Patent Grant
11214841
Valley fever or coccidioidomycosis is a systemic fungal infection that is endemic to the Southwestern United States and occurs with the highest frequency in the state of Arizona, with over 50,000 reported cases from 2010 to 2014 according to the CDC. The illness is caused by two different fungal species: Coccidioides immitis, which is limited to California's San Joaquin valley; and Coccidioides posadasii, which is distributed throughout semi-arid regions in the U.S., Mexico, and Central and South America. Although the two species are genetically distinct, both cause very similar symptoms in infected patients.
Valley fever is currently detected through three principal methods in the United States: culture, microscopy, and serology. Both species of Coccidioides grow readily in culture media at 35° C. and can be detected in 2-7 days. Cells from the culture can then be identified in microscopy through their appearance. Alternatively, chemiluminescent nucleic acid probes (AccuProbe, Hologic, Inc.) that target ribosomal RNAs (rRNAs) of the fungi can be used for more specific molecular identification. Beyond the time required for the assay, the critical drawback of these diagnostic methods is the danger associated with culturing Coccidioides. These fungi were listed as Select Agents of bioterrorism up until 2012 and require biosafety level 3 containment. When cultured to substantial quantities, Coccidioides species pose a significant risk for unintended infection for laboratory workers. Microscopy can be applied directly to respiratory samples for identification albeit with poor sensitivity. Serology is currently the most commonly used method of detecting Valley fever using immunodiffusion assays. These assays provide sensitivity ranging from 75 to 91%. However, they can yield false negatives, particularly in immunocompromised patients who are unable to mount an effective immune response.
Survey of conventional diagnostics currently approved for use in the United States reveals that they pose substantial safety concerns to laboratory workers and require multiple days to return results, or they offer poor sensitivity, particularly for immunocompromised patients most likely to suffer through serious bouts of the illness. Of the diagnostics in development, PCR- and immunosignature-based assays are highly sensitive and can be specific; however, they require substantial investment in equipment and trained personnel for running the tests. These infrastructure requirements substantially increase both the cost and time required to return assay results. Accordingly, there remains a need in the art for rapid, inexpensive, and highly sensitive diagnostic tests for Valley fever that require neither sophisticated laboratory equipment nor biosafety level 3 containment.
This disclosure is related to methods and compositions for rapid, highly sensitive detection of the causative agents of Valley fever. As described herein, the methods and compositions are useful for early detection of Valley fever and, consequently, improved health outcomes.
In a first aspect, provided herein is a method of detecting a target Valley Fever (VF) nucleic acid in a sample. The method can comprise or consist essentially of the steps of (a) amplifying nucleic acids obtained from a biological sample of a subject, wherein amplifying comprises isothermal amplification; (b) contacting the amplified nucleic acid to a toehold switch, wherein the toehold switch encodes at least a portion of a reporter protein and comprises one or more single-stranded toehold sequence domains that are complementary to a target VF nucleic acid or the reverse complement thereof, wherein the contacting occurs under conditions that allow translation of the coding domain in the presence of the target nucleic acid but not in the absence of the target nucleic acid; and (c) detecting the reporter protein as an indicator that the target VF nucleic acid is present in the amplified nucleic acids. The target VF nucleic acid can be a C. immitis DNA or a C. posadasii DNA. The target nucleic acid can detectable at a concentration as low as 1 fM. The reporter protein, if present, can be detectable in less than 4 hours. The reporter protein, if present, can be detectable in less than 2 hours. The isothermal amplification can be a method selected from the group consisting of NASBA, LAMP, and RPA. The toehold switch can comprise SEQ ID NO:1.
In another aspect, provided herein is a method of detecting presence of pathogen-associated nucleic acid in a sample. The method can comprise or consist essentially of the steps of: (a) amplifying nucleic acids obtained from a biological sample of a subject, wherein amplifying comprises isothermal amplification; and (b) contacting the amplified nucleic acids to an aptamer-based sensor, wherein the aptamer-based sensor is a nucleic acid sequence comprising one or more single-stranded toehold sequence domains that are complementary to the target Valley Fever-associated nucleic acid, a fully or partially double-stranded stem domain, a loop domain, and an aptamer-ligand complex, and wherein the contacting occurs under conditions that promote activation of the aptamer-ligand complex in the presence of the target Valley Fever-associated nucleic acid but not in the absence of the Valley Fever-associated nucleic acid. The aptamer-ligand complex can comprise a fluorescent aptamer selected from the group consisting of Broccoli, Spinach2, Carrot, Radish, a G-quadruplex-containing aptamer, and a malachite green binding aptamer. Fluorescence, if present, can be detectable in less than 4 hours. Fluorescence, if present, can be detectable in less than 2 hours. The target VF nucleic acid can be a C. immitis DNA or a C. posadasii DNA. The isothermal amplification can be method selected from the group consisting of NASBA, LAMP, and RPA. The target nucleic acid can be detectable at a concentration as low as 1 fM.
