WO2021229486A1 - Point-of-care device for detection of genetic material - Google Patents

Abstract

Provided are systems and methods for detecting target genetic material based on a smartphone devise and application software for the fluorescence detection obtained from the CRISPR-based assay in a low-cost and low-resource matter. Also provided is an example of the system for sensitive and specific detection of SARS-CoV-2.

Description

POINT-OF-CARE DEVICE FOR DETECTION OF GENETIC MATERIAL

CROSS-REFERENCE TO RELATED APPLICATION This application claims priority to U.S. Provisional Application 63/024,710, filed May

14, 2020 the content of which is hereby incorporated herein by reference for all purposes.

REFERENCE TO A SEQUENCE LISTING The present application makes reference to a Sequence Listing (submitted electronically as a .txt file named “C2741_4_IVPCT.ST25.txt”. The .txt file was generated on April. 4, 2021 and is 1,356 bytes in size. The entire contents of the Sequence Listing are herein incorporated by reference.

TECHNICAL FIELD

The present disclosure relates to systems and devices that may be used for detection of the presence of specific nucleic acids in point-of-care conditions. BACKGROUND OF THE INVENTION

This section is intended to introduce the reader to various aspects of the art that may be related to aspects of the embodiments that are described or claimed below. The discussion is provided as background information to facilitate the understanding of the aspects of the embodiments of the present disclosure. As such, while the statements include useful background, these statements should not be construed as admissions of prior art.

Several medical conditions may be associated with the presence of specific genetic sequence as disease markers. For example, certain viral infections may be associated with the presence of genetic material from the viruses ( e.g ., RNA from RNA or retrovirus, DNA from the virus). In another example, microbial infections (e.g., bacterial infections, protozoan infections, fungal infections, worm infections) may be associated with the presence of genetic material from the responsible pathogen. As a further example, certain types of cancers are associated with specific genetic mutations, which may be found in the genome of the cancer cells of the patient.

In particular situations, fast detection of these genetic sequences in a point-of-care environment may be of great importance. As an example, the Corona Virus Disease of 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), became a world-wide public health emergency due to its particularly fast human-to-human transmission. As such, quick testing and treatment of patients at large scale may be of particular importance for epidemiologic management. However, existing detection methods for SARS-CoV- 2 being deployed may limit its use. A classic method based on virus isolation required up to 3 weeks of turn-around time. Plaque assays that quantify viral titers in patient samples may have a similarly slow turn-around time. These slow techniques may prevent quick identification and treatment of patients. Faster techniques, including the quantitative reverse transcription- polymerase chain reaction (qRT-PCR) techniques, yield results in up to 6 hours required specialized equipment, costly reagents, and technical experts to carry out the procedure. These requirements effectively limit qRT-PCR from its use in low-resource areas. As such, epidemiologic control of certain diseases may be substantially improved by point-of-care systems or devices that may quickly verify the presence of specific disease markers in a low-resource environment. More generally, there is a need for a low-cost, novel system or device to detect specific genetic materials that may provide results quickly in low-resource environments. SUMMARY OF THE INVENTION

The invention is directed to, but not limited to, the following embodiments.

One object of the present invention is to provide a method for identifying a first genetic sequence in a biological sample, the method comprising: collecting the biological sample containing target nucleic acid comprising ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), wherein the target nucleic acid comprises the first genetic sequence; adding at least one synthetic Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) ribonucleic acid (crRNA), wherein the at least one crRNA is designed to hybridize with the first genetic sequence; adding a light emitting probe, comprising a fluorescent probe or a luminescent probe or both; adding a nucleic acid-guided endonuclease, wherein the endonuclease that cuts the target nucleic acid that hybridizes with the at least one crRNA at the first genetic sequence and subsequently activates the light emitting probe; detecting light emitted, thereby identifying the first genetic sequence; wherein, optionally, detecting light emitted comprise, wherein said detecting light emitted optionally comprises_placing the biological sample in a detection device comprising: a sample housing configured to house a sample holder and wherein the sample housing comprises a dark enclosure; and an optical detector configured to record a light emission from the sample holder disposed within the sample housing; and determining identifying the first genetic sequence on the target nucleic acid based, in part, on the light emission from the sample holder comprising the biological sample. In one embodiment, the first enzymatic site of the nucleic-acid guided endonuclease cuts double-stranded deoxyribonucleic acid (dsDNA), the target nucleic acid comprises target RNA, and wherein the method comprises performing a reverse transcriptase reaction on the target RNA to obtain complementary deoxyribonucleic acid (cDNA) corresponding to the target RNA. In another embodiment, amplifying the cDNA is conducted to produce the dsDNA employing an enzymatic amplification method that comprises a recombinase polymerase amplification (RPA) or a polymerase chain reaction (PCR).

