System and method for detecting the presence of analytes such as SARS-CoV-2 in a sample

Abstract

A method for detecting an analyte in a sample is disclosed. The method can include placing the sample in an instrument, such as a loop-mediated isothermal amplification (LAMP) instrument, configured to selectively amplify an analyte, such as a characteristic portion of a pathogen genome. A moving average of the amount of the analyte at a time point can be compared to the sum of (1) the moving average at the previous time point and (2) a multiple of the moving standard deviation at the previous time point. If the amount of the analyte at a time point is greater than the sum of (1) the moving average at the previous time point and (2) a multiple of the moving standard deviation at the previous time point, it can indicate that the sample is positive for the analyte.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to and benefit from U.S. Provisional Patent Application No. 63 / 146,259, the entire disclosure of which is incorporated herein by reference.

[0002] The embodiments described herein generally relate to systems and methods for selectively amplifying a target analyte and determining whether the target analyte is present in a sample based on changes in the signal related to the amount of the analyte. Some embodiments described herein are particularly suited to diagnostic tests configured to determine whether a sample taken from a patient contains severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the pathogen that caused the global COVID-19 pandemic. [Background technology]

[0003] Several diagnostic and analytical techniques have been developed to detect the presence of proteins, DNA, or other suitable biomarkers, such as those associated with SARS-CoV-2. Many such techniques are designed to amplify a target analyte over a predetermined period and then determine whether the amount of the target analyte is detectable and / or exceeds a predetermined threshold indicating a “positive” result. Such techniques are generally time-consuming, as they must be run until completion, or at least until the signal associated with the target analyte exceeds a predetermined threshold. The long run times of such techniques contribute to significant delays in obtaining test results. For example, the waiting time to obtain COVID-19 test results is often 7–10 days. Therefore, there is a need for systems and methods that can reduce the run time required to determine the presence of an analyte in a sample. The systems and methods described herein are well suited for “rapid” testing and may provide results in less than 30 minutes while the patient is waiting, which can significantly contribute to curbing the spread of COVID-19. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] “PCR Amplification - Acomprehensive instruction to PCR and qPCR methods, including video tutorials and example protocols.” <URL: https: / / www.promega.com / resources / guides / nucleic-acid-analysis / pcr-amplification / > [Non-Patent Document 2] OL Bodulev and I. Yu. Sakharov,“Isothermal Nucleic Acid Amplification Techniques and Their Use inBioanalysis”, Vol. 85, Biochemistry (Moscow), 2020, Vol. 85, No.2, pp.147-166 [Overview of the project] [Problems that the invention aims to solve]

[0005] Some embodiments described herein relate to methods for detecting the presence of an analyte in a sample. These methods may include placing the sample in an instrument such as a loop-mediated isothermal amplification (LAMP) instrument configured to selectively amplify an analyte, such as a characteristic portion of the genome of a pathogen. The amount of the analyte can be continuously monitored by receiving a signal, such as a fluorescence signal, associated with the analyte. A moving average and a moving standard deviation of the analyte can be calculated. The moving average of the analyte at a given time can be compared to the sum of (1) the moving average at a previous time and (2) multiples of the moving standard deviation at a previous time. If the amount of analyte at a given time is greater than the sum of (1) the moving average at a previous time and (2) multiples of the moving standard deviation at a previous time, it may suggest that the sample is positive for the analyte.

[0006] Some embodiments described herein relate to systems for evaluating a sample to determine whether it contains an analyte. The system includes the following: A well configured to receive a reaction tube containing a sample; A light source configured to emit excitation light at a wavelength that irradiates the sample inside the reaction tube; A photodetector configured to receive an optical signal in response to a sample being irradiated with excitation light; and, A processor operably coupled to a light source and a photodetector. The processor is, Activate the light source, Multiple signals are received from the photodetector, and each signal from the multiple signals is associated with an optical signal, indicating the amount of analyte at a given time. At each point in time, the moving average and moving standard deviation of the analyte quantities are calculated based on a subset of signals associated with the period ending at that point. The moving average of the amount of analyte at the first time point can be configured to compare (1) the moving average for the second time point with (2) the sum of multiples of the moving standard deviation at the second time point, where the second time point is the time point prior to the first time point.

[0007] Some embodiments described herein relate to methods executed on a computer (for example, a non-temporary computer-readable medium for storing instructions configured to cause a processor to execute the method). The method performed by the computer may involve receiving multiple signals, each signal from the multiple signals being associated with the quantity of the analyte at a given time. The moving average and moving standard deviation of the quantity of the analyte can be calculated for each time point based on a subset of the multiple signals associated with the period ending at that time. The moving average of the quantity of the analyte at the first time point can be compared to (1) the sum of the moving average at the second time point, which is the quantity prior to the first time point, and (2) multiples of the moving standard deviation at the second time point. Based on the fact that the moving average of the quantity of the analyte at the first time point is greater than (1) the sum of the moving average at the second time point and (2) multiples of the moving standard deviation at the second time point, a signal indicating a positive result can be generated and / or transmitted.

[0008] Some embodiments described herein relate to methods for determining the presence or absence of an analyte in a biological sample. These methods may include placing the biological sample in an instrument configured to selectively amplify the analyte. Multiple signals may be received, and each signal from these multiple signals may be associated with the amount of the analyte at a given time. A moving average and a moving standard deviation of the analyte amount may be calculated for each time point based on a subset of the multiple signals associated with the period ending at that time. The moving average of the amount of analyte at the first time point can be compared with (1) the moving average of the amount at the second time point, which is the amount of time prior to the first time point, and (2) the multiple of the moving standard deviation at the second time point. If the moving average of the amount of analyte at the first time point is greater than (1) the sum of the moving average of the amount of analyte at the second time point and (2) a multiple of the moving standard deviation of the amount of analyte at the second time point, then the biological sample can be determined to contain more analyte than the threshold amount. (a) If the moving average of the control amount at the first time point is greater than the sum of (1) the moving average of the control amount at the second time point and (2) a multiple of the moving standard deviation of the control amount at the second time point, and, (b) If the moving average of the control amount at the first time point is less than the sum of (1) the moving average of the analyte amount at the second time point and (2) a multiple of the moving standard deviation of the analyte amount at the second time point, the biological sample can be determined to contain more than a threshold amount of analyte.

Brief Description of the Drawings

[0009] [Figure 1A] Shows a device operable to amplify an analyte and measure a signal related to the amount of the analyte, according to one embodiment. [Figure 1B] Shows a device operable to amplify an analyte and measure a signal related to the amount of the analyte, according to one embodiment. [Figure 1C] Shows a device operable to amplify an analyte and measure a signal related to the amount of the analyte, according to one embodiment. [Figure 1D] Shows a device operable to amplify an analyte and measure a signal related to the amount of the analyte, according to one embodiment. [Figure 2] Is a flowchart of a method for detecting an analyte, according to one embodiment. [Figure 3] Is experimental data from the FLOS-LAMP analysis of the example samples.

