Patent Application
20210291176
The present invention generally relates to diagnostic devices, systems, and methods for detecting the presence of a target nucleic acid sequence.
The ability to rapidly diagnose diseases—particularly highly infectious diseases—is critical to preserving human health. As one example, the high level of contagiousness, the high mortality rate, and the lack of a treatment or vaccine for the coronavirus disease 2019 (COVID-19) have resulted in a pandemic that has already infected millions and killed hundreds of thousands of people. The existence of rapid, accurate COVID-19 diagnostic tests could allow infected individuals to be quickly identified and isolated, which could assist with containment of the disease. In the absence of such diagnostic tests, COVID-19 may continue to spread unchecked throughout communities.
In some embodiments, a diagnostic test includes a housing, a first blister chamber formed in the housing containing a first reagent and a lateral flow assay strip disposed in the housing. In some embodiments, the diagnostic test also includes a fluidic channel between the blister chamber and the lateral flow assay strip, and a seal positioned between the first blister chamber and the lateral flow assay strip. Applying a threshold force to the first blister chamber is configured to open the seal to fluidly connect the first blister chamber to the fluidic channel.
In some embodiments, a method of performing a diagnostic test includes depositing a sample into a first blister chamber through a sample port formed in a housing, allowing the sample to react with a first reagent in the first blister chamber to form a first solution, applying a threshold force to a first blister chamber to break a first frangible seal holding the first solution in the first blister chamber, and allowing the first solution to flow toward a lateral flow assay strip disposed in the housing.
In some embodiments, a method of making a diagnostic test includes placing a first reagent in a first blister chamber, placing a second reagent in a second blister chamber, positioning a first seal between the first blister chamber and the second blister chamber, and placing a lateral flow assay strip in a third chamber. In some embodiments, the method also includes positioning a second seal between the second blister chamber and the lateral flow assay strip.
It should be appreciated that the foregoing concepts, and additional concepts discussed below, may be arranged in any suitable combination, as the present disclosure is not limited in this respect. Further, other advantages and novel features of the present disclosure will become apparent from the following detailed description of various non-limiting embodiments when considered in conjunction with the accompanying figures.
The accompanying drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component that is illustrated in various figures may be represented by a like numeral. For purposes of clarity, not every component may be labeled in every drawing. In the drawings:
Conventional nucleic acid tests for various diseases requires trained medical professional to collect samples and process those samples in a sterile environment in a laboratory. Such a process is time consuming, resulting in a delay in providing results to patients. Additionally, such tests require a patient to visit a location where a sample may be collected and transported in a sterile manner to an appropriate processing location. Travel to and from locations may risk spread of the disease being tested for and may inadvertently expose medical personnel to the disease.
As the COVID-19 pandemic has highlighted, there is a critical need for rapid, accurate systems and methods for diagnosing diseases—particularly infectious diseases. In the absence of diagnostic testing, asymptomatic infected individuals may unknowingly spread the disease to others, and symptomatic infected individuals may not receive appropriate treatment. With testing, however, infected individuals may take appropriate precautions (e.g., self-quarantine) to reduce the risk of infecting others and may receive targeted treatment as helpful.
While diagnostic tests for various diseases, including COVID-19, are known, such tests often require specialized knowledge of laboratory techniques and/or expensive laboratory equipment. For example, polymerase chain reaction (PCR) tests generally require skilled technicians and expensive, bulky thermocyclers. In addition, there is a need for diagnostic tests that are both rapid and highly accurate. Known diagnostic tests with high levels of accuracy often take hours, or even days, to return results, and more rapid tests generally have low levels of accuracy. Many rapid diagnostic tests detect antibodies, which generally can only reveal whether a person has previously had a disease, not whether the person has an active infection. In contrast, nucleic acid tests (i.e., tests that detect one or more target nucleic acid sequences) may indicate that a person has an active infection.
In view of the above, the inventors have recognized the benefits of a rapid diagnostic test that is usable by user who may not be medical professionals. In particular, the inventors have recognized the benefits of a rapid diagnostic tests employing fluid reservoirs having blister chambers and seals that may be easily punctured to fluidly connect various elements of the rapid diagnostic test in sequence while maintaining sterility. Such a rapid diagnostic test including blister chambers may allow users to perform tests and receive results in a rapid manner without necessarily requiring input from trained medical staff. Telemedicine, or applications may be employed to further enhance the usability of the rapid diagnostic test, such that a variety of diseases such as COVID-19, influenza, (or any target nucleic acid) may be tested for in an at-home or point-of-care environment.
