CN113203715A - Portable fluorescence detection device and diagnostic method - Google Patents

Abstract

The invention relates to a portable fluorescence detection device and a diagnosis method. Provided herein are devices and systems for detecting fluorescent signals from a sample using a camera module of a mobile computing device. Also provided are methods of detecting target nucleic acids and methods of diagnosing microbial infections using the devices and systems described herein.

Description

Portable fluorescence detection device and diagnostic method

Technical Field

The present application relates to a low cost, portable fluorescence detection device for diagnosing microbial infections.

Background

Coronaviruses are enveloped RNA viruses that are widely distributed among humans, other mammals and birds and cause respiratory, intestinal, liver, and nervous system diseases. Although most human coronary virus infections are mild, both strains have been associated with sometimes fatal diseases. One of them is SARS-CoV, which is the cause of the outbreak of severe acute respiratory syndrome in 2002 and 2003; the other is MERS-CoV, which is the causative agent of the 2012 outbreak of severe respiratory disease. Recently, a new coronavirus strain named 2019-nCoV, SARS-CoV2, was identified in hospitalized patients. Phylogenetic analyses revealed that 2019-nCoV, SARS-CoV2, forms branches within the sub-genus sarbecovirus of the subfamily orthocoronaviridae (Orthoconovirinae). The novel strain is also associated with more severe and sometimes fatal diseases (Zhu et al (2020),2019, NEJM, DOI: 10.1056/NEJMoa 2001017).

Several methods have been developed to diagnose 2019-nCoV, SARS-CoV2, including high throughput sequencing and real-time reverse transcription PCR (Zhu et al (2020)). However, these methods are time consuming and require specialized equipment. In order to better diagnose and control the spread of microbial infections like 2019-nCoV, SARS-CoV2, there is a need to develop fast and low cost diagnostic devices that can be used in various field testing environments such as local clinics, train stations, airports and homes.

All references, including patent applications and publications, cited herein are hereby incorporated by reference in their entirety.

Disclosure of Invention

The present application provides fluorescence detection devices and systems, and methods of using such devices and systems.

One aspect of the present application provides a system for detecting a fluorescent signal from a detection panel, comprising: (a) a base structure comprising a support surface; (b) a Light Emitting Diode (LED) unit attached to a base structure, wherein the LED unit comprises: an LED light source, and a conical reflector configured to focus a light beam emitted by the LED light source such that the light beam travels along the support surface and through a detector plate disposed on the support surface; and (c) a housing configured to enclose the base structure and the LED unit, wherein the housing comprises an opening; a mobile computing device including a camera module therein may be attached to the housing to capture an image of the detector board through the opening. In some embodiments, the system is portable.

In some embodiments according to any of the above systems, the conical reflector is configured to focus the light beam such that the light beam is at an angle of about 10 ° or less relative to the support surface. In some embodiments, the conical reflector is configured to focus the light beam such that the light beam is parallel to the support surface.

In some embodiments of any of the systems above, the system further comprises a filter configured to be placed over the opening. In some embodiments, the LED light source has a wavelength of about 470nm to about 510nm, for example about 490 nm. In some embodiments, the optical filter selectively transmits light having a wavelength of about 530 to about 580nm, such as about 550 nm.

In some embodiments of any of the systems above, the housing is a cartridge (e.g., a cassette). In some embodiments, the case includes a case having an open surface, wherein the mobile computing device can be attached to the surface. In some embodiments, the system further comprises a battery for the LED light source enclosed in the housing. In some embodiments, the base structure is detachable from the LED unit.

In some embodiments according to any of the above systems, the system further comprises a mobile computing device configured to position the camera module over the opening. In some embodiments, the mobile computing device is a smartphone.

In some embodiments of any of the systems above, the system further comprises a detection plate configured to be positioned on the support surface. In some embodiments, the detection panel comprises two or more reaction cells.

Another aspect of the present application provides a test plate comprising two or more reaction wells, wherein each reaction well comprises a dry powder composition comprising: (a) a Cas nuclease; (b) a guide RNA comprising a sequence that binds to a Cas nuclease and a guide sequence that hybridizes to a target nucleic acid of interest; and (c) a detection DNA comprising a fluorescent tag, wherein the detection DNA is single stranded and does not hybridize to the guide sequence of the guide RNA, and wherein a fluorescent signal from the fluorescent tag is detectable upon cleavage of the detection DNA. In some embodiments, the fluorescent tag is FAM (5(6) -carboxyfluorescein). In some embodiments, the Cas nuclease is a type V CRISPR/Cas nuclease. In some embodiments, the Cas nuclease is Cas12a nuclease. In some embodiments, the Cas nuclease is Cas12b nuclease. In some embodiments, the Cas nuclease is a Cas12i nuclease (e.g., Cas12i1 or Cas12i 2). In some embodiments, the Cas nuclease is Cas13 nuclease.

In some embodiments of the assay plate according to any of the above, wherein the dry powder composition further comprises one or more reagents for Recombinase Polymerase Amplification (RPA). In some embodiments, the dry powder composition further comprises: (a) a recombinase; (b) single-stranded DNA binding protein (SSB); (c) a strand displacing polymerase; (d) a forward primer and a reverse primer for amplification of the target nucleic acid; and (e) dNTPs.

In some embodiments of the assay plate according to any of the above, wherein the dry powder composition further comprises one or more reagents for reverse transcription. In some embodiments, the dry powder composition further comprises: (a) a reverse transcriptase; and (b) a reverse transcription primer.

In some embodiments of the assay plate according to any one of the above, each reaction cell has a volume of about 20 μ l to about 500 μ l, for example about 20 μ l to about 200 μ l, about 50 μ l, or about 100 μ l. In some embodiments, each reaction cell is about 3mm by about 3mm in size.

One aspect of the present application provides a method of detecting a target nucleic acid in a sample using any of the systems or assay plates described above. In some embodiments, there is provided a method of detecting a target nucleic acid in a sample, comprising: (a) applying a sample to a reaction cell of a detection plate including any one of the detection plates described above; (b) incubating the detection plate at a temperature suitable for Cas cleavage of the target nucleic acid or amplicon thereof; (c) placing the test panel in a system comprising: (i) a base structure comprising a support surface, wherein the sensing plate is disposed on the support surface; (ii) a Light Emitting Diode (LED) unit attached to the base structure, wherein the LED unit comprises: an LED light source, and a conical reflector configured to focus a light beam emitted by the LED light source such that the light beam travels along the support surface and through a detector plate disposed on the support surface; and (iii) a housing configured to enclose the base structure and the LED unit, wherein the housing comprises an opening; (d) attaching a mobile computing device including a camera module to a housing of a system to place the camera module over an opening of the system; and (e) capturing an image of the reaction cell using a camera module of the mobile computing device, thereby detecting the presence or absence of the target nucleic acid in the sample. In some embodiments, step (b) comprises incubating the test plate at about 42 ℃ for about 5 minutes to about 40 minutes. In some embodiments, the method further comprises analyzing the captured image in the mobile computer device.