[0010] 〔Definitions〕 Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings commonly understood by those skilled in the art. Generally, the nomenclature and techniques associated with chemistry, molecular biology, cell biology and cancer biology, immunology, microbiology, pharmacology, as well as protein chemistry and nucleic acid chemistry described herein are well known and commonly used in the art.

[0011] As used herein, the following terms have their respective meanings unless otherwise specified.

[0012] The term "includes" is used to mean "includes but not limited to." "Includes" and "includes but not limited to these" are used interchangeably.

[0013] The words "a" and "an" mean one or more unless otherwise specified.

[0014] "Approximately" means a quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length that varies by 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% relative to the quantity, level, value, number, size, volume, weight, or length of reference. In any embodiment discussed in the context of a numerical value used in conjunction with the term "approximately," it is specifically intended that the term "approximately" may be omitted.

[0015] Unless the context requires otherwise, the word “comprise” and its variations, such as “comprises” and “comprising,” throughout this specification and the claims should be interpreted in an open and comprehensive sense, that is, “including, but not limited to.”

[0016] "To consist of" means that everything that precedes the phrase "to consist of" is included and limited to it. Therefore, the phrase "to consist of" indicates that the listed elements are necessary or essential, and that other elements cannot exist.

[0017] "Essentially consisting of" means including any of the elements listed before that phrase, but limited to other elements that do not interfere with or contribute to the activity or action of the listed elements as identified in this disclosure. Thus, the phrase "essentially consisting of" indicates that the listed elements are necessary or essential, while the other elements are optional and may or may not be present, depending on whether they affect the activity or action of the listed elements.

[0018] Throughout this specification, any reference to “one embodiment” or “a particular embodiment” means that a specific feature, structure, or characteristic described in relation to that embodiment is included in at least one embodiment of the present invention. Therefore, occurrences of the phrase “in one embodiment” or “in a particular embodiment” in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, specific features, structures, or characteristics can be combined in any suitable manner in one or more embodiments.

[0019] As used herein, the term “sample” refers to a composition containing one or more analytes. A sample may be heterogeneous, containing various components, or homogeneous, containing only one component. In some examples, a sample may be naturally occurring, biological, and / or artificial.

[0020] In some examples, the sample is a biological sample. In some cases, the sample may be a single cell (or single cell contents) or a multicellular (or multicellular contents), a saliva sample, a mucus sample, a blood sample, a tissue sample, a skin sample, a urine sample, a water sample, and / or a soil sample. In some cases, the sample may be derived from a living organism such as a eukaryote, a prokaryote, a mammal, a human, a yeast, and / or a bacterium, or it may be derived from a virus. In some embodiments, the sample may be a food or beverage product. In some embodiments, the sample may be a surface swab, e.g., a swab of a food preparation surface or container. Biological samples include, but are not limited to, tissues, cells, and bodily fluids obtained from the subject. For example, biological samples include, but are not limited to, blood, as well as parts or components of blood such as serum, plasma, or lymph, saliva, nasal fluid, etc. In certain embodiments, the biological sample is a blood sample, a serum sample, a saliva sample, a mucous membrane sample, a tissue sample, a skin sample, or a urine sample. In one embodiment, the biological sample contains a virus or protein molecule derived from the test subject. The biological sample may be a peripheral blood leukocyte sample isolated from the subject by conventional means. In certain embodiments, the biological sample may be serum, blood, salivary secretions (e.g., saliva), tear secretions (e.g., tears), respiratory secretions (e.g., mucus), nasal secretions, nasal swabs, oral swabs, mucus samples, and intestinal secretions (e.g., mucus).

[0021] As used herein, the term “analyte” refers to any molecule or compound detected as described herein. Suitable analytes include, but are not limited to, small chemical molecules and / or biomolecules such as environmental molecules, clinical molecules, chemicals, and contaminants. More specifically, such chemical molecules and / or biomolecules may include, but are not limited to, pesticides, insecticides, toxins, therapeutic and / or abuse drugs, hormones, antibiotics, antibodies, organic materials, proteins (e.g., enzymes, immunoglobulins, and / or glycoproteins), nucleic acids (e.g., DNA and / or RNA), lipids, lectins, carbohydrates, whole cells (e.g., prokaryotic cells such as pathogenic bacteria and / or eukaryotic cells such as mammalian tumor cells), viruses, spores, polysaccharides, glycoproteins, metabolites, cofactors, nucleotides, polynucleotides, transition state analogs, inhibitors, nutrients, electrolytes, growth factors, and other biomolecules and / or non-biomolecules, as well as fragments and combinations thereof. Some of the analytes described herein may be proteins such as enzymes, drugs, cells, antibodies, antigens, cell membrane antigens, and / or receptors or their ligands (e.g., nerve receptors or their ligands, hormone receptors or their ligands, nutrient receptors or their ligands, and / or cell surface receptors or their ligands). In certain embodiments, the analyte may be infectious or pathological factors such as bacteria, viruses, yeast, or fungi.

[0022] As used herein, the term “protein” means proteins, polypeptides, oligopeptides, peptides, and analogs (including proteins containing amino acids and amino acid analogs that do not exist in nature), as well as peptide-mimicking structures. The term “protein” also means proteins, polypeptides, oligopeptides, peptides, and analogs. [Modes for carrying out the invention]

[0023] Figures 1A to 1C show a FLOS-LAMP (fluorescence of loop primers during self-quenching loop-mediated isothermal amplification) instrument 100 according to one embodiment, which is capable of operating to amplify an analyte and measure a signal related to the amount of analyte. Figure 1B shows the instrument 100 in an open, empty configuration. Figure 1C shows a reaction tube 110 containing a sample placed inside the instrument 100. Figure 1A shows the instrument 100 in a closed configuration. With the cover 104 closed, the instrument may be configured to selectively amplify polynucleotide sequences. For example, the reaction tube 110 may contain suitable primers for selectively amplifying one or more analytes and / or one or more controls in a sample according to known techniques (e.g., loop-mediated isothermal amplification).

[0024] The apparatus 100 includes a housing 102 configured to receive a reaction tube 110. A cover 104 can be coupled to the housing 102, as shown in Figure 1A. In exemplary embodiments, the cover 104 may be a hinged cover (or any other suitable cover attached to the apparatus 100 in any other suitable way) configured to move over the top of the reaction tube 110. In some cases, the cover 104 is configured to house (and possibly lock) the reaction tube 110 within the housing 102.

