Patent Application
20220290261
Polymerase chain reaction (PCR) is a molecular biology technique that allows amplification of nucleotides for various analytical purposes. Quantitative PCR (qPCR) is an adaptation of PCR which allows monitoring of the amplification of a targeted nucleotide during the PCR. Diagnostic qPCR has been applied to detect nucleotides that are diagnostic of infectious diseases, cancer, and genetic abnormalities. Reverse transcriptase qPCR (RT-qPCR) is an adaptation of qPCR which allows detection of a target RNA nucleotide. Because of this ability, RT-qPCR is well-suited for detecting virus pathogens. However, RT-qPCR requires sizeable conventional equipment which may not be available in certain point of care settings, and additionally requires significant sample preparation and time to perform and obtain results.
By contrast, Loop-Mediated Isothermal Amplification (LAMP) is a more simplistic approach to diagnostic identification of target nucleotides. In particular, LAMP is a one-operation nucleic acid amplification method to multiply specific target nucleotide sequences. In addition to use of an isothermal heating process, LAMP can use a visual output test indicator, such as a simple color change rather than a more complicated fluorescent indicator required by PCR. Reverse-transcriptase LAMP (RT-LAMP) can be used like RT-qPCR in order to identify the presence or absence target nucleotides from RNA, and as such, can be used in a diagnostic capacity to identify the presence or absence of viral pathogens in a test subject. Because LAMP is a more simplistic, it can be performed with less equipment and sample preparation and therefore is more accessible for use in point of care settings, such as clinics, emergency rooms, and even on a mobile basis.
The present disclosure is drawn to technology (e.g., cDNA, primer sets, and methods) for reverse transcription loop-mediated isothermal amplification (RT-LAMP) analysis and detecting a Sarbecovirus target pathogen in a subject.
In some disclosure embodiments, an isolated complementary DNA (cDNA) of a nucleic acid molecule can include a nucleotide sequence that is at least 85% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or a combination thereof. In one aspect, the nucleotide sequence can be at least 85% identical to SEQ ID NO: 9 (e.g., SEQ ID NO: 1 joined to SEQ ID NO: 2). In yet another aspect, the nucleotide sequence can comprise SEQ ID NO: 1 joined to SEQ ID NO: 2 by a linking sequence selected from Table 11. In a further aspect, the nucleotide sequence can be at least 85% identical to SEQ ID NO: 10 (e.g., SEQ ID NO:3 joined to SEQ ID NO: 4). In yet another aspect, the nucleotide sequence can comprise SEQ ID NO: 3 joined to SEQ ID NO: 4 by a linking sequence selected from Table 11.
In one aspect, the guanine and cytosine (GC) content of the nucleotide sequence can be 50% or less. In another aspect, the guanine and cytosine (GC) content of the nucleotide sequence can be 40% or less. In another aspect, an end stability of the nucleotide sequence can be less than −2.5 kcal/mol. In another aspect, the nucleotide sequence can have a melting temperature of from about 40° C. to about 62° C. In yet another aspect, the nucleotide sequence can have a minimum primer dimerization energy of less than −1.0 kcal/mol. In yet another aspect, the nucleotide sequence can be less than 50% identical to nucleotide sequences associated with non-target agents (commensal microorganisms, other pathogens, and human genome).
In another aspect, the nucleotide sequence can be at least 90% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or a combination thereof. In another aspect, the nucleotide sequence can be at least 95% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or a combination thereof. In another aspect, the nucleotide sequence can be 100% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or a combination thereof.
In some disclosure embodiments, a primer set for reverse transcription loop-mediated isothermal amplification (RT-LAMP) analysis can include: a forward inner primer (FIP) sequence that is at least 85% identical to a combination of SEQ ID NO: 1 coupled to SEQ ID NO: 2 (e.g. SEQ ID NO: 9); a backward inner primer (BIP) sequence that is at least 85% identical to a combination of seq ID NO: 3 coupled to SEQ ID NO: 4 (e.g. SEQ ID NO: 10); a forward outer primer (F3) sequence that is at least 85% identical to SEQ ID NO: 5; a backward outer primer (B3) sequence that is at least 85% identical to SEQ ID NO: 6; a forward loop primer (LF) sequence that is at least 85% identical to SEQ ID NO: 7; and a backward loop primer (LB) sequence that is at least 85% identical to SEQ ID NO: 8.