In some embodiments according to any of the above methods, the target nucleic acid is viral DNA, bacterial DNA, fungal DNA, protozoan DNA, or algal DNA. In some embodiments, the target nucleic acid is a viral RNA, a bacterial RNA, a fungal RNA, a protozoan RNA, or an algal RNA. In some embodiments, the target nucleic acid is RNA from a coronavirus. In some embodiments, the target nucleic acid is RNA from 2019-nCoV, SARS-CoV 2. In some embodiments, the sample is a nasal or pharyngeal swab sample.

One aspect of the present application provides a method of diagnosing a microbial infection in an individual, comprising detecting a target nucleic acid of a microbe associated with a microbial infection in a sample from the individual using any of the detection methods described above. In some embodiments, the microbial infection is a bacterial infection. In some embodiments, the microbial infection is a protozoan infection. In some embodiments, the microbial infection is an algal infection. In some embodiments, the microbial infection is a viral infection. In some embodiments, the microbial infection is a coronavirus infection, such as a 2019-nCoV or SARS-CoV2 infection. In some embodiments, the subject is a human subject.

Another aspect of the present application provides a dry powder composition comprising: (a) a Cas nuclease, (b) a guide RNA comprising a sequence that binds to the Cas nuclease and a guide sequence that hybridizes to a target nucleic acid of interest; (c) a detection DNA comprising a fluorescent tag, wherein the detection DNA is single-stranded and does not hybridize to a guide sequence of a guide RNA, and wherein a fluorescent signal from the fluorescent tag is detectable upon cleavage of the detection DNA; (d) a recombinase; (e) single-stranded DNA binding protein (SSB); (f) a strand displacing polymerase; (g) a forward primer and a reverse primer for amplifying the target nucleic acid; (h) dNTP; (i) a reverse transcriptase; and (j) a reverse transcription primer. In some embodiments, the dry powder composition is a lyophilized composition.

Kits and articles of manufacture (articles of manufacture) are also provided, which include a system according to any of the above systems, or a test plate according to any of the above test plates. In some embodiments, the kit or article further comprises said system and said detection panel. In some embodiments, a kit or article of manufacture comprises a plurality of said detection panels. In some embodiments, the kit or article of manufacture further comprises a dry powder composition according to any one of the dry powder compositions described above. In some embodiments, the kit or article of manufacture comprises instructions for use in any of the detection methods or diagnostic methods described above.

Drawings

FIG. 1 shows a schematic diagram of an exemplary workflow for diagnosing viral infections.

Fig. 2 shows a schematic illustration of a portable fluorescence detection system.

Fig. 3-5 show schematic diagrams of the system of fig. 2 from three perspectives (A, B, and C). In fig. 5, the mobile computing device (e.g., smartphone) is placed on top of the imaging cover (surface C) such that the lens of the mobile computing device is positioned on top of the imaging opening.

Fig. 6 shows a schematic perspective view of the system of fig. 2.

Fig. 7 shows a schematic cross-sectional view of the system of fig. 2.

Fig. 8 shows a schematic cross-sectional view of the system of fig. 2.

Fig. 9 shows two views of the detection plate.

Fig. 10-13 show different views of a prototype of a portable fluorescence detection system. In fig. 10, the LED unit (including the cone mirror) (left) and the drawer (right) are detachable from each other. In fig. 11, the LED unit (including the conical mirror) and the drawer are attached to each other. In fig. 12, the imaging cover of the system is removed to show the LED units and drawers embedded within the dark box. Fig. 13 shows a smartphone placed on top of the imaging cover of the system to capture an image of the detection plate.

Fig. 14A-14B show images of a detector board captured by a smartphone using the system of fig. 13.

FIG. 15 schematically shows a flow chart of an exemplary method for detecting viral infection using a portable fluorescence detection device.

Detailed Description

The present application provides fluorescence detection devices and systems that are portable and attachable to a smartphone for data capture and analysis using camera functionality on the phone. The devices and systems described herein can be used to detect the fluorescence intensity of any fluorescent liquid or reaction mixture. In some embodiments, the fluorescence detection system is used in conjunction with a CRISPR/Cas-based fluorescence detection system. Also provided are assays, methods, and kits for detecting a target nucleic acid using the devices and systems described herein. The device and system enable rapid diagnosis of microbial infections (e.g., 2019-nCoV or SARS-CoV2 infections) at low cost in non-professional institutions including local clinics, airports, train stations, schools, offices, and patient homes.

I. Definition of

Unless otherwise defined below, terms are used herein as commonly used in the art.

The terms "polynucleotide" and "nucleic acid," used interchangeably herein, refer to a polymeric form of nucleotides of any length, whether ribonucleotides or deoxyribonucleotides. Thus, the terms "polynucleotide" and "nucleic acid" include single-stranded DNA; double-stranded DNA; a multi-stranded DNA; single-stranded RNA; double-stranded RNA; a multi-stranded RNA; genomic DNA; cDNA; a DNA-RNA hybrid; and polymers comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derivatized nucleotide bases.

As used herein, the terms "individual," "patient," or "subject" are used interchangeably and refer to any single animal, e.g., a mammal (including non-human animals, such as, e.g., dogs, cats, horses, rabbits, zoo animals, cattle, pigs, sheep, and non-human primates) for which treatment is desired. In some embodiments, the subject is a human.

It is to be understood that embodiments of the invention described herein include embodiments "consisting of …" and/or "consisting essentially of …".

Reference herein to a "value or parameter" about "includes (and describes) variations that are directed to that value or parameter itself. For example, a description referring to "about X" includes a description of "X".

As used herein, reference to a "non" value or parameter generally means and describes a value or parameter that is "different from". For example, the method is not used to treat type X cancer means the method is used to treat a cancer other than type X.

The term "about X-Y" as used herein has the same meaning as "about X to about Y".

As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

It is to be understood that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of embodiments relating to the apparatus, systems, and methods described herein are specifically contemplated by the present invention and are disclosed herein as if each and every combination were individually and explicitly disclosed. Moreover, all subcombinations of the features and characteristics of the apparatus, systems, and methods of the embodiments describing such variables are also specifically embraced by the present invention and are disclosed herein just as if each and every such subcombination were individually and specifically disclosed herein.

Fluorescence detection System

The present invention provides optical (e.g., fluorescence) detection devices and systems.