[0025] Figure 1A also shows a display screen 111 configured to display information during and after the assay. Furthermore, the instrument 100 may have a cover button 121 for opening the cover 104. Additionally, there may be selection buttons for selecting options displayed on the screen 111, and up and down buttons 122A and 122B, respectively, for moving up and down between options on the screen. In various embodiments, the options may be associated with the type of assay being performed.

[0026] Beneath the cover 104, the housing 102 may include a well 107 for arranging the reaction tube 110, as shown in Figure 1B. Figure 1C shows the reaction tube 110 positioned within the well 107 of compartment 102. In various embodiments, the instrument 100 is configured to amplify an analyte using polymer chain reaction (PCR), loop-mediated isothermal amplification (LAMP), real-time FLOS (RT-LAMP), fluorescence of loop primers upon self-quenching (FLOS LAMP), or other suitable techniques. According to one embodiment, the instrument 100 can further be configured to measure a signal related to the amount of analyte. For example, the instrument 100 may be configured to selectively amplify polynucleotide sequences. For example, the reaction tube 110 may include suitable primers for selectively amplifying one or more analytes and / or one or more controls in a sample according to known techniques (e.g., LAMP).

[0027] In various embodiments, the reaction tube 110 includes a body portion that is closed at the bottom, and the bottom is at least partially transparent to excitation light at the excitation wavelength and emission light at the emission wavelength.

[0028] Figure 1D is a cross-sectional view of the apparatus 100 in a closed configuration in which the reaction tube 110 is located inside. The heating block 130 is configured to control the temperature of the reaction tube and the sample, for example, by maintaining the temperature of the analyte in the sample (containing a suitable primer) to allow the analyte to undergo isothermal amplification. The heating block 130 includes holes that provide an optical path so that a light source 140 (e.g., a light-emitting diode, laser, etc.) can irradiate the sample at a predetermined wavelength and / or excite the fluorescent dye contained in the reaction tube 110. Another hole passing through the heating block 130 provides an optical path so that a sensor 150 (e.g., a photodiode, photomultiplier tube, charge-coupled device (CCD), and / or any other suitable optical detector) can detect an optical signal, such as a fluorescent signal emitted by the fluorescent dye and associated with the amount and / or concentration of the analyte and / or control. In other embodiments, the instrument 100 may include any other suitable sensor that can operate to detect signals characteristic of the amount and / or concentration of the analyte and / or control, such as an optical sensor, electrochemical sensor, pH sensor, or any other sensor that can operate to detect signals indicating the concentration and / or amount of the analyte, which are configured to detect intrinsic fluorescence, absorbance, and / or color (change).

[0029] The device 100 includes a processor 162 and / or memory 164. The processor 162 may be, for example, a general-purpose processor, a field-programmable gate array (FPGA), an application-specific integrated circuit (ASIC), a digital signal processor (DSP), and / or similar. The processor 162 may be configured to retrieve data from and / or write data to memory, for example, memory 164. Memory 164 may be, for example, random access memory (RAM), a memory buffer, a hard drive, a database, erasable programmable read-only memory (EPROM), electrically erasable programmable read-only memory (EEPROM), read-only memory (ROM), flash memory, a hard disk, a floppy disk, cloud storage, and / or other.

[0030] The processor 162 and memory 164 may be communicatively coupled to the heating block 130, the light source 140, and / or the sensor 150, and configured to control the run in which the analytes and / or controls placed in the reaction tube 110 are selectively amplified. The processor and memory may be operable to receive, process, and / or record signals related to the concentrations of the analytes and / or controls. The processor and / or memory may be configured to determine whether the sample contained in the reaction tube 110 is “positive” or “negative” for one or more analytes, according to a method described in further detail herein. Although shown within the housing of the instrument 110, in other embodiments the processor 162 and / or memory 164 may be located in a separate device. Similarly, the instrument 110 may be communicatively coupled to an external computing device configured to control the run and / or determine whether the sample is positive or negative.

[0031] Figure 2 is a flowchart of a method for detecting an analyte according to one embodiment. The method shown and described in Figure 2 can be performed by instrument 100 illustrated and described with reference to Figure 1, or by any other suitable instrument configured to selectively amplify an analyte. Throughout the method, instrument 100 can analyze the sample over time. For example, instrument 100 can be configured to perform LAMP to amplify an analyte such as a characteristic sequence of the SARS-CoV-2 genome. During the analysis, instrument 100 can continuously receive signals related to the amount of analyte at 220. Instrument 100 can be configured to receive signals related to the amount of analyte at every second, every 5 seconds, every 10 seconds, every 20 seconds, or at any other suitable sampling rate. For example, in the FLOS-LAMP technique, a labeled loop probe can be configured to fluoresce when bound to an analyte, so that the intensity (L) of light emitted from the fluorescent label can be used to determine the amount and / or concentration of the analyte when the sample is selectively amplified. The signal received at 220 can represent time-series data of the intensity of the fluorescent dye molecule and / or the concentration of the analyte.

[0032] The instrument (or a computing device coupled to the instrument) may be capable of processing the signal related to the analyte received at 220. This instrument may calculate the intensity moving average (μ) at 230. L ) and the standard deviation of intensity (σ L ) can be calculated. Typically, the same window is applied to the moving average and the standard deviation. The width of the moving average and moving standard deviation windows can be predetermined and / or determined dynamically. For a FLOS-LAMP signal, a suitable fixed window for calculating the moving average and / or standard deviation can be at least or about 3 minutes, at least or about 2 minutes, at least or about 60 seconds, at least or about 30 seconds, at least or about 20 seconds, or any suitable time length. In some embodiments, the window for calculating the moving average and / or standard deviation may be a function of the elapsed time and / or temperature of the amplification reaction, for example, such that the length of the window decreases as the run progresses.

[0033] In other embodiments, the instrument can further process the intensity measurements to calculate the amount of analyte. Moving averages and moving standard deviations can be calculated based on the amount of analyte rather than the intensity of the fluorescent dye molecules.