In yet another embodiment, the nucleic-acid guided endonuclease comprises a CRISPR associated endonuclease 12a (Casl2a).

In another embodiment, the first enzymatic site of the nucleic-acid guided endonuclease cuts ribonucleic acid.

In another embodiment, the nucleic-acid guided endonuclease comprises CRISPR associated endonuclease 13 (Casl3). In further embodiment, a second genetic sequence is provided, and the method comprises adding a second crRNA designed to hybridize with a second genetic sequence.

In a further embodiment, the light emitting probe comprises the fluorescent probe, and the detection device comprises a light source configured to excite the fluorescent probe.

In another embodiment, the light emitting probe comprises the luminescent probe, and the method comprises adding a substrate for the luminescent probe.

In another embodiment, provided is the first genetic sequence on the target nucleic acid comparing the light emission from the sample holder comprising the biological sample with a second light emission from a second sample holder comprising a control sample, wherein the control sample comprises a positive control sample or a negative control sample. In another embodiment, the method comprises quantifying an amount of the first genetic sequence on the target nucleic acid based on the light emission from the sample holder.

In a further embodiment, the target nucleic acid comprises a genetic sequence present in a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome. A further objective of the present invention is to provide a system comprising: a housing that comprises a dark enclosure; a light source configured to emit light at a sample holder disposed within the housing, wherein the sample holder is configured to hold a fluorescent probe; an optical detector configured to record a light emission from the sample holder disposed within the housing; and a carrier that carries the sample holder, wherein the carrier is configured to travel along the housing.

In another embodiment, the system comprises the housing coupled to a portable computing device or a mobile phone.

In another embodiment, the system comprises the portable computing device or the mobile phone comprising a camera that comprises the optical detector.

In another embodiment, the system comprises the portable computing device or the mobile phone communicatively coupled to the light source or the optical detector or both.

In further embodiment, the system comprises the portable computing device or the mobile phone comprising a non-transitory memory device comprising a series of instructions that, when executed by the portable computing device or the mobile phone, cause the portable computing device or the mobile phone to capture the light emission from the fluorescent probe within the sample holder, wherein the second instruction is simultaneously or subsequently to the first instruction.

In further embodiment the system comprises the series of instructions causing the portable computing device or the mobile phone to compare the captured light emission from the fluorescent probe with a threshold value disposed in the non-transitory memory device.

In another embodiment, the system comprises the series of instructions causing the portable computing device or the mobile phone to compare the captured light emission from the fluorescent probe with a previously captured light emission from a control sample disposed in a second sample holder disposed in the carrier.

In another embodiment, the system comprises the series of instructions causing the portable computing device or the mobile phone to quantify the fluorescent probe within the sample holder based on the captured light emission.

In further embodiment, the system comprises the fluorescent probe comprising an emission wavelength and wherein the optical detector comprises an emission filter specific for the emission wavelength.

In a further embodiment, the system comprises the fluorescent probe comprising an excitation wavelength, and wherein the light source comprises a light emission diode (LED) specific for the excitation wavelength.

In a further embodiment, the system comprises the optical detector comprising a lens.

In another embodiment, the system comprises the housing and the carrier comprising a 3D- printed apparatus. In still a further embodiment, the system comprises the housing and the carrier comprising a black material.

BRIEF DESCRIPTION OF DRAWINGS

A more complete appreciation of the invention and many of the attendant advantages thereof will be readily obtained as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings, wherein:

Fig. 1 depicts the overall workflow of the specific genetic material detection using the CRISPR-based diagnostic and smartphone-based fluorescence readout. Fig. 2 depicts schematic representation of specific primers and crRNAs for spike gene of SARS-CoV-2.