In some embodiments, there is provided an apparatus for detecting an optical (e.g., fluorescent) signal from a detection panel, comprising: (a) a base structure comprising a support surface; (b) an LED unit attached to a base structure, the LED unit comprising: an LED light source, and a conical reflector (e.g., a conical mirror) configured to focus a light beam emitted by the LED light source such that the light beam is transmitted along the support surface. In some embodiments, the apparatus includes a detection plate configured to be placed on the support surface. In some embodiments, the light beam is transmitted along the support surface. In some embodiments, the device may be placed in a dark box with an opening, where a mobile computing device (e.g., a smartphone) may be attached to the dark box, placing a camera module over the opening to capture an image of the detection panel.

In some embodiments, a system for detecting an optical (e.g., fluorescent) signal from a detection panel is provided, comprising: (a) a base structure comprising a support surface; (b) an LED unit attached to the base structure, the LED unit comprising: an LED light source, and a conical reflector (e.g., a conical mirror) configured to focus a light beam emitted by the LED light source such that the light beam is transmitted along a support surface; and (c) a housing configured to enclose the base structure and the LED unit, wherein the housing includes an opening, and wherein a mobile computing device (e.g., a smartphone) including a camera module may be attached to the housing to capture an image of a detector board passing through the opening. In some embodiments, the system includes a detection plate configured to be placed on the support surface. In some implementations, a system includes a mobile computing device attached to a housing of the system, wherein a camera module is disposed over an opening.

In some embodiments, a system for detecting an optical (e.g., fluorescent) signal from a detection panel is provided, comprising: (a) a base structure comprising a support surface; (b) a detection plate configured to be placed on the support surface; (c) an LED unit attached to the base structure, wherein the LED unit comprises: an LED light source; a conical mirror configured to focus the light beam emitted by the LED light source such that the light beam travels along the support surface and through the detection plate; (d) a housing configured to enclose a chip and an LED unit, wherein the housing includes an opening; and (e) an optical sensor configured to capture an image of the chip through the opening. In some embodiments, the optical sensor is a lens of a mobile computing device, such as a smartphone.

The fluorescence detection devices and systems described herein include components such as an LED unit including an LED light source, a conical reflector, a detection plate (also referred to as a sample block), a base structure having a bearing surface (e.g., a "drawer"), a housing (e.g., a cassette), and an opening (e.g., an opening in a cover of the cassette). The LED light source provides excitation light to illuminate the fluorophores in the sample. For example, when the sample contains FAM (5(6) -carboxyfluorescein), a blue LED light source (e.g., in the wavelength range of about 470nm to 510nm, such as about 490nm) can be used. While fluorophores with other excitation wavelengths may be used, LED light sources with appropriate wavelength ranges may be selected. LED light sources with specific wavelengths are readily available in the market. The LED light source may be attached to the housing, or to the conical reflector.

A cone reflector, such as a cone mirror, is used to prevent flare and direct the LED light into a beam that is uniform across the detection panel. The conical reflector may be positioned, for example, at a small angle, for example, no more than about 30, 20, 15, 10, 5, or less, with respect to the support surface or a detection plate on the support surface, for example, of the light beam emitted by the LED light. In some embodiments, the light beam emitted by the LED light is parallel to the support surface and passes through a detector plate on the support surface.

The detection plate is designed to be easy to use and image. In some embodiments, the detection panel is a flat square or rectangle. The flat surface ensures that the test panel is not prone to tipping over. In some embodiments, the detection plate comprises two or more reaction cells. The reaction cell may be of any suitable shape, for example circular or square. The reaction cell may have a small volume to allow the use of small amounts of reagents and samples. In some embodiments, each reaction cell has a cross-section of about 5mm x 5mm, 4mm x 4mm, 3mm x 3mm, 2mm x 2mm, or 1mm x 1mm, including any values and ranges between these values. In some embodiments, the depth of each reaction cell is any one of about 2mm, 2.5mm, 3mm, 4mm, or 5mm, including any values and ranges between these values. In some embodiments, the volume of each reaction cell is no greater than any of about 1mL, 500 μ L, 400 μ L, 300 μ L, 200 μ L, 100 μ L, 50 μ L, 30 μ L, 20 μ L, or lower, including any values and ranges between these values. In some embodiments, the detection plate includes 3 reaction wells for a positive control, a negative control, and a test sample (e.g., a patient sample), respectively. In some embodiments, the test panel is transparent. For example, the detection plate may be made of PDMS (polydimethylsiloxane) or other transparent material. In some embodiments, the plate may be flicked to ensure that the reagents are mixed evenly with the sample in the reaction cell. In some embodiments, the reagent is preloaded in the detection plate. In some embodiments, the detection plate is covered by a cover (e.g., another PDMS plate, or a glass cover slip, or a plastic sheet, or a transparent label) that prevents the reaction mixture from evaporating in the reaction cell. In some embodiments, the test panel is covered with scotch tape.

In some embodiments, the detection plate comprises reagents (e.g., reagents for reverse transcription, RPA, and/or CRISPR/Cas-based nucleic acid detection) in each reaction cell. In some embodiments, the agent is in the form of a dry powder. In some embodiments, the reagents are lyophilized in a reaction cell on a detection plate.

In some embodiments, each reaction cell of the detection plate comprises reagents for CRISPR/Cas-based nucleic acid detection. In some embodiments, reagents for CRISPR/Cas-based nucleic acid detection comprise: (a) a Cas nuclease; (b) a guide RNA comprising a sequence that binds to a Cas nuclease and a guide sequence that hybridizes to a target nucleic acid of interest; and (c) a detection DNA comprising a fluorescent tag, wherein the detection DNA is single-stranded and does not hybridize to the guide sequence of the guide RNA, and wherein a fluorescent signal from the fluorescent tag is detectable upon cleavage of the detection DNA.

In some embodiments, each reaction cell of the detection plate further comprises a reagent for amplification of the target nucleic acid. In some embodiments, the reagents are used for isothermal amplification, such as Recombinase Polymerase Amplification (RPA). In some embodiments, the agent for RPA comprises (a) a recombinase; (b) single-stranded DNA binding protein (SSB); (c) a strand displacing polymerase; (d) a forward primer and a reverse primer for amplifying a target nucleic acid; and (e) dNTPs.

In some embodiments, each reaction cell of the detection plate further comprises a reagent for reverse transcription of a target RNA molecule. In some embodiments, the reagent for reverse transcription comprises: (a) a reverse transcriptase; (b) reverse transcription primers (e.g., poly-dT, random hexamer, or target specific primers); and (c) dNTPs.