In a further aspect, provided herein is a synthetic Valley Fever (VF)-specific toehold switch sensor comprising a fully or partially double-stranded stem domain, a loop domain, a toehold domain, and at least a portion of a coding sequence of a reporter gene, wherein the toehold domain and at least a portion of the stem domain are complementary to a target VF RNA sequence. The toehold switch sensor can comprise the RNA sequence of SEQ ID NO:1.
In another aspect, provided herein is a device for identifying a Valley Fever (VF)-associated nucleic acid, comprising a preserved paper test article, wherein the method is performed using the preserved paper test article. The paper test article can be preserved by freeze-drying.
In a further aspect, provided herein is a kit for detecting a Valley Fever (VF)-associated nucleic acid, comprising a plurality of preserved test articles, a VF detection agent, a plurality of toehold switches that encode at least a portion of a reporter protein and comprise one or more single-stranded toehold sequence domains that are complementary to a target VF nucleic acid or the reverse complement thereof, and an electronic optical reader. Also provided herein is a kit for detecting a Valley Fever (VF)-associated nucleic acid, comprising a plurality of aptamer-based sensors and an electronic optical reader, wherein the aptamer-based sensor is a nucleic acid sequence comprising one or more single-stranded toehold sequence domains that are complementary to the target Valley Fever-associated nucleic acid, a fully or partially double-stranded stem domain, a loop domain, and an aptamer-ligand complex. The kit may further comprise instructions for performing a detection method as described herein.
All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as though set forth in their entirety in the present application.
The methods and compositions provided herein are based at least in part on the inventors' development of a rapid, highly sensitive assay for detecting the causative agents of Valley fever in biological samples. Advantages of the methods and compositions provided herein are multifold. For example, results can be obtained in under 4 hours, and the assays require limited laboratory infrastructure (i.e., pipettes, hot plate, two heating blocks). Consequently, they do not need to take place at centralized labs and can provide same-day results for patients who are in desperate need of care. Second, the assays are nucleic acid based so they can provide improved specificity over antibody-based tests and can be rapidly repurposed for other pathogens, for instance if a new, more virulent Coccidioides strain emerges. Third, they are extremely low cost at $1 per test presently and potential for scaling down to ˜$0.10 per test, and they do not require a cold-chain. This technology could substantially reduce overall costs for Valley fever testing in affected states and improve patient outcomes. Fourth, the liquid-based reactions in which amplification and detection are combined in a single test tube enable rapid detection of Valley fever using aptasensors. Tests results with aptasensors can be detected by eye using simple and low-cost instrumentation.
Accordingly, in a first aspect, provided herein are two diagnostic platforms for detecting a Valley Fever-specific nucleic acids (e.g., Coccidioides posadasii DNAs, Coccidioides immitis DNAs) in a sample. As illustrated in
Methods of the first platform can comprise or consist essentially of the following steps: (a) amplifying nucleic acids obtained from a biological sample of a subject, wherein amplifying comprises isothermal amplification; (b) contacting the amplified nucleic acid to a toehold switch, wherein the toehold switch encodes at least a portion of a reporter protein and comprises one or more single-stranded toehold sequence domains that are complementary to a target VF nucleic acid or the reverse complement thereof, wherein the contacting occurs under conditions that allow translation of the coding domain in the presence of the target nucleic acid but not in the absence of the target nucleic acid; and (c) detecting the reporter protein as an indicator that the target VF nucleic acid is present in the amplified nucleic acids.
