Devices and applications linking nucleic acid amplification testing and CRISPR systems

Abstract

This disclosure relates to a device and application of a nucleic acid amplification test (NAAT) and CRISPR system integrated into a single system.

Description

[Technical Field]

[0001] The present invention generally relates to devices, systems and methods for coupling nucleic acid amplification tests (NAAT) with a CRISPR system and integrating them into one system to improve target detection of microorganisms in a sample. [Background Art]

[0002] Rapid and accurate diagnosis of diseases is critical for effective treatment and prevention of long-term complications. Nucleic acid-based biomarkers associated with diseases play a central role in diagnosis because DNA and RNA can be amplified from minute amounts, facilitating their highly specific detection through complementary nucleotide pairing. Indeed, nucleic acid-based diagnostics have emerged as the gold standard for a variety of acute and chronic conditions, particularly those caused by infectious diseases.

[0003] Nucleic acid diagnostics based on quantitative polymerase chain reaction (qPCR) and sequencing are widely accepted and frequently used in clinical laboratories. Nevertheless, conventional PCR techniques require trained personnel to prepare samples and set up amplification reactions, and thermocyclers to cycle between different temperatures. Isothermal nucleic acid amplification strategies eliminate the need for thermal cyclers, but on the other hand, can lead to reduced detection specificity due to non-specific amplification. Certain additional readouts incorporating several fluorescent probes, oligonucleotide displacement probes or molecular beacons can improve specificity, but still rely on amplification of the template.

[0004] Diagnostics based on clustered, regularly arranged short palindromic sequence repeats (CRISPR) can address some of these limitations. While various CRISPR-Cas systems exist within different species of archaea and bacteria, they share a common reliance on crRNA for inducing CRISPR proteins in the identification and cleavage of specific nucleic acid targets. crRNA can be customized to target specific DNA or RNA regions by binding to complementary sequences, which in certain systems are limited to regions near protospacer facies (PAMs) or protospacer facies.

[0005] There is an unprecedented need for systems and methods that can improve the ability to practically, timely, and accurately identify disease-causing microorganisms. This disclosure addresses this need by devising a dual detection system that combines separate technologies for detecting microorganisms, and thus provides output that significantly improves the performance of existing technologies.

[0006] [Sequence List] This application includes the sequence listing provided in Table 1. The sequence listing related to this application is further provided in XML format. The name of the text file containing the sequence listing is P38755-WO_Sequence_Listing.xml. The XML file is 57.143 bytes and was created on July 24, 2024. [Overview of the project]

[0007] This summary is provided to present a simplified selection of concepts that will be further described in the detailed description below. This summary is not intended to identify any important or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. Other features, details, usefulness, and advantages of the claimed subject matter will become apparent from the detailed description below, including the embodiments shown in the accompanying drawings and defined in the accompanying claims.

[0008] In some embodiments, devices, systems, and methods are provided hereby for linking nucleic acid amplification testing with a CRISPR system, thereby improving the targeted detection of microorganisms in a sample.

[0009] In some embodiments, the Disclosure relates to a device comprising an assay tube subdivided into a plurality of subsections, wherein a first subsection is connected to an openable cap, and an opening accessible from the openable cap of the first subsection is fitted to receive a sample, the first subsection is sealed along the tube from subsequent subsections, a second subsection contains a solution including an internal control, the second subsection is sealed along the tube from subsequent subsections, a third subsection contains a solution including magnetic beads in the solution, the third subsection is sealed along the tube from subsequent subsections, and a fourth subsection contains a solution including a lysis buffer, the fourth subsection is sealed along the tube from subsequent subsections The device provides a 5th subsection sealed from the end of the assay tube, the 5th subsection containing a solution including a wash buffer, the 6th subsection containing a solution including an elution buffer, the 6th subsection sealed from the end of the assay tube, the 7th subsection containing a solution including a first PCR master mix, the 7th subsection sealed from the end of the tube, the 8th subsection containing a solution including a second PCR master mix, the 8th subsection sealed from the end of the tube, and the 9th tube containing a solution including a CRISPR enzyme, the 9th subsection sealed from the end of the assay tube. The assay tube typically has a length of 10⁶ ± 21 mm and is subdivided into sections for receiving appropriate volumes of reagents for one or more steps of a NAAT and / or CRISPR reaction.In some configurations, the first subsection is adapted to accept a sample volume of approximately 200 ± 40 microliters, the second subsection contains 12.5 ± 2.5 microliters of a solution containing an internal control reagent, the third subsection contains 12.5 ± 2.5 microliters of a solution containing magnetic beads, the fourth subsection contains 215 ± 43 microliters of a solution containing lysis buffer, the fifth subsection contains 240 ± 48 microliters of a solution containing washing buffer, the sixth subsection contains 50 ± 10 microliters of a solution containing elution buffer, the seventh subsection contains 30 ± 6 microliters of a solution containing the first PCR master mix, the eighth subsection contains 15 ± 3 microliters of a solution containing the second PCR master mix, and the ninth subsection contains 60 ± 12 microliters of a solution containing CRISPR enzyme. The present invention envisions scenarios in which certain configurations of the device may include overlapping sections and scenarios in which certain buffering reagents may be combined. In certain configurations, the internal control reagent is a control material for the reverse transcription polymerase chain reaction (RT-PCR) reagent. In certain configurations, the magnetic bead reagent is Liat Magnetic Particles (Roche®) used for sample preparation and nucleic acid extraction. In certain configurations, the lysis buffer is Liat® Generic Lysis Buffer 1. In certain configurations, the washing buffer is cobas® Omni Wash Buffer. In certain configurations, the elution buffer is FRTA Elution Buffer (Roche®). In certain configurations, the device contains two or more PCR master mixes. The first PCR master mix may be a solution containing reaction buffer, dNTPs, RT-PCR primers, MMLV RT polymerase, and uracil DNA glycosylase. The second PCR master mix may be a solution containing reaction buffer, Z05 polymerase and its aptamer, and RT-PCR primers. The assay tube further comprises a tenth subsection containing a solution comprising the CRISPR enzyme, the tenth subsection being sealed from the end of the assay tube.A CRISPR enzyme may be part of a CRISPR system adapted to specifically cleave multiple nucleic acids amplified by a PCR reaction. In certain configurations, the CRISPR enzyme is a CRISPR-Cas enzyme having trans-cleavage activity, such as Cas12 and Cas13. The CRISPR master mix described herein includes guide RNA, substrates, and metal ion cofactors for targeting target sequences in microorganisms. In some embodiments, the assay tube is mounted on a frame, e.g., an open frame. In certain configurations, either the left or right side of the frame may include a guide groove adapted to facilitate the sliding insertion of the device into a system that provides signal readout, for example. In certain configurations, a sample preparation reagent filled in the device extracts nucleic acid material from a sample such as influenza A virus, influenza B virus, respiratory syncytial virus (RSV), and SARS-CoV-2 virus. The assay tube may be a thermoplastic tube, e.g., a tube made from polypropylene. In some embodiments, this disclosure provides a method for detecting target nucleic acid sequences using the assay tube described herein.

[0010] In some embodiments, the Disclosure provides a method for dual detection of a target microorganism, comprising the steps of (a) amplifying a first target nucleic acid sequence in a nucleic acid amplification reaction and detecting a signal from the nucleic acid amplification of the first target nucleic acid sequence; (b) detecting a second target nucleic acid sequence using a CRISPR system that targets the second target nucleic acid sequence and detecting a signal from the activation and substrate cleavage of the second target nucleic acid by the CRISPR system; and (c) comparing the signal detected from the nucleic acid amplification of the first target nucleic acid sequence with an amplification threshold and comparing the signal detected from the activation and substrate cleavage of the second target nucleic acid by the CRISPR system with a CRISPR threshold, wherein the first and second target nucleic acid sequences are sequences derived from a target microorganism, and the target nucleic acid sequence is dually detected if both the signal detected from the first target nucleic acid amplification reaction and the signal detected from the activation and substrate cleavage of the second target nucleic acid by the CRISPR system exceed thresholds. This disclosure envisions embodiments in which the nucleic acid amplification reaction is a polymerase chain reaction (PCR) loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), rolling circle amplification (RCA), strand substitution amplification (SDA), nicking and extension amplification (NEAR), exponential amplification (EXPAR), multiple substitution amplification (MDA), helicase-dependent amplification (HAD), or hybridization chain reaction (HCR). This disclosure envisions embodiments in which the CRISPR system is a system having trans-cleavage nuclease activity, such as a Cas9 system, a Cas12 system, or a Cas13 system. In some embodiments, the first target nucleic acid sequence and the second target nucleic acid sequence are FluA. In some embodiments, a region of the first target nucleic acid amplification sequence is amplified during the nucleic acid amplification reaction, thereby producing an amplicon containing the second target nucleic acid sequence. In some examples, the first target nucleic acid sequence contains multiple regions that include the second target nucleic acid sequence.In some examples, activation and substrate cleavage of multiple secondary target nucleic acid sequences further amplifies the signal detected from the secondary target nucleic acid at a rate directly proportional to the number of secondary target nucleic acid sequences. In some examples, the method includes a step of amplifying multiple primary target nucleic acid sequences for the multiple detection of multiple microorganisms; for example, if the primary nucleic acid amplification is 3plex (3 channels), CRISPR would result in 6plex (original 3 channels + additional 3 channels). In some examples, the method includes a step of amplifying multiple primary target nucleic acid sequences for the multiple detection of the same microorganism, thereby increasing sensitivity by amplifying different regions of the same target. In some examples, the amplification threshold provides a sensitivity of at least 120 copies / mL. In some examples, the CRISPR threshold provides a sensitivity of at least 120 copies / mL (same sensitivity but with improved signal intensity). In some examples, the method further includes a cleavage step, after detection of the amplification threshold and the CRISPR threshold, of cleaving multiple nucleic acids using a nonspecific nuclease, thereby reducing nucleic acid contamination in the system. [Brief explanation of the drawing]

[0011] The above and other features and advantages of the present invention will be more clearly understood from the following detailed description of exemplary embodiments, which are taken into account together with the accompanying drawings.

