200 to 500 nm has a 300nm wavelength transmit range). All three sensors 120 may be connected in a geometrically aligned line, as are their diodes 122.

[0037] The sensors 120 may be dimensioned to fit tightly into the outer wall of the measuring kit. In between it may be possible to position a sample tube that does not absorb the wavelengths in which sensors 120 and transmitting diodes 124 operate (PET).

[0038] From the teaching herein, the skilled person will readily appreciate that the computer 200 or micro-controller may be responsible for collecting data and controlling the sensors 120. On the circuit, there are a module and thermistors, or temperature sensors, next to the computer 200 or micro-controller.

[0039] The entire circuit may be battery-powered or plugged in to an external power source. The battery may be located in housing with a circuit. The device 10 may be connected to a another computer 200, which may be in the form of a smartphone, on which the measurement capture app is installed. All computer operations and data display and storage may be carried out in the application's measurement interface. The device 100 can work independently with a smartphone 200 and it doesn't need to be connected to the web. When connected to the Web, all data is transferred from your phone to the cloud. It is also possible to correct the data calculation, as the cloudy service allows for more precise and faster calculation.

[0040] In one embodiment, the device's 100 Ohio system is designed to prevent external light, as it would interfere with the sensor's performance. This can be made of various materials such as plastics, aluminum, other metals, or a combination of metal and plastic. This also has a test fit for a 125 pl single-use sample tube. The sample tube is made of plastic and is intended for measurements in the spectrophotometer. At the top of the sample tube is a lid that protects the device from spilling the measuring material. The preparation of a sample that we want to analyze takes place in more than one a few steps. Technically, the detection of, for example, SARS-CoV-2 viral RNA and human biological samples by reverse transcription (RT) coupled to a Loop-mediated isothermal AMPlification (LAMP) method is a three-step process.

[0041] First, a commercial (Qiagen) lysis buffer is used to lyse viral particles and human cells in a biological sample. The biological sample can be a nasal, buccal, or skin swab, blood, saliva, urine, etc. Biological samples are incubated in the 300 pL of lysis buffer for 10 min at room temperature. Fresh biological samples (taken within 30 min or 1 hour for swabs and body fluids, respectively) are preferably used to prevent/minimize the RNA losses due to lytic processes.

[0042] In the second step viral and human RNA are extracted from lysed samples using a commercial kit for isolation of viral RNA (Qiagen). One volume (300 pL) of 100% ethanol is added to the 300 pL of lysed sample and mixed on a vortex mixer (can be any mixer) for 15 s to precipitate the RNA in the sample. The whole sample (600 pL) is then loaded on a column (a column consists of two parts; the upper part containing the silica membrane and the bottom collection tube) and centrifuged for 30 s at no more than 6000 x g. At this step precipitated RNA is caught on a silica membrane. The upper part of the column is then transferred into a clean collection tube, and the used collection tube containing the filtrate is discarded. To wash the RNA, 500 pL of wash buffer #1 is added to the column followed by centrifugation for 30 s at no more than 6000 x g. Once more the upper part of the column is transferred into a clean collection tube, and the used collection tube containing the filtrate is discarded. In the second wash step, 500 pL of a wash buffer #2 is added to the column followed by centrifugation for 3 min at 14000 x g or less. The upper part of the column is transferred into a clean collection tube, and the used collection tube containing the filtrate is discarded. In the last step, 60 pl of DNAse- free water is added to the column to dissolve the RNA. After 1 min incubation at room temperature, the column is centrifuged for 30 seconds at no more than 6000 x g to elute the RNA. The end product of the isolation protocol is purified viral and human RNA dissolved in DNAse-free water. Purified RNA species in DNAse-free water are very unstable at room temperature so it is recommended to perform a detection (third) step as soon as possible for optimal assay performance. All steps of the extraction protocol are performed at room temperature.

[0043] In the last third step, one-step enzyme assays may be used to detect the RNA of interest in a sample.