In certain embodiments, the method employs programmable riboregulators known as toehold switches. As used herein, the term “toehold switch” generally refers to a nucleic acid-based regulator of gene expression, configured to repress or activate translation of an open reading frame and thus production of a protein. Toehold switches, which are a type of prokaryotic riboregulator, activate gene expression in response to cognate RNAs with essentially arbitrary sequences. Gene regulation is achieved through the presence of a regulatory nucleic acid element (the cis-repressive RNA or crRNA) within the 5′ untranslated region (5′ UTR) of an mRNA molecule. The cis-repressive nucleic acid element (crRNA) forms a hairpin structure comprising a stem domain and a loop domain through complementary base pairing. The hairpin structure blocks access to the mRNA transcript by the ribosome, thereby preventing translation. In some embodiments, the stem domain of the hairpin structure sequesters the ribosome binding site (RBS). In some embodiments, including, for example, embodiments involving eukaryotic cells, the stem domain of the hairpin structure is positioned upstream of the start (or initiation) codon. As described in the Examples, that follow, toehold switches particularly useful for the methods provided herein are configured for lower leakage relative to previously described riboregulators. As illustrated in
In some cases, toehold switches are synthetic (engineered) molecules. In other cases, toehold switches comprise endogenous, naturally occurring RNAs or regions thereof. See, for example, U.S. 2015/0275203. The stem domain can be as small as 12 bps, but in some cases will be longer than 12 bps, including 13, 14, 15, 16, 17, 18, 19, 20, or more base pairs in length. In other cases, the loop domain is complementary to a non-naturally occurring RNA. The toehold domain can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleotides in length.
The toehold switch further comprises a fully or partially double-stranded stem domain comprising an initiation codon, a loop domain comprising a RBS, and a coding domain. The unpaired region upstream of the RBS in a toehold switch can be shortened or lengthened to modulate protein output and, in turn, device dynamic range. In some cases, the toehold and stem domains are complementary in sequence to a naturally occurring RNA. In other cases, the sequence detected can also be the complement of the naturally occurring RNA. For example, after isothermal amplification, it is possible to transcribe the antisense of the RNA rather than the sense.
The toehold switch can further comprise a thermodynamically stable double-stranded stem domain, a loop domain comprising a ribosome binding site, and a coding domain. Preferably, the loop domain is 11 nucleotides or 12 nucleotides in length. In some cases, the length of loop domains can be increased or decreased, for example, to alter reaction thermodynamics.
In certain embodiments, the toehold switch is configured to detect a portion of a pathogen genome that is conserved among two or more species or strains of the pathogen. For example, the Examples that follow describe identifying conserved sequence regions of Valley Fever specific nucleic acids suitable for isothermal amplification and toehold-switch-based detection. In some cases, toehold switches useful for the methods provided herein include, without limitation, synthetic norovirus-specific toehold switches that comprise a fully or partially double-stranded stem domain, a loop domain, a toehold domain, and at least a portion of a coding sequence of a reporter gene, wherein the toehold domain and at least a portion of the stem domain are complementary to a target norovirus RNA sequence. In some cases, synthetic Valley Fever-specific toehold switches comprise RNA sequence set forth as SEQ ID NO:1.
In another aspect, provided herein is a method of detecting presence of pathogen-associated nucleic acid in a sample. The method can comprise or consist essentially of the following steps: (a) amplifying nucleic acids obtained from a biological sample of a subject, wherein amplifying comprises isothermal amplification; and (b) contacting the amplified nucleic acids to an aptamer-based sensor, wherein the aptamer-based sensor is a nucleic acid sequence comprising one or more single-stranded toehold sequence domains that are complementary to the target Valley Fever-associated nucleic acid, a fully or partially double-stranded stem domain, a loop domain, and an aptamer-ligand complex, and wherein the contacting occurs under conditions that promote activation of the aptamer-ligand complex in the presence of the target Valley Fever-associated nucleic acid but not in the absence of the Valley Fever-associated nucleic acid.