The present disclosure provides diagnostic devices, systems, and methods for rapidly detecting one or more target nucleic acid sequences (e.g., a nucleic acid sequence of a pathogen, such as SARS-CoV-2 or an influenza virus). A diagnostic system, as described herein, may be self-administrable and comprise a sample-collecting component (e.g., a swab) and a diagnostic device. The diagnostic device may comprise a blister pack detection device, according to some embodiments. In some cases, the diagnostic device comprises a detection component (e.g., a lateral flow assay strip), results of which are self-readable, or automatically read by a computer algorithm. In certain embodiments, the diagnostic device further comprises one or more reagents (e.g., lysis reagents, nucleic acid amplification reagents, CRISPR/Cas detection reagents). In certain other embodiments, the diagnostic system separately includes one or more reaction tubes comprising the one or more reagents. The diagnostic device may also comprise an integrated heater, or the diagnostic system may comprise a separate heater. The isothermal amplification technique employed yields not only fast but very accurate results.
Provided herein are a number of diagnostic tests useful for detecting target nucleic acid sequences. According to exemplary embodiments described herein, it may be desirable to selectively move solutions contained in different chambers of a diagnostic test. In particular, the inventors have recognized that moving reagents through a diagnostic test at specific times in a sterile manner may provide rapid, accurate results. Accordingly, the inventors have recognized the benefits of employing in a diagnostic test a blister pack including one or more blister chambers. The diagnostic tests including one or more blister chambers, as described herein, are able to be performed in a point-of-care (POC) setting or home setting without specialized equipment. In some aspects, a cartridge or housing includes one or more blister chambers that enable reliable, sterile transmission of one or more solutions throughout a testing process. The blister chambers may allow a user to apply a threshold force to an exterior of a blister chamber to break a frangible seal and/or transfer the contents of the blister chamber to another portion of the diagnostic test. As the force may be applied externally and no tools may be required to transfer solutions through the diagnostic test, the internal sterility of the diagnostic test may be maintained throughout a testing process. Multiple blister chambers may be arranged in sequence so that the steps of performing a diagnostic test, including fluid transfers to a lateral flow assay strip, are simple for an at-home user.
In some embodiments of the present technology, a blister pack is described, which blister pack may be used as part of a diagnostic test. In some embodiments, a blister pack may comprise one or more chambers, in which each chamber may be a “blister” of the blister pack. In some cases, each chamber may comprise one or more reagents (e.g., lysis reagents, nucleic acid amplification reagents, and the like) and/or one or more buffers (e.g., a dilution buffer). In certain, a chamber may be separated from an adjacent chamber by a breakable seal (e.g., a frangible seal) or a valve (e.g., a rotary valve). The blister pack described may be used in any diagnostic test with which it can be advantageous, including the exemplary test described herein.
Diagnostic devices and systems described herein may comprise any number of blister packs, arranged in such a way so as to process a sample as described herein. In some embodiments, the blister packs may comprise one or more seals (e.g., differential seals, frangible seals) that allow reagents to be delivered in a controlled manner (e.g., using differential seal technology). In some embodiments, a frangible seal may be formed of a metal foil, elastomeric film, flexible plastic, or any other suitable breakable material. In some embodiments, the blister packs may comprise one or more chambers, where each chamber may comprise one or more reagents. In certain embodiments, one or more chambers may store one or more reagents in solid form (e.g., lyophilized, dried, crystallized, air jetted, etc.), and one or more chambers may store one or more reagents and/or buffers in liquid form. In some cases, a chamber comprising one or more reagents in solid form may be separated from a chamber comprising one or more reagents and/or buffers in liquid form by a seal (e.g., a frangible seal). In some cases, breaking the frangible seal may result in the one or more solid reagents being suspended in the one or more liquid reagents and/or buffers. In some cases, the suspended solid reagent(s) may be added to a sample.
In some embodiments of the present technology, the delivery of each reagent in a blister pack may be fully automated. For example, the user may insert a sample in a sample collection region of the blister pack and then activate the blister pack. Upon activation, all of the reagents may be added to the sample in the correct amount and at the appropriate time, such that the sample is processed as described herein. In some embodiments, the blister pack may further comprise a detection component (e.g., a lateral flow assay strip). The detection component may alert the user as to whether the sample was positive or negative for the target nucleic acid sequence.