Fig. 3 depicts a limit of detection for SARS-CoV-2 based on RT-RPA and CRISPR- Casl2a assay. Fig. 4 depicts cross-reactivity testing of CRISPR-based assay against several respiratory viruses.

Figs. 5A and 5B show the exploded view of smartphone-based assemblies for the fluorescence imaging of the CRISPR-based assay.

Figs. 6A and 6B show different views of a housing of the smartphone-based device for fluorescence imaging.

Figs. 7A and 7B depict a step-by-step summary of the software for fluorescence detection. These two figures connect at the position of the two asterisks (**).

Figs. 8A-8C show, in combination, a flowchart depicting an application that may interface with the apparatus and perform data analysis in the process of detection of genetic material. Connections between the portions of the flowchart are indicated at numbers 1, 2 and 3.

DETAILED DESCRIPTION OF THE EMBODIMENTS

One or more embodiments of the present disclosure are described below. To provide a concise description of these embodiments, certain features of an actual implementation may be omitted. It should be appreciated that in an actual implementation of the embodiments, as in any engineering or design project, several implementation-specific decisions are made to achieve the developers’ specific goals and, as a result, implementations may vary from one another. Moreover, while a development effort might be complex and time consuming, certain developments would, nevertheless, be a routine undertaking of design, fabrication, and manufacture for those of ordinary skill having the benefit of this disclosure.

When introducing elements of various embodiments of the present disclosure, the articles “a,” “an,” and “the” are intended to mean that there are one or more of the elements. The terms “comprising,” “including,” and “having” are intended to be inclusive and mean that there may be additional elements other than the listed elements. Additionally, references to “one embodiment” or “an embodiment” of the present disclosure are not intended to be interpreted as excluding the existence of additional embodiments that also incorporate the recited features. Furthermore, the phrase A “based on” B is intended to mean that A is at least partially based on B. As discussed above, there is a need for low-cost systems and devices, and method of operation thereof, to determine and/or quantify the presence of specific nucleic acid sequences ( e.g ., deoxyribonucleic acid (DNA) sequences, ribonucleic acid (RNA) sequences) in biological samples. The present disclosure describes, among other things, methods and systems for point-of- care detection of nucleic acids from biological samples that may minimize the resources needed for deployment. To that end, the method and/or the systems may employ enzymatic reactions on a biological sample that may generate fluorescent or luminescent activity on the sample based on the presence of a target sequence, as detailed below.

In some of the following descriptions, systems and methods that may be used to the detection of presence of SARS-CoV-2 in biological samples is used as an illustrative embodiment. It should be noted that these descriptions should not be construed as a limitation of the embodiments described herein to a single application and, instead, should be considered an illustration of the tailoring of the useful and innovative concepts of the embodiments to a single application. Modifications of the embodiments described for other applications are, thus, within of the scope of this application.

In some embodiments, the method may diminish or eliminate the need for purification of the biological samples obtained in the field by employing amplification steps and/or employing a high sensitivity detection system. In some embodiments, the method or system may employ simple equipment for preparation of the samples by employing isothermal reactions at relatively low temperatures. In some embodiments, the system may facilitate adoption and widespread use of the method by employing a low-cost fabrication method and a modular design to obtain a detection apparatus that may be easily coupled to widely available portable computing devices ( e.g ., mobile phones, tablets, portable media players, portable digital cameras). An example of the method is illustrated in Figure 1 which shows the overall workflow of the specific genetic material detection using the CRISPR-based diagnostic and the smartphone-based fluorescence readout.

In some embodiments, the enzymatic reactions may employ nucleic acid-guided endonucleases, such as the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated proteins with a secondary non-specific activity. The CRISPR associated endonuclease may be used along with a synthetic CRISPR RNA (crRNA) that may be used to target the CRISPR associated endonuclease activity to a specific nucleic acid sequence. Specifically, the crRNA may be synthesized to be capable of hybridizing with a nucleic sequence of interest. In such situation, when a CRISPR associated endonuclease forms a complex with a crRNA, it is capable of binding to its primary target, a nucleic acid containing the nucleic sequence of interest.