[0034] When the moving average and moving standard deviation are calculated, the dataset is obtained as follows: for each time point (t), instantaneous intensity (L(t)) and average intensity (μ) over the period ending at that point. L (t)), and the standard deviation (σ) of the intensity measurements taken over the period ending at that point. L Generate a dataset that can be stored in memory, including (t). The moving average and moving standard deviation can be calculated virtually in real time (e.g., within less than 1 second) as the intensity measurement is performed. Once calculated, the moving average of intensity is the sum of the moving average of intensity calculated for the previous time point in time and a multiple of the standard deviation of intensity calculated for that previous time point in time: μ L (tx)+y*σ L (tx) (Formula 1) It can be compared to that. Here, x represents the time difference between the current time and the previous time. y is a constant or function that is multiplied by the moving standard deviation. For FLOS-LAMP analysis, preferred x values ​​are approximately 8 minutes, 6 minutes, 4 minutes, 2 minutes, or any other preferred time. Preferred y values ​​are 1.2, 1.5, 1.8, 2, 2.5, 3, 4, or any other preferred value. Similarly, for FLOS-LAMP analysis, the current (e.g., most recently calculated) moving average of intensity can be compared to the moving average of intensity calculated 4 minutes ago plus twice the standard deviation of intensity calculated 4 minutes ago. The current moving average of intensity being greater than the sum of the moving average of intensity calculated for the previous point in time and multiples of the standard deviation of intensity calculated for that previous point in time can be called the "target index". μ L (t)>μ L(t - x)+y*σ L (t - x) (Equation 2) The target indicator can represent a positive result or the presence of an analyte in the sample. At some points in time, when it is determined that the sample is positive, at 250, a signal indicating a positive result can be immediately transmitted (e.g., within 3 seconds) (e.g., to the user or technician), and / or at 260, the execution of the sample can be terminated. At other points in time, an indicator of a positive result can be transmitted based on the target indicator for a certain period (e.g., 5 seconds, 10 seconds, 15 seconds, 30 seconds, etc.). In this way, the positivity of the sample can be continuously evaluated during amplification, and since the execution can be terminated when a positive result is detected, it is possible to eliminate the need to amplify the sample over a predetermined period and evaluate the sample after processing. Such techniques can significantly reduce the execution time compared to known methods at many points in time.

[0035] In some cases, similar techniques can be applied to control signals to detect negative results (e.g., samples containing the absence of the analyte and / or an amount of the analyte below the detection threshold). A sample may include one or more internal controls and a fluorescent tag configured to produce a luminescence signal indicating the amount of the control. Typically, a sample includes a control with a known initial amount and / or concentration. The instrument can be configured to selectively amplify the control simultaneously with the analyte so that, given a control with a known initial amount / concentration, the time the control will exhibit can be predicted. As will be discussed in more detail herein, the difference in the sequence exhibited by the control and / or the time the control exhibits can be used to determine whether a sample is positive or negative. Thus, the initial amount / concentration of the control can be correlated with the detection threshold of the analyte. A sample may also include additional controls configured to indicate whether the sample run failed for various reasons. For example, controls can be used to determine whether a sufficient amount of sample was obtained. RNaseP is known to be present in human nasal mucus at predictable concentrations and can be used to assess whether a sufficient amount of human nasal mucus sample is present. Therefore, if the RNaseP control fails to display (for example, before a control with a known concentration shows it), the instrument may send a signal indicating that the test was not conclusive regarding insufficient sample.

[0036] In some embodiments, the internal control may be endogenous to the sample, e.g., a biological sample, or the internal control may be added to the sample. In a non-limiting example, when detecting the presence of viral DNA or RNA in a biological sample, the internal control may be RNA or ribosomal RNA expressed from a housekeeping gene. Typically, the luminescence signal indicating the amount of control has different spectral and / or temporal characteristics than the luminescence signal indicating the amount of analyte. At other points in time, the sample may be subdivided into two or more subsamples. Each subsample may be configured to be analyzed for one or more different analytes and / or to function as a control for one or more different analytes. In such embodiments, each subsample is usually amplified simultaneously. During the analysis of the sample, the instrument can continuously receive signals related to the amount of control at 225.

[0037] The instrument (or a computing device coupled to the instrument) may be capable of processing the signal associated with the reference received at 225. The instrument processes the reference signal (μ) at 235. C Moving average of the intensity of the control signal (σ) C The standard deviation of the intensity can be calculated. In other embodiments, the instrument can further process the intensity measurements to calculate the control quantity. In other embodiments, the instrument can further process the intensity measurements to calculate the control quantity. The moving average and moving standard deviation can be calculated against the control quantity, rather than against the intensity associated with the control quantity.

[0038] Calculating the moving average and moving standard deviation of a dataset means that for each time point (t), you get the instantaneous intensity of the control signal (C(t)) and the average intensity of the control signal over the period ending at that point (μ). C (t)), and the standard deviation (σ) of the intensity of the control signal acquired over the period ending at that point. CGenerate a dataset that can be stored in memory, including (t). The current moving average of the intensity of the control signal can be called a "control index" if it is greater than the sum of the moving average of the intensity of the control signal calculated for the previous time point and a multiple of the standard deviation of the intensity of the control signal calculated for the time point before that. μ C (t)>μ C (ts)+v*σ C (ts) (Equation 3) (s represents the time difference between the current time and the previous time; v is a constant or function that multiplies the moving standard deviation. For FLOS-LAMP analysis, preferred s are approximately 8 minutes, 6 minutes, 4 minutes, 2 minutes, or any other preferred time. Preferred v are 1.2, 1.5, 1.8, 2, 2.5, 3, 4, or any other preferred value. In some examples, s may be equal to x (from Equation 1 and / or 2), and / or v may be equal to y (from Equation 1 and / or 2). At other points in time, the constants / functions used to determine whether something is a control indicator may differ from those used to determine whether it is a target indicator. For example, x may be equal to 240 seconds, y may be equal to 2, s may be equal to 360 seconds, and v may be equal to 1.5. In addition, or alternatively, the windows over which the moving average and standard deviation are applied for the target and control may be the same or different.

[0039] In some examples, if there is a control index and no target index, a signal indicating a negative result can be transmitted at 255, and / or the run of the sample can be terminated at 260. In some embodiments, a negative result can be transmitted at 255, and / or the run of the sample can be terminated at 260 immediately after (e.g., within 3 seconds) a control index without a sample. In other embodiments, based on the index, the run can continue over a fixed period (e.g., 3 minutes, 5 minutes, or any other preferred period) or over a dynamically determined period that is a function of time from the start of the run. If the target index is present during the period after the control index, a signal indicating a positive test result can be transmitted at 250, and / or the run can be terminated at 260. In yet another embodiment, based on the target index, the run can continue over a fixed period (e.g., 3 minutes, 5 minutes, or any other preferred period) or over a dynamically determined period that is a function of time from the start of the run. If the control index is present during the period after the target index, a signal indicating a negative test result can be transmitted at 255, and / or the run can be terminated at 260. When two or more controls are used, the absolute and / or relative timing of each control and / or target indicator can be used to determine whether to send a positive or negative result indication.