In some embodiments, each reaction cell of the detection panel contains a dry powder composition comprising: (a) a Cas nuclease, (b) a guide RNA comprising a sequence that binds to the Cas nuclease and a guide sequence that hybridizes to a target nucleic acid of interest; (c) a detection DNA comprising a fluorescent tag, wherein the detection DNA is single-stranded and does not hybridize to the guide sequence of the guide RNA, and wherein a fluorescent signal from the fluorescent tag is detectable upon cleavage of the detection DNA; (d) a recombinase; (e) single-stranded DNA binding protein (SSB); (f) a strand displacement polymerase; (g) a forward primer and a reverse primer for amplifying a target nucleic acid; (h) dNTP; (i) a reverse transcriptase; and (j) a reverse transcription primer.

In some embodiments, each reaction cell of the detection panel contains a dry powder composition comprising: (a) a Cas nuclease, (b) a guide RNA comprising a sequence that binds to the Cas nuclease and a guide sequence that hybridizes to a target nucleic acid of interest; (c) a detection DNA comprising a fluorescent tag, wherein the detection DNA is single-stranded and does not hybridize to the guide sequence of the guide RNA, and wherein a fluorescent signal from the fluorescent tag is detectable upon cleavage of the detection DNA; (d) a recombinase; (e) single-stranded DNA binding protein (SSB); (f) a strand displacing polymerase; (g) a forward primer and a reverse primer for amplifying a target nucleic acid; and (h) dNTPs.

The base structure includes a support surface that can hold the sensing plate. The base structure is connected to the LED unit during fluorescence detection, but may be detachable from the LED unit to load or unload the detection plate. The base structure may be in the form of a drawer that can be slid into a slot in the housing to attach to the LED unit. The support surface holding the sensing plate may have a structure such as a short shaft (short draft) to hold the sensing plate in place so that the sample does not move. The drawer design allows a user to easily and quickly place samples into and out of the system with minimal access. The drawer is precisely positioned so that the detection plate is aligned with the camera pin hole (i.e., the "opening") on the top cover of the cassette so that the detection plate is precisely flashed at the same level as the LED light source.

The housing may be a cartridge such as a cassette. The cassette avoids stray light. In some embodiments, the surfaces of the housing, including the inner and outer surfaces of the housing, are frosted black to minimize noise signals from light scattering. The housing is designed to allow a mobile computing device, such as a smartphone, to be stably placed during data collection and analysis. The housing may have one or more removable sides, and one or more openings, allowing a user to place various components in the housing. The housing encloses a base structure (e.g., a drawer) and an LED unit. In some embodiments, the housing may contain a battery that powers the LED light source.

The housing may have a cover or surface that includes an opening for capturing images by the mobile computing device. The opening may be a pinhole. The cover covers the cartridge to ensure complete darkness during imaging. The aperture is designed to accommodate a primary camera lens for data collection. The opening is designed with suitable dimensions to fit the lens of the camera on most mobile phones. The cover is placed at a suitable distance from the detection plate to facilitate image capture by the mobile computing device. In some embodiments, the lid is at least about 8cm, 9cm, 10cm, 12cm, 15cm, or more from the test plate, including any values and ranges between these values.

A filter configured to selectively transmit light at the emission wavelength of the fluorophore in the sample may be positioned over the opening. Any fluorophore having an emission wavelength in the visible spectrum may be used and a filter suitable for the fluorophore may be selected accordingly. For example, a green filter selective for a wavelength of about 530 and 580nm, such as about 550nm, may be used to allow only positive signals from FAM (5(6) -carboxyfluorescein) in the sample to be detected. The filter may be attached on either side of the opening, such as between the opening and a camera lens of the mobile device, or under a cover or surface such that the filter is placed on the opposite side relative to the mobile device.

The devices and systems described herein may be used with various mobile computing devices, such as smart phones. Almost all smart phones on the market today include a built-in camera that can use conventional imaging setup and does not require further amplification by an additional lens to detect the fluorescent signal from the detection plate. Various brands and designs of smart phones may be used, as well as other mobile devices with suitable imaging capabilities, including, but not limited to, tablet and laptop computers. Typically, the mobile computing device includes a processor configured to process the captured image to analyze a fluorescence signal from the sample. Additionally or alternatively, the mobile computing device includes a display screen on which the processor is configured to present an assessment of the fluorescent signal (e.g., the presence or absence of the target nucleic acid, or whether the individual is diagnosed as having a microbial infection).

The systems described herein may have a small amount of space (footprint) so that they are portable. In some embodiments, the length, width, and/or height of the shell is no more than about any of 50cm, 40cm, 30cm, 20cm, 15cm, 10cm, or lower, including any values and ranges between these values.

Fig. 2-5 provide schematic illustrations of the exterior of an exemplary fluorescence detection system (100). 116 is a handle of a drawer that can be slid into the cassette holding the test panels. 140 is a cover containing an opening (142) over which a camera of the mobile device (150) can be placed to allow image capture. A filter (144) may be placed either above the opening (142), below the lid (140), or between the lid (140) and the mobile device (150), which selectively transmits the fluorescent signal from the sample.

A perspective view of an exemplary fluorescence detection system (100) is shown in fig. 6. The drawer/base structure (110) is attached to an LED unit comprising a conical reflector (120), said conical reflector (120) guiding a light beam emitted by an LED light source (not shown, attached to the inside of the conical reflector) to a detection plate (200) placed above the plate holder (114). The LED light source (130) is powered by a battery (300). Fig. 7 and 8 provide cross-sectional views of the drawer (110) and LED unit (120).

FIG. 9 shows a schematic of a detection plate (200) with 3 reaction cells (212, 214, 216) and corresponding sample addition parts (222, 224, 226).

Figures 10-13 show pictures of a prototype of a fluorescence detection system. The system has dimensions of 15cm x10cm x10 cm. The distance from the cover having the imaging opening to the detection plate is at least about 8 cm. The detection plates have dimensions of 2cm x 2cm and each reaction cell is 3mm x 3mm x about 2.2mm (smallest).

Detection or diagnostic method

The present application further provides methods of detecting a target nucleic acid in a sample, and methods of diagnosing a microbial infection (e.g., a viral or bacterial infection) in an individual using any of the fluorescent detection devices and systems described herein. The methods described herein may be used for point-of-care testing (point-of-care), field testing, or home testing.

In some embodiments, there is provided a method of detecting a target nucleic acid in a sample, comprising: (a) loading a sample into a reaction cell of any of the detection plates described herein; (b) incubating the detection plate at a temperature suitable for Cas cleavage of the target nucleic acid or its amplicon; (c) placing a detection plate on a support surface in any of the fluorescence detection systems described herein; (d) attaching a mobile computing device including a camera module to the system such that the camera module is positioned over an opening of the system; and (e) capturing an image of the reaction cell using a camera module of the mobile computing device, thereby detecting the presence or absence of the target nucleic acid in the sample. In some embodiments, step (b) comprises incubating the test plate at about 42 ℃ for about 5 minutes to about 40 minutes.