In the embodiments described herein, the CRISPR associated endonucleases employed may have a secondary non-specific activity, which may be leveraged to activate a light emission reaction or a fluorophore activity. For example, embodiments may employ the CRISPR associated endonuclease 12a (Casl2a) or the CRISPR associated endonuclease 13 (Casl3). Both Casl2a and Casl3 may be capable of non-specific and/or indiscriminate digestion of single stranded DNA (ssDNA) upon binding to its primary target sequence. In this example, by having a fluorescent probe that is quenched by the ssDNA, the secondary activity of Casl2a or Casl3 may activate the fluorescence of the probe.

As the primary target for Casl3 is RNA, Casl3 may be used to detect RNA in a sample. For example, some embodiments targeting the SARS-CoV-2 virus, which is an RNA virus, may employ Casl 3. In contrast, as the primary target for Casl2a is DNA, Casl2a may be used to detect DNA in a sample. Embodiments targeting the SARS-CoV-2 virus may employ reverse transcription reactions to employ Casl 2a. In situations where the target DNA may be small, DNA amplification methods, such as polymerase chain reactions (PCRs) and recombinase polymerase amplification (RPA), may be used to increase the sensitivity. As such, a reverse transcriptase may be used in combination with a DNA polymerase ( e.g ., for PCR or RPA) to detect, for example, the SARS-CoV-2 virus with high sensitivity, using Casl2a. As discussed above, synthetic crRNA that is capable to hybridize with the target nucleic acid may be designed. For example, in a system or method for detection of SARS-CoV-2, two crRNAs may be designed, as detailed in Figure 2 and Table 1, below. A first crRNA may be specific to a conserved sequence universal to the family of coronaviruses (e.g., SARS-CoV-1, MERS-CoV). A second crRNA may be specific to the SARS-CoV-2. A suitable target sequence used for designing the crRNA may be a sequence from the virus genome located in the vicinity of a protospacer adjacent motif (PAM) sequence (e.g., 5’-TTTV-3’) or of a PAM sequence complement (5’-BAAA-3’). The crRNA may be produced by placing the designed sequence in a DNA form, near a T7 promotor. Upon an in vitro transcription reaction, the crRNA for the application may be created. The two sequences for the crRNA may be located in relative proximity in a common region of interest of the viral RNA, and the amplification primers employed may be chosen to amplify the region of interest as detailed in Figure 2 and Table 1.

EXAMPLES

Example 1

Primers and crRNA specific to SARS-CoV-2

The invention is further described in Example lfor SARS-CoV-2 detection [PMID: 33153299] using primers and crRNA specific to SARS-CoV-2. As shown in Table 1 and Figure 2, the primers and crRNA specific to SARS-CoV-2 were designed to target the Spike gene.

Oligonucleotides for crRNA template (4 mM) were annealed to T7 promoter primer (4 mM) in T4 DNA Ligase Buffer and incubated in the following condition; 95°C for 3 min, 65°C for 3 min, 42°C for 5 min, and 37°C for 45 min. Then crRNA was transcribed by using Riboprobe In Vitro Transcription Systems according to the manufacturer protocol. Transcribed crRNA (approximately 40 nucleotides) was purified and quantified for further use. The process for detecting specific genetic materials illustrated in Figure 1 above may begin with reverse transcription to converse RNA into cDNA. The target genetic material may also be amplified using RPA or PCR. In some embodiments using the RPA amplification, the sample may be incubated at 39 °C, whereas in embodiments using PCR amplification, the sample may be placed in a thermocycler. The biological sample treated with the amplification process, the crRNA, the Casl2a, and the fluorescent probe may be tested in the dark for fluorescence using a fluorescent detection apparatus. In some embodiments, the results may be assessed by comparing the fluorescence of the biological sample being tested with positive and negative controls. If both the sample and positive control tubes provide comparable fluorescence, the patient is tested positive for the target genetic material.