[0012] [Figure 1A] This device shows an assay tube (elongated in shape) subdivided into multiple subsections, each subsection containing reagents for performing nucleic acid amplification testing. [Figure 1B] This device features an assay tube (elongated in shape) subdivided into multiple subsections, each containing reagents for performing nucleic acid amplification tests and CRISPR assays. [Figure 2] This disclosure describes a system for providing a readout of a signal generated by a nucleic acid amplification test or CRISPR assay in conjunction with the device of this disclosure. [Figure 3] The present disclosure shows guide grooves adapted to facilitate the sliding insertion of the device into a system that provides signal readout together with the device. [Figure 4] This schematic diagram illustrates three distinct steps of the method of the present disclosure for combining signals detected from a NAAT system and a CRISPR system into a single integrated process. Step 1 shows the NAAT process with NAAT signal readout. Step 2 shows the liquid handling process to enable amplicon transfer and interaction with the CRISPR system. Step 3 shows the CRISPR process with CRISPR signal readout. [Figure 5] This chart shows the results from an experiment that detected a series of single-sequence mutations by referencing the wild-type FluA sequence. [Figure 6A] This chart shows the generalized linear model fit detected in CRISPR channels (Cas). [Figure 6B] This chart shows the inverse prediction of generalized linear model fitting detected in CRISPR channels (Cas). [Figure 6C] This chart shows the generalized linear model fit detected in CRISPR channels (Cas). [Figure 6D] This chart shows the inverse prediction of generalized linear model fitting detected in CRISPR channels (Cas). [Figure 7] This chart shows the results of the CRISPR variation test.

[0013] Please understand that drawings are not necessarily proportional to actual size, and that similar reference numbers refer to similar features.

[0014] [Integration by reference] All publications, patents, and patent applications referenced herein are incorporated by reference to the same extent as each individual publication, patent, or patent application is incorporated by reference specifically and individually.

[0015] [Technical Terms] When used herein, terms such as “first,” “second,” “third,” “fourth,” “fifth,” “sixth,” “seventh,” “eighth,” “ninth,” “tenth,” and “nth” merely identify one of several subsections, components, steps, operations, functions, and / or reference points disclosed herein, and similarly do not necessarily limit embodiments of the disclosure to any particular configuration or orientation. Furthermore, terms such as “preceding,” “following,” “left,” “right,” “top,” “bottom,” “front,” “rear,” “side,” “height,” “length,” “width,” “upper,” “lower,” “internal,” “external,” “inside,” and “outside” merely describe reference points and do not necessarily limit embodiments of the disclosure to any particular orientation or configuration.

[0016] Terms such as "target nucleic acid sequence" or "target sequence" refer to any gene locus of a microorganism or microbial population that is targeted for cleavage or amplification by a CRISPR system or a nucleic acid amplification test (NAAT) system.

[0017] As used herein, the abbreviation "NAAT" generally refers to nucleic acid amplification testing.

[0018] As used herein, the term "Ct" or "ct" refers to "cycle threshold", defined as the number of cycles required for a fluorescent signal to exceed the threshold (i.e., exceed background level). Ct levels are generally inversely proportional to the amount of target nucleic acid in a sample (i.e., the lower the Ct level, the greater the amount of target nucleic acid in the sample). In a real-time PCR assay, a positive reaction is detected by accumulation of a fluorescent signal. Real-time assays generally undergo 40 cycles of amplification. Cts <29 represent a strong positive reaction indicating abundant target nucleic acid in the sample. Cts of 30 to 37 are positive reactions indicating a moderate amount of target nucleic acid, and Cts of 38 to 40 are weak reactions indicating a minimal amount of target nucleic acid that may represent an infection status or environmental contamination.

[0019] As used herein, the abbreviation "CRISPR" generally refers to clustered regularly interspaced short palindromic repeats. Although diverse CRISPR-Cas systems exist among different species of archaea and bacteria, these systems are generally linked by their dependence on a single-stranded RNA molecule (crRNA) that guides the CRISPR protein to recognize and cleave a nucleic acid target. crRNA can be programmed to target a specific DNA or RNA region of interest through hybridization to a complementary sequence that is restricted to the vicinity of a protospacer adjacent motif (PAM) or protospacer adjacent sequence in certain systems.

[0020] As used herein, the abbreviation "RSV" generally refers to respiratory syncytial virus (RSV).

[0021] As used herein, the abbreviations "FluA" and "FluB" generally refer to influenza A and influenza B, the two main types of influenza virus that cause seasonal influenza epidemics. As used herein, the term "Flu" refers to all viruses of the influenza family.

[0022] As used herein, the abbreviation "SARS-CoV-2" refers to the virus that causes COVID-19.

[0023] As used herein, certain terms may have the following defined meanings. As used herein and in the claims, the singular forms “a,” “an,” and “the” include singular and plural referents unless otherwise explicitly indicated by the context. For example, the term “nucleic acid” includes a single nucleic acid molecule and multiple nucleic acids, and mixtures thereof.

[0024] Where used herein, “approximately” means the exact stated quantity and a small variation within a limited range that includes plus or minus 10% of the stated quantity. In other words, the limited range that is included may include ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.5%, ±0.2%, ±0.1%, ±0.05%, or less, and the stated value itself. Therefore, for example, “approximately 10” should be understood to mean the range of “10” and “9 to 11” or less.

[0025] Where used herein, the term “comprising” is intended to mean that a composition and method includes the enumerated elements but does not exclude others. “Consisting essentially of,” when used to define a composition and method, means excluding other elements that are essentially important to the composition or method. “Consisting of” means excluding elements and substantial method steps that are in trace amounts of other components of the claimed composition. Examples and embodiments defined by each of these transitional terms are within the scope of this disclosure. Accordingly, the methods and compositions may include additional steps and components (including ~), or alternatively, they may include non-significant steps and compositions (essentially ~), or alternatively, they may intend to consist only of the described method steps or compositions (~).

[0026] As used herein, “percent (%) sequence identity” with respect to a reference nucleic acid sequence is defined as the percentage of nucleic acid bases in the target sequence that are identical to those in the reference sequence after the sequences have been aligned and gaps introduced as necessary to achieve the maximum percentage of sequence identity. Methods for sequence alignment are well known in the art. Optimal sequence alignment can be performed by the methods described in Needleman and Wunsch, 1970, J.Mol.Biol.48:443; Pearson and Lipman, 1988, PNAS 85:2444, or by computerized embodiments of these algorithms. Alignment can be performed using publicly available computer software such as BLASTp, BLASTn, BLAST-2, ALIGN, or MegAlign Pro (DNASTAR) software.

[0027] As used herein, the term “sample” generally refers to any source of nucleic acids from a specimen, from an object that “hosts” the specimen, or both, which may provide information about the environment. It may refer to a sample derived from an object such as a nasal swab, blood, plasma, urine, tissue, face, bone marrow, saliva, cerebrospinal fluid, or any other suitable tissue sample. It may refer to a swab sample containing nucleic acids collected from a food processing facility, long-term care facility, hospital, restaurant, or any suitable surface. It may refer to a sample containing biological tissue, soil, water, air, air filter material, animal production, feed, fertilizer, crop production, manufacturing plant, or any other suitable sample. Such samples may originate from a hospital or clinic, and they may be analyzed on a mobile platform.

[0028] As used herein, the term “subject” may refer to a human or another animal. The animal may be a mouse, rat, guinea pig, dog, cat, horse, rabbit, and various other animals. The subject may be of any age; for example, the subject may be an infant, toddler, child, pre-adolescent, adolescent, adult, or elderly individual. [Modes for carrying out the invention]

[0029] The following description includes numerous specific details to provide a more complete understanding of the invention. However, it will be apparent to those skilled in the art that the invention can be carried out without one or more of these specific details. In other examples, features and procedures well known to those skilled in the art are omitted to avoid obscuring the invention. The terms used herein are intended to have plain, ordinary meanings as understood by those skilled in the art.

[0030] Nucleic acid amplification tests (NAATs) are widely used diagnostic methods for infectious microorganisms worldwide, including SARS-CoV-2, FluA, FluB, and RSV. The most well-known and widely used NAATs are polymerase chain reaction (PCR) and / or reverse transcription polymerase chain reaction (RT-PCR). However, several other NAATs have been developed over the years, each with its own unique characteristics and applications. Generally, however, most NAAT techniques rely on the principle that nucleic acid sequences with specific complementarity to a target region can be used to amplify the target. As a result, NAATs typically produce a series of amplicons of sequences specifically targeted for amplification.

[0031] In contrast to NAATs, CRISPR systems generally consist of two main components: a ribonucleotide (RNA) sequence that is sequence-complementary to the target DNA, and a CRISPR enzyme that works with the RNA sequence to perform gene editing. In biotechnology, most CRISPR systems deploy a guide RNA (gRNA; either "crRNA" or "gRNA") as a single molecule, which acts as a guide to direct the CRISPR enzyme (e.g., Cas9, Cas12) to a specific target DNA sequence, usually for gene editing or gene detection. Generally, one region of the gRNA contains a sequence complementary to the target sequence of interest, which is typically the target sequence adjacent to a protospacer fringe motif (PAM). Another target region acts as a scaffold that helps stabilize the complex between the gRNA and the CRISPR enzyme. This interacts with both the gRNA and the CRISPR enzyme to form a stable ribonucleoprotein complex that guides the CRISPR enzyme to the target DNA to cleave the target.

[0032] Therefore, while NAAT methods produce amplicons in the target region, CRISPR methods, when deployed for diagnostic purposes, tend to primarily cleave the target sequence. This disclosure provides novel devices, systems, and methods for integrating the outputs of NAAT and CRISPR assays to increase the specificity and comprehensiveness of targeted nucleic acid detection.

[0033] device

[0034] Suboptimal oligo design or challenging reaction conditions can lead to false positive (specificity concern) or false negative (inclusivity concern) signals in NAAT or CRISPR strategies for detecting microorganisms(s). This can lead to misidentification of the target pathogen. Due to differences in the target recognition mechanisms of the two systems, the dual NAAT / CRISPR nature of the devices described herein effectively eliminates the possibility of the system detecting a defective signal twice. This disclosure provides a NAAT+CRISPR system that improves the specificity and inclusivity of assays for target microorganism detection.