[0040] In some embodiments, the indication of a positive test result and / or negative test result is ignored (e.g., not analyzed, not suppressed, not transmitted, not reported, not recorded, and / or not a basis for terminating the run) if it occurs in the initial part of the analysis. In LAMP analysis, the sample is typically inserted into a preheated heater block. Typically, due to the properties of the fluorescent dye molecule, the target and control signals are weaker at low temperatures (e.g., before the sample reaches thermal equilibrium with the heater block). Such weak signals may not be reliable indicators of the sample's positive / negative state. In addition, the rate at which the intensity of the fluorescent dye molecule increases as the sample approaches thermal equilibrium usually decreases. Similarly, during the initial part of the sample run when the sample is approaching thermal equilibrium with the heater block, the target and control signals typically rise and fall. Therefore, in some examples, if the target signal and / or control signal have a positive slope and a negative dip (dip downwards), respectively, the positive test result and / or negative test result can be ignored. The measurement of slopes and dips in target and / or control signals can be based on time window cues, similar to the moving average and moving standard deviation discussed above. When a negative slope or positive dip (downward dip) is detected in the target and / or control signal, the indication of that signal can no longer be ignored. In other examples, positive and / or negative test results can be ignored over a predetermined fixed period. For example, the target indicator can be ignored for the first 180, 240, 300, 360, 420 seconds of the run, or any other appropriate period. In another example, the control indicator can be ignored for the first 630, 690, 750, 810, 870 seconds, or any other appropriate period.

[0041] Figure 3 shows experimental data from FLOS-LAMP analysis of the sample in the example. Line 310 represents the moving average of the intensity of the fluorescent dye molecule relative to the amount of target analyte. Lines 320 and 322 represent the moving average of the intensity of the fluorescent dye molecule offset by time ± a multiple of the standard deviation of the intensity of the fluorescent dye molecule, respectively. In this example, the offset is 240 seconds and the multiple of the standard deviation is 3. Therefore, line 320 is μ L (t-240)+3σ L (t-240) and line 322 is μ L (t-240)-3σ L (t-240) This represents approximately from the start of execution. 1800 In seconds, line 310 and line 320 intersect, μ L ( 1800 )>μ L ( 1800 -240)+3σ L ( 1800 This becomes -240), which represents the target index. Therefore, approximately 1800 Within seconds, a signal indicating a positive result can be sent, and the execution can be optionally terminated. Alternatively, the execution can be continued for an additional period to ensure that the target indicator remains present.

[0042] While various embodiments have been described above, it should be understood that they are presented only as examples and not as limitations. Where the schematic diagrams and / or embodiments above show components arranged in a certain orientation or position, the arrangement of components may be modified. While embodiments have been specifically shown and described, it will be understood that various changes in form and detail may be made. Various embodiments have been described as having specific features and / or combinations of components, but other embodiments having any combination of features and / or components from any of the embodiments described above are also possible.

[0043] For example, while the methods described herein generally relate to FLOS-LAMP analysis and are particularly well suited for SARS-CoV-2 detection, it should be understood that the techniques described herein can be applied to many other analytes and / or targeted detection schemes. Similarly, the embodiments described herein are not limited to SARS-CoV-2 detection and can be applied to any analyte that can be selectively amplified or concentrated by, for example, LAMP, polymerase chain reaction (PCR), chemosynthesis, electrochemistry, bioproduction, chromatography, electrophoresis, isoelectric focusing, weight separation, etc. The embodiments described herein generally describe fluorescence signals that are related to or correlated with the amount or concentration of the analyte, but the analyte can be detected by any suitable means such as, for example, a pH-driven colorimetric signal from real-time loop-mediated isothermal amplification (RT-LAMP), or other suitable colorimetric signals, electrical signals, electrochemical signals, or photoabsorbance signals. Similarly, the methods illustrated and described with reference to Figure 2 are very well suited to any suitable analysis in which the "positive" result is characterized by an exponential or other rapid increase in the signal from a relatively low baseline.

[0044] In certain embodiments, the methods disclosed herein may be used to determine the presence or absence of an analyte by detecting and / or measuring the signal generated via PCR. A variety of different PCR methods may be used, including, but not limited to, basic PCR, reverse transcriptase (RT)-PCR, hot-start PCR, competitive PCR, or quantitative real-time (qRT)-PCR, as described, for example, in Non-Patent Document 1 and the references discussed therein.

[0045] In certain embodiments, the methods disclosed herein may be used to determine the presence or absence of an analyte by detecting and / or measuring a signal generated via isothermal nucleic acid amplification. Isothermal amplification of nucleic acids is an alternative to polymerase chain reaction (PCR). The advantage of these methods is that, unlike PCR which requires periodic temperature changes, nucleic acid amplification can be performed at a constant temperature. In certain embodiments, isothermal nucleic acid amplification is performed using, for example, loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), helicase-dependent amplification (HDA), exponential amplification of nucleic acids (EXPAR), strand displacement amplification (SDA), and recombinase polymerase amplification (RPA), which are described, for example, in Non-Patent Document 2 and the references cited therein, the whole of which is incorporated herein by reference.

[0046] As shown in Figure 2, the parameters described above (e.g., moving average window, standard deviation window, time offset (x), and standard deviation multiple (y)) are generally explained in the context of FLOS-LAMP and selected based on the characteristic shapes of the positive target signal and / or control signal. A skilled data scientist can take the above into consideration and easily select other appropriate parameters for signals with different characteristics.

[0047] In certain embodiments, the methods disclosed herein may be used to determine the presence of an analyte (e.g., (1) a detectable amount and / or concentration, and / or (2) an amount and / or concentration exceeding a threshold) or the absence of an analyte (e.g., (1) the absence of a detectable amount and / or concentration, and / or (2) an amount and / or concentration below a threshold). For example, in certain embodiments, the method determines the presence or absence of the analyte in a sample by measuring the presence or absence of nucleic acid components of the analyte, for example, using PCR. In certain embodiments, the analyte is an infectious agent or pathogen, or a component thereof. In certain embodiments, the infectious agent or pathogen is a virus, bacterium, or fungus. In certain embodiments, the infectious agent is an influenza virus or coronavirus, for example, SARS-CoV-2. In some embodiments, the methods disclosed herein are used to determine the presence of an infectious agent or pathogen by, for example, detecting the presence of the infectious agent's DNA or RNA in a sample. In some embodiments, the sample is a biological sample obtained from a subject diagnosed with an infection, or a subject considered to have an infection or be at risk of developing one. In other embodiments, the sample is a food or beverage product. In some embodiments, the sample is obtained from a surface, for example, a food preparation surface, a food or beverage packaging surface, or a surface in a home, rental property, or hotel, for example, but not limited to, a kitchen counter surface, a bathroom counter surface, a toilet, shower, or bathtub surface, or a table or dresser surface.

[0048] In certain embodiments, the analyte is a virus or a component thereof. In some embodiments, the sample is a biological sample obtained from a subject diagnosed with, suspected of, or at risk of being infected with a virus. In certain embodiments, the virus is norovirus, rotavirus, adenovirus, astrovirus, influenza virus, coronavirus, parainfluenza virus, respiratory syncytial virus, human immunodeficiency virus (HIV), human T lymphotropic virus (HTLV), rhinovirus, hepatitis A virus, hepatitis B virus, Epstein-Barr virus, or West Nile virus. In certain embodiments, the virus is SARS-CoV-2.