Example 2

Isothermal amplification and CRISPR-based detection of SARS-CoV-2 The invention is further described with the example for SARS-CoV-2 detection [PMID: 33153299] Isothermal reverse transcription and amplification were conducted by the RT-RPA technique. Briefly, each detection assay consisting of 0.48 mM forward primer, 0.48 mM reverse primer (Table 1), 200 U RevertAid Reverse Transcriptase, and rehydration buffer was mixed with the lyophilized reaction of the TwistAmp Basic Kit. Then 1-5 pL of RNA template and 14 mM MgOAC were added before incubating at 39°C for 30 min followed by heat inactivation at 75°C for 5 min. The reaction of CRISPR-Casl2a-based- nucleic acid detection consisted of 30 nM crRNA, 330 nM of EnGen^® Lba Casl2a (Cpfl), 200 nM fluorescent reporter/quencher probe, 1 x reaction buffer and 1 pL of RPA product in a final volume of 15 pL. The reaction was incubated at 39°C for 15 min, and then the fluorescent signal was measured. Limit of detection (LOD) testing can be used to indicate the sensitivity of the detection method by determining the lowest concentration of viral RNA that may produce a positive result. The detection limit testing may be done by carrying out an in vitro transcription on a positive control to create an RNA standard. From the RNA standard, serial dilution may be carried out, followed by the reverse transcription process, RPA and viral detection via Casl2a, in a process similar to that illustrated in Figure 1.

Example 3

Limit of detection for SARS-CoV-2 based on RT-RPA and CRISPR-Casl2a The invention is further described with the example for SARS-CoV-2 detection [PMID: 33153299] Standard RNA was prepared by using T7 Riboprobe^® in vitro transcription systems.

The limit of detection was performed in triplicate by using a 10-fold serial dilution of each standard in vitro transcribed RNA (ranging from 10⁷ to 10 copies/reaction) as templates for RT-RPA with CRISPR-Casl2a. The limit of detection was observed from reaction tubes containing the lowest concentration of RNA template that yielded fluorescent signal. The results revealed that the limit of detection for SARS-CoV-2 was approximately 10 copies/reaction (Figure 3).

Cross-reactivity testing may be used to indicate the specificity of the detection method. Such method may be done by testing, using a process similar to that illustrated in Figure 1 with SARS-CoV-2-specific crRNA and then tested against a sample containing human DNA samples extracted from the respiratory tract via nasal swab. The cross-reactivity test may also include samples of different viruses or human tissue infected with other respiratory viruses. In some embodiments, at least 3 other viral samples are used to determine adequate specificity of the method to detect SARS-CoV-2. Example 4

Cross-reactivity testing for SARS-CoV-2 detection The invention is further described with the example for SARS-CoV-2 detection [PMID: 33153299] The assays were tested against several human respiratory viruses, including the Human coronavirus (strain 229E, HKU1, OC43 and NL63), Respiratory Syncytial Virus (RSV), Human Bocavirus (HBoV), Human Rhinovirus (HRV), Human Parainfluenza virus (HPIV), Human Adenovirus (HAdV), Human metapneumovirus (hMPV), Influenza A virus (H1N1 and H3N2 subtypes) and Influenza B virus (Victoria and Yamagata lineages). The results demonstrated that the CRISPR-based assay against several respiratory viruses yielded very high specificity without any cross-reactivity with other respiratory viruses (Figure 4).

Clinical performance can be used to indicate the sensitivity, specificity and accuracy of the detection method for testing with clinical samples. Such method may be done by testing against clinical samples, using a process similar to that illustrated in Figure 1 with SARS-CoV-2-specific crRNA and then compare the results with the standard technique (real-time PCR). Example 5

Clinical performance of the CRISPR-based assay for SARS-CoV-2 detection The invention is further described with the example for SARS-CoV-2 detection [PMID: 33153299] Nasopharyngeal swab samples were collected from patients with influenza-like illness. The viral RNA was extracted from 200 pL of swab samples using a viral RNA extraction kit following manufacturing instruction. To evaluate the clinical performance of the CRISPR- based assay for SARS-CoV-2 detection, 164 RNA extracted from clinical samples were tested. The results of the assay were compared with the gold standard real-time PCR assay. The performances of the assay were calculated by a web-based calculator (hypertext transfer protocol secure://www.medcalc.org/calc/diagnostic_test.php). The clinical performance of the CRISPR- based assay for SARS-CoV-2 detection yielded very high specificity (100%), sensitivity (96.23%) and diagnostic accuracy (98.78%), as summarized in Table 2.