[0035] The devices of the present disclosure generally include multiple subsections. For example, the devices of the present disclosure may have one subsection, two subsections, three subsections, four subsections, five subsections, six subsections, seven subsections, eight subsections, nine subsections, ten subsections, eleven subsections, twelve subsections, thirteen subsections, fourteen subsections, fifteen subsections, sixteen subsections, seventeen subsections, eighteen subsections, nineteen subsections, twenty subsections, or another appropriate number of subsections. In some configurations, the multiple subsections are hermetically sealed together with specific reagents for sample preparation and isolation, purification, extraction, target nucleic acid amplification, and target nucleic acid cleavage of target analytes.

[0036] In some configurations, the elongated assay tubes of this disclosure are 70±20 mm, 71±20 mm, 72±20 mm, 73±20 mm, 74±20 mm, 75±20 mm, 76±20 mm, 77±20 mm, 78±20 mm, 79±20 mm, 80±20 mm, 81±20 mm, 82±20 mm, 83±20 mm, 84±20 mm, 85±20 mm, 86±20 mm, 87±20 mm, 88±20 mm millimeters, 89±20 mm, 90±20 mm, 91±20 mm, 92±20 mm, 93±20 mm, 94±20 mm, 95±20 mm, 96±20 mm, 97±20 mm, 98±20 mm, 99±20 mm, 100±20 mm, 101±20 mm, 102±20 mm, 103±20 mm, 104±20 mm, 105±20 mm, 106±20 mm, 107±20 mm, 108±2 0 mm, 109 ± 20 mm, 110 ± 20 mm, 111 ± 20 mm, 112 ± 20 mm, 113 ± 20 mm, 114 ± 20 mm, 115 ± 20 mm, 116 ± 20 mm, 117 ± 20 mm, 118 ± 20 mm, 119 ± 20 mm, 120 ± 20 mm, 121 ± 20 mm, 122 ± 20 mm, 123 ± 20 mm, 124 ± 20 mm, 125 ± 20 mm, 126 ± 20 mm, 127 ± 20 millimeters, 128±20 mm, 129±20 mm, 130±20 mm, 131±20 mm, 132±20 mm, 133±20 mm, 134±20 mm, 135±20 mm, 136±20 mm, 137±20 mm, 138±20 mm, 139±20 mm, 140±20 mm, 70±21 mm, 71±21 mm, 72±21 mm, 73±21 mm, 74±21 mm, 75±21 mm,76±21 mm, 77±21 mm, 78±21 mm, 79±21 mm, 80±21 mm, 81±21 mm, 82±21 mm, 83±21 mm, 84±21 mm, 85±21 mm, 86±21 mm, 87±21 mm, 88±21 mm, 89±21 mm, 90±21 mm, 91±21 mm, 92±21 mm, 93±21 mm, 94±21 mm, 95±21 mm, 96±21 mm 97±21 mm, 98±21 mm, 99±21 mm, 100±21 mm, 101±21 mm, 102±21 mm, 103±21 mm, 104±21 mm, 105±21 mm, 106±21 mm, 107±21 mm, 108±21 mm, 109±21 mm, 110±21 mm, 111±21 mm, 112±21 mm, 113±21 mm, 114±21 mm, 115±21 mm, 116± 21 mm, 117 ± 21 mm, 118 ± 21 mm, 119 ± 21 mm, 120 ± 21 mm, 121 ± 21 mm, 122 ± 21 mm, 123 ± 21 mm, 124 ± 21 mm, 125 ± 21 mm, 126 ± 21 mm, 127 ± 21 mm, 128 ± 21 mm, 129 ± 21 mm, 130 ± 21 mm, 131 ± 21 mm, 132 ± 21 mm, 133 ± 21 mm, 134 ± 21 mm, 135 ± 21 mm Remometers: 136±21 mm, 137±21 mm, 138±21 mm, 139±21 mm, 140±21 mm, 70±22 mm, 71±22 mm, 72±22 mm, 73±22 mm, 74±22 mm, 75±22 mm, 76±22 mm, 77±22 mm, 78±22 mm, 79±22 mm, 80±22 mm, 81±22 mm, 82±22 mm, 83±22 mm, 84±22 mm,85±22 mm, 86±22 mm, 87±22 mm, 88±22 mm, 89±22 mm, 90±22 mm, 91±22 mm, 92±22 mm, 93±22 mm, 94±22 mm, 95±22 mm, 96±22 mm, 97±22 mm, 98±22 mm, 99±22 mm, 100±22 mm, 101±22 mm, 102±22 mm, 103±22 mm, 104±22 mm, 105±22 mm, 106±22 mm, 107±22 mm, 108±22 mm, 109±22 mm, 110±22 mm, 111±22 mm, 112±22 mm, 113±22 mm, 11 4±22 mm, 115±22 mm, 116±22 mm, 117±22 mm, 118±22 mm, 119±22 mm, 120±22 mm, 121±22 mm, 122±22 mm, 123±22 mm, 124±22 mm, 125±22 mm, 126±22 mm, 127±22 mm, 128±22 mm, 129±22 mm, 130±22 mm, 131±22 mm, 132±22 mm, 133±22 mm, 134±22 mm, 135±22 mm, 136±22 mm, 137±22 mm, 138±22 mm, 139±22 mm, 140±22 mm, or another suitable length.

[0037] In some configurations, the elongated assay tubes of the present disclosure have widths of 2±1 mm, 3±1 mm, 4±1 mm, 5±1 mm, 6±1 mm, 7±1 mm, 8±1 mm, 9±1 mm, 10±1 mm, 11±1 mm, 12±1 mm, 13±1 mm, 14±1 mm, 15±1 mm, 16±1 mm, 17±1 mm, 18±1 mm, 19±1 mm, 20±1 mm, or another suitable width, or another suitable width.

[0038] In most configurations, the length of each subsection is determined by the amount (volume) of reagent required for each step of the reaction. In some configurations, each subsection of multiple subsections of the device may have a different length as needed to accommodate the reagents required for each subsection. In some configurations, two or more subsections, three or more subsections, four or more subsections, five or more subsections, six or more subsections, seven or more subsections, eight or more subsections, nine or more subsections, and ten or more subsections may have the same or different lengths. For example, see Figures 1A, 1B, 2, and 3, which show exemplary lengths of subsections in the device.

[0039] In some configurations, the device is manufactured from a polymer. Non-limiting examples of polymers contemplated by this disclosure for the manufacture of the device include thermoplastic polymers, because thermoplastic polymers can be repeatedly heated during a thermal cycling reaction process without undergoing significant chemical changes. Examples of thermoplastic polymers contemplated by this disclosure include polyethylene (PE), including high-density polyethylene (HDPE) and low-density polyethylene (LDPE); polypropylene (PP); polyvinyl chloride (PVC); polystyrene (PS); polyethylene terephthalate (PET); polyamide (nylon); polycarbonate (PC); acrylonitrile butadiene styrene (ABS); poly(methyl methacrylate) (PMMA); or polyvinylidene fluoride (PVDF): including PVDF.

[0040] In some configurations, the devices of the present disclosure provide reagents for the dual detection of one or more target microorganisms by combining a NAAT strategy with CRISPR. One or more segments of the devices of the present disclosure typically include reagents for one or more of the following: sample preparation and target analyte isolation, purification, extraction, target nucleic acid amplification, and target nucleic acid cleavage, depending on the NAAT technique selected for amplification and / or the CRISPR system selected for cleavage.

[0041] In certain device configurations, the first subsection is fitted to accept a sample such as saliva or sputum. The first subsection most typically does not contain reagents and is accessible via a cap, such as a screw-on cap or pressure-type lid. The first subsection is generally fitted to accept a fixed volume of sample to be analyzed in an assay. In certain configurations, the first subsection is fitted to receive a specific volume of sample.Examples of sample volumes that are not limited include 50±40 microliters, 55±40 microliters, 60±40 microliters, 65±40 microliters, 70±40 microliters, 75±40 microliters, 80±40 microliters, 85±40 microliters, 90±40 microliters, 95±40 microliters, 100±40 microliters, 105±40 microliters, 110±40 microliters, 115±40 microliters, 120±40 microliters, and 125±40 microliters. 0 microliters, 130±40 microliters, 135±40 microliters, 140±40 microliters, 145±40 microliters, 150±40 microliters, 155±40 microliters, 160±40 microliters, 165±40 microliters, 170±40 microliters, 175±40 microliters, 180±40 microliters, 185±40 microliters, 190±40 microliters, 195±40 microliters, 200±40 microliters 205±40 microliters, 210±40 microliters, 215±40 microliters, 220±40 microliters, 225±40 microliters, 230±40 microliters, 235±40 microliters, 240±40 microliters, 245±40 microliters, 250±40 microliters, 255±40 microliters, 260±40 microliters, 265±40 microliters, 270±40 microliters, 275±40 microliters, 280±40 microliters Includes samples in liters, 285±40 microliters, 290±40 microliters, 295±40 microliters, 300±40 microliters, 305±40 microliters, 310±40 microliters, 315±40 microliters, 320±40 microliters, 325±40 microliters, 330±40 microliters, 335±40 microliters, 340±40 microliters, 345±40 microliters, 350±40 microliters, or other appropriate volumes of samples.

[0042] In certain configurations of the device, the second subsection is typically adapted to provide an internal control reagent, such as a positive or negative control. A positive control might be, for example, a known sequence of a known target nucleic acid. While in many device configurations the internal control is located in the second subsection, it will become apparent that it may be conceivable that the internal control could be located in an alternative subsection. In many configurations of the device, the subsection containing the internal control has a volume of 5±2.5 microliters, 6±2.5 microliters, 7±2.5 microliters, 8±2.5 microliters, 9±2.5 microliters, 10±2.5 microliters, 11±2.5 microliters, 12±2.5 microliters, 13±2.5 microliters, 14±2.5 microliters, 15±2.5 microliters, 16±2.5 microliters, 17±2.5 microliters, 18±2.5 microliters, 19±2.5 microliters, 20±2.5 microliters, 21±2.5 microliters, 22±2.5 microliters, 23±2.5 microliters, 24±2.5 microliters, 25±2.5 microliters, or another suitable volume.