[0049] In one embodiment, the virus is an influenza virus including, but not limited to, any of the types, subtypes, lineages, or clades disclosed herein. There are four types of influenza viruses: A, B, C, and D. Human influenza A and B viruses cause seasonal illness outbreaks (known as flu season) in the United States almost every winter. Influenza A virus is the only influenza virus known to cause an influenza pandemic, i.e., a global epidemic of influenza disease. Influenza C infection generally causes mild symptoms and is not thought to cause human influenza outbreaks. Influenza D virus primarily infects cattle and is not known to infect or cause illness in humans.

[0050] Influenza A viruses are classified into subtypes based on two proteins on the surface of the virus: hemagglutinin (H) and neuraminidase (N). There are 18 different hemagglutinin subtypes and 11 different neuraminidase subtypes (H1-H18 and N1-N11, respectively). Current subtypes of influenza A viruses that routinely circulate among people include A(H1N1) and A(H3N2). A particular circulating influenza A(H1N1) virus is associated with the pandemic 2009 H1N1 virus, which emerged in the spring of 2009 and caused an influenza pandemic. Scientifically called the "A(H1N1)pdm09 virus," and more commonly called "2009 H1N1," this virus has continued to circulate seasonally ever since. Influenza A(H3N2) viruses have recently formed many separate genetically distinct lineages and continue to co-circulate.

[0051] Influenza B viruses cannot be divided into subtypes, but instead are further classified into two lineages: B / Yamagata and B / Victoria.

[0052] In certain embodiments, the virus is a coronavirus, including but not limited to any of the types, subtypes, or groupings disclosed herein. Coronaviruses are named after the crown-like spikes on their surface. There are four main subgroups of coronaviruses, known as alpha, beta, gamma, and delta. Seven coronaviruses that can infect humans are common human coronaviruses: 229E (alpha coronavirus); NL63 (alpha coronavirus); OC43 (beta coronavirus); HKU1 (beta coronavirus); and other human coronaviruses: MERS-CoV (beta coronavirus that causes Middle East Respiratory Syndrome or MERS); SARS-CoV (beta coronavirus that causes Severe Acute Respiratory Syndrome or SARS); and SARS-CoV-2 (novel coronavirus that causes Coronavirus Disease 2019 or COVID-19). Humans are commonly infected with human coronaviruses 229E, NL63, OC43, and HKU1.

[0053] In certain embodiments, the virus is SARS-CoV-2. A new novel coronavirus called coronavirus disease 2019 (COVID-19) has been reported. COVID-19 is caused by infection with the novel coronavirus, SARS-CoV-2, or 2019-nCoV. In some embodiments, the analyte is detected in biological samples obtained from subjects diagnosed with COVID-19 or subjects considered to be at risk of contracting or developing COVID-19.

[0054] In certain embodiments, the analyte is a bacterium or a component thereof. In some embodiments, the sample is a biological sample obtained from a subject diagnosed with or considered to be at risk of having a bacterial infection. In certain embodiments, the bacteria are any of the following: Acinetobacter, Bacteroides, Burkholderia, Clostridium, Enterobacteriaceae, Enterococcus, Klebsiella, Staphylococcus, Streptococcus, Morganela, Mycobacterium, Neisseria, Pseudomonas, or Stenotrophomonas (including any of Acinetobacter baumannii, Bacteroides fragilis, Burkholderia cepacia, Clostridium difficile, Clostridium sordellii, or carbapenem-resistant Enterobacteriaceae); Enterococcus faecalis, Klebsiella pneumonia, Staphylococcus aureus (including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Staphylococcus aureus), Morganella morganii, Mycobacterium abscessus, or Pseudomonas. aeruginosa, Stenotrophomonas maltophilia, Mycobacterium tuberculosis, Streptococcus pneumonia, Neisseria meningitidis, or vancomycin-resistant Enterococci.

[0055] In certain embodiments, the analyte is a fungus. In certain embodiments, the sample is a biological sample obtained from a subject diagnosed with a fungal infection, or a subject considered to have or be at risk of developing a fungal infection. In certain embodiments, the fungus is one of the following: Aspergillus, Candida (including Candida auris), Cryptococcus neoformans, Pneumocystis (including Pneumocystis girobesi), Mucormycetes, Taromyces, Candida, Blastomyces, Coccidioides, Histoplasma, Cryptococcus (including Cryptococcus gattii), or Paracoccidioides.

[0056] In addition, some methods described herein describe ending the sample run when the target or control is indicated (optionally, after a waiting period). However, it should be understood that in other embodiments, the sample can be run for a maximum duration (e.g., 60 minutes, 90 minutes, etc.) (e.g., selective amplification of the target analyte). In such embodiments, if the target is indicated during that period, a positive result indication can be transmitted (optionally, the indication is accepted during the excluded initial period). If the control is indicated within that period, a negative result indication can be transmitted. In other scenarios, a signal can be transmitted indicating that the test failed or is inconclusive.

[0057] If the methods and / or events described above indicate specific events and / or procedures occurring in a particular order, the order of the specific events and / or procedures may be changed. In addition, the specific events and / or procedures may be executed not only sequentially as described above, but also simultaneously in parallel processes if possible.

Claims

1. The sample is placed in an instrument configured to selectively amplify the analyte, Each signal receives multiple signals related to the amount of analyte at a given time point. For each point in time, the moving average and moving standard deviation of the amount of the analyte are calculated based on a subset of the multiple signals associated with the period ending at that point in time. Moving average of the amount of the analyte at the first time point (t): μ L (t) of, (1) Moving average with respect to the second time point (t-x), which is the amount of time before the first time point (t): μ L (t-x) and, (2) The sum of the moving standard deviation σ L (t-x) at the second time point (t-x) and y times that: μ L (t-x)+y*σ L (t-x) Compared with, μ L (t)>μ L (t-x)+y*σ L (t-x) Here, x represents the time difference between the first time point and the second time point. The aforementioned y is a constant that is multiplied by the moving standard deviation. When the conditions are met, a signal indicating a positive result is sent. A method that includes the act of doing so.

2. The method according to claim 1, wherein the sample is a biological sample.

3. The method according to claim 2, wherein the biological sample is selected from the group consisting of serum, blood, salivary secretions, tear secretions, respiratory secretions, nasal fluid, mucus samples, and intestinal secretions.

4. The method according to any one of claims 1 to 3, wherein the analyte is a polynucleotide sequence.

5. The method according to claim 4, wherein the polynucleotide sequence is a polynucleotide sequence of a virus.

6. A method according to any one of claims 1 to 3. The aforementioned device is a FLOS-LAMP device. The analyte is a characteristic sequence of the SARS-CoV-2 genome, according to the method.