Table 2. The clinical performance of CRISPR-based assay for SARS-CoV-2 detection.

Modular detection. The methods disclosed herein may employ a modular detection apparatus that may be coupled ( e.g ., mounted) to portable electronic devices (e.g., smartphones, mobile phones, media players, tablets) that are widely available. The wide availability of these electronic devices may substantially decrease the cost of the nucleic acid detection method. For example, a result of such use may include the increased access to diagnostic tests from smaller hospitals with limited budgets. Moreover, the ability to employ mobile phone processing or cloud computing to process signals and/or images obtained from the mobile cameras may facilitate triaging patients with low levels of infection from patients with high level of infection, and the knowledge of the virus quantity can allow staging the patient’s infection progression, and guide the standard of care of the patient

Figures 5A-5B illustrate an embodiment of an apparatus that may be used to detect fluorescent samples. The detection apparatus illustrated includes three parts: 1) a light source, 2) a camera detector equipped with an optical filter specific for assay fluorescence, and 3) sample housing. Specifically, the light source may be a light emitting diode (LED) light source. The light source illuminates a sample housing that may include one or more sample holders with transparent or translucid walls ( e.g ., test tubes, conical tubes, centrifuge tubes, thermocycler tubes, Eppendorf tubes). The light source and the optical filter may be specific to the fluorescent probe employed in the sample. A focusing lens, a camera housing, and mobile phone camera may be aligned to image fluorescent emissions from the sample. The light source and the optical filter may be suited based on the fluorescent probe. In one embodiment, in which the fluorophore is 6-carboxyfluorescein (6-FAM), which has excitation maximum at 495 nm and emission maximum at 520 nm. In this example, the LED light source employed may emit light in a narrow bandwidth around wavelengths near 495 nm. The filter may also have a narrow bandwidth around 520 nm. Such choice decreases diffuse light from the LED from reaching the camera. In addition, the light source and the detector may be positioned such that the excitation path is perpendicular to the emission path, in order to further mitigate bleed- through. An optical diffuser may also be installed between the light source and sample housing in order to ensure reliable excitation intensity. In some embodiments, a battery-powered LEDs may facilitate the modularity of the system. In other embodiments, the light source may draw power from the coupled portable electronic device.

The detector system may include an optical filter, a focusing lens, and a mobile phone camera. Upon excitation, 6-FAM fluoresces at 520 nm emission maximum. The emission may be discriminated through the optical filter, concentrated by the focusing lens, and detected by the mobile phone camera, as illustrated in Figures 5A-5B. The optical filter minimizes background and other interfering lights to maximize accuracy and precision of the system. Mobile phone camera was chosen for this project due to its prevalence and compatibility with many existing image processer software. Figure 6 illustrates a perspective view of the housing of the smartphone-based device for fluorescence imaging. The housing may have a dark enclosure to protect samples from ambient light. The illustrated device shows a carrier capable of carrying 8-sample holders. The carrier may travel along the housing, as illustrated by the red arrows. The design illustrated in Figures 6A and 6B may be 3D-printed to facilitate widespread adoption and to reduce production costs. It should be understood that other designs that may be used to increase the number of samples in the carrier or to house different types of sample carriers are also embodiments of this disclosure.

The images and/or data obtained from the camera may be stored and/or processed automatically on servers ( e.g ., cloud-based servers). The images and/or data may be used to train a software that may employ artificial intelligence methods or techniques. In some embodiments, image processing may employ computer vision and/or machine learning programs or modules. For example, automatic segmentation, localization, edge detection, and/or background subtraction may be performed, as illustrated in Figure 7 A and 7B. The processing method may employ over 100 test images to improve accuracy and precision. Viral titers may be calculated for each sample based on standard curves previously determined. The results may be communicated directly to health care providers over the internet or through cellular networks.