[0043] In certain configurations of the device, a third subsection is typically adapted to provide reagents for sample preparation and isolation, purification, and extraction of target analytes. In many configurations, the third subsection is adapted to provide adsorption extraction techniques, i.e., magnetic separation or size separation, for isolating target nucleic acids from a sample, either through the inherent properties of the sample or by labeling with a magnetic material. In certain configurations, this disclosure envisions adsorption extraction techniques utilizing magnetic particles consisting of iron oxides (e.g., magnetite or maghemite), nickel, and cobalt, or other elements in combination with several metals such as zinc, copper, strontium, and barium. The magnetic particles may be of various shapes, including, but are not limited to, spherical particles with a narrow size distribution prepared by ball milling, coprecipitation, hydrothermal synthesis, pyrolysis, laser ablation, microemulsion, chemical vapor deposition, arc discharge, flame spray synthesis, and biosynthesis. In some configurations, the magnetic particles contemplated by this disclosure may be functionalized with different compounds to provide functional groups for further binding of bioactive molecules through different techniques such as direct binding, the Hong method, or bioremediation. Specifically, the functionalization of magnetic particles enables the attachment of ligands and receptors to their surface, as well as the binding of biomolecules such as monoclonal antibodies, nucleic acids, streptavidin, proteins, and peptides, to ensure specific interactions with target molecules.

[0044] In some configurations, the disclosure envisions one or more subsections containing magnetic particles for selectively capturing, concentrating, transferring, and / or labeling a target analyte and carrying out stringency and washing steps of the target analyte (target nucleic acid sequence). Often, the target nucleic acid sequence is a FluA, FluB, RSV, or SARS-CoV-2 target sequence. In the case of magnetic separation, the sample is allowed to come into contact with the magnetic particles, for example, by sequentially exposing the sample to a series of reagents in multiple subsections of an assay tube, and is incubated for a certain period of time to allow interaction with the target analyte through affinity adsorption and / or antibody-antigen or hydrophobic interactions. Since nucleic acids are polyanionic molecules with numerous phosphate groups, electrostatic interactions can be amplified by functionalization with positively charged species such as aminosilanes, and similarly, immobilization with specific oligonucleotide sequences can allow affinity capture of nucleic acids by hybridization of complementary sequences. Next, the particles can be separated from the sample by applying a magnet to the outside of the container wall (see, for example, Figure 3, which shows an analytical system in which such a magnetic field can be applied to the assay tube of this disclosure). The obtained analyte is eluted from the magnetic particles and subjected to further analysis. In some configurations of the device, the subsection containing magnetic particles has a solution containing a magnet in a volume of 5±2.5 microliters, 6±2.5 microliters, 7±2.5 microliters, 8±2.5 microliters, 9±2.5 microliters, 10±2.5 microliters, 11±2.5 microliters, 12±2.5 microliters, 13±2.5 microliters, 14±2.5 microliters, 15±2.5 microliters, 16±2.5 microliters, 17±2.5 microliters, 18±2.5 microliters, 19±2.5 microliters, 20±2.5 microliters, 21±2.5 microliters, 22±2.5 microliters, 23±2.5 microliters, 24±2.5 microliters, 25±2.5 microliters, or another appropriate volume.

[0045] In some configurations, this disclosure intends to include one or more subsections, for example, lysis buffers for disrupting cell or tissue membranes. The specific composition of the lysis buffer may vary significantly depending, for example, the sample being analyzed, reagents suitable for a NAAT reaction, or reagents suitable for a CRISPR reaction. In many cases, this disclosure intends to include lysis buffers comprising surfactants, salts, buffers, protease inhibitors, and / or reducing agents. Non-limiting examples of surfactants include triton X-100, NP-40, or Tween 20. Non-limiting examples of salts include sodium chloride (NaCl), a commonly used salt. Non-limiting examples of buffers include Tris-HCl or phosphate buffer. Non-limiting examples of protease inhibitors include PMSF (phenylmethylsulfonyl fluoride), aprotinin, or EDTA. Non-limiting examples of reducing agents include dithiothreitol (DTT) or 2-mercaptoethanol.In some configurations of the device, the subsection containing the lysis buffer is available in 50±43 microliters, 55±43 microliters, 60±43 microliters, 65±43 microliters, 70±43 microliters, 75±43 microliters, 80±43 microliters, 85±43 microliters, 90±40 microliters, 95±43 microliters, 100±43 microliters, 105±43 microliters, 110±43 microliters, 115±43 microliters, and 120±43 microliters. Chlorol, 125±43 microliters, 130±43 microliters, 135±43 microliters, 140±43 microliters, 145±43 microliters, 150±43 microliters, 155±43 microliters, 160±43 microliters, 165±43 microliters, 170±43 microliters, 175±43 microliters, 180±43 microliters, 185±43 microliters, 190±43 microliters, 195±43 microliters, 200±43 microliters 10 liters, 205±43 microliters, 210±43 microliters, 215±43 microliters, 220±43 microliters, 225±43 microliters, 230±43 microliters, 235±43 microliters, 240±43 microliters, 245±43 microliters, 250±43 microliters, 255±43 microliters, 260±43 microliters, 265±43 microliters, 270±43 microliters, 275±43 microliters, 280±43 microliters The solution contains liters, 285±43 microliters, 290±43 microliters, 295±43 microliters, 300±43 microliters, 305±43 microliters, 310±43 microliters, 315±43 microliters, 320±43 microliters, 325±43 microliters, 330±43 microliters, 335±43 microliters, 340±43 microliters, 345±43 microliters, 350±43 microliters of lysis buffer, or another appropriate volume of lysis buffer.

[0046] In some configurations, this disclosure envisions one or more subsections containing a washing buffer for removing unbound or nonspecifically bound substances while retaining the target molecule of interest. The specific composition of the washing buffer may vary significantly depending, for example, the sample to be analyzed, the reagents suitable for the NAAT reaction, or the reagents suitable for the CRISPR reaction. In many cases, this disclosure envisions a washing buffer containing one or more buffers, salts, surfactants, blocking agents, and / or other additives, such as chelating agents, reducing agents, and / or stabilizers. A non-limiting example of a buffer is Tris-HCl. A non-limiting example of a salt is NaCl. A non-limiting example of a surfactant is Tween 20 and Triton X-100. A non-limiting example of a blocking agent is bovine serum albumin (BSA). The number of segments containing the washing buffer on the device may vary, for example, depending on the target analyte being analyzed. The number of segments containing the cleaning buffer within the device itself and the cleaning conditions implemented in this method (e.g., incubation time, temperature, and agitation) may vary depending on the sensitivity of the target molecule.In some configurations of the device, the subsection containing the washing buffer is available in the following sizes: 100±48 microliters, 105±48 microliters, 110±48 microliters, 115±48 microliters, 120±48 microliters, 125±48 microliters, 130±48 microliters, 135±48 microliters, 140±48 microliters, 145±48 microliters, 150±48 microliters, 155±48 microliters, 160±48 microliters, 165±48 microliters, and 1 70±48 microliters, 175±48 microliters, 180±48 microliters, 185±48 microliters, 190±48 microliters, 195±48 microliters, 200±48 microliters, 205±48 microliters, 210±48 microliters, 215±48 microliters, 220±48 microliters, 225±48 microliters, 230±48 microliters, 235±48 microliters, 240±48 microliters, 245±48 microliters, 250± 48 microliters, 255±48 microliters, 260±48 microliters, 265±48 microliters, 270±48 microliters, 275±48 microliters, 280±48 microliters, 285±48 microliters, 290±48 microliters, 295±48 microliters, 300±48 microliters, 305±48 microliters, 310±48 microliters, 315±48 microliters, 320±48 microliters, 325±48 microliters, 330±48 microliters The solution contains 1 / 2 microliters, 335±48 microliters, 340±48 microliters, 345±48 microliters, 350±48 microliters, 355±48 microliters, 360±48 microliters, 365±48 microliters, 370±48 microliters, 375±48 microliters, 380±48 microliters, 385±48 microliters, 390±48 microliters, 395±48 microliters, 400±48 microliters of wash buffer, or another appropriate volume of wash buffer.

[0047] In some configurations, this disclosure envisions one or more subsections comprising an elution buffer for releasing or "eluting" target nucleic acids of interest from magnetic beads, solid supports, or other binding matrices. Elution is the process of removing specifically bound nucleic acids from the stationary phase (magnetic, resin, or matrix) and transferring them to the liquid phase (elution buffer). The specific composition of the elution buffer may vary significantly depending, for example, the sample being analyzed, reagents suitable for a NAAT reaction, or reagents suitable for a CRISPR reaction. In many cases, this disclosure envisions an elution buffer comprising one or more of the following: salts, pH-adjusted buffers, competing ligands, organic solvents, and / or chelating agents. In some configurations of the device, the subsection containing the elution buffer contains 20±10 microliters, 25±10 microliters, 30±10 microliters, 35±10 microliters, 40±10 microliters, 45±10 microliters, 50±10 microliters, 55±10 microliters, 60±10 microliters, 65±10 microliters, 70±10 microliters, 75±10 microliters, 80±10 microliters, 85±10 microliters, 90±10 microliters, 95±10 microliters, 100±10 microliters, 135±10 microliters, 105±10 microliters, 110±10 microliters, 115±10 microliters, 120±10 microliters, or another appropriate volume of elution buffer.

[0048] Nucleic acid amplification test (NAAT)

[0049] In a preferred configuration, the Disclosure intends to comprise one or more, two or more, or three or more subsections comprising reagents for carrying out nucleic acid amplification reactions (i.e., NAATs). The Disclosure intends to comprise a variety of mechanistically different strategies for amplifying target nucleic acids, and in some embodiments, the Disclosure intends for one or more segments of the device to comprise a combination of strategies including polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), rolling circle amplification (RCA), strand substitution amplification (SDA), nicking and extension amplification (NEAR), exponential amplification (EXPAR), multiple substitution amplification (MDA), helicase-dependent amplification (HAD), or hybridization chain reaction (HCR).

[0050] In some configurations, the nucleic acid amplification reaction is polymerase chain reaction (PCR), such as RT-PCR (reverse transcription polymerase chain reaction). Amplification of nucleic acid sequences by PCR is described in U.S. Patents 4,683,195, 4,683,202, and 4,965,188. PCR is now well-known in the art and is widely described in the scientific literature. See PCR Applications ((1999) Innis et al., eds., Academic Press, San Diego), PCR Strategies ((1995) Innis et al., eds., Academic Press, San Diego); PCR Protocols ((1990) Innis et al., eds., Academic Press, San Diego), and PCR Technology ((1989) Erlich, ed., Stockton Press, New York). "Real-time" PCR assays can simultaneously amplify and detect / quantify the starting amount of a target sequence. A basic TaqMan real-time PCR assay using the 5' to 3' nuclease activity of DNA polymerase is described in Holland et al., (1991) Proc. Natl. Acad. Sci. 88:7276-7280 and U.S. Patent No. 5210015. Real-time PCR without nuclease activity (nuclease-free assay) is described in U.S. Patent Application Publication No. 20100143901. The use of fluorescent probes in real-time PCR is described in U.S. Patent No. 5,538,848. Rapid one-step reverse transcriptase PCR (RT-PCR) is described in U.S. Patent No. 8,119,353.