In one aspect, the FIP sequence can include a linking sequence joining SEQ ID NO: 1 and SEQ ID NO: 2. In one aspect, the linking sequence can be selected from Table 11. In another aspect, the BIP sequence can include a linking sequence joining SEQ ID NO: 3 and SEQ ID NO: 4. In one aspect, the linking sequence can be selected from Table 11.
In another aspect, the guanine and cytosine (GC) content of the FIP, the BIP, the F3, the B3, the LF, the LB, or a combination thereof can be 50% or less. In another aspect, the guanine and cytosine (GC) content of the FIP, the BIP, the F3, the B3, the LF, the LB, or a combination thereof can be 40% or less. In yet another aspect, an end stability of the FIP, the BIP, the F3, the B3, the LF, the LB, or a combination thereof can be less than −2.5 kcal/mol. In another aspect, the FIP, the BIP, the F3, the B3, the LF, the LB, or a combination thereof can have a melting temperature of from about 40° C. to about 62° C. In yet another aspect, the FIP, the BIP, the F3, the B3, the LF, the LB, or a combination thereof can have a minimum primer dimerization energy of less than −1.0 kcal/mol. In another aspect, the FIP, the BIP, the F3, the B3, the LF, the LB, or a combination thereof can have less than 50% identical to nucleotide sequences associated with non-target agents (commensal microorganisms, other pathogens, and human genome).
In another aspect, the FIP sequence can be at least 90% identical to a combination of SEQ ID NO: 1 and SEQ ID NO: 2. In another aspect, the BIP sequence can be at least 90% identical to a combination of seq ID NO: 3 and SEQ ID NO: 4. In another aspect, the F3 sequence can be at least 90% identical to SEQ ID NO: 5. In another aspect, the B3 sequence can be at least 90% identical to SEQ ID NO: 6. In another aspect, the LF sequence can be at least 90% identical to SEQ ID NO: 7. In another aspect, the LB sequence can be at least 90% identical to SEQ ID NO: 8.
In another aspect, the FIP sequence can be at least 95% identical to a combination of SEQ ID NO: 1 and SEQ ID NO: 2. In another aspect, the BIP sequence can be at least 95% identical to a combination of seq ID NO: 3 and SEQ ID NO: 4. In another aspect, the F3 sequence can be at least 95% identical to SEQ ID NO: 5. In another aspect, the B3 sequence can be at least 95% identical to SEQ ID NO: 6. In another aspect, the LF sequence can be at least 95% identical to SEQ ID NO: 7. In another aspect, the LB sequence can be at least 95% identical to SEQ ID NO: 8.
In yet another aspect, the FIP sequence can be at least 100% identical to a combination of SEQ ID NO: 1 and SEQ ID NO: 2, which is equivalent to SEQ ID NO: 9. In another aspect, the BIP sequence can be at least 100% identical to a combination of seq ID NO: 3 and SEQ ID NO: 4, which is equivalent to SEQ ID NO: 10. In another aspect, the F3 sequence can be at least 100% identical to SEQ ID NO: 5. In another aspect, the B3 sequence can be at least 100% identical to SEQ ID NO: 6. In another aspect, the LF sequence can be at least 100% identical to SEQ ID NO: 7. In another aspect, the LB sequence can be at least 100% identical to SEQ ID NO: 8.