As discussed above, the device may be mounted to a portable electronic device. In some embodiments, the portable electronic device may be communicatively coupled to one or more modules (e.g., light source, camera detector, filters) of the detection apparatus. The coupling may take place through a wired interface (e.g., Ethernet, Universal Serial Bus (USB), Serial Interface, Parallel Interface, Firewire, etc.), or through a wireless interface (e.g., Wi-Fi (IEEE 802.11), Bluetooth, etc.) either directly or through a hub, router, a network, or any other bridging interface. Moreover, the portable electronic device may include a memory that contains software (e.g., an application, an app) to control one or more modules of the detection apparatus, to collect images or light emissions, and to process it. Figures 8A-8C, in combination, depict a flow chart that illustrates an example of operation for an application that may interface with the apparatus and perform data analysis in the process of detection of genetic material disclosed herein. Once captured by the phone camera, the images are processed either on the phone or sent to be processed on the server. Taking advantage of the spatial stability of the system, the image is first cropped around the expected position of the test tube at its center.

This image acquired from the device may be processed by a segmentation algorithm that will extract the pixels corresponding to the liquid inside the tube. This may be achieved in three steps: (1) the image is passed into an edge detector model that uses pixel wise forest classification to create an edge map, enhancing the border of the tube as well as the surface line of the liquid; (2) the edge map is then segmented into a fixed number of regions using morphological Tree operations; and (3) the combination of shapes corresponding to the liquid is extracted using shape matching methods and intensity thresholding. The fluorescence of the tube is then analyzed by computing descriptors in the HSV color space. The color angles between the background and the foreground pixels are computed as well as the fluorescence levels. Those descriptors are combined and transformed into a probability of virus infection. An appropriate probability threshold is then determined using a gallery of images taken with different known concentration of virus copies. The embodiments described in the present disclosure may be susceptible to various modifications and alternative forms, and specific embodiments have been shown by way of example in the drawings and have been described in detail herein. It should, however, be understood that the disclosure is not intended to be limited to the particular embodiments disclosed and that the disclosure covers all modifications, equivalents, and alternatives falling within the spirit and scope of the embodiments as defined by the following appended claims. In addition, the techniques and systems claimed herein refer to practical and useful embodiments and may be applied to concrete examples of a practical nature that demonstrably improve the present technical field and, as such, are not abstract, intangible or purely theoretical. Moreover, in claims that are appended to the end of this specification and contain one or more elements designated as “means for [performing a function] ... ” or “step for [performing a function] ... ,” it is intended that such elements are to be interpreted under 35 U.S.C. 112(f). For any claims containing elements designated in any other manner, however, it is intended that such elements are not to be interpreted under 35 U.S.C. 112(f).

This application contains a sequence listing which forms an integral part of this disclosure. The .txt file named “C2741_4_IVPCT.ST25.txt" was generated on April. 4, 2021 and is 1,356 bytes in size.

Claims

CLAIMS What is claimed is:

1. A method for identifying a first genetic sequence in a biological sample, the method comprising: collecting the biological sample containing a target nucleic acid comprising ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), wherein the target nucleic acid comprises the first genetic sequence; adding at least one synthetic Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) ribonucleic acid (crRNA), wherein the at least one crRNA is designed to hybridize with the first genetic sequence; adding a light emitting probe, comprising a fluorescent probe or a luminescent probe or both; adding a nucleic acid-guided endonuclease that cuts the target nucleic acid, which hybridizes with the at least one crRNA at the first genetic sequence, and subsequently activates the light emitting probe; and detecting light emitted, thereby identifying the first genetic sequence; wherein, optionally, detecting light emitted comprises: placing the biological sample in a detection device comprising: a sample housing configured to house a sample holder and wherein the sample housing comprises a dark enclosure; and an optical detector configured to record a light emission from the sample holder disposed within the sample housing; and identifying the first genetic sequence on the target nucleic acid based, in part, on the light emission from the sample holder comprising the biological sample.

2. The method of claim 1, wherein the first enzymatic site of the nucleic-acid guided endonuclease cuts double-stranded deoxyribonucleic acid (dsDNA), the target nucleic acid comprises target RNA, and wherein the method comprises performing a reverse transcriptase reaction on the target RNA to obtain complementary deoxyribonucleic acid (cDNA) corresponding to the target RNA.