In some embodiments, a diagnostic test includes a housing having a first blister chamber containing a first reagent. The diagnostic test also includes a lateral flow assay strip disposed in the housing. A fluidic channel selectively connects the blister chamber and the lateral flow assay strip, such that a solution may flow from the blister chamber to the lateral flow assay strip. A seal positioned between the blister chamber and the lateral flow assay strip (e.g., in the fluidic channel) is configured to prevent the solution from flowing to the lateral flow assay strip until the seal is opened. The blister chamber may be configured to receive an external force, where when the external force reaches a threshold force the seal opens and allows a solution inside of the first blister chamber to flow toward the lateral flow assay strip. In some embodiments, the diagnostic test may include additional blister chambers positioned between the first blister chamber and the lateral flow assay strip. In such an embodiment, application of the threshold force to the first blister chamber may transfer a solution contained therein to an adjacent blister chamber between the first blister chamber and the lateral flow assay strip. Accordingly, a threshold force may then be applied to the next blister chamber and so on until a final solution is transferred to the lateral flow assay strip. Of course, in some embodiments, one or more blister chambers may not be sequentially connected. For example, in one embodiment a plurality of blister chambers may be connected to an amplification blister chamber, where solutions from the plurality of blister chambers are pooled in the amplification blister chamber. Of course, combinations of the above arrangements are also contemplated, where some blister chambers are arranged in sequence and pool with at least one non-sequential chamber in a separate central chamber.
According to exemplary embodiments described herein, multiple blister chambers may be employed in a diagnostic test. In some embodiments, a diagnostic test may include two blister chambers. In other embodiments, a diagnostic test may include three blister chambers, four blister chambers, or five blister chambers. A diagnostic test may also include any suitable number of fluid chambers that are not arranged as blisters or are otherwise not configured to receive an external force. Non-blister chambers and blister chambers may be combined in any suitable number and arrangement in a diagnostic test. In some embodiments, a lateral flow assay strip of a diagnostic test may be disposed in a blister chamber. In other embodiments, a lateral flow assay strip may be disposed in a non-blister chamber.
According to exemplary embodiments described herein, a blister chamber of a diagnostic test may include a sample port configured to receive a sample from a patient. The sample port may be configured to receive a sample from various testing arrangements. For example, the sample port may be configured to receive a swab. In some embodiments, the sample port may be configured as a septum configured to open when force is applied to the septum with the sample. Accordingly, the sample may be taken from a patient and then easily inserted into the blister chamber through the septum. In other embodiments, a blister chamber may include a removable cap which is removed to allow a sample to be deposited in the blister chamber. In some embodiments, the sample port may include a frangible seal that is broken by the sample or another puncturing tool. Of course, any suitable cap or seal may be employed to form a sample port through which a sample may be deposited in a blister chamber, as the present disclosure is not so limited.
According to exemplary embodiments described herein, a blister chamber may include a reagent forming a component of a diagnostic test. A reagent may be a liquid solution or may be in solid form. For example, in some embodiments a reagent may be buffer solution or an amplification solution. In some embodiments, a reagent may be a lyophilized solid, where the solid is configured to dissolve in a solution contained in a connected blister chamber. The reagent may be a solid or liquid amplification reagent, buffer reagent, lysis reagent, or another desired reagent.
In some embodiments, a blister chamber may be configured to receive an external force to open a seal separating the blister chamber from a fluidic channel or lateral flow assay strip. Depending on the particular reagent and/or feeling for the end user, a blister chamber may be formed of a material that provides feedback to the user in combination with a seal. In some embodiments, the blister chamber may be formed of a rigid film material (e.g., metal foil), which provides a rigid feeling when applying force to the blister chamber. In other embodiments, the blister chamber may be formed of a flexible film material (e.g., an elastomeric or flexible plastic material). Of course, a blister chamber may be formed of any suitable material, as the present disclosure is not so limited.
According to exemplary embodiments described herein, a method of performing a diagnostic test includes depositing a sample into a blister chamber through a sample port formed in a housing. In some embodiments, depositing the sample in the blister chamber may include pushing the sample through a septum forming the sample port. In the method may also include allowing the sample the react with a first reagent in the blister chamber to form a first solution. The first reagent may be a sample buffer solution. The method may also include applying a threshold force to an external portion of the blister chamber to break a frangible seal holding the first solution inside the blister chamber. Once the seal is broken, the solution may be forced and/or allowed to flow toward a lateral flow assay strip disposed in the housing. In some embodiments, once the seal of the first blister chamber is opened, the first solution may flow into a second blister chamber. The second blister chamber may include a second reagent configured to react with the first solution. Like the first blister chamber, the second blister chamber may be configured to receive an external force. When a second threshold force is applied to the second blister chamber, a second seal may be opened to allow the solution contained inside of the second blister back. In embodiments where more than two blister packs are employed, the process of applying a threshold force to the additional blister chambers may be repeated to sequentially release solutions and/or solid reagents.