In certain embodiments, aptasensors suitable for the methods described herein comprise aptamers that can bind to conditionally fluorescent dye molecules (e.g., Broccoli/DFHBI-1T). See
Any appropriate fluorescent aptamer can be used for aptamer-based sensors (“aptasensors”) described herein. For example, the fluorescent RNA aptamer can be Broccoli. As used herein, the term “Broccoli” or “Broccoli aptamer” refers to a 49-nt fluorescent RNA aptamer-fluorophore complex (see Filonov et al., J. Am. Chem. Soc. 2014, 136(46):16299-16308) that confers fluorescence to a target analyte (e.g., target RNA) of interest via activation of the bound fluorophore DFHBI or a DFHBI-derived fluorophore such as (Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-2-methyl-1-(2,2,2-trifluoroethyl)-1H-imidazol-5(4H)-one) (DFHBI-1T) as described by Song et al., J. Am. Chem. Soc. 2014, 136:1198. Other fluorescent RNA aptamers that can be used include, without limitation, Spinach and Spinach2 (Strack et al., Nature Methods 2013, 10:1219-1224), Carrot and Radish (Paige et al., Science 2011, 333:642-646), RT aptamer (Sato et al., Angew. Chem. Int. Ed. 2014, 54:1855-1858), hemin-binding G-quadruplex DNA and RNA aptamers, and malachite green binding aptamer (Babendure et al., J. Am. Chem. Soc. 2003). Several new alternatives to the Broccoli aptamer were recently reported by Song et al., Nature Chemical Biology 13, 1187 (2017). These aptamers all bind to the molecule 3,5-difluoro-4-hydroxybenzylidene-imidazolinone-2-oxime (DFHO), which resembles the fluorophore of red fluorescent protein (RFP), and thus provide red-shifted fluorescence compared to the green emission from Broccoli when it binds to DFHBI-1T. The new DFHO-binding aptamers are named Corn, Red Broccoli, and Orange Broccoli. As will be understood by practitioners in the art, selection of a fluorescent RNA aptamer-fluorophore complex for use in an aptasensor described herein will depend on fundamental properties of the aptamer such as brightness (or enzymatic output), folding properties, and amenability to sequence modifications.
In other cases, the aptasensors provided herein comprise colorimetric aptamers. In such cases, the presence and location of the target nucleic acid is indicated by a color change. Any appropriate colorimetric aptamer can be used. The term “colorimetric” is defined as an analysis where the reagent or reagents constituting the aptasensors system produce a color change in the presence or absence of an analyte. The degree the color changes in response to the analyte (e.g., target nucleic acid) may be quantified by colorimetric quantification methods known to those of ordinary skill in the art in. In some cases, standards containing known amounts of the selected analyte may be analyzed in addition to the sample to increase the accuracy of the comparison.
An advantage of the methods described herein is that they can be applied for the detection and identification of essentially any nucleic acid-containing organism. Accordingly, the pathogen can be virtually any pathogen or infectious agent (e.g., viruses, parasites, bacteria, fungi, prions) for which genetic information is available.
The term “detect” or “detection” as used herein indicates the determination of the existence, presence or fact of a target or signal in a limited portion of space, including but not limited to a sample, a reaction mixture, a molecular complex and a substrate including a platform and an array. Detection is “quantitative” when it refers, relates to, or involves the measurement of quantity or amount of the target or signal (also referred as quantitation), which includes but is not limited to any analysis designed to determine the amounts or proportions of the target or signal. Detection is “qualitative” when it refers, relates to, or involves identification of a quality or kind of the target or signal in terms of relative abundance to another target or signal, which is not quantified. An “optical detection” indicates detection performed through visually detectable signals: fluorescence, spectra, or images from a target of interest or a probe attached to the target.
In some cases, the method includes detecting pathogen-associated nucleic acids in a biological sample obtained from a subject, where identifying comprises: (i) amplifying nucleic acid obtained from the biological sample; (ii) contacting the amplified nucleic acid of (i) to a unimolecular aptamer-based sensor under conditions that allow for sequence-specific activation of the aptamer-based sensor when a pathogen-specific nucleic acid is present; and (iii) detecting activation of the aptamer-based sensor by detecting fluorescence of the bound fluorophore, where fluorescence is not detectable in the absence of the pathogen-specific target nucleic acid, thereby indicating the presence of the pathogen-specific nucleic acid.
As shown in
Any isothermal amplification protocol can be used according to the methods provided herein. In some cases, isothermal amplification comprises NASBA (nucleic acid sequence-based amplification). Other isothermal amplification methods include: loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), helicase-dependent amplification (HDA), nicking enzyme amplification reaction (NEAR), signal mediated amplification of RNA technology (SMART), rolling circle amplification (RCA), isothermal multiple displacement amplification (IMDA), single primer isothermal amplification (SPIA), recombinase polymerase amplification (RPA), and polymerase spiral reaction (PSR), which is described at nature.com/articles/srep12723 on the World Wide Web. In some cases, recombinase polymerase amplification (RPA) is used with the “one-pot” amplification and detection methods provided herein. In such cases, the methods comprise performing reverse transcription (RT), RPA, and transcription (TX) methods in a single test tube. In other cases, LAMP (loop-mediated isothermal amplification) is performed. As described in the Examples that follow, the unimolecular aptamer-based sensors described herein can bind directly to DNA LAMP amplification products. Alternatively, the amplification protocol is configured to add promoter sites to DNA LAMP amplification products such that each LAMP DNA can generate multiple RNA copies for improved assay effectiveness.