[0051] In some configurations, the nucleic acid amplification reaction is a loop-mediated isothermal amplification (LAMP) reaction. The LAMP method deploys multiple primers that target several regions of the target DNA, thus resulting in high amplification efficiency of the target nucleic acid. LAMP can amplify DNA at a constant temperature of typically 60°C to 65°C. Nucleic acid sequence amplification by LAMP is described in U.S. Patent No. 6,410,278 and is widely documented in the literature.Notomi T,Okayama H,Masubuchi H,Yonekawa T,Watanabe K,Amino N,Hase T(2000)”Loop-mediated isothermal amplification of DNA”.Nucleic Acids Res.28(12):63e-63;Shirshikov,Fedor V.;Pekov,Yuri PeerJ.7:e6801;Mori Y,Kitao M,Tomita N,Notomi T(2004).”MorphoCatcher:a multiple-alignment based web tool for target selection and designing taxon-specific primers”. turbidimetry of LAMP reaction for quantifying template DNA”.J.Biochem.Biophys.Methods.59(2):145-57; urine specimens by reverse transcription loop-mediated isothermal amplification(RT-LAMP)”.

[0052] In some configurations, nucleic acid amplification reactions are nucleic acid sequence-based amplification (NASBA) reactions for generating multiple copies of single-stranded RNA. NASBA is a primer-dependent technique that can be used for sequential amplification of nucleic acids in a single mixture at one or two temperatures. Nucleic acid sequence amplification by NASBA is described, for example, in Deiman, Birgit; van Aarle, Pierre; Sillekens, Peter (2002). "Characteristics and Applications of Nucleic Acid Sequence-Based Amplification (NASBA)". Molecular Biotechnology. 20(2):163-180; Malek, L.; Sooknanan, R.; Compton, J. (1994). Nucleic acid sequence-based amplification (NASBA). Methods in Molecular Biology. Vol.28.pp.253-260; Compton, J (1991). "Nucleic acid sequence-based amplification". Nature. 350(6313):91-2.

[0053] In some configurations, nucleic acid amplification reactions are transcription-mediated amplification (TMA) reactions, i.e., isothermal amplification systems utilizing two enzymes: RNA polymerase and reverse transcriptase. Amplification of nucleic acid sequences by TMA is described, for example, in Daniel L. Kacian, Timothy J. Fultz: Nucleic acid sequence amplification methods. In: Biotechnology Advances 1995, 13.3, pp. 569-569.

[0054] In some configurations, the nucleic acid amplification reaction is a rolling circle amplification (RCA) reaction. RCA is an isothermal nucleic acid amplification technique in which polymerase sequentially adds single nucleotides to primers annealed to a circular template, resulting in long concatemer ssDNA containing tens to hundreds of tandem repeats (complementary to the circular template). RCA is now well-known in the field and is widely described in the scientific literature. For example, Ali, M. Monsur; Li, Feng; Zhang, Zhiqing; Zhang, Kaixiang; Kang, Dong-Ku; Ankrum, James A.; Le, See M.; Huang, Xiaohua; Zhu, Zhengrong; Bray-Ward, Patricia; Thomas, David C.; Ward, David C. (July 1998). “Mutation detection and single-molecule counting using isothermal rolling-circle amplification”. Nature Genetics. 19(3):225-232.

[0055] In some configurations, nucleic acid amplification reactions are either strand substitution amplification (SDA) or multiple substitution amplification (MDA). SDA and MDA are isothermal amplification techniques based on the ability of a nicking enzyme, such as HincII, to nick an unmodified strand of a chain (e.g., a hemiphosphorothioate form of the HincII recognition site), and the ability of an exonuclease-deficient klenow (exoclenow) to extend the 3' end of the nick and substitute a downstream DNA strand. Exponential amplification is achieved by combining a sense reaction and an antisense reaction, where the strand substituted in the sense reaction acts as the target of the antisense reaction, and vice versa. SDA is currently well known in the art. For example, GTWalker, MCLittle, JGNadeau and DDShank(1992)Proc.Natl.Acad.Sci 89,392-396;Spits;Le Caignec,C;De Rycke,M;Van Haute,L;Van Steirteghem,A;Liebaers,I;Sermon,K(2006).”Whole-genome multiple displacement amplification from single Nature Protocols.1(4):1965-70.

[0056] In some configurations, nucleic acid amplification reactions are Nicking enzyme amplification reactions (NEAR). NEAR is isothermal, using polymerase (and Nicking enzyme) to replicate DNA at a constant temperature and exponentially amplifying the DNA over a temperature range of 55°C to 59°C. NEAR is described in U.S. Patents 6,191,267 and 6,660,475. NEAR is further described in the art in Biochemistry. 2008 Sep 23;47(38):9987-99.

[0057] In some configurations, the nucleic acid amplification reaction is a helicase-dependent amplification (HAD) reaction. HAD utilizes a DNA helicase to generate a single-strand template for primer hybridization and subsequent primer extension by DNA polymerase. HAD is described in the literature Saiki RK, et al. (1988) "Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase". Science. 239(4839):487-491; and Kornberg A, Baker T (1992). DNA Replication, 2nd edn. WH Freeman and Company: New York.

[0058] In some configurations, nucleic acid amplification reactions are hybridization chain reactions (HCRs). HCRs provide multiplexed, isothermal, enzyme-free molecular signal amplification in a variety of situations.

[0059] In a preferred configuration, the disclosure intends to comprise one or more, two or more, or three or more subsections comprising reagents for carrying out a series of nucleic acid amplification reactions (i.e., NAAT). In some examples, one segment of the device comprises reagents for an RT-PCR reaction, e.g., reaction buffer, dNTPs, RT-PCR primers (e.g., primers for amplifying one or more target sequences from FluA, FluB, RSV, and / or SARS-CoV-2, etc.), reverse transcription polymerase (e.g., MMLV RT polymerase), and / or uracil DNA glycosylase. In some examples, one segment of the device comprises reagents for a PCR reaction, e.g., reaction buffer, polymerase, and necessary functional components (Z05 polymerase and its aptamers), and / or RT-PCR primers. In some configurations of the device, the subsection containing the NAAT reaction mixture ("NAAT Master Mix") is available in quantities of 5±3 microliters, 10±3 microliters, 15±3 microliters, 20±3 microliters, 25±3 microliters, 30±3 microliters, 35±3 microliters, 40±3 microliters, 45±3 microliters, 50±3 microliters, 55±3 microliters, 60±3 microliters, 65±3 microliters, 70±3 microliters, 75±3 microliters, and 80±3 microliters. This includes 10±6 microliters, 15±6 microliters, 20±6 microliters, 25±6 microliters, 30±6 microliters, 35±6 microliters, 40±6 microliters, 45±6 microliters, 50±6 microliters, 55±6 microliters, 60±6 microliters, 65±6 microliters, 70±6 microliters, 75±6 microliters, and 80±6 microliters of NAAT master mix, or another appropriate volume containing functional reagents for a NAAT methodology selected for production on a device.

[0060] CRISPR system

[0061] Since their initial discovery, the number of different CRISPR-Cas systems has rapidly expanded. Currently, CRISPR-Cas systems can be divided into two classes, six types, and several subtypes according to their evolutionary relationships. The classes of CRISPR-Cas systems are defined by the nature of their ribonucleoprotein effector complexes; Class 1 systems are characterized by complexes of multiple effector proteins, while Class 2 systems encompass a single crRNA-binding protein. The main difference between CRISPR type II (Cas9) systems and type V (Cas12) and type VI (Cas13) systems is the latter two systems' ability to induce nonspecific collateral cleavage (trans cleavage) in target recognition. Collateral activity involves cleaving non-target single-stranded DNA (ssDNA; Cas12) or single-stranded RNA (ssRNA; Cas13) in solution, which enables the sensing of nucleic acids through signal amplification and allows for various readouts through the addition of functionalized reporter nucleic acids, which are generally cleaved by collateral activity.

[0062] In CRISPR-based diagnosis, quantification is performed by comparison with a standard curve, ranging from picomolar to micromolar concentrations (10 -12 M~10 -6 The collateral cleavage activity based on CRISPR can be achieved within M) and correlates with the target concentration.

[0063] In some configurations, this disclosure intends one or more, two or more, or three or more subsections comprising a CRISPR reagent (enzyme, guide RNA, substrate, cofactor) having trans-cleavage activity. When activated by a target nucleic acid, a CRISPR enzyme having trans-cleavage activity (e.g., Cas12 and Cas13) indiscriminately cleaves a short single-stranded reporter oligo. If the target nucleotide is labeled with a reporter molecule, such cleavage typically separates the labeled quencher and fluorophore at both ends of the reporter molecule, generating a measurable fluorescent signal.

[0064] In some configurations, the disclosed device for a NAAT+CRISPR dual detection system can be used to target different pathogens from the same patient sample input. Since the signals are generated at different stages (see, e.g., Figure 3), two different pathogens can be read out from the same optical channel, thus increasing the multiplexing capability of NAAT without the need to increase the physical analyzer system setup (optical channels). Non-limiting examples of suitable analyzer systems and existing sample processing tubes in the art are described in U.S. Patents 6,036,920, 7,718,421, 9,005,551, 9,708,599, and 10,443,050.