In some embodiments, a method of making a diagnostic test includes placing a first reagent in a first blister chamber and placing a second reagent in the second blister chamber. In some embodiments, the first and second reagents may be liquid solutions, lyophilized solids. In some cases, one of the first blister chamber and second blister chamber may include a liquid solution, and the other may include a lyophilized solid. In some embodiments, the liquid solution in one of the blister chambers may be configured to hydrate a lyophilized solid in another blister chamber. The method may also include positioning a first seal between the first blister chamber and the second blister chamber. When the first seal is opened (e.g., by application of an external threshold force the first blister chamber and/or second blister chamber) the first blister chamber may be fluidly connected to the second blister chamber. The method may also include placing a lateral flow assay strip in a third chamber and placing a second seal between the second blister chamber and the lateral flow assay strip Like the first seal, opening the second seal may fluidly connect the second blister chamber to the lateral flow assay strip.
It should be noted that while in exemplary embodiments described herein a threshold force is externally applied to a blister chamber to open a seal, in other embodiments a puncturing tool or external force may instead be applied to the seal itself to open the seal. In this regard, a diagnostic test is not limited to external force being applied to the blister chamber to open the seal, and any suitable opening arrangement may be employed.
In some embodiments, a diagnostic device comprises one or more blister packs. In some embodiments, a blister pack comprises one or more chambers. In some cases, each chamber may comprise one or more reagents (e.g., lysis reagents, nucleic acid amplification reagents) and/or one or more buffers (e.g., dilution buffer). In certain, a chamber may be separated from an adjacent chamber by a breakable seal (e.g., a frangible seal) or a valve (e.g., a rotary valve).
Diagnostic devices and systems described herein may comprise any number of blister packs, arranged in such a way so as to process a sample as described herein. In some embodiments, the blister packs comprise one or more seals (e.g., differential seals, frangible seals) that allow reagents to be delivered in a controlled manner (e.g., using differential seal technology) or an uncontrolled manner (e.g., using a burstable, frangible seal). In some embodiments, the blister packs comprise one or more chambers, where each chamber comprises one or more reagents. In certain embodiments, one or more chambers store one or more reagents in solid form (e.g., lyophilized, dried, crystallized, air jetted), and one or more chambers store one or more reagents and/or buffers in liquid form. In some cases, a chamber comprising one or more reagents in solid form may be separated from a chamber comprising one or more reagents and/or buffers in liquid form by a seal (e.g., a frangible seal). In some cases, breaking the frangible seal may result in the solid reagents being suspended in the one or more liquid reagents and/or buffers. In some cases, the suspended solid reagents may be added to a sample.
In some embodiments, the delivery of each reagent in a blister pack is fully automated. For example, the user may insert a sample in a sample collection region of the blister pack and then activate the blister pack. Upon activation, all of the reagents may be added to the sample in the correct amount and at the appropriate time, such that the sample is processed as described herein. In some embodiments, the blister pack further comprises a detection component (e.g., a lateral flow assay strip). The detection component may alert the user as to whether the sample was positive or negative for the target nucleic acid sequence.
Turning to the figures, specific non-limiting embodiments of diagnostic tests including one or more blister chambers are described in further detail. It should be understood that the various features and methods of the blister chambers described relative to these embodiments may be used either individually and/or in any desired combination, as the disclosure is not limited to only the specific embodiments described herein.
One embodiment is shown in
In operation, a sample may be added through sample port 1006. A first blister pack 1008 comprising one or more lysis and/or decontamination reagents (e.g., UDG) are released from blister pack 1008 into tube 1002. In some embodiments, tube 1002 may be heated by a heater (not shown in
An exemplary blister pack is shown in
In another embodiment, the sample is processed initially in a sample tube, and then injected into a sample port of the cartridge (blister pack), where it undergoes amplification (e.g., RPA, LAMP, NEAR, or other isothermal amplification process) and then is added to a lateral flow device to be analyzed. In a further embodiment, the swab is mixed with the sample buffer and a lyophilized lysis mix is added when a frangible seal is broken. The sample is then moved to a lyophilized amplification mix comprising the reagents necessary for RPA, LAMP, or other isothermal amplification techniques. Similarly, a dilution buffer is added to the lyophilized mixture when its frangible seal is broken. The sample, after processing, is then added to a lateral flow device to be analyzed. In some embodiments, the lysis is accomplished by enzymatic and/or detergent lysis mechanisms. In a further embodiment, heat lysis is used. That is, the sample is added to the sample buffer and then heat is applied to lyse the sample. After the sample has been lysed, it is then moved to a lyophilized amplification mix chamber (blister). Similarly, a dilution buffer is added to the lyophilized mixture when its frangible seal is broken. The sample, after processing, is then added to a lateral flow device to be analyzed. In some embodiments, each of the steps is separated by a rotary valve, which controls the flow of the sample into the next chamber (blister).