Nucleic acids and/or other moieties of the invention may be isolated. As used herein, “isolated” means to separate from at least some of the components with which it is usually associated whether it is derived from a naturally occurring source or made synthetically, in whole or in part.
Nucleic acids and/or other moieties of the invention may be purified. As used herein, purified means separate from the majority of other compounds or entities. A compound or moiety may be partially purified or substantially purified. Purity may be denoted by a weight by weight measure and may be determined using a variety of analytical techniques such as but not limited to mass spectrometry, HPLC, etc.
In some cases, it may be advantageous to adapt the methods described herein for high-throughput, reproducible, and rapid detection, for example in a clinical setting or in the field. When aptasensor output is coupled to a reporter element, such as fluorescence emission or a color-change through enzymatic activity, the aptasensors act as genetically encodable sensors and imaging probes for endogenous virus RNAs in a sample. For example, such aptasensors can be provided in a device configured for rapid, reproducible detection in a clinical setting. In some cases, the device comprises a preserved paper test article, upon which any step(s) of the method provided herein can be performed. In some cases, the device comprises a preserved paper test article, upon which any step(s) of the method provided herein can be performed. In preferred embodiments, the paper test article is preserved by freeze-drying. In such cases, aptasensors and methods provided herein can be performed at a cost of less than $1 per assay and do not require translation to produce reporters for the diagnostic test. In other embodiments, nucleic acids encoding the aptasensors can be freeze-dried in test tubes to render them stable at room temperature. These freeze-dried components can be reactivated upon addition of a sample and water, and can report on the presence of an endogenous nucleic acid of interest in the sample.
Any appropriate sample can be used according to the methods provided herein. In some cases, the sample is a biological sample obtained from an individual (e.g., a human subject, a non-human mammal). The sample is, in some cases, a diagnostic sample. The sample type will vary depending on the target pathogen. For example, Valley Fever can be detected in serum or blood samples or in sputum samples. Accordingly, a diagnostic sample for detecting Valley Fever can be a serum sample or a blood sample or a sputum sample. In some cases, serum samples have been frozen (e.g., at −80° C.) prior to testing since freezing is known to kill Coccidioides. Samples appropriate for use according to the methods provided herein can also include, without limitation, food samples, drinking water, environmental samples, and agricultural products. In some cases, samples appropriate for use according to the methods provided herein are “non-biological” in whole or in part. Non-biological samples include, without limitation, plastic and packaging materials, paper, clothing fibers, and metal surfaces. In certain embodiments, the methods provided herein are used in food safety and food biosecurity applications, such as screening food products and materials used in food processing or packaging for the presence of pathogens in biological and/or non-biological samples.
Other applications for which the methods provided herein include, without limitation, profiling species in an environment (e.g., water); profiling species in an human or animal microbiome; food safety applications (e.g., detecting the presence of a pathogenic species, determining or confirming food source/origin such as type of animal or crop plant); obtaining patient expression profiles (e.g., detecting expression of a gene or panel of genes (e.g., biomarkers) to monitor the patient's response to a therapeutic regimen, to select a therapeutic regimen suitable for the patient, or to detect exposure of the patient to a toxin or environmental agent that affects expression of the gene or a panel of genes.
In some cases, the device is used with a portable electronic reader. In this manner, the electronic reader serves as companion technology that provides robust and quantitative measurements of device outputs. In some embodiments, the electronic reader comprises readily available consumer components, open-source code, and laser-cut acrylic housing, and is powered by a rechargeable lithium ion battery. The electronic reader can further comprise an onboard data storage unit. In some cases, to achieve sensitive detection of toehold switch signal output, an acrylic chip that holds the freeze-dried, paper-based reactions is placed into the reader between a light source (e.g., to read optical density at excitation and emission wavelengths of light appropriate for and characteristic of a particular detectable reporter) and electronic sensors. In some cases, the light source is a light emitting diode (LED) light source. Samples can be read using onboard electronics. In this manner, a portable electronic reader can provide low-noise measurements of changes associated with the reporter element including changes in light transmission due to LacZ-mediated color change.
In certain embodiments, provided herein is a device for identifying a pathogen-associated nucleic acid, comprising a preserved paper test article, wherein the methods described herein are performed using the preserved paper test article. In some cases, the paper test article is preserved by freeze-drying.