3. The method of claim 2, comprising amplifying the cDNA to produce the dsDNA employing an enzymatic amplification method that comprises a recombinase polymerase amplification (RPA) or a polymerase chain reaction (PCR).

4. The method of claim 2, wherein the nucleic-acid guided endonuclease comprises a CRISPR associated endonuclease 12a (Casl2a).

5. The method of claim 1, wherein the first enzymatic site of the nucleic-acid guided endonuclease cuts ribonucleic acid.

6. The method of claim 5, wherein the nucleic-acid guided endonuclease comprises

CRISPR associated endonuclease 13 (Casl3).

7. The method of claim 1, wherein the target nucleic acid comprises a second genetic sequence, and wherein the method comprises adding a second crRNA designed to hybridize with a second genetic sequence.

8. The method of claim 1, wherein the light emitting probe comprises the fluorescent probe, and wherein the detection device comprises a light source configured to excite the fluorescent probe.

9. The method of claim 1, wherein the light emitting probe comprises the luminescent probe, and wherein the method comprises adding a substrate for the luminescent probe.

10. The method of claim 1, wherein identifying the first genetic sequence on the target nucleic acid comprises comparing the light emission from the sample holder comprising the biological sample with a second light emission from a second sample holder comprising a control sample, wherein the control sample comprises a positive control sample or a negative control sample.

11. The method of claim 1 , comprising quantifying an amount of the first genetic sequence on the target nucleic acid based on the light emission from the sample holder.

12. The method of claim 1, wherein the target nucleic acid comprises a genetic sequence present in a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome.

13. A system comprising: a housing that comprises a dark enclosure; a light source configured to emit light at a sample holder disposed within the housing, wherein the sample holder is configured to hold a fluorescent probe; an optical detector configured to record a light emission from the sample holder disposed within the housing; and a carrier that carries the sample holder, wherein the carrier is configured to travel along the housing.

14. The system of claim 13, wherein the housing is coupled to a portable computing device or a mobile phone.

15. The system of claim 14, wherein the portable computing device or the mobile phone comprises a camera that comprises the optical detector.

16. The system of claim 13, wherein the portable computing device or the mobile phone is communicatively coupled to the light source or the optical detector or both.

17. The system of claim 16, wherein the portable computing device or the mobile phone comprises a non-transitory memory device comprising a series of instructions that, when executed by the portable computing device or the mobile phone, cause the portable computing device or the mobile phone to capture the light emission from the fluorescent probe within the sample holder, wherein the second instruction is simultaneously or subsequently to the first instruction.

18. The system of claim 17, wherein the series of instructions cause the portable computing device or the mobile phone to compare the captured light emission from the fluorescent probe with a threshold value disposed in the non-transitory memory device.

19. The system of claim 17, wherein the series of instructions cause the portable computing device or the mobile phone to compare the captured light emission from the fluorescent probe with a previously captured light emission from a control sample disposed in a second sample holder disposed in the carrier.

20. The system of claim 17, wherein the series of instructions cause the portable computing device or the mobile phone to quantify the fluorescent probe within the sample holder based on the captured light emission.

21. The system of claim 13, wherein the fluorescent probe comprises an emission wavelength, and wherein the optical detector comprises an emission filter specific for the emission wavelength.

22. The system of claim 13, wherein the fluorescent probe comprises an excitation wavelength, and wherein the light source comprises a light emission diode (LED) specific for the excitation wavelength.

23. The system of claim 13, wherein the optical detector comprises a lens.

24. The system of claim 13, wherein the housing and the carrier comprises a 3D- printed apparatus.

25. The system of claim 13, wherein the housing and the carrier comprises a black material.

26. The method of claim 1 , wherein the primers comprise the nucleic acid sequences of

SEQ ID NOS: 1 and 2.

27. The method of claim 1 , wherein the primers comprise the nucleic acid sequences of SEQ ID NOS: 3 and 4.

28. The method of claim 1, wherein the crRNA comprises the nucleic acid sequence of SEQ ID NO: 5

29. The method of claim 1 , wherein the crRNA comprises the nucleic acid sequence of SEQ ID NO: 6.