[0065] In some configurations of the device, the subsection containing the CRISPR reaction mixture ("CRISPR Master Mix") is available in 20±12 microliters, 25±12 microliters, 30±12 microliters, 35±12 microliters, 40±12 microliters, 45±12 microliters, 50±12 microliters, 55±12 microliters, 60±12 microliters, 65±12 microliters, 70±12 microliters, 75±12 microliters, 80±12 microliters, 10±12 microliters, 15±12 microliters, 20±12 microliters, 25±12 microliters, 30±12 microliters, and 35±12 microliters. Includes chlorites, 40±12 microliters, 45±12 microliters, 50±12 microliters, 55±12 microliters, 60±12 microliters, 65±12 microliters, 70±12 microliters, 75±12 microliters, 80±12 microliters, 85±12 microliters, 90±12 microliters, 95±12 microliters, 100±12 microliters, 105±12 microliters, 110±12 microliters, 115±12 microliters, 120±12 microliters of CRISPR master mix, or another appropriate volume containing functional reagents for the CRISPR enzyme selected for inclusion on the device.

[0066] method

[0067] All functions described herein in relation to the devices, systems, and processes are intended to be applicable to the detection of at least one target microorganism from a sample. The sample may include a mixture of viral nucleic acids, mammalian nucleic acids, and bacterial nucleic acids.

[0068] When pathogen concentrations in a sample are low or close to the detection limit, specific signals generated from nucleic acid amplification tests may be weak and difficult to distinguish from negative control signals. In certain embodiments, this disclosure addresses this challenge by developing a CRISPR system assay that can further amplify amplicon signals from NAATs using their side-cleavage activity. That is, a single NAAT amplicon can generate multiple signal molecules (e.g., fluorescent labels). In contrast, in devices or analyzer systems that detect only NAAT signals, a single NAAT amplicon typically generates only one signal molecule. Therefore, the devices, systems, and methods of this disclosure that integrate signals from NAATs and CRISPR systems can result in an amplified true signal and improve the robustness of assays for detecting microorganisms.

[0069] In some embodiments, the disclosure provides a method for the dual detection of a target microorganism, comprising the steps of: amplifying a first target nucleic acid sequence in a nucleic acid amplification reaction and detecting a signal from the nucleic acid amplification of the first target nucleic acid sequence; detecting a second target nucleic acid sequence using a CRISPR system that targets the second target nucleic acid sequence and detecting a signal from the activation and substrate cleavage of the second target nucleic acid by the CRISPR system; and comparing the signal detected from the nucleic acid amplification of the first target nucleic acid sequence with an amplification threshold and comparing the signal detected from the activation and substrate cleavage of the second target nucleic acid by the CRISPR system with a CRISPR threshold, wherein the first and second target nucleic acid sequences are sequences derived from a target microorganism, and the target nucleic acid sequence is dually detected if both the signal detected from the first target nucleic acid amplification reaction and the signal detected from the activation and substrate cleavage of the second target nucleic acid by the CRISPR system exceed thresholds. In some embodiments, the first target nucleic acid sequence is amplified with a primer selected from the group consisting of SEQ ID NOs: 2 to 5. In some embodiments, the CRISPR system that targets the second target nucleic acid has the guide sequence of SEQ ID NO: 1.

[0070] In some embodiments, the present disclosure provides a method for detecting a target nucleic acid sequence, comprising the steps of (a) adding a sample into a device, the device comprising an assay tube subdivided into a plurality of subsections, the first subsection being connected to an openable cap, the opening accessible from the openable cap of the first subsection being fitted to receive a sample, the first subsection being sealed along the assay tube from subsequent subsections, the second subsection containing a solution comprising an internal control reagent, the second subsection being sealed along the assay tube from subsequent subsections, the third subsection containing a solution comprising magnetic beads in the solution, the third subsection being sealed along the assay tube from subsequent subsections, the fourth subsection containing a solution comprising a lysis buffer, the fourth subsection being sealed along the assay tube from subsequent subsections, and the fifth subsection containing a solution comprising a wash buffer, the fifth subsection being sealed along the assay tube from subsequent subsections (b) a device containing the sample is sealed, the sixth subsection containing a solution containing elution buffer, the sixth subsection being sealed along the assay tube away from subsequent subsections, the seventh subsection containing a solution containing a first PCR master mix (part 1), the seventh subsection being sealed along the tube away from subsequent subsections, the eighth subsection containing a solution containing a second PCR master mix (part 2), the eighth subsection being sealed along the assay tube away from subsequent subsections, the ninth tube containing a solution containing a CRISPR enzyme, the ninth subsection being sealed from the end of the assay tube, the steps of adding reagents, (b) incorporating a device containing the sample into a system that enables sequential rupture of one subsection from subsequent subsections, thereby enabling sequential reaction of reagents in one subsection with reagents in subsequent subsections, and (b) detecting a target nucleic acid by detecting a signal from the last subsection.Methods are provided. Non-limiting examples of suitable analyzer systems and methods for use with the devices of this disclosure are described in U.S. Patents 6,036,920, 7,718,421, 8,936,933, 9,005,551, 9,708,599, and 10,443,050.

[0071] In certain embodiments of this method, the device described herein is configured to distinguish at least one microorganism(s) from multiple microorganisms selected from the group consisting of SARS-CoV-2, influenza A, influenza B, and human respiratory syncytial virus (RSV). In other cases, at least one sample in multiple nucleic acids is selected from the group consisting of SARS-CoV-2, influenza A, influenza B, human respiratory syncytial virus (RSV), adenovirus, coronavirus 229E, coronavirus HKU1, coronavirus NL63, human metapneumovirus, human rhinovirus / enterovirus, parainfluenza virus 1, parainfluenza virus 2, parainfluenza virus 3, and parainfluenza virus 4.

[0072] In some configurations of the device, the device comprises one or more subsections having CRISPR enzymes with trans-cleavage activity such as Cas12 and Cas13, guide RNA, substrates, and metal ion cofactors, targeting a region of the target sequence (e.g., FluA, FluB, RSV, and / or SARS-CoV-2). In some embodiments of the device, the device comprises additional subsections, each subsection comprising a set of CRISPR system reagents for detecting one or more of FluA, FluB, RSV, and / or SARS-CoV-2. Non-limiting examples of Corona-derived microorganisms that can be distinguished by the methods of this disclosure include both viruses with low lethality rates (CFR), namely HCoV-NL63, HCoV-229E, HCoV-OC43, and HCoV-HKU1, and viruses with high CFR, namely MERS-CoV, SARS-CoV, and SARS-CoV-2.

[0073] [array] Table 1 lists non-exclusive examples of guide RNA sequences(s), primers, and probes that can be used to detect Flu type A. [Table 1] TIFF2026529663000002.tif219164 TIFF2026529663000003.tif219164 TIFF2026529663000004.tif219164 TIFF2026529663000005.tif172164

[0074] The guide nucleic acid comprises a guide sequence, which is a polynucleotide sequence that hybridizes with the target sequence and has sufficient complementarity to the target sequence to direct sequence-specific binding of the nucleic acid-induced nuclease to the target sequence. The degree of complementarity between the guide sequence and its corresponding target sequence is about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more, when optimally aligned using a suitable alignment algorithm. Optimal alignment can be determined by using any suitable algorithm for aligning sequences. In some embodiments, the guide sequence has a nucleotide length of about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more. In some embodiments, the guide sequence has a nucleotide length of less than approximately 75, 50, 45, 40, 35, 30, 25, or 20 nucleotides. Preferably, the guide sequence has a nucleotide length of 10 to 30 or 15 to 20 nucleotides, or 15, 16, 17, 18, 19, or 20 nucleotides.

[0075] [Non-limiting examples] The following embodiments are provided to those skilled in the art to provide a complete disclosure and explanation of how to construct and use the present invention, and are not intended to limit the scope of what the inventors consider to be the present invention, nor are they intended to represent or imply that the following experiments are all or only experiments in which the invention has been performed. Those skilled in the art will understand that numerous variations and / or modifications can be made to the present invention as shown in particular embodiments, without departing from the spirit or scope of the invention as broadly described. For example, without altering the spirit of the invention, certain master mixes may be combined in principle, certain enzymes may be replaced by other enzymes having similar functionality, certain buffers may be modified depending on the enzyme being performed, and the number of segments may be changed (e.g., overlapping segments may be implemented in certain configurations of the device, and / or certain segments may be combined in principle). Therefore, embodiments of the present invention should be construed in all respects as illustrative rather than restrictive.

[0076] Example 1 - Device and system for detecting target microorganisms A device having a tube with multiple subsections for linking nucleic acid amplification testing with a CRISPR system was manufactured as described below:

[0077] A series of master mixes containing reagents for detecting FluA were prepared. For this particular embodiment of the device, the first PCR master mix was prepared as follows: [Table 2]

[0078] For this particular embodiment of the device, a second PCR master mix was prepared as follows: [Table 3]

[0079] For various embodiments of the device, several mixtures containing CRISPR enzymes were prepared. In the specific embodiments described herein, the CRISPR master mix was prepared as follows: [Table 4]

[0080] An internal control was prepared in the quality control hood as follows: [Table 5]

[0081] The remaining reagents were prepared as described below: [Table 6]

[0082] Each assay tube contains all the reagents for a single test, packaged in a continuous segment. Figure 1A is a schematic diagram showing one such device prepared using NAAT and CRISPR reagents. Figure 1B is a schematic diagram showing one such state-of-the-art device, i.e., prepared using the reagents described herein, but containing only the reagents for nucleic acid amplification testing (NAAT) and no CRISPR enzyme or CRISPR system whatsoever. The devices are shown side by side for comparison.

[0083] Assay tubes made from thermoplastic materials suitable for use in amplification reactions were assembled with approximately the following volumes of reagents: [Table 7] [Table 8]

[0084] Next, the assay tube, i.e., the device, was mounted to the frame. In this particular embodiment, the frame was an open frame, such as the frame shown in Figures 2 and 3. Figure 2 shows an open frame next to the system for detecting signals from the assay. In this particular embodiment, the assay tube and frame are configured for use with Roche Diagnostics® cobas® omni utility channels, which integrate in vitro diagnostics and open channel assays into a single platform for detecting and reporting signals detected by the assay. Figure 3 shows a device configuration with a guide groove on the right side of the frame. In certain systems, such as the cobas® 6800 / 8800 system, the guide groove is adapted to facilitate the sliding insertion of the device into the system that provides the signal readout.