Articles of Manufacture
In another aspect, the present invention provides articles of manufacture useful for detecting a pathogen in a sample according to the methods provided herein. In certain embodiments, the article of manufacture is a kit for detecting Valley Fever, where the kit comprises a Valley Fever detecting agent, a plurality of preserved paper test articles as described herein, and an electronic optical reader. Optionally, a kit can further include instructions for performing the Valley Fever detection methods provided herein.
In certain embodiments, provided herein is a kit for detecting a Valley Fever-associated nucleic acid, where the kit comprises a plurality of preserved paper test articles, a Valley Fever detection agent, a plurality of toehold switches that encode at least a portion of a reporter protein and comprise one or more single-stranded toehold sequence domains that are complementary to a target Valley Fever nucleic acid or the reverse complement thereof, and an electronic optical reader. In some cases, the kit also comprises instructions for performing the Valley Fever detection methods provided herein.
In other embodiments, provided herein is a kit for detecting a Valley Fever-associated nucleic acid, where the kit comprises a plurality of preserved test tube test articles, a Valley Fever detection agent, a plurality of toehold switches that encode at least a portion of a reporter protein and comprise one or more single-stranded toehold sequence domains that are complementary to a Valley Fever pathogen nucleic acid or the reverse complement thereof, and an electronic optical reader. In some cases, the kit also comprises instructions for performing the Valley Fever detection methods provided herein.
In other embodiments, provided herein is a kit for detecting a Valley Fever-associated nucleic acid, where the kit comprises a plurality of preserved test tube test articles, a Valley Fever detection agent, a plurality of aptasensors that encode at least a portion of a reporter aptamer and comprise one or more single-stranded toehold sequence domains that are complementary to a Valley Fever pathogen nucleic acid or the reverse complement thereof, and an electronic optical reader. In some cases, the kit also comprises instructions for performing the Valley Fever detection methods provided herein.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.
All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.
The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”
The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.
As used herein, the terms “approximately” or “about” in reference to a number are generally taken to include numbers that fall within a range of 5% in either direction (greater than or less than) the number unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). Where ranges are stated, the endpoints are included within the range unless otherwise stated or otherwise evident from the context.
The present embodiments have been described in terms of one or more preferred embodiments, and it should be appreciated that many equivalents, alternatives, variations, and modifications, aside from those expressly stated, are possible and within the scope of the embodiments.
This section demonstrates rapid, low-cost, sensitive platforms for detection of Valley Fever. As illustrated in
Toehold switches were designed to detect specific Coccidioides posadasii DNAs. Using these toehold switches, we developed sensors that enable direct visual detection of synthetic Valley fever DNA within hours (
The isothermal amplification method Recombinase Polymerase Amplification (RPA) was used with RPA forward and reverse primers (Table 1) to amplify DNA sequences. The RPA amplification products were added to paper-based cell-free reactions containing the VF-specific toehold switches, which provide a visual reaction readout. As shown in
We designed a library of Broccoli aptasensors targeting the same Coccidioides posadasii nucleic acids and evaluated them for sensitivity. Sequences used for Broccoli aptamer Valley Fever diagnostic assays are provided in Table 2. As shown in
As shown in
The experiments and data described herein demonstrate development and use of paper-based assays for detection of Valley Fever nucleic acids that do not require expensive thermal cycling equipment, provide test results that can be read directly by eye, and employ toehold switch riboregulators to eliminate false positives caused by non-specific amplification. Toehold switches and Broccoli aptasensors for detection of Valley fever have been validated using synthetic DNA samples. When coupled with isothermal amplification via RPA, these toehold switches and aptasensors enable simple visual detection of Valley fever DNA down to at least 50 fM and 20 fM, respectively. We expect that further optimization of amplification primers and sensors will enable additional improvements in assay sensitivity. One-pot reactions that combine isothermal amplification and detection using Broccoli aptasensors have the potential to substantially decrease assay time and complexity.
This application claims priority to U.S. Provisional Application No. 62/639,623, filed Mar. 7, 2018, which is hereby incorporated by reference in its entirety for all purposes.
This invention was made with government support under GM126892 awarded by the National Institutes of Health. The government has certain rights in the invention.
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| Number | Date | Country | |
|---|---|---|---|
| 20190276901 A1 | Sep 2019 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62639623 | Mar 2018 | US |