In some embodiments, a method of diagnosing a microbial infection in an individual is provided, comprising: (a) obtaining a sample from an individual; (b) loading a sample into a reaction cell of any of the assay plates described herein; (c) incubating the detection plate at a temperature suitable for Cas cleavage of the target nucleic acid or its amplicon in a microorganism associated with a microbial infection; (d) placing a detection plate on a support surface in any of the fluorescence detection systems described herein; (e) attaching a mobile computing device including a camera module to the system such that the camera module is positioned over an opening of the system; and (f) capturing an image of the reaction cell using a camera module of the mobile computing device, thereby providing a diagnosis based on the presence or absence of the target nucleic acid in the sample. In some embodiments, step (b) comprises incubating the test plate at about 42 ℃ for about 10 minutes to about 40 minutes. It is understood that the detection methods described herein can be used to diagnose other diseases or conditions associated with biomarker nucleic acids.

Fig. 15 provides a schematic flow diagram of an exemplary method of diagnosing a viral infection in a patient. Samples are collected from patients containing a virus (e.g., a coronavirus such as 2019-nCoV, SARS-CoV 2). The sample is heated at high temperature (e.g., 56 ℃ for 1 minute) to inactivate the virus and release nucleic acids from the virus into the sample. Alternatively, a commercially available kit can be used to extract nucleic acids from a sample. The sample is then applied to the reaction cell of the assay plate that has been pre-loaded with reagent (e.g., dry powder). After loading the sample, the test plate was flicked to mix the sample with the reagent. The sample detection plate is then placed in an incubator at a temperature, e.g., 42 ℃, suitable for reactions (e.g., reverse transcription, RPA, and Cas-based nucleic acid detection). Multiple assay plates may be incubated simultaneously, which may include repetition of the experiment. After about 5-40 minutes of incubation, the test plate was loaded into a drawer, which was then placed in the fluorescent detection system. The smartphone is then placed on top of the fluorescence detection system to capture an image of the detection plate. The images are analyzed by the mobile app, which provides diagnostic results to the user.

The methods described herein relate to CRISPR/Cas based detection. CRISPR/Cas based detection methods have been described, for example, in US10,253,365B1, CN202011414384.3 and PCT/CN2018/118457, the entire contents of which are incorporated herein by reference. For example, V-type CRISPR/Cas nucleases, Cas12 proteins such as Cpfl (Cas12a) and C2C1(Cas12b), can promiscuously cleave non-targeted single-stranded DNA (ssdna) once activated by detection of target DNA (double-stranded or single-stranded). Once a V-type CRISPR/Cas nuclease (e.g., Cas12 proteins such as Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas12 i) is activated by a guide RNA, which occurs when the guide RNA hybridizes to a target sequence of a target DNA (i.e., the sample includes the targeted DNA), the nuclease can promiscuously cleave ssDNA (i.e., the nuclease cleaves non-target ssDNA, i.e., ssDNA to which the guide sequence of the guide RNA is not hybridized). Thus, when the target DNA is present in the sample (e.g., above a threshold amount in some cases), the result is cleavage of ssDNA in the sample, which can be detected using fluorescently labeled single stranded detector DNA. In some embodiments, the methods described herein use Cas12a, Cas12b, Cas12i (e.g., Cas12i1, Cas12i2), or Cas13 nucleases, including functional variants and mutants thereof.

The V-type CRISPR/Cas effector protein binds to the target DNA at a target sequence defined by a complementary region between the RNA of the target DNA and the target DNA. Like many CRISPR/Cas endonucleases, site-specific binding (and/or cleavage) of double-stranded target DNA occurs at positions determined by both: (i) base-pairing complementarity between the guide RNA and the target DNA; and (ii) a short motif in the target DNA [ referred to as a prepro-spacer sequence adjacent motif (PAM) ]. Guide RNAs comprising a sequence that binds to a Cas nuclease and a guide sequence that hybridizes to a target nucleic acid can be designed using methods known in the art.

By "hybridizable" or "complementary" or "substantially complementary" is meant that the nucleic acid (e.g., RNA, DNA) comprises a sequence of nucleotides that is capable of allowing non-covalent binding, i.e., formation of watson-crick base pairs and/or G/U base pairs, "annealing", or "hybridizing", to another nucleic acid in a sequence-specific, antiparallel manner (i.e., the nucleic acid specifically binds to the complementary nucleic acid) under in vitro and/or in vivo conditions of suitable temperature and solution ionic strength. Standard watson-crick base pairing includes: adenine/adenosine (A) pairs with thymine/thymidine (T), A pairs with uracil/uridine (U), and guanine/guanosine (G) pairs with cytosine/cytidine (C). In addition, for hybridization between two RNA molecules (e.g., dsRNA), and for hybridization of a DNA molecule to an RNA molecule (e.g., when a DNA target nucleic acid is paired with a guide RNA base, etc.): g may also base pair with U.

Hybridization requires that the two nucleic acids contain complementary sequences, although mismatches between bases are possible. The conditions suitable for hybridization between two nucleic acids depend on the length and degree of complementarity of the nucleic acids, variables known in the art. The greater the degree of complementarity between two nucleotide sequences, the greater the value of the melting temperature (Tm) for hybrids of nucleic acids having those sequences. Typically, the length of the hybridizable nucleic acid is 8 nucleotides or more (e.g., 10 nucleotides or more, 12 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, 22 nucleotides or more, 25 nucleotides or more, or 30 nucleotides or more).

In some cases, a detection DNA that is single stranded (ssDNA) and does not hybridize to the guide sequence of the guide RNA is used (i.e., the detection ssDNA is a non-target ssDNA). A CRISPR/Cas-based detection method comprising (a) contacting a sample with: (i) cas nuclease (e.g., Cas12a, Cas12b, Cas12i, or Cas 13); (ii) a guide RNA comprising: a region that binds to a Cas nuclease, and a guide sequence that hybridizes to a target DNA; and (iii) a fluorescently labeled detector DNA that is single stranded and that is not hybridized to the guide sequence of the guide RNA; wherein the fluorescent signal is generated by cleavage of the single-stranded detection DNA by the Cas nuclease, thereby allowing detection of the target nucleic acid using the fluorescent detection apparatus and systems described herein. CRISPR/Cas based detection methods have attomolar (aM) detection sensitivity.