[0044] In one embodiment, the enzyme assay contains commercially available 1 x Isothermal Amplification Buffer (New England Biolabs, NEB) (IX Buffer Components: 20 mM Tris-HCl, 10 mM (NH4)2SO4, 50 mM KC1, 2 mM MgSO4, 0.1% Tween® 20 pH 8.8 and 25°C), 6.5 mM MgSO4, a mixture of deoxynucleotides (dNTPs) dNTA, dNTT, dNTC and dNTG (1.4 mM each), 0.2 pM F3 and B3 primers, 1.6 pM FIP and BIP primers, 0.8 pM LF and LB primers, 7.5 U of Warm Start recombinant reverse transcriptase (NEB), 8 U of recombinant Bst 2.0 DNA Polymerase (NEB) and DNAse- free water. The final volume of an enzyme assay is 25 pL. Assay specificity (detection of RNA of interest) is defined by three pairs of primers. Detection of SARS-CoV-2 is based on primers that detect the viral N-gene. An enzyme assay containing primers that detect the human gene POP7 is used for internal positive control.

[0045] Reverse transcriptase is used to reverse transcribe the RNA of interest into the complementary DNA (cDNA). cDNA is then a substrate for DNA polymerase that amplifies a select region in the RNA of interest. The amplification of cDNA by DNA polymerase but not the reverse transcription determines the final result (positive/negative) of an essay.

[0046] To detect, for example, SARS-CoV-2 in human samples, 5 pL of extracted purified RNA from the previous (second) step is added to the 20 pL of enzyme reaction (SARS-CoV-2 Assay) in a microcentrifuge. The enzyme reaction is allowed to proceed at 65 C (149 F) for 30 min in a temperature regulator or heater such as, a VIROCHECK PLUS thermoblock. After 30 min, the result of an enzyme reaction (presence or absence of RNA of interest) is evaluated by measuring the fluorescence of deoxynucleotides in the enzyme reaction at 300 to 900 nm using a spectrophotometer such as, a VIROCHECK PLUS spectrophotometer.

[0047] Internal controls are used to validate, for example, the SARS-CoV-2 assay. A positive internal control (to check the effectiveness of isolation protocol and assay sensitivity) contains three pairs of primers that detect the human gene POP7. A negative internal control (to check a non-specific enzyme reaction and false-positive results) uses DNAse-free water instead of an RNA sample.

[0048] Advantageously, in one embodiment, the measuring system utilizes a calibration curve with a known concentration of the measured high molecular weight amplicons. This may be done in a way to measure an unknown sample and the known reference of this sample, compare them mathematically, and determine the mathematical function that best matches the reference sample using an approximation. Calibration may be done by a comparative measurement or a reference purchased for which the concentration is known. Each system may consist of a meter, a data capture device (an app on a computer such as, computer 200), and an operational server that takes care of the accuracy of the measurement. The measuring system must be calibrated appropriately for the type of measurement during production.

[0049] Data capture may take place in an app that is installed on a computer 200 which may be in the form of a smartphone, tablet or pc.

[0050] Advantageously, in addition to extracting the measurement results, the application may also allows analysis of individual sample parameters, data storage, measurement history, etc.

[0051] While, the device is primarily intended for the detection of viruses and bacteria, it is possible to use the device for measurements of other substances using appropriate clearances and additional calibration and knowledge of reference values.

[0052] The light travels from the photodiode through the tube such as Eppendorf tube with a measured pattern, bounces off the reflective element, and returns to the spectral sensors 120. Each of the three sensors 120 captures its own set of wavelengths using filters and a photodetector, the output of which is converted into a digital record via an analogdigital converter. The intensity of the light on the sensor 120 depends on the optical characteristics of the substance in the sample, which is then read by the master controller from each of the three sensors 120. The readings may be transmitted via Bluetooth connection to the computer 200 (smartphone, pc, tablet...), which partially processes, stores, sends the data to the operating server, and displays the results of the measurement after calculation. New calibration curves or algorithms for the detection of new substances may also be added through the cloud service. The user is therefore not required to update the application as the measurement may also be made online. The device also allows only calibration or calibration. Additionally, a user may add their own calibration curve or add those provided by others.

[0053] As part of the calculation, all data read may be filtered and aggregated into the spectral response function of the measured sample. Data processing on the operational server adds the calibration values of the meter and corrects the sample parameters, taking into account the baseline factory parameters and the specific characteristics of the individual sensors. The treated spectral response of the measured sample goes through a model algorithm that calculates the final results of the individual samples. The algorithm on the server changes and adjusts the calculation algorithms during calculation using all data stored in the central database and all previous successfully executed measurements, allowing for troubleshooting during measurement and consequently improving the accuracy of the final results.