7. The method according to claim 1, wherein if the signal is obtained within a predetermined period after the analyte has begun to be selectively amplified, the user is not informed of the signal indicating that a positive result has been obtained.

8. The method according to claim 1, wherein the signal indicating a positive result is not reported to the user while the amount of the analyte has a positive slope as a function of time within a predetermined period after the start of selective amplification of the analyte and is concave downwards.

9. Moving average of the amount of the analyte at the first time point (t): μ L (t) is, (1) The moving average μ L (t-x) for the second time point (t-x), (2) The sum of the number of multiples of the moving standard deviation σ L (t-x) at the second time point (t-x): μ L (t-x)+y*σ L (t-x) Larger than: μ L (t)>μ L (t-x)+y*σ L (t-x) The method according to claim 1, wherein the analysis of the analyte is completed within a predetermined time after the determination of the analyte.

10. The method according to claim 1, Multiple signals are the first multiple signals, The above method further, Receive a second set of signals, each associated with a control quantity at a given time; For each point in time, calculate the moving average and moving standard deviation of the control quantity based on a subset of second signals related to the period ending at that point in time; The moving average μC(t) of the control quantity at the first time point (t) is, (1) The moving average of the control quantity at the second time point (t-s), which is a time quantity prior to the first time point (t): μ C (t-s), (2) The sum of v times the moving standard deviation σ C (t-s) of the control quantity at the second time point (t-s): μ C (t-s)+y*σ c (t-s) Compared to, The moving average μC(t) of the control quantity at the first time point (t) is, (1) The moving average μC(t-s) of the control quantity at the second time point (t-s) and (2) The sum of the moving standard deviation σ C (t-s) of the control quantity at the second time point (t-s) and v times that: μ C (t-s)+v*σ C (t-s) Larger than μ C (t)>μ C (ts)+v*σ c (ts) Here The aforementioned s represents the time difference between the first time point and the second time point. The aforementioned v is a constant that is multiplied by the moving standard deviation, And, The moving average μL(t) of the amount of analyte at the first time point (t) is, (1) The moving average μL (t-x) of the amount of analyte at the second time point (t-x) and (2) The sum of the amount of analyte at the second time point (t-x) and y times the moving standard deviation σ L (t-x): μ L (t-x)+y*σ L (t-x) Smaller than μ L (t)<μ L (t-x)+y*σ L (t-x) Based on this, it sends a signal indicating a negative result. A method that includes doing so.

11. The method according to claim 10, wherein if a signal indicating a negative result is obtained within a predetermined period after the analyte has begun to be selectively amplified, the signal indicating a negative result is not reported to the user.

12. The method according to claim 10, wherein the signal is not reported to the user while the amount of the analyte, as a function of time, has a positive slope and is concave downwards within a predetermined period after the analyte has begun to be selectively amplified.

13. The method according to claim 1, The aforementioned plurality of signals are the first plurality of signals, The aforementioned method, Each signal receives a second set of signals, each associated with a control quantity at a given time point. For each point in time, the moving average and moving standard deviation of the control quantity are calculated based on a subset of the second set of signals associated with the period ending at that point in time. The moving average μC(t) of the control quantity at the first time point (t) is, (1) The moving average of the control quantity with respect to the second time point (t-s), which is the time quantity before the first time point (t): μ C (t-s), (2) The sum of the number of v multiples of the moving standard deviation σ C (t-s) of the control quantity at the second time point (t-s): μ C (t-s)+y*σ c (t-s) Compared with, The moving average μC(t) of the control quantity at the first time point (t) is, (1) The moving average μC(t-s) of the control quantity at the second time point (t-s) and (2) The sum of the moving standard deviation σ C (t-s) of the control quantity at the second time point (t-s) and v times that: μ C (t-s)+v*σ C (t-s) Smaller than μ C (t)<μ C (t-s)+v*σ C (t-s) Here, s represents the time difference between the first time point and the second time point. The aforementioned v is a constant that is multiplied by the moving standard deviation. and The moving average μL(t) of the amount of analyte at the first time point (t) is, (1) The moving average μL (t-x) of the amount of analyte at the second time point (t-x) and (2) The sum of the amount of analyte at the second time point (t-x) and y times the moving standard deviation σ L (t-x): μ L (t-x)+y*σ L (t-x) Larger than: μ L (t)>μ L (t-x)+y*σ L (t-x) Based on this, it sends a signal indicating a positive result. A method that further includes the following.

14. The method according to claim 1, wherein the period ending at the first point in time is at least 20 seconds long.

15. The method according to claim 1, wherein the period ending at the first point in time is of a predetermined fixed length of time.

16. The method according to claim 1, wherein the length of the period ending at the first point in time is dynamically determined based on a function of elapsed time.

17. The method according to claim 1, wherein the plurality of signals is at least one of a plurality of electrochemical signals or a plurality of fluorescent signals indicating the amount of polynucleotide being amplified.

18. The method according to claim 1, The aforementioned multiple signals indicate the amount of polynucleotides undergoing amplification. A method in which the length of the period ending at the first point in time is dynamically determined based on a function of the temperature of the amplification reaction.

19. The method according to claim 1, wherein the second time point is at least 180 seconds before the first time point.

20. The method according to claim 1, The aforementioned plurality of signals are a plurality of first signals associated with the intensity of the first fluorescent dye, The aforementioned method, Each signal receives a second set of signals associated with the intensity of a second fluorescent dye. For each point in time, the moving average and moving standard deviation of the intensity of the second fluorescent dye are calculated based on a subset of the second multiple signals associated with the period ending at that point in time. The moving average of the intensity of the second fluorescent dye at the first time point (t): μL(t) (1) Moving average of the second time point (t-x), which is the amount of time before the first time point (t): μ L (t-x) and (2) The sum of the moving standard deviation σ L (t-x) at the second time point (t-x) and y times that: μ L (t-x)+y*σ L (t-x) Here, x represents the time difference between the first time point and the second time point. The aforementioned y is a constant that is multiplied by the moving standard deviation. A method that further includes comparing with

21. The method according to claim 1, The aforementioned plurality of signals are the first plurality of signals, The aforementioned method, The system receives a second set of signals, each of which is associated with the amount of RNaseP in the sample at a given time point. For each point in time, the moving average and moving standard deviation of the amount of RNaseP are calculated based on a subset of the second RNaseP signals associated with the period ending at that point in time. The moving average μC(t) of the amount of RNaseP at the first time point (t) is, (1) Moving average of the amount of RNaseP at the second time point (t-s), which is the time period before the first time point (t): μ C (t-s) and (2) The sum of the amount of RNaseP at the second time point (t-s) and v times the moving standard deviation σ C (t-s): μ Ｃ (t-s)+v*σ ｃ (t-s) Here, s represents the time difference between the first time point and the second time point. The aforementioned v is a constant that is multiplied by the moving standard deviation. Compared with, The moving average of the amount of RNaseP at the first time point (t) is greater than (1) the sum of the moving average of the amount of RNaseP at the second time point (t-s) and (2) the multiple of the moving standard deviation of the amount of RNaseP at the second time point (t-s), and Moving average of the amount of analyte at the first time point (t): μ L (t) is, (1) Moving average of the amount of analyte at the second time point (t-x): μL (t-x) and (2) The sum of the amount of analyte at the second time point (t-x) and y times the moving standard deviation σ L (t-x): μ L (t-x)+y*σ L (t-x) Smaller than μ L (t)< μ L (t-x)+y*σ L (t-x) Based on this, a signal indicating an insufficient volume of the sample is transmitted. A method that includes doing so.