A further embodiment of the blister pack configuration comprises a swab in conjunction with a blister pack. A sample is taken using a swab. The swab is added to a tube comprising buffer and incubated for 10 minutes at room temperature. Then, a cap comprising one or more lysis reagents is added to the tube. Adding the cap dispenses the lysis reagents into the buffer and sample. The mixture is then heated at 95° C. for three minutes but the invention is not so limited. Other temperatures are envisioned. In some embodiments, the heating is accomplished with any heater described herein (e.g., boiling water, a fixed heat source). The reaction mixture is then allowed to cool for 1 minute, but this time period is not limiting as other time periods are envisioned. The resulting reaction mixture is then injected into a sample port of the blister pack (e.g., using a pipette). The blister pack is then sealed with seal tape and then shaken or otherwise agitated (e.g., shaken) for 10 seconds but this time period is not limiting. The blister pack is heated for 20 minutes but this time period also is not limiting. In some embodiments, the blister pack may be placed in a user's clothing pocket (e.g., back pocket of pants, front pocket of pants, front pocket of shirt) to heat the blister pack using the user's body heat. The user then pushes on a first blister to release a one or more amplification reagents (e.g., one or more reagents for LAMP, RPA, NEAR, or other isothermal amplification methods). The user presses on a second blister to release the dilution buffer and turns a valve to permit the mixture to proceed to a lateral flow strip after the appropriate amount of processing. The lateral flow strip may indicate whether one or more target nucleic acid sequences are present in the sample. In some embodiments, the results on the lateral flow strip are interpreted using a mobile software-based application, downloadable to a smart device, such as that described herein.
From the state shown in
As shown in
In step 1404 of the process shown in
In step 1406 of the process shown in
It should be noted that while in some embodiments a detection component of a diagnostic test may be formed as a blister pack including multiple blisters, in other embodiments detection component may have any suitable form including one or more blisters. In some cases, one or more blisters may be employed in any portion of a diagnostic test to facilitate release and combination of different fluid volumes. For example, in some embodiments a blister may be integrated into a cap of a sample tube and may be configured to release lyophilized reagents for amplifying a sample. As another example, in some embodiments a blister pack may be employed to release a diluent to rehydrate one or more reagents inside of a cartridge. Thus, any suitable arrangement employing one or more blisters as described herein may be employed, as the present disclosure is not so limited.
Diagnostic devices, systems, and methods described herein may be safely and easily operated or conducted by untrained individuals. Unlike prior art diagnostic tests, some embodiments described herein may not require knowledge of even basic laboratory techniques (e.g., pipetting). Similarly, some embodiments described herein may not require expensive laboratory equipment (e.g., thermocyclers). In some embodiments, reagents are contained within a reaction tube and/or a blister pack, such that users are not exposed to any potentially harmful chemicals.
Diagnostic devices, systems, and methods described herein are also highly sensitive and accurate. In some embodiments, the diagnostic devices, systems, and methods are configured to detect one or more target nucleic acid sequences using nucleic acid amplification (e.g., an isothermal nucleic acid amplification method). Through nucleic acid amplification, the diagnostic devices, systems, and methods are able to accurately detect the presence of extremely small amounts of a target nucleic acid. In certain cases, for example, the diagnostic devices, systems, and methods can detect 1 pM or less, or 10 aM or less.
As a result, the diagnostic devices, systems, and methods described herein may be useful in a wide variety of contexts. For example, in some cases, the diagnostic devices and systems may be available over the counter for use by consumers. In such cases, untrained consumers may be able to self-administer the diagnostic test (or administer the test to friends and family members) in their own homes (or any other location of their choosing). In some cases, the diagnostic devices, systems, or methods may be operated or performed by employees or volunteers of an organization (e.g., a school, a medical office, a business). For example, a school (e.g., an elementary school, a high school, a university) may test its students, teachers, and/or administrators, a medical office (e.g., a doctor's office, a dentist's office) may test its patients, or a business may test its employees for a particular disease. In each case, the diagnostic devices, systems, or methods may be operated or performed by the test subjects (e.g., students, teachers, patients, employees) or by designated individuals (e.g., a school nurse, a teacher, a school administrator, a receptionist).