[0054] The algorithm is designed to qualify the sample dissolved in the dissolute.

[0055] From the teaching herein, the skilled person readily understand that the data may be saved and analyzed in the cloud

[0056] Measurement data may be stored locally on a users computer 200, which may be their smartphone 200. In the presence of a data connection on the phone, they are simultaneously synchronized to the operating server, where they are further processed to improve algorithms and thus a more accurate reading of the result at the following measurements.

[0057] Advantageously, the invention provides a portable device 100 for quantifying individual viruses or bacteria and the problem of a simple and affordable tester for detection of a virus or bacteria in an unknown sample. The device 100 may be used multiple times, and it may connected to an app on a user’s smartphone to see the result. The device 100 may consist of a printed circuit board space, a battery compartment, an sample holder, and an appropriate cover that prevents the effects of external light. The device 100 may operate spectrophotometrically and may have at least three detectors and three odors in it, along with his. The sample tube may suitable for single use with a suitable solvent. The device 100 may connected to a smartphone via low energy blue tooth (BLE). [0058] One embodiment of a personal device 200 suitable for use in the present invention is shown in Figs. 2A and 2B. In the embodiment shown personal device 200 comprises a computer module 201 comprising input devices such as a keyboard 202, a mouse pointer device 203, a scanner 226, an external hard drive 227, and a microphone 280; and output devices including a printer 215, a display device 214 and loudspeakers 217. In some embodiments video display 214 may comprise a touchscreen.

[0059] A Modulator-Demodulator (Modem) transceiver device 216 may be used by the computer module 201 for communicating to and from a communications network 220 via a connection 221. The network 220 may be a wide-area network (WAN), such as the Internet, a cellular telecommunications network, or a private WAN. Through the network 220, computer module 201 may be connected to other similar personal devices 290 or server computers 291. Where the connection 221 is a telephone line, the modem 216 may be a traditional “dial-up” modem. Alternatively, where the connection 221 is a high capacity (e.g. : cable) connection, the modem 216 may be a broadband modem. A wireless modem may also be used for wireless connection to network 220.

[0060] The computer module 201 typically includes at least one processor 205, and a memory 206 for example formed from semiconductor random access memory (RAM) and semiconductor read only memory (ROM). The module 201 also includes a number of input/output (I/O) interfaces including: an audio-video interface 207 that couples to the video display 214, loudspeakers 217 and microphone 280; an I/O interface 213 for the keyboard 202, mouse 203, scanner 226 and external hard drive 227; and an interface 208 for the external modem 216 and printer 215. In some implementations, modem 216 may be incorporated within the computer module 201, for example within the interface 208. The computer module 201 also has a local network interface 211 which, via a connection 223, permits coupling of the personal device 200 to a local computer network 222, known as a Local Area Network (LAN).

[0061] As also illustrated, the local network 222 may also couple to the wide network 220 via a connection 224, which would typically include a so-called “firewall” device or device of similar functionality. The interface 211 may be formed by an Ethernet circuit card, , WiFi, including WiFi HaLow, a Bluetooth wireless arrangement or an IEEE 802.11 wireless arrangement or other suitable interface, such as Zigbee and Morse Micro, which may be implemented in (Industrial) Internet of Things ((I)IoT) or home automation technology.

[0062] The I/O interfaces 208 and 213 may afford either or both of serial and parallel connectivity, the former typically being implemented according to the Universal Serial Bus (USB) standards and having corresponding USB connectors (not illustrated).

[0063] Storage devices 209 are provided and typically include a hard disk drive (HDD) 210. Other storage devices such as, an external HD 227, a disk drive (not shown) and a magnetic tape drive (not shown) may also be used. An optical disk drive 212 is typically provided to act as a non-volatile source of data. Portable memory devices, such as optical disks (e.g.: CD-ROM, DVD, Blu-Ray Disc), USB-RAM, external hard drives and floppy disks for example, may be used as appropriate sources of data to the personal device 200. Another source of data to personal device 200 is provided by the at least one server computer 291 through network 220.