In some embodiments, the labeled detection ssDNA comprises a quencher/fluorophore pair comprising a quencher moiety and a fluorescent tag. The quencher moiety quenches the signal from the fluorescent tag (e.g., by absorbing energy in the emission spectrum of the tag). Thus, when the quencher moiety is not in the vicinity of the signal moiety (e.g., when the detection DNA is cleaved by the Cas nuclease), the (emitted) signal from the fluorescent tag is detectable because the signal is not absorbed by the quencher moiety. Any conventional fluorophore/quencher moiety pair can be used, and many suitable pairs are known in the art.

In some embodiments, the sample is subjected to one or more additional steps prior to loading the sample into the reaction cell. For example, the sample can be incubated at an elevated temperature to inactivate a microorganism (e.g., a virus), or to release the target nucleic acid from the microorganism. The sample may also be subjected to a step of extracting and/or fragmenting the target nucleic acid molecule. In some embodiments, wherein the target nucleic acid is an RNA molecule, the sample is subjected to a reverse transcription step. In some embodiments, the sample is subjected to an amplification step. In some embodiments, the sample is first subjected to a reverse transcription step followed by an amplification step.

In some embodiments, the sample is simultaneously amplified and the CRISPR/Cas-based nucleic acid detection reaction is performed within a reaction cell of the detection plate. In some embodiments, the sample is subjected to simultaneous reverse transcription, amplification, and CRISPR/Cas-based nucleic acid detection reactions within the reaction cell of the detection plate.

The methods described herein are suitable for rapid detection and diagnosis. In some embodiments, the incubation time allowed for reverse transcription, amplification, and/or CRISPR/Cas-based nucleic acid detection reactions is no more than any of about 40 minutes, 35 minutes, 30 minutes, 25 minutes, 20 minutes, 15 minutes, 10 minutes, 5 minutes, or less, including any values and ranges therebetween. In some embodiments, the total incubation time is from about 5 minutes to about 40 minutes, such as from about 5 minutes to about 10 minutes, from about 10 minutes to about 15 minutes, from about 15 minutes to about 20 minutes, from about 20 minutes to about 30 minutes, from about 30 minutes to about 40 minutes, from about 5 minutes to about 20 minutes, from about 10 minutes to about 20 minutes, or from about 10 minutes to about 40 minutes.

In some embodiments, the amplification step is an isothermal amplification step. The term "isothermal amplification" refers to a method of nucleic acid (e.g., DNA) amplification (e.g., using the enzyme chain reaction) that can use a single temperature incubation, thereby avoiding the need for thermal cycling. Isothermal amplification is a form of nucleic acid amplification that is not dependent on thermal denaturation of the target nucleic acid during the amplification reaction and thus may not require multiple rapid changes in temperature. Isothermal nucleic acid amplification methods can therefore be performed in a laboratory environment or outside. These amplification methods can be used for isothermal amplification of RNA by combination with a reverse transcription step.

Examples of isothermal amplification methods include, but are not limited to: loop-mediated isothermal amplification (LAMP), Helicase Dependent Amplification (HDA), Recombinase Polymerase Amplification (RPA), Strand Displacement Amplification (SDA), Nucleic Acid Sequence Based Amplification (NASBA), Transcription Mediated Amplification (TMA), Nicking Enzyme Amplification Reaction (NEAR), Rolling Circle Amplification (RCA), Multiple Displacement Amplification (MDA), reticulo-branch amplification (RAM), helicase dependent amplification (cHDA), Single Primer Isothermal Amplification (SPIA), Signal Mediated Amplification of RNA Technology (SMART), self-sustained sequence amplification (35R), Genomic Exponential Amplification Reaction (GEAR), and isothermal displacement amplification (IMDA).

In some cases, the amplification is Recombinase Polymerase Amplification (RPA) (see, e.g., U.S. Pat. Nos. 8,030,000; 8,426,134; 8,945,845; 9,309,502; and 9,663,820, the entire contents of which are incorporated herein by reference). Recombinase Polymerase Amplification (RPA) uses two opposing primers (much like PCR) and employs three enzymes, recombinase, single-stranded DNA binding protein (SSB) and strand-displacing polymerase. The recombinase pairs the oligonucleotide primer with a homologous sequence in the duplex DNA, SSB binds to the displaced strand of DNA to prevent the primer from being displaced, and strand displacing polymerase initiates DNA synthesis at the location where the primer has bound to the target DNA. The addition of reverse transcriptase to the RPA reaction can facilitate RNA as well as DNA detection without the need for a separate step to generate cDNA. An example of components for the RPA reaction is as follows: 50mM Tris pH 8.4, 80mM potassium acetate, 10mM magnesium acetate, 2mM DTT, 5% PEG compound (Carbowax-20M), 3mM ATP, 30mM phosphocreatine, 100 ng/. mu.l creatine kinase, 420 ng/. mu.l gp32, 140 ng/. mu.l UvsX, 35 ng/. mu.l UvsY, 2000M dNTPs, 300nM of each oligonucleotide, 35 ng/. mu.l Bsu polymerase, and nucleic acid containing samples.

The methods described herein may further comprise one or more data analysis steps. In some embodiments, the mobile computing device is used to process captured images, for example, using a mobile application running on a processor in the mobile device. In some embodiments, binary data is reported, i.e., the presence or absence of a target nucleic acid, or the presence or absence of a microbial infection. In some implementations, a user may download mobile application software on a mobile computing device from a website or "application store" (app store). The mobile application software includes a user interface that guides the user through the test and accepts user input as needed, and a driver component to manipulate the camera module and display screen of the mobile computing device as needed. The application software also includes an image processing and analysis component that analyzes images of the sample captured by the camera module and calculates test results.

The target nucleic acid can be DNA or RNA. In some embodiments, the target nucleic acid is single stranded. In some embodiments, the target nucleic acid is double stranded. In some embodiments, the target nucleic acid is genomic DNA. In some embodiments, the target nucleic acid is genomic RNA.