22. A system for analyzing a sample for the presence of an analyte, A well configured to receive a reaction tube containing the analyte; A light source configured to emit excitation light at a wavelength for irradiating the analyte in the reaction tube; an optical detector configured to receive an optical signal corresponding to the analyte being irradiated by the excitation light; and, A processor operably coupled to the light source and the optical detector, The aforementioned light source is activated, Each signal is associated with the optical signal, and multiple signals indicating the amount of analyte at a given time are received from the optical detector. For each point in time, the moving average and moving standard deviation of the amount of the analyte are calculated based on a subset of signals associated with the period ending at that point in time. Moving average of the amount of the analyte at the first time point (t): μ L (t) of, (1) Moving average with respect to the second time point (t-x), which is the amount of time before the first time point (t): μ L (t-x) and (2) The sum of the moving standard deviation σ L (t-x) at the second time point (t-x) and y times that: μ L (t-x)+y*σ L (t-x) Compared to, Moving average of the amount of analyte at the first time point (t): μL (t-x) but, (1) Moving average with respect to the second time point (t-x): μ L (t-x) and (2) The sum of the moving standard deviation σ L (t-x) at the second time point (t-x) and y times that: μ L (t-x)+y*σ L (t-x) Larger than μ L (t)>μ L (t-x)+y*σ L (t-x) Here, x is the time difference between the first time point and the second time point. The aforementioned y is a constant that is multiplied by the moving standard deviation. Based on this, a signal indicating a positive result is sent. A processor configured in such a way, A system equipped with these features.

23. Each signal receives multiple signals associated with the amount of analyte at a given time; For each point in time, the moving average and moving standard deviation of the analyte quantities are calculated based on a subset of the multiple signals associated with the period ending at that point; Moving average of the amount of analyte at the first time point (t): μ L (t) of, (1) The moving average μ L (t-x) at the second time point (t-x), which is the amount of time before the first time point (t). and (2) Compare with the sum of the moving standard deviation σ L (t-x) at the second time point (t-x) above and y times that; Moving average of the amount of analyte at the first time point (t): μ L (t) but, (1) Moving average at the second time point (t-x): μ L (t-x) and (2) The sum of the moving standard deviation σ L (t-x) at the second time point (t-x) and y times that: μ L (t-x)+y*σ L (t-x) Larger than μ L (t)>μ L (t-x)+y*σ L (t-x) Here, x is the time difference between the first time point and the second time point. The aforementioned y is a constant that is multiplied by the moving standard deviation. Based on this, a signal indicating a positive result is sent. A non-temporary, computer-readable medium that stores instructions configured to be executed by a processor.

24. A method for determining the presence or absence of an analyte in a biological sample, A biological sample is placed in an instrument configured to selectively amplify the analyte; Each signal receives multiple signals associated with the amount of analyte at a given time; For each point in time, the moving average and moving standard deviation of the amount of the analyte are calculated based on a subset of the multiple signals associated with the period ending at that point; Moving average of the amount of analyte at the first time point (t): μ L (t) of, (1) Moving average of the second time point (t-x), which is the amount of time before the first time point (t): μ L (t-x) and (2) The sum of the moving standard deviation σ L (t-x) at the second time point (t-x) and y times that: μ L (t-x)+y*σ L (t-x) Compare to, This includes, Moving average of the amount of analyte at the first time point (t): μ L (t) but, (1) Moving average with respect to the second time point (t-x): μ L (t-x) and (2) The sum of the moving standard deviation σ L (t-x) at the second time point (t-x) and y times that: μ L (t-x)+y*σ L (t-x) Larger than: μ L (t)>μ L (t-x)+y*σ L (t-x) Here, x is the time difference between the first time point and the second time point. The aforementioned y is a constant that is multiplied by the moving standard deviation. At that time, it is determined that the biological sample contains an amount of analyte greater than the threshold amount, The above method further, Receive a second set of signals, each associated with a control quantity at a given time; For each point in time, calculate the moving average and moving standard deviation of the control quantity based on a subset of second signals related to the period ending at that point in time; Moving average of the control quantity at the first time point (t): μ C (t) is, (1) The moving average of the control quantity at the second time point (t-s), which is the time quantity prior to the first time point (t): μ C (t-s) and (2) The sum of the control quantity at the second time point (t-s) and a multiple of the moving standard deviation. μ C (t-s)+v*σ C (t-s) Larger than: μ C (t)>μ C (ts)+v*σ c (ts) ,and, The moving average μL(t) of the amount of analyte at the first time point (t) is, (1) The moving average μL (t-s) of the amount of analyte at the second time point (t-s) and (2) The sum of the amount of analyte at the second time point (t-s) and v times the moving standard deviation σ C (t-s): μ L (t-x)+v*σ L (t-x) Smaller than: μ L (t)<μ L (t-x)+v*σ L (t-x) Here, s is the time difference between the first time point and the second time point. The aforementioned v is a constant that is multiplied by the moving standard deviation. At that time, A method for determining that the aforementioned biological sample contains an analyte in an amount less than a threshold amount.

25. The method according to claim 24, A method wherein the biological sample is selected from the group consisting of serum, blood, salivary secretions, tear secretions, respiratory secretions, nasal fluid, nasal swab fluid, oral swab fluid, mucus samples, and intestinal secretions.

26. The method according to claim 24 or 25, wherein the analyte is a polynucleotide sequence.

27. The method according to claim 26, wherein the polynucleotide sequence is a polynucleotide sequence of a virus.

28. The method according to claim 27, wherein the virus is the SARS-CoV2 virus or a variant thereof.

29. The method according to claim 24 or 25, wherein the device is a FLOS-LAMP device.

30. The method according to any one of claims 1 to 3, 7 to 21, 24, or 25, wherein the amount of analyte is measured by a method comprising a polymerase chain reaction.

Images