[0064] The components 205 to 213 of the computer module 201 typically communicate via an interconnected bus 204 in a manner that results in a conventional mode of operation of personal device 200. In the embodiment shown in FIGS. 2A and 2B, processor 205 is coupled to system bus 204 through connections 218. Similarly, memory 206 and optical disk drive 212 are coupled to the system bus 204 by connections 219. Examples of personal devices 200 on which the described arrangements can be practiced include IBM- PC's and compatibles, Sun Sparc stations, Apple computers; smart phones; tablet computers or like a device comprising a computer module like computer module 201 or (Industrial) Internet of Things ((I)IoT) home automation technology such as, zigbee and morse micro access points and/or connected devices. It is to be understood that when personal device 200 comprises a smart phone or a tablet computer, display device 214 may comprise a touchscreen and other input and output devices may not be included such as, mouse pointer device 203; keyboard 202; scanner 226; and printer 215.

[0065] Fig. 2B is a detailed schematic block diagram of processor 205 and a memory 234. The memory 234 represents a logical aggregation of all the memory modules, including the storage device 209 and semiconductor memory 206, which can be accessed by the computer module 201 in Fig. 2A.

[0066] The methods of the invention may be implemented using personal device 200 wherein the methods may be implemented as one or more software application programs 233 executable within computer module 201. In particular, the steps of the methods of the invention may be effected by instructions 231 in the software carried out within the computer module 201

[0067] The software instructions 231 may be formed as one or more code modules, each for performing one or more particular tasks. The software 233 may also be divided into two separate parts, in which a first part and the corresponding code modules performs the method of the invention and a second part and the corresponding code modules manage a graphical user interface between the first part and the user.

[0068] The software 233 may be stored in a computer readable medium, including in a storage device of a type described herein. The software is loaded into the personal device 200 from the computer readable medium or through network 221 or 223, and then executed by personal device 200. In one example the software 233 is stored on storage medium 225 that is read by optical disk drive 212. Software 233 is typically stored in the HDD 210 or the memory 206.

[0069] A computer readable medium having such software 233 or computer program recorded on it is a computer program product. The use of the computer program product in the personal device 200 preferably effects a device or apparatus for implementing the methods of the invention.

[0070] In some instances, the software application programs 233 may be supplied to the user encoded on one or more disk storage medium 225 such as a CD-ROM, DVD or Blu- Ray disc, and read via the corresponding drive 212, or alternatively may be read by the user from the networks 220 or 222. Still further, the software can also be loaded into the personal device 200 from other computer readable media. Computer readable storage media refers to any non-transitory tangible storage medium that provides recorded instructions and/or data to the computer module 201 or personal device 200 for execution and/or processing. Examples of such storage media include floppy disks, magnetic tape, CD-ROM, DVD, Blu-ray Disc, a hard disk drive, a ROM or integrated circuit, USB memory, a magneto-optical disk, or a computer readable card such as a PCMCIA card and the like, whether or not such devices are internal or external of the computer module 201. Examples of transitory or non-tangible computer readable transmission media that may also participate in the provision of software application programs 233, instructions 231 and/or data to the computer module 201 include radio or infra-red transmission channels as well as a network connection 221, 223, 334, to another computer or networked device 290, 291 and the Internet or an Intranet including email transmissions and information recorded on Websites and the like.

[0071] The second part of the application programs 233 and the corresponding code modules mentioned above may be executed to implement one or more graphical user interfaces (GUIs) to be rendered or otherwise represented upon display 214. Through manipulation of, typically, keyboard 202, mouse 203 and/or screen 214 when comprising a touchscreen, a user of personal device 200 and the methods of the invention may manipulate the interface in a functionally adaptable manner to provide controlling commands and/or input to the applications associated with the GUI(s). Other forms of functionally adaptable user interfaces may also be implemented, such as an audio interface utilizing speech prompts output via loudspeakers 217 and user voice commands input via microphone 280. The manipulations including mouse clicks, screen touches, speech prompts and/or user voice commands may be transmitted via network 220 or 222.