In some embodiments, diagnostic devices described herein are relatively small. In certain cases, for example, a blister pack may be approximately the size of a pen or a marker. Thus, unlike diagnostic tests that require bulky equipment, diagnostic devices and systems described herein may be easily transported and/or easily stored in homes and businesses. In some embodiments, the diagnostic devices and systems are relatively inexpensive. Since no expensive laboratory equipment (e.g., a thermocycler) is required, diagnostic devices, systems, and methods described herein may be more cost effective than known diagnostic tests.
In some embodiments, any reagents contained within a diagnostic device or system described herein may be thermostabilized, and the diagnostic device or system may be shelf stable for a relatively long period of time. In certain embodiments, for example, a blister pack may be stored at room temperature (e.g., 20° C. to 25° C.) for a relatively long period of time (e.g., at least 1 month, at least 3 months, at least 6 months, at least 9 months, at least 1 year, at least 5 years, at least 10 years). In certain embodiments, the blister pack may be stored across a range of temperatures (e.g., 0° C. to 20° C., 0° C. to 37° C., 0° C. to 60° C., 0° C. to 90° C., 20° C. to 37° C., 20° C. to 60° C., 20° C. to 90° C., 37° C. to 60° C., 37° C. to 90° C., 60° C. to 90° C.) for a relatively long period of time (e.g., at least 1 month, at least 3 months, at least 6 months, at least 9 months, at least 1 year, at least 5 years, at least 10 years).
The diagnostic devices, systems, and methods described herein may be used to detect the presence or absence of any target nucleic acid sequence (e.g., from any pathogen of interest) or multiple target nucleic acid sequences. Target nucleic acid sequences may be associated with a variety of diseases or disorders. In some embodiments, the diagnostic devices, systems, and methods are used to diagnose at least one disease or disorder caused by a pathogen. In certain instances, the diagnostic devices, systems, and methods are configured to detect a nucleic acid encoding a protein (e.g., a nucleocapsid protein) of SARS-CoV-2, which is the virus that causes COVID-19. In some embodiments, the diagnostic devices, systems, and methods are used to diagnose at least one disease or disorder caused by a virus, bacteria, fungus, protozoan, parasite, and/or cancer cell. Of course, a diagnostic test according to exemplary embodiments described herein (e.g., a blister pack) may be employed to detect any desired target nucleic acid sequence, as the present disclosure is not so limited.
According to some embodiments, diagnostic systems comprise a sample-collecting component (e.g., a swab) and a diagnostic device. In certain cases, the diagnostic device comprises a blister pack. In some cases, the diagnostic device comprises a detection component (e.g., a lateral flow assay strip). In certain embodiments, the diagnostic device further comprises one or more reagents (e.g., lysis reagents, nucleic acid amplification reagents, CRISPR/Cas detection reagents). Each of the one or more reagents may be in liquid form (e.g., in solution) or in solid form (e.g., lyophilized, dried, crystallized, air jetted). The diagnostic device may also comprise an integrated heater, or the diagnostic system may comprise a separate heater configured to heat one or more chambers or reservoirs of a blister pack or other portion of a diagnostic system. In some embodiments, a heater may be a printed circuit board (PCB) heater that may be integrated into a blister pack.
In some embodiments, a diagnostic method comprises collecting a sample from a subject (e.g., a human subject, an animal subject). In some embodiments, a diagnostic system comprises a sample-collecting component configured to collect a sample from a subject (e.g., a human subject, an animal subject). Exemplary samples include bodily fluids (e.g. mucus, saliva, blood, serum, plasma, amniotic fluid, sputum, urine, cerebrospinal fluid, lymph, tear fluid, feces, or gastric fluid), cell scrapings (e.g., a scraping from the mouth or interior cheek), exhaled breath particles, tissue extracts, culture media (e.g., a liquid in which a cell, such as a pathogen cell, has been grown), environmental samples, agricultural products or other foodstuffs, and their extracts. In some embodiments, the sample comprises a nasal secretion. In certain instances, for example, the sample is an anterior nares specimen. An anterior nares specimen may be collected from a subject by inserting a swab element of a sample-collecting component into one or both nostrils of the subject for a period of time. In some embodiments, the sample comprises a cell scraping. In certain embodiments, the cell scraping is collected from the mouth or interior cheek. The cell scraping may be collected using a brush or scraping device formulated for this purpose. The sample may be self-collected by the subject or may be collected by another individual (e.g., a family member, a friend, a coworker, a health care professional) using a sample-collecting component described herein.