[0085] Example 2 - Verification of a device and system for detecting target microorganisms Comprehensive testing

[0086] Several devices were fabricated as described in Example 1. To test the device's performance in detecting FluA viruses that may contain specific nucleic acid sequence changes, multiple FluA test synthetic ssDNA target sequences (position 37 + WT) with a single mismatch were synthesized. These correspond to the sequences listed in SEQ ID NOs. 1 and SEQ ID NOs. 9-47. Four copies of each FluA DNA target sequence (position 37 + WT) with a single mismatch were tested as follows:

[0087] Negative control validation workflow:

[0088] The user pressed the power on / off button to start the cobas®Liat®Analyzer system.

[0089] The user selected "Login" on the screen of the cobas®Liat®Analyzer system and entered the relevant authentication information.

[0090] The user selected "Assay Menu" on the main menu of the cobas®Liat®Analyzer system and then selected "New Lot" at the bottom of the list.

[0091] When prompted to scan the insert ID, the user selected "Scan" and scanned the negative control package and negative control barcode card, typically included in the control kit. They ensured that the red scan light covered the entire barcode. The cobas® Liat® analyzer system then displayed the message, "Add a negative control and scan the tube ID."

[0092] The user held the negative control tube upright and gently tapped the flat surface to collect the liquid at the bottom of the tube. The user visually confirmed that the diluent had accumulated at the bottom of the tube.

[0093] The user carefully removed the cap from the device under test and inserted the pipette into the opening. The user slowly squeezed the valve to empty the negative control sample from the pipette and dispense it into the device assay tube. The user took care to avoid creating air bubbles in the sample.

[0094] The user replaced the cap on the assay device tube and screwed it in, then disposed of the transfer pipette as a biological hazard. Next, the user selected "Scan" and positioned the assay tube horizontally on the table under the barcode reader so that the red scanning light covered the entire barcode. Once the barcode was read, the tube inlet door on top of the system (i.e., the analyzer system) automatically opened.

[0095] The user removed the assay tube sleeve and immediately inserted the assay tube into the cobas®Liat®Analyzer until the tube clicked into place. In the cobas®Liat®Analyzer system, the guide groove or groove of the assay tube engages only to the left while the cap is in the upper position.

[0096] Once the assay tube was properly inserted, the cobas® Liat® analyzer system automatically closed its door and started the test.

[0097] During the test, the cobas® Liat® Analyzer displayed the operational status and estimated remaining time. Upon completion of the test, the cobas® Liat® analyzer system displayed the message "Please remove the tube slowly and carefully" and automatically opened the tube inlet door. At the end of the run, the message "Negative control result approved" was displayed, which the user confirmed.

[0098] Positive control validation workflow:

[0099] For analysis using the devices and systems of this disclosure, several positive controls were prepared, each having a single mismatch and possessing various FluA test synthetic ssDNA target sequences (position 37 + WT) (see, for example, SEQ ID NOs. 9 to SEQ ID NOs. 47 and SEQ ID NOs. 1).

[0100] In short, the user redissolved the synthetic sequence in the positive control tube(s) and then performed the same procedure as for the negative control tube(s). Next, the positive control sample was tested.

[0101] In this particular embodiment, the detection of FluA as a test microorganism was tested using a device for dual detection of FluA via NAAT and CRISPR detection. This particular experiment evaluated the performance of the device described in Example 1 for the detection of FluA wild-type and single mismatch sequences.

[0102] result:

[0103] Figure 4 is a schematic diagram showing three distinct stages in which a signal is generated from the device of this disclosure and detected by a system analyzer in a single integrated process. As shown in Figure 4, the fluorescent signals for the NAAT and CRISPR stages of the process are detected at different time points. Stage 1 shows the NAAT process with NAAT signal readout. The fluorescent signal from NAAT is detected before the reaction from the CRISPR enzyme takes place. Stage 2 shows a liquid handling process to allow amplicon transfer and interaction with the CRISPR system. Stage 3 shows the CRISPR process with CRISPR signal readout, which takes place after the signal from Stage 1 has been generated.

[0104] Figure 5 provides the results of inclusion experiments evaluating the ability of systems and methods to comprehensively detect FluA test synthetic ssDNA target sequences (position 37 + WT) with a single mismatch (see, e.g., SEQ ID NOs. 9–47 and SEQ ID NO. 1). As shown in Figure 5, generally good inclusion was observed for both the CRISPR system and the NAAT detection mode (demonstrated by PCR Taqman probes), and thus the devices and systems described herein were validated for the detection of FluA in wild-type sequences and several single mismatch sequences.

[0105] Example 3 - Verification of a device and system for detecting target microorganisms Sensitivity test

[0106] NAAT sensitivity refers to its ability to amplify small amounts of target nucleic acids (DNA or RNA) in a sample. CRISPR sensitivity refers to the ability of an enzyme to accurately and specifically target a particular target sequence.

[0107] To investigate the sensitivity of the devices described herein, several devices were fabricated as described in Example 1. The same program as described in Example 2 was executed, except that only wild-type sequences were used for sensitivity evaluation. For consistency, the devices were tested on 10 different cobas® Liat® analyzer systems. Briefly, a generalized linear model fit was used to fit known concentrations of FluA synthetic WT sequences (SEQ ID NO: 1) detectable by CRISPR channels and NAAT channels.

[0108] The results of this experiment are listed in Figures 6A to 6D. The figures are charts showing (A) the generalized linear model fit detected in the CRISPR channel (Cas) and its inverse prediction (B), and (C) the generalized linear model fit detected in the CRISPR channel (Cas) and its inverse prediction (D). An overview of the sensitivity evaluation is reproduced below. [Table 9]

[0109] The data indicates that detection in both NAAT and CRISPR modes was performed with similar sensitivity.

[0110] Example 4 - Verification of a device and system for detecting target microorganisms Cas variation test

[0111] To experimentally evaluate the CRISPR variation reaction, the following master mixes were prepared and fabricated in the device: [Table 10]

[0112] For this particular embodiment of the device, the first core PCR master mix was prepared as follows: [Table 11]

[0113] The following reagents were added to this first core master mix to form the first master mix. [Table 12]

[0114] For this particular embodiment of the device, a second PCR master mix was prepared as follows: [Table 13]

[0115] For this particular embodiment of the device, a third core PCR master mix was prepared as follows: [Table 14]

[0116] The following reagents were added to this third core master mix to form a third master mix. [Table 15]

[0117] In the quality control hood, the quality control reagents were prepared as follows: [Table 16]

[0118] The remaining reagents were prepared as described below: [Table 17]

[0119] Each assay tube contains all the reagents for a single test, packaged in a continuous segment as shown earlier in Figure 1A.

[0120] Assay tubes made from thermoplastic materials suitable for use in amplification reactions were assembled with approximately the following volumes of reagents: [Table 18]

[0121] To evaluate any potential inconsistencies within a device, the device was tested with 10 different analyzer systems. As shown in Figure 7, the CRISPR variability observed with the 10 different analyzers provides acceptable ranges for NTC and FluA. The mean endpoint-start difference for CRISPR gg of cas in 10-fold diluted FluA was 1130, and the cv was 15.6% across the 10 analyzers. The mean endpoint-start difference for CRISPR gg in NTC of cas was 2.9 from the 10 analyzers. The results shown in Figure 7 were obtained using a device fabricated with CRISPR enzyme master mix 1 of the last segment.

[0122] Example 5 - Verification of a device and system for detecting multiple target microorganisms After validating a dual detection system (NAAT+CRISPR) for the detection of FluA, this disclosure considers the validation of a device having an assay tube with multiple subsections for linking nucleic acid amplification tests and CRISPR systems for the detection of multiple target microorganisms.

[0123] Rapid and accurate diagnosis and differentiation of SARS-CoV-2, RSV, and influenza (types A and B) infections are crucial in individuals suspected of having a respiratory infection. The seasonal ranges of COVID-19 and influenza overlap, and the clinical manifestations of these diseases can be similar, ranging from asymptomatic or mild "flu-like" illnesses (fever, cough, shortness of breath, or muscle aches, etc.) in the majority of individuals to more severe and life-threatening illnesses. The current widespread implementation of rapid point-of-care (POC) testing for influenza highlights the importance of speed and accuracy.

[0124] Suboptimal oligo design or challenging reaction conditions can lead to false-positive (specificity concern) or false-negative (inclusivity concern) signals in NAAT or CRISPR for detecting microorganisms(s), potentially causing misidentification of the target pathogen. Due to differences in the target recognition mechanisms of the two systems, integrated devices and methods employing both NAAT and CRISPR detection systems are less likely to detect simultaneous defective signals when targeting the same pathogen compared to a single system.

[0125] Therefore, the NAAT+CRISPR dual detection device, system, and methodology developed and described in this system can improve the specificity and comprehensiveness of the assay.

[0126] A series of master mixes containing reagents for detecting FluA, FluB, RSV-A, and SARS-CoV-2 were prepared. For this particular embodiment of a device targeting FluA, the first PCR master mix was prepared as follows: [Table 19]

[0127] For this particular embodiment of the device targeting FluA, a second PCR master mix is ​​prepared as follows: [Table 20]

[0128] For this particular embodiment of the device targeting FluA, a third PCR master mix is ​​prepared as follows: [Table 21]

[0129] A CRISPR master mix may include a Cas enzyme / guide RNA system that targets multiple pathogens in a multiplexed setup. In this particular embodiment, a CRISPR master mix targeting FluA may be prepared as follows: [Table 22]

[0130] If necessary, an internal control may be added to tube segment 2. In this particular embodiment, no internal control was used.

[0131] The remaining reagents were prepared as described below: [Table 23]

[0132] Each assay tube contains all the reagents for a single test, packaged in a continuous segment. [Table 24]

[0133] This technology should not be limited to the specific embodiments described in this application, which are intended as single examples of individual aspects of this technology. As will be apparent to those skilled in the art, many modifications and variations of this technology can be made without departing from its spirit and scope. In addition to those enumerated herein, functionally equivalent methods and apparatus within the scope of this technology will be apparent to those skilled in the art from the foregoing description. Such modifications and variations are intended to be within the scope of this technology. It should be understood that this technology is not limited to specific methods, reagents, compound compositions or biological systems, and is naturally subject to change. It should also be understood that the terminology used herein is intended solely to describe specific embodiments and is not intended to limit them.