[0072] When the computer module 201 is initially powered up, a power-on self-test [0073] (POST) program 250 may execute. The POST program 250 is typically stored in a ROM 249 of the semiconductor memory 206. A hardware device such as the ROM 249 is sometimes referred to as firmware. The POST program 250 examines hardware within the computer module 201 to ensure proper functioning, and typically checks processor 205, memory 234 (209, 206), and a basic input-output systems software (BIOS) module 251, also typically stored in ROM 249, for correct operation. Once the POST program 250 has run successfully, BIOS 251 activates hard disk drive 210. Activation of hard disk drive 210 causes a bootstrap loader program 252 that is resident on hard disk drive 210 to execute via processor 205. This loads an operating system 253 into RAM memory 206 upon which operating system 253 commences operation. Operating system 253 is a system level application, executable by processor 205, to fulfill various high level functions, including processor management, memory management, device management, storage management, software application interface, and generic user interface.

[0074] Operating system 253 manages memory 234 (209, 206) in order to ensure that each process or application running on computer module 201 has sufficient memory in which to execute without colliding with memory allocated to another process. Furthermore, the different types of memory available in the personal device 200 must be used properly so that each process can run effectively. Accordingly, the aggregated memory 234 is not intended to illustrate how particular segments of memory are allocated, but rather to provide a general view of the memory accessible by computer module 201 and how such is used.

[0075] Processor 205 includes a number of functional modules including a control unit 239, an arithmetic logic unit (ALU) 240, and a local or internal memory 248, sometimes called a cache memory. The cache memory 248 typically includes a number of storage registers 244, 245, 246 in a register section storing data 247. One or more internal busses 241 functionally interconnect these functional modules. The processor 205 typically also has one or more interfaces 242 for communicating with external devices via the system bus 204, using a connection 218. The memory 234 is connected to the bus 204 by connection 219.

[0076] Application program 233 includes a sequence of instructions 231 that may include conditional branch and loop instructions. Program 233 may also include data 232 which is used in execution of the program 233. The instructions 231 and the data 232 are stored in memory locations 228, 229, 230 and 235, 236, 237, respectively. Depending upon the relative size of the instructions 231 and the memory locations 228-230, a particular instruction may be stored in a single memory location as depicted by the instruction shown in the memory location 230. Alternately, an instruction may be segmented into a number of parts each of which is stored in a separate memory location, as depicted by the instruction segments shown in the memory locations 228 and 229.

[0077] In general, processor 205 is given a set of instructions 243 which are executed therein. The processor 205 then waits for a subsequent input, to which processor 205 reacts by executing another set of instructions. Each input may be provided from one or more of a number of sources, including data generated by one or more of the input devices 202, 203, or 214 when comprising a touchscreen, data received from an external source across one of the networks 220, 222, data retrieved from one of the storage devices 206, 209 or data retrieved from a storage medium 225 inserted into the corresponding reader 212. The execution of a set of the instructions may in some cases result in output of data. Execution may also involve storing data or variables to the memory 234.

[0078] The disclosed arrangements use input variables 254 that are stored in the memory 234 in corresponding memory locations 255, 256, 257, 258. The described arrangements produce output variables 261 that are stored in the memory 234 in corresponding memory locations 262, 263, 264, 265. Intermediate variables 268 may be stored in memory locations 259, 260, 266 and 267.

[0079] The register section 244, 245, 246, the arithmetic logic unit (ALU) 240, and the control unit 239 of the processor 205 work together to perform sequences of microoperations needed to perform "fetch, decode, and execute" cycles for every instruction in the instruction set making up the program 233. Each fetch, decode, and execute cycle comprises:

(a) a fetch operation, which fetches or reads an instruction 231 from memory location 228, 229, 230;

(b) a decode operation in which control unit 239 determines which instruction has been fetched; and

(c) an execute operation in which the control unit 239 and/or the ALU 240 execute the instruction.

[0080] Thereafter, a further fetch, decode, and execute cycle for the next instruction may be executed. Similarly, a store cycle may be performed by which the control unit 239 stores or writes a value to a memory location 232.

[0081] Each step or sub-process in the methods of the invention may be associated with one or more segments of the program 233, and may be performed by register section 244- 246, the ALU 240, and the control unit 239 in the processor 205 working together to perform the fetch, decode, and execute cycles for every instruction in the instruction set for the noted segments of program 233.