The source of the target nucleic acid can be from any source. In some embodiments, the target nucleic acid is a genomic nucleic acid (e.g., DNA or RNA) of a microorganism, such as a pathogenic microorganism, including but not limited to, a viral bacterium, a fungus, a protozoan, an algae. In some embodiments, the target nucleic acid is genomic DNA of a bacterium, such as a pathogenic bacterium. In some embodiments, the target nucleic acid is genomic DNA of a DNA virus. In some embodiments, the target nucleic acid is a genomic RNA of an RNA virus. Exemplary viruses that can be detected include, but are not limited to, coronaviruses (e.g., SARS, MERS, and 2019-nCoV, SARS-CoV2), papovaviruses (e.g., Human Papilloma Virus (HPV), polyoma virus); alpha influenza viruses (e.g., influenza a virus subtype H1N 1); liver viruses (e.g., Hepatitis B Virus (HBV)); herpes viruses (e.g., Herpes Simplex Virus (HSV)), Varicella Zoster Virus (VZV), epstein-barr virus (EBV), Cytomegalovirus (CMV)); adenoviruses (e.g., ataadenovirus, avian adenovirus, fish adenovirus, mammalian adenovirus, monkey adenovirus); poxviruses (e.g., smallpox, vaccinia virus (vaccinia virus), vaccinia virus, monkeypox virus, capripox virus, pseudovaccinia virus, bovine papular stomatitis virus; tanapoxvirus, yamusa tumor virus; Molluscum Contagiosum Virus (MCV)); parvoviruses (e.g., adeno-associated virus (AAV), parvovirus B19, human bocavirus, bunavirus, human parv4 Gi); arboviruses (e.g., Hepatitis C Virus (HCV), zika virus, dengue virus); lentiviruses (e.g., Human Immunodeficiency Virus (HIV)); geminiviridae; dwarf virus family; algal deoxyribonuclease family (Phycodnaviridae); and the like. In some embodiments, the target nucleic acid is from a parasite.

The term "sample" is used herein to mean any sample containing nucleic acids. The sample may be derived from any source, for example, the sample may be a synthetic combination of purified nucleic acids, or the sample may be a cell lysate. The sample may be from a patient (e.g., for diagnostic purposes). Suitable samples include, but are not limited to, saliva, blood, serum, plasma, urine, aspirate, and biopsy samples. In some embodiments, the sample obtained from the patient is selected from the group consisting of whole blood, plasma, serum, or a combination thereof. In some embodiments, the sample is an archived blood sample, a fresh blood sample, or a frozen blood sample. In some embodiments, the sample of the present disclosure is a tissue sample. The sample of the present disclosure is from or comprises: amniotic fluid, blood, plasma, serum, semen (semen), lymph, cerebrospinal fluid, ocular fluid (oculus fluid), urine, saliva, stool, mucus, sweat, blood, skin, hair follicle, saliva, oral mucus, vaginal mucus, sweat, tears, epithelial tissue, urine, semen (semen), semen (seminal fluid), seminal plasma, prostatic fluid, Cowper's fluid, fecal matter, vital tissue, ascites fluid, cerebrospinal fluid, and/or lymph. In some embodiments, the sample is a solid sample. In some embodiments, the sample is a liquid sample.

In some cases, the source of the sample is from a cell, liquid, tissue, or organ that is diseased or suspected to be diseased. In some embodiments, the source of the sample is a normal (non-diseased) cell, a liquid, a tissue, or an organ. In some embodiments, the sample is from an individual suspected of having a microbial infection, e.g., a viral infection. For example, the source of the sample can be an individual who may or may not be infected-and the sample can be any biological sample collected from the individual (e.g., blood, saliva, biopsy, plasma, serum, bronchoalveolar lavage, sputum, stool sample, cerebrospinal fluid, fine needle aspirate, swab sample (e.g., oral swab, cervical swab, nasal swab), interstitial fluid, synovial fluid (synovial fluid), nasal discharge, tears, buffy coat (buffy coat), mucosal sample, epithelial cell sample (e.g., epithelial cell scrapings), etc.). In some cases, the sample is a cell-free liquid sample. In some cases, the sample is a liquid sample that may contain cells. Pathogens include viruses, fungi, bacteria, protozoa, and microbial parasites.

Kit or article of manufacture

Also provided herein are kits and articles of manufacture for use in any of the detection and diagnostic methods described herein.

In some embodiments, a kit is provided comprising any of the dry powder compositions described herein. The kit may be useful for performing reverse transcription, RPA, and/or CRISPR/Cas-based nucleic acid detection reactions in a single reaction tube.

In some embodiments, a kit is provided that includes any one of the detection plates described herein. The detection plate may comprise reagents designed to detect one or more target nucleic acids of interest, such as a viral or bacterial genomic nucleic acid sequence.

In some embodiments, kits are provided that include any of the fluorescence detection devices or systems described herein. In some embodiments, the kit further comprises one or more assay plates described herein. The fluorescence detection system can be reused.

Also provided are articles of manufacture comprising any of the compositions, devices, systems, or kits described herein. The article of manufacture may include, for example, a container and a label or package insert on or associated with the container. The article of manufacture may also include other materials desirable from a commercial or user standpoint, including sample collection tubes, capillaries, gloves, buffers, solvents, diluents, filters, needles, injectables, and the like.

The kits and articles of the present application may also include information, for example, in the form of a package insert. The insert or label may take any form, such as paper or on electronic media such as magnetic recording media (e.g., floppy disks), CD-ROMs, and Universal Serial Bus (USB) flash drives. In some embodiments, the kit further comprises instructions for using the kit in any of the detection methods and diagnostic methods described herein.

Examples

The following examples are intended only as illustrations of the present application and therefore should not be construed as limiting the invention in any way. The following examples and detailed description are provided by way of illustration and not by way of limitation.

Example 1 diagnosis of viral infection Using an exemplary Rapid test kit

This example describes the use of a kit comprising an exemplary fluorescence detection device of the invention for the detection of viral infection. The kit is suitable for diagnosis of viral infections (e.g., 2019-nCoV or SARS-CoV2) by medical personnel in secondary hospitals, community hospitals or clinics, airports, and train stations. The kit contains disposable consumables for preparation, sampling, and incubation. The fluorescence detection device can be reused.

The kit comprises: 1.75% ethanol was used for decontamination before and after testing; PE gloves; 3. a mask; 4, cotton swabs; 5. virus Sampling Kit (Virus Sampling Kit, Yocon); 6.20 μ l capillary (including rubber tip); 7. lyophilizing the reagent; 8.3V LED, wavelength 490 nm; 9. biohazard waste bags; 10. detecting a plate; 11. tape or 3D printed PDMS cover; 12. a thermometer; 13. flask and forceps; 14. mobile phone software; CR2032 batteries, battery cases, switches, and electrical wires.

Fig. 1 shows a schematic of an exemplary overall workflow for using the kit. FIG. 15 provides a flow chart summarizing various steps in a workflow.

Samples were obtained from patients suspected of having a viral infection by throat swabs and stored in Virus Sampling tubes (Virus Sampling Kit, Yocon) containing Virus preservation solution. Prior to testing, the samples were incubated at 56 ℃ for 10 minutes to inactivate the virus and release the viral genome (e.g., viral RNA) into the samples.