Claims

1. It is a device, The assay tube is subdivided into multiple subsections, A first subsection is connected to a retractable cap, and an opening of the first subsection accessible from the retractable cap is fitted to receive a sample, and the first subsection is sealed along the assay tube from subsequent subsections. A second subsection contains a solution including an internal control, and the second subsection is sealed along the assay tube away from subsequent subsections. A third subsection contains a solution containing magnetic beads in the solution, and the third subsection is sealed along the assay tube from subsequent subsections. A fourth subsection contains a solution comprising a lysis buffer, and the fourth subsection is sealed along the assay tube from subsequent subsections. A fifth subsection contains a solution including a washing buffer, and the fifth subsection is sealed along the assay tube from subsequent subsections. A sixth subsection contains a solution comprising an elution buffer, and the sixth subsection is sealed along the assay tube from subsequent subsections. A seventh subsection contains a solution comprising a first PCR master mix, and the seventh subsection is sealed along the assay tube from subsequent subsections. The eighth subsection contains a solution comprising the second PCR master mix, and the eighth subsection is sealed along the assay tube from the subsequent subsections. A ninth tube contains a solution containing the CRISPR enzyme, and the ninth subsection is sealed from the end of the assay tube. device.

2. The device according to claim 1, wherein the tube has a length of 106 ± 21 millimeters.

3. The device according to claim 1 or 2, wherein the first subsection is adapted to accept a volume of 200 ± 40 microliters.

4. The device according to any one of claims 1 to 3, wherein the second subsection comprises 12.5 ± 2.5 microliters of the solution containing the internal control reagent.

5. The device according to any one of claims 1 to 4, wherein the third subsection comprises 12.5 ± 2.5 microliters of the solution containing the magnetic beads.

6. The device according to any one of claims 1 to 5, wherein the fourth subsection comprises 215 ± 43 microliters of the solution containing the lysis buffer.

7. The device according to any one of claims 1 to 6, wherein the fifth subsection comprises 240 ± 48 microliters of the solution containing the washing buffer.

8. The device according to any one of claims 1 to 7, wherein the sixth subsection comprises 50 ± 10 microliters of the solution containing the elution buffer.

9. The device according to any one of claims 1 to 8, wherein the seventh subsection comprises 30 ± 6 microliters of a solution containing the first PCR master mix.

10. The device according to any one of claims 1 to 9, wherein the eighth subsection comprises 15 ± 3 microliters of a solution containing the second PCR master mix.

11. The device according to any one of claims 1 to 10, wherein the ninth subsection comprises 60 ± 12 microliters of the solution containing the CRISPR enzyme.

12. The device according to any one of claims 1 to 11, wherein the internal control reagent is a control material for a reverse transcription polymerase chain reaction (RT-PCR) reagent.

13. The device according to any one of claims 1 to 12, wherein the magnetic beads are Liat Magnetic Particles.

14. The device according to any one of claims 1 to 13, wherein the lysis buffer is Liat Lysis Buffer.

15. The device according to any one of claims 1 to 14, wherein the washing buffer is cobas Omni Wash Buffer.

16. The device according to any one of claims 1 to 15, wherein the elution buffer is FRTA Elution Buffer.

17. The device according to any one of claims 1 to 16, wherein the first PCR master mix is ​​a solution comprising a reaction buffer, dNTPs, RT-PCR primers, MMLV RT polymerase, and uracil DNA glycosylase.

18. The device according to any one of claims 1 to 17, wherein the second PCR master mix is ​​a solution comprising a reaction buffer, Z05 polymerase and its aptamer, and an RT-PCR primer.

19. The device according to any one of claims 1 to 18, wherein the CRISPR enzyme is a solution comprising a CRISPR-Cas enzyme having trans-cleavage activity such as Cas12 and Cas13, guide RNA, a substrate, and a metal ion cofactor.

20. The device according to any one of claims 1 to 19, wherein the assay tube is attached to a frame.

21. The device according to claim 20, wherein the frame is an open frame.

22. The device according to claim 21, wherein the right side of the frame is provided with a scale.

23. The device according to claim 22, wherein the scale is adapted to facilitate sliding the device into a system that provides a signal readout.

24. The device according to any one of claims 1 to 23, wherein the sample preparation reagent extracts nucleic acid material from influenza A virus, influenza B virus, respiratory syncytial virus (RSV), and SARS-CoV-2 virus.

25. The device according to any one of claims 1 to 24, wherein the assay tube is a thermoplastic tube.

26. The device according to claim 25, wherein the thermoplastic tube is made from polypropylene.

27. The device according to any one of claims 1 to 26, wherein the tube further comprises a tenth subsection containing a solution comprising the CRISPR enzyme, and the tenth subsection is sealed from the end of the tube.

28. The device according to claim 27, wherein the CRISPR enzyme is part of a CRISPR system adapted to specifically cleave a plurality of nucleic acids amplified by a PCR reaction.

29. A method for the dual detection of target microorganisms, (a) A step of amplifying a first target nucleic acid sequence in a nucleic acid amplification reaction and detecting a signal from the nucleic acid amplification of the first target nucleic acid sequence, (b) The steps of detecting the second target nucleic acid sequence using a CRISPR system that targets the second target nucleic acid sequence, and detecting signals from the activation of the second target nucleic acid and substrate cleavage by the CRISPR system, (c) The step of comparing the signal detected from the nucleic acid amplification of the first target nucleic acid sequence with an amplification threshold, and comparing the signal detected from the activation and substrate cleavage of the second target nucleic acid by the CRISPR system with a CRISPR threshold. Includes, The first target nucleic acid sequence and the second target nucleic acid sequence are sequences derived from the target microorganism. A method for double detection of a target nucleic acid sequence when both the signal detected from a first target nucleic acid amplification reaction and the signal detected from the activation and substrate cleavage of the second target nucleic acid by the CRISPR system exceed the threshold.

30. The method according to claim 29, wherein the nucleic acid amplification reaction is a polymerase chain reaction (PCR).

31. The method according to claim 29, wherein the nucleic acid amplification reaction is a loop-mediated isothermal amplification (LAMP) reaction.

32. The method according to claim 29, wherein the nucleic acid amplification reaction is a recombinase polymerase amplification (RPA) reaction.

33. The method according to claim 29, wherein the nucleic acid amplification reaction is a nucleic acid sequence-based amplification (NASBS) reaction.

34. The method according to claim 29, wherein the nucleic acid amplification reaction is a transcription-mediated amplification (TMA) reaction.

35. The method according to claim 29, wherein the nucleic acid amplification reaction is a rolling circle amplification (RCA) reaction.

36. The method according to claim 29, wherein the nucleic acid amplification reaction is a strand substitution amplification (SDA) reaction.

37. The method according to claim 29, wherein the nucleic acid amplification reaction is a nickeling and extension amplification reaction (NEAR).

38. The method according to claim 29, wherein the nucleic acid amplification reaction is an exponential amplification reaction (EXPAR).

39. The method according to claim 29, wherein the nucleic acid amplification reaction is a multiple substitution amplification (MDA) reaction.

40. The method according to claim 29, wherein the nucleic acid amplification reaction is a helicase-dependent amplification (HAD) reaction.

41. The method according to claim 29, wherein the nucleic acid amplification reaction is a hybridization chain reaction (HCR).

42. The method according to any one of claims 29 to 41, wherein the CRISPR system is a system having trans-cleavage nuclease activity.

43. The method according to claim 42, wherein the system having the trans-cleavage activity is a Cas12 system.

44. The method according to claim 42, wherein the system having the trans-cleavage activity is a Cas13 system.

45. The method according to any one of claims 29 to 44, wherein the first target nucleic acid sequence is a FluA sequence.

46. The method according to any one of claims 29 to 44, wherein the second target nucleic acid sequence is a FluA sequence.

47. The method according to any one of claims 29 to 46, wherein the region of the first target nucleic acid amplification sequence includes the second target nucleic acid sequence.

48. The method according to claim 47, wherein the first target nucleic acid sequence includes a plurality of regions including the second target nucleic acid sequence(s).

49. The method according to claim 48, wherein the activation and substrate cleavage of the plurality of second target nucleic acid sequences further amplifies the signal detected from the second target nucleic acid at a rate directly proportional to the number of the plurality of second target nucleic acid sequences.

50. The method according to any one of claims 29 to 49, comprising the step of amplifying a plurality of first target nucleic acid sequences for the multiple detection of a plurality of microorganisms.

51. The method according to any one of claims 29 to 49, comprising the step of amplifying a plurality of first target nucleic acid sequences for the multiple detection of the same microorganism.

52. The method according to any one of claims 29 to 51, wherein the amplification threshold provides a sensitivity of at least 120 copies / mL.

53. The method according to any one of claims 29 to 52, wherein the CRISPR threshold provides a sensitivity of at least 120 copies / mL.

54. The method according to any one of claims 29 to 53, further comprising the step of cleaving a plurality of nucleic acids using a nonspecific nuclease after detection of the amplification threshold and the CRISPR threshold, thereby reducing nucleic acid contamination in the system.

55. A method for detecting a target nucleic acid sequence, (a) A step of adding a sample into a device, wherein the device is The assay tube comprises a plurality of subsections, the first subsection being connected to a retractable cap, the opening of the first subsection accessible from the retractable cap being fitted to receive a sample, and the first subsection being sealed along the tube from subsequent subsections. A second subsection contains a solution containing an internal control reagent, and the second subsection is sealed along the tube away from subsequent subsections. A third subsection contains a solution containing magnetic beads, and the third subsection is sealed along the tube away from subsequent subsections. A fourth subsection contains a solution comprising a lysis buffer, and the fourth subsection is sealed along the tube away from subsequent subsections. A fifth subsection contains a solution comprising a washing buffer, and the fifth subsection is sealed along the tube away from subsequent subsections. A sixth subsection contains a solution comprising an elution buffer, and the sixth subsection is sealed along the tube from subsequent subsections. The seventh subsection contains a solution comprising PCR master mix part 1, and the seventh subsection is sealed along the tube from the subsequent subsections. The eighth subsection contains a solution comprising PCR master mix part 2, and the eighth subsection is sealed along the tube from the subsequent subsections. A step of adding a sample, wherein a ninth tube contains a solution containing the CRISPR enzyme, and the ninth subsection is sealed from the end of the tube, (b) A step of incorporating the device containing the sample into a system that enables sequential rupture of one subsection in subsequent subsections, thereby enabling a reagent in one subsection to react sequentially with a reagent in subsequent subsections; (c) The step of detecting the target nucleic acid by detecting the signal from the last subsection Methods that include...

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