[0082] One or more other computers 290 may be connected to the communications network 220 as seen in Fig. 2A. Each such computer 290 may have a similar configuration to the computer module 201 and corresponding peripherals.

[0083] One or more other server computer 291 may be connected to the communications network 220. These server computers 291 response to requests from the personal device or other server computers to provide information.

[0084] Method 100 may alternatively be implemented in dedicated hardware such as one or more integrated circuits performing the functions or sub functions of the described methods. Such dedicated hardware may include graphic processors, digital signal processors, or one or more microprocessors and associated memories.

[0085] It will be understood that in order to practice the methods of the invention as described above, it is not necessary that the processors and/or the memories of the processing machine be physically located in the same geographical place. That is, each of the processors and the memories used in the invention may be located in geographically distinct locations and connected so as to communicate in any suitable manner. Additionally, it will be understood that each of the processor and/or the memory may be composed of different physical pieces of equipment. Accordingly, it is not necessary that a processor be one single piece of equipment in one location and that the memory be another single piece of equipment in another location. That is, it is contemplated that the processor may be two pieces of equipment in two different physical locations. The two distinct pieces of equipment may be connected in any suitable manner. Additionally, the memory may include two or more portions of memory in two or more physical locations.

[0086] To explain further, processing as described above is performed by various components and various memories. It will be understood, however, that the processing performed by two distinct components as described above may, in accordance with a further embodiment of the invention be performed by a single component. Further, the processing performed by one distinct component as described above may be performed by two distinct components. In a similar manner, the memory storage performed by two distinct memory portions as described above may, in accordance with a further embodiment of the invention, be performed by a single memory portion. Further, the memory storage performed by one distinct memory portion as described above may be performed by two memory portions.

[0087] Further, various technologies may be used to provide communication between the various processors and/or memories, as well as to allow the processors and/or the memories of the invention to communicate with any other entity, i.e., so as to obtain further instructions or to access and use remote memory stores, for example. Such technologies used to provide such communication might include a network, the Internet, Intranet, Extranet, LAN, an Ethernet, a telecommunications network (e.g., a cellular or wireless network) or any client server system that provides communication, for example. Such communications technologies may use any suitable protocol such as TCP/IP, UDP, or OSI, for example.

[0088] While this invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modification(s). This application is intended to cover any variations uses or adaptations of the invention following in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth.

[0089] As the present invention may be embodied in several forms without departing from the spirit of the essential characteristics of the invention, it should be understood that the above-described embodiments are not to limit the present invention unless otherwise specified, but rather should be construed broadly within the spirit and scope of the invention as defined in the appended claims. The described embodiments are to be considered in all respects as illustrative only and not restrictive.

[0090] Various modifications and equivalent arrangements are intended to be included within the spirit and scope of the invention and appended claims. Therefore, the specific embodiments are to be understood to be illustrative of the many ways in which the principles of the present invention may be practiced. In the following claims, means-plus- function clauses are intended to cover structures as performing the defined function and not only structural equivalents, but also equivalent structures.

[0091] Whenever a range is given in the specification, for example, a temperature range, a time range, or a composition or concentration range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure. It will be understood that any subranges or individual values in a range or subrange that are included in the description herein can be excluded from the claims herein.

[0092] As used herein, “comprising” is synonymous with “including”, “containing” or "characterized by" and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, “consisting of’ excludes any element, step, or ingredient not specified in the claim element. As used herein, “consisting essentially of’ does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. The broad term “comprising” is intended to encompass the narrower “consisting essentially of’ and the even narrower “consisting of’ Thus, in any recitation herein of a phrase “comprising one or more claim element” (e.g., “comprising A”), the phrase is intended to encompass the narrower, for example, “consisting essentially of A” and “consisting of A” Thus, the broader word “comprising” is intended to provide specific support in each use herein for either “consisting essentially of’ or “consisting of’. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein.

[0093] One of ordinary skill in the art will appreciate that materials and methods, other than those specifically exemplified can be employed in the practice of the invention without resort to undue experimentation. All art-known functional equivalents, of any such materials and methods are intended to be included in this invention. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by examples, preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

[0094] Each reference cited herein is incorporated in its entirety. Such references may provide sources of materials; alternative materials, details of methods, as well as additional uses of the invention.