Mu.l of a sample from a virus stock is added to a reaction cell (212 or 214) containing reagent powder in a test plate (210) using a 20. mu.l capillary tube with a rubber bulb (rubber bulb) on top. The test plate included a reaction cell for negative control to which 20. mu.l of water or virus preservation solution without virus was added. The test plate may also include a reaction cell for a positive control. The reagent powder includes reagents for performing reverse transcription (e.g., reverse transcriptase, target-specific primers, deoxynucleotides), recombinase polymerase amplification (forward and reverse RPA primers for amplification of viral nucleic acid, recombinase, SSB, and strand-displacing polymerase), and CRISPR/Cas-based nucleic acid detection reagents (e.g., Cas12b, guide RNA, FAM-labeled single-stranded detection DNA). The plate was covered by a lid (220) and incubated in an incubator (300) at 42 ℃ for 40 minutes.

A fluorescent signal from the detection plate is detected in a fluorescent detection device (100). The light source of the fluorescence detection device is 3V LED light (120) with a wavelength of 490 nm. The light emitted by the sample is filtered by a filter that selectively transmits light at 550 nm. Images of the reaction cells on the plate were obtained by the mobile phone and analyzed by the mobile application on the phone.

The detection plate, the tweezers, the throat swab, the gloves, the mask, the virus sampling tube and the capillary tube are disposed in biohazard waste and decontaminated by ethanol.

FIGS. 14A-14B show images of two assay plates containing samples with a predetermined concentration of viral RNA. In FIG. 14A, the top and middle pools are negative controls, and the bottom pool contains 1 copy of the target viral RNA per μ L. In FIG. 14B, the top pool is a negative control, the middle pool contains 1 copy of the target viral RNA per μ L, and the bottom pool contains 10 copies per μ L⁸Copies of the target viral RNA.

Claims (29)

1. A system for detecting a fluorescent signal from a detection plate, comprising: a base structure including a support surface; A light emitting diode (LED) unit attached to the base structure, wherein the LED unit includes: LED light source, a conical reflector configured to focus the light beam emitted by the LED light source for transmission along the support surface and through a detection plate placed on the support surface; and a housing configured to enclose the base structure and the LED unit, wherein the housing includes an opening; A mobile computing device including a camera module therein may be attached to the housing to capture images of the detection plate through the opening. 2. The system of claim 1, wherein the conical reflector is configured to focus the light beam such that the light beam is at an angle of less than about 10° relative to the support surface. 3. The system of claim 2, wherein the conical reflector is configured to focus the light beam such that the light beam is parallel to the support surface. 4. The system of any of claims 1-3, further comprising a filter configured to be positioned over the opening. 5. The system of any of claims 1-4, wherein the LED light source has a wavelength of about 470 nm to about 510 nm. 6. The system of claim 4 or 5, wherein the filter selectively transmits light having a wavelength of about 530 to about 580 nm. 7. The system of any of claims 1-6, wherein the housing is a case including a surface having the opening, wherein the mobile computing device can be attached to the surface. 8. The system of any of claims 1-7, further comprising a battery for an LED light source enclosed in the housing. 9. The system of any of claims 1-8, wherein the base structure is detachable from the LED unit. 10. The system of any of claims 1-9, further comprising the detection plate configured to rest on the support surface. 11. The system of any of claims 1-10, wherein the detection plate comprises two or more reaction cells. 12. The system of any of claims 1-11, further comprising the mobile computing device configured to place the camera module over the opening. 13. A detection plate comprising two or more reaction cells, wherein each reaction cell comprises a dry powder composition comprising: (a) Cas nuclease, (b) a guide RNA comprising a sequence that binds to the Cas nuclease and a guide sequence that hybridizes to a target nucleic acid of interest; and (c) a detection DNA comprising a fluorescent tag, wherein the detection DNA is single-stranded and does not hybridize to a guide sequence of a guide RNA, and wherein the fluorescent signal from the fluorescent tag is detectable upon cleavage of the detection DNA . 14. The assay plate of claim 13, wherein the fluorescent label is FAM. 15. The detection plate of claim 14, wherein the dry powder composition further comprises: (a) a recombinase, (b) single-stranded DNA binding protein (SSB); (c) a strand displacement polymerase; (d) forward primers and reverse primers for amplifying the target nucleic acid; and (e) dNTPs. 16. The detection plate of any one of claims 13-15, wherein the dry powder composition further comprises: (a) reverse transcriptase; and (b) Reverse transcription primer. 17. The assay plate of any one of claims 13-16, wherein the volume of each reaction cell is about 20 [mu]l to about 200 [mu]l. 18. A method for detecting target nucleic acid in a sample, comprising: (a) adding the sample to the reaction cell of the detection plate according to any one of claims 13-17; (b) incubating the detection plate at a temperature suitable for Cas cleavage of the target nucleic acid or its amplicon; (c) placing the test plate on the support surface in the system of any one of claims 1-9; (d) attaching a mobile computing device including a camera module to the system to place the camera module over the opening of the system; and (e) capturing an image of the reaction cell using the camera module of the mobile computing device to detect the presence or absence of the target nucleic acid in the sample. 19. The method of claim 18, wherein step (b) comprises incubating the assay plate at about 42°C for about 5 minutes to about 40 minutes. 20. The method of claim 18 or 19, wherein the target nucleic acid is viral DNA, bacterial DNA, fungal DNA, protozoan DNA or algal DNA. 21. The method of claim 18 or 19, wherein the target nucleic acid is viral RNA. 22. The method of claim 21, wherein the viral RNA is RNA from a coronavirus. 23. The method of any one of claims 18-22, wherein the sample is a nasal or throat swab sample. 24. A method of diagnosing a microbial infection in an individual comprising using the method of any one of claims 18-23 to detect a target nucleic acid of a microorganism associated with the microbial infection in a sample from the individual. 25. The method of claim 24, wherein the microbial infection is a bacterial infection or a viral infection. 26. The method of claim 25, wherein the microbial infection is a coronavirus infection. 27. A dry powder composition comprising: (a) a Cas nuclease, (b) a guide RNA, which includes a sequence that binds to the Cas nuclease and a guide sequence that hybridizes to a target nucleic acid of interest; (c) a detection DNA, which includes a fluorescent tag, wherein the detection DNA is a single stranded and does not hybridize to the guide sequence of the guide RNA, and wherein the fluorescent signal from the fluorescent tag is detectable after the detection DNA cleavage; (d) recombinase; (e) single-stranded DNA binding protein (SSB); (f) strand displacement polymerase; (g) forward and reverse primers for amplifying the target nucleic acid; (h) dNTPs; (i) reverse transcriptase; and (j) reverse Record primers. 28. A kit comprising the system of any of claims 1-9 and/or the assay plate of any of claims 13-17. 29. The kit of claim 28, further comprising instructions for detecting a target nucleic acid in a sample or diagnosing a microbial infection in an individual.

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