In some embodiments, lysis is performed by chemical lysis (e.g., exposing a sample to one or more lysis reagents) and/or thermal lysis (e.g., heating a sample). Chemical lysis may be performed by one or more lysis reagents. In some embodiments, the one or more lysis reagents comprise one or more enzymes. In some embodiments, the one or more lysis reagents comprise one or more detergents. In some embodiments, cell lysis is accomplished by applying heat to a sample (thermal lysis). In certain instances, thermal lysis is performed by applying a lysis heating protocol comprising heating the sample at one or more temperatures for one or more time periods using any heater described herein. In some embodiments, a lysis heating protocol comprises heating the sample at a first temperature for a first time period.
Following lysis, one or more target nucleic acids (e.g., a nucleic acid of a target pathogen) may be amplified. In some cases, a target pathogen has RNA as its genetic material. In certain instances, for example, a target pathogen is an RNA virus (e.g., a coronavirus, an influenza virus). In some such cases, the target pathogen's RNA may need to be reverse transcribed to DNA prior to amplification. In some embodiments, reverse transcription is performed by exposing lysate to one or more reverse transcription reagents. In certain instances, the one or more reverse transcription reagents comprise a reverse transcriptase, a DNA-dependent polymerase, and/or a ribonuclease (RNase). In some embodiments, DNA may be amplified according to any nucleic acid amplification method known in the art.
In some embodiments, the nucleic acid amplification reagents are LAMP reagents. LAMP refers to a method of amplifying a target nucleic acid using at least four primers through the creation of a series of stem-loop structures. Due to its use of multiple primers, LAMP may be highly specific for a target nucleic acid sequence.
In some embodiments, the nucleic acid amplification reagents are RPA reagents. RPA generally refers to a method of amplifying a target nucleic acid using a recombinase, a single-stranded DNA binding protein, and a strand-displacing polymerase.
In some embodiments, amplification of one or more target nucleic acids is accomplished through the use of a nicking enzyme amplification reaction (NEAR) reaction. NEAR generally refers to a method for amplifying a target nucleic acid using a nicking endonuclease and a strand displacing DNA polymerase. In some cases, NEAR may allow for amplification of very small amplicons.
As described herein, a sample undergoes lysis and amplification prior to detection. In certain embodiments, one or more (and, in some cases, all) of the reagents necessary for lysis and/or amplification are present in a single pellet or tablet. In some embodiments, a pellet or tablet may comprise two or more enzymes, and it may be necessary for the enzymes to be activated in a particular order. Therefore, in some embodiments, the enzyme tablet further comprises one or more molecular switches. Molecular switches, as described herein, are molecules that, in response to certain conditions, reversibly switch between two or more stable states. In some embodiments, the condition that causes the molecular switch to change its configuration is pH, light, temperature, an electric current, microenvironment, or the presence of ions and other ligands. In one embodiment, the condition is heat. In some embodiments, the molecular switches described herein are aptamers. Aptamers generally refer to oligonucleotides or peptides that bind to specific target molecules (e.g., the enzymes described herein). The aptamers, upon exposure to heat or other conditions, may dissociate from the enzymes. With the use of molecular switches, the processes described herein (e.g., lysis, decontamination, reverse transcription, and amplification) may be performed in a single test tube with a single enzymatic tablet.
In some embodiments, amplified nucleic acids (i.e., amplicons) may be detected using any suitable methods. In some embodiments, one or more target nucleic acid sequences are detected using a lateral flow assay strip (e.g., disposed in a blister pack). In some embodiments, a fluidic sample (e.g., comprising a particle-amplicon conjugate) is configured to flow through a region of the lateral flow assay strip (e.g., a test pad) comprising one or more test lines. In some embodiments, a first test line comprises a capture reagent (e.g., an immobilized antibody) configured to detect a first target nucleic acid and an opaque marking may appear if the target nucleic acid is present in the fluidic sample. The marking may have any suitable shape or pattern (e.g., one or more straight lines, curved lines, dots, squares, check marks, x marks). In certain embodiments, the lateral flow assay strip comprises one or more additional test lines. In some instances, each test line of the lateral flow assay strip is configured to detect a different target nucleic acid. In certain embodiments, the region (e.g., the test pad) of the lateral flow assay strip generating an opaque marking further comprises one or more control lines to indicate that a human (or animal) sample was successfully collected, nucleic acids from the sample were amplified, and that amplicons were transported through the lateral flow assay strip.