Claims

1. A method of detecting a microbial or viral infection in a putative host, the method comprising: reverse transcribing RNA determinative of the microbial or viral infection into cDNA and amplifying the reverse transcribed cDNA in an assay solution comprising a buffer, deoxynucleotides, primers, reverse transcriptase, DNA polymerase and a biological sample extracted from the putative host to produce high molecular weight amplicons; measuring with a spectrophotometer the amplified assay solution to detect the high molecular weight amplicons; and comparing the measurement with a reference measurement, optionally a calibration curve for the high molecular weight amplicons, to provide a result of the measurement to thereby detect the microbial or viral infection.

2. A device for detecting a microbial or viral infection in a putative host, the method comprising: a reaction chamber for holding a reaction vessel in which RNA determinative of the microbial or viral infection is reverse transcribed into cDNA and amplified in an assay solution comprising a buffer, deoxynucleotides, primers, reverse transcriptase, DNA polymerase and a biological sample extracted from the putative host to produce high molecular weight amplicons; a spectrophotometer to measure the amplified assay solution to detect the high molecular weight amplicons; and a processor for comparing the measurement with a reference measurement, optionally a calibration curve for the high molecular weight amplicons, to provide a result of the measurement to thereby detect the microbial or viral infection.

3. A system for detecting a microbial or viral infection in a putative host, the method comprising: a reaction chamber for holding a reaction vessel in which RNA determinative of the microbial or viral infection is reverse transcribe into cDNA and amplified in an assay solution comprising a buffer, deoxynucleotides, primers, reverse transcriptase, DNA polymerase and a biological sample extracted from the putative host to produce high molecular weight amplicons; a spectrophotometer for measuring the amplified assay solution to detect the high molecular weight amplicons; a processor for comparing the measurement with a reference measurement, optionally a calibration curve for the high molecular weight amplicons, to provide a result of the measurement to thereby detect the microbial or viral infection.

4. The method, device or system of any one of the preceding claims wherein the comparison is conducted locally on a personal computing device such as, a smart phone.

5. The method, device or system of any one of claims 1 to 3 wherein the comparison is conducted by a server operatively connected to a database.

6. The method, device or system of claim 5 wherein the personal computing device is operatively connected through a network to the server and/or database.

7. The method, device or system of any one of claims 3 to 5 wherein the personal computing device is wirelessly connected to the spectrophotometer such as, through Bluetooth.

8. The method, device or system of any one of the preceding claims wherein the calibration curve is prepared with (i) one or more measurements of one or more known concentrations of high molecular weight amplicons associated with the microbial or viral infectious agent.

9. The method, device or system of claim 8 wherein the calibration further comprises (ii) one or more measurement of one or more unknown samples and/or (iii) one or more measurement of one or more reference samples of known concentration.

10. The method, device or system of claim 9 wherein the calibration is prepared by comparing one or more of (i); (ii) and (iii).

11. The method, device or system of any one of the preceding claims wherein the database comprises measurement comparison parameters which may comprise one or more of calibration values of the spectrophotometer; one or more baseline factory parameters; and one or more specific characteristics of sensors comprised on the spectrophotometer.

12. The method, device or system of any one of the preceding claims wherein the database comprises a corpus of previous measurements.

13. The method, device or system of any one of the preceding claims wherein the comparison comprises application of a model algorithm.

14. The method, device or system of claim 13 wherein the model algorithm calculates the result of the detection.

15. The method, device or system of claim 13 or claim 14 wherein the model algorithm changes and/or adjusts the comparison using one or more of the measurement comparison parameters.

16. The method, device or system of any one of claims 13 to 15 wherein the model algorithm qualifies the measurement.

17. The method, device or system of any one of claims 4 to 16 wherein the algorithm is applied by the personal computing device and/or server.

18. The method, device or system of any one of claims 13 to 17 wherein the model algorithm is specific to the particular microbe or virus.

19. The method, device or system of any one claims 13 to 17 wherein the model algorithm and/or calibration curve are updated.

SUBSTITUTE SHEET (RULE 26)