In some embodiments, a diagnostic system comprises instructions for using a diagnostic device and/or otherwise performing a diagnostic test method. The instructions may include instructions for the use, assembly, and/or storage of the diagnostic device and any other components associated with the diagnostic system. The instructions may be provided in any form recognizable by one of ordinary skill in the art as a suitable vehicle for containing such instructions. For example, the instructions may be written or published, verbal, audible (e.g., telephonic), digital, optical, visual (e.g., videotape, DVD, etc.) or electronic communications (including Internet or web-based communications). In some embodiments, the instructions are provided as part of a software-based application. In certain cases, the application can be downloaded to a smartphone or device, and then guides a user through steps to use the diagnostic device.
In some embodiments, a software-based application may be connected (e.g., via a wired or wireless connection) to one or more components of a diagnostic system. In certain embodiments, for example, a heater may be controlled by a software-based application. In some cases, a user may select an appropriate heating protocol through the software-based application. In some cases, an appropriate heating protocol may be selected remotely (e.g., not by the immediate user). In some cases, the software-based application may store information (e.g., regarding temperatures used during the processing steps) from the heater.
In some embodiments, a diagnostic system comprises or is associated with software to read and/or analyze test results. In some embodiments, a device (e.g., a camera, a smartphone) is used to generate an image of a test result (e.g., one or more lines detectable on a lateral flow assay strip). In some embodiments, a user may use an electronic device (e.g., a smartphone, a tablet, a camera) to acquire an image of the visible portion of the lateral flow assay strip. In some embodiments, software running on the electronic device may be used to analyze the image (e.g., by comparing any lines or other markings that appear on the lateral flow assay strip with known patterns of markings). That result may be communicated directly to a user or to a medical professional. In some cases, the test result may be further communicated to a remote database server. In some embodiments, the remote database server stores test results as well as user information such as at least one of name, social security number, date of birth, address, phone number, email address, medical history, and medications.
While the present teachings have been described in conjunction with various embodiments and examples, it is not intended that the present teachings be limited to such embodiments or examples. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art. Accordingly, the foregoing description and drawings are by way of example only.
This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application Ser. No. 62/991,039, filed Mar. 17, 2020, U.S. Provisional Application Ser. No. 63/002,209, filed Mar. 30, 2020, U.S. Provisional Application Ser. No. 63/010,578, filed Apr. 15, 2020, U.S. Provisional Application Ser. No. 63/010,626, filed Apr. 15, 2020, U.S. Provisional Application Ser. No. 63/013,450, filed Apr. 21, 2020, U.S. Provisional Application Ser. No. 63/022,534, filed May 10, 2020, U.S. Provisional Application Ser. No. 63/022,533, filed May 10, 2020, U.S. Provisional Application Ser. No. 63/036,887, filed Jun. 9, 2020, U.S. Provisional Application Ser. No. 63/081,201, filed Sep. 21, 2020, U.S. Provisional Application Ser. No. 63/065,131, filed Aug. 13, 2020, U.S. Provisional Application Ser. No. 63/059,928, filed Jul. 31, 2020, U.S. Provisional Application Ser. No. 63/068,303, filed Aug. 20, 2020, U.S. Provisional Application Ser. No. 63/027,859, filed May 20, 2020, U.S. Provisional Application Ser. No. 63/027,874, filed May 20, 2020, U.S. Provisional Application Ser. No. 63/027,890, filed May 20, 2020, U.S. Provisional Application Ser. No. 63/027,864, filed May 20, 2020, U.S. Provisional Application Ser. No. 63/027,878, filed May 20, 2020, U.S. Provisional Application Ser. No. 63/027,886, filed May 20, 2020, and U.S. Provisional Application Ser. No. 63/053,534, filed Jul. 17, 2020, each of which is hereby incorporated by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63081201 | Sep 2020 | US | |
| 63068303 | Aug 2020 | US | |
| 63065131 | Aug 2020 | US | |
| 63059928 | Jul 2020 | US | |
| 63053534 | Jul 2020 | US | |
| 63036887 | Jun 2020 | US | |
| 63027859 | May 2020 | US | |
| 63027874 | May 2020 | US | |
| 63027890 | May 2020 | US | |
| 63027864 | May 2020 | US | |
| 63027878 | May 2020 | US | |
| 63027886 | May 2020 | US | |
| 63022534 | May 2020 | US | |
| 63022533 | May 2020 | US | |
| 63013450 | Apr 2020 | US | |
| 63010578 | Apr 2020 | US | |
| 63010626 | Apr 2020 | US | |
| 63002209 | Mar 2020 | US | |
| 62991039 | Mar 2020 | US |