In some disclosure embodiments, a method of detecting a target Sarbecovirus pathogen in a subject can include providing a primer set. In one aspect, the primer set can include: a forward inner primer (FIP) sequence that is at least 85% identical to a combination of SEQ ID NO: 1 and SEQ ID NO: 2; a backward inner primer (BIP) sequence that is at least 85% identical to a combination of seq ID NO: 3 and SEQ ID NO: 4; a forward outer primer (F3) sequence that is at least 85% identical to SEQ ID NO: 5; a backward outer primer (B3) sequence that is at least 85% identical to SEQ ID NO: 6; a forward loop primer (LF) sequence that is at least 85% identical to SEQ ID NO: 7; and a backward loop primer (LB) sequence that is at least 85% identical to SEQ ID NO: 8.
In one aspect, the target pathogen can be a human coronavirus selected from: Severe Acute Respiratory Syndrome (SARS)-CoV (SARS-CoV), Severe Acute Respiratory Syndrome (SARS)-CoV 2 (SARS-CoV-2), Middle East Respiratory Syndrome (MERS)-CoV (MERS-CoV), SARS-CoV hCoV-HKU1, hCoV-0C43, hCoV-NL63, and hCoV-229E. In one aspect, the subject can be a human subject. In yet another aspect, the target pathogen can be Severe Acute Respiratory Syndrome (SARS)-CoV 2 (SARS-CoV-2).
Features and advantages of the disclosure will be apparent from the detailed description which follows, taken in conjunction with the accompanying drawings, which together illustrate, by way of example, features of the disclosure; and, wherein:
Reference will now be made to the exemplary embodiments illustrated, and specific language will be used herein to describe the same. It will nevertheless be understood that no limitation of the scope of the technology is thereby intended.
Before invention embodiments are described, it is to be understood that this disclosure is not limited to the particular structures, process steps, or materials disclosed herein, but is extended to equivalents thereof as would be recognized by those ordinarily skilled in the relevant arts. It should also be understood that terminology employed herein is used for the purpose of describing particular examples or embodiments only and is not intended to be limiting. The same reference numerals in different drawings represent the same element. Numbers provided in flow charts and processes are provided for clarity in illustrating steps and operations and do not necessarily indicate a particular order or sequence.
Furthermore, the described features, structures, or characteristics can be combined in any suitable manner in one or more embodiments. In the following description, numerous specific details are provided, such as examples of compositions, storage, administration etc., to provide a thorough understanding of various invention embodiments. One skilled in the relevant art will recognize, however, that such detailed embodiments do not limit the overall inventive concepts articulated herein, but are merely representative thereof.
It should be noted that as used herein, the singular forms “a,” “an,” and, “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “an excipient” includes reference to one or more of such excipients, and reference to “the carrier” includes reference to one or more of such carriers.
As used herein, the terms “formulation” and “composition” are used interchangeably and refer to a mixture of two or more compounds, elements, or molecules. In some aspects, the terms “formulation” and “composition” may be used to refer to a mixture of one or more active agents with a carrier or other excipients.
As used herein, the term “soluble” is a measure or characteristic of a substance or agent with regards to its ability to dissolve in a given solvent. The solubility of a substance or agent in a particular component of the composition refers to the amount of the substance or agent dissolved to form a visibly clear solution at a specified temperature such as about 25° C. or about 37° C.
As used herein, a “subject” refers to an animal. In one aspect the animal may be a mammal. In another aspect, the mammal may be a human.
As used herein, “non-liquid” when used to refer to the state of a composition disclosed herein refers to the physical state of the composition as being a semi-solid or solid. In this written description, the use of the term “solid” shall provide express support for the term “semisolid” and vice versa.
As used herein, “solid” and “semi-solid” refers to the physical state of a composition that supports its own weight at standard temperature and pressure and has adequate viscosity or structure to not freely flow. Semi-solid materials may conform to the shape of a container under applied pressure.
As used herein, a “solid phase medium” refers to a non-liquid medium. In one example, the non-liquid medium can be a material with a porous surface. In another example, the non-liquid medium can be a material with a fibrous surface. In yet another example, the non-liquid medium can be paper.
As used herein, a first nucleotide sequence can be joined to a second nucleotide sequence by a “linking sequence” when the first nucleotide sequence is coupled to a first end (e.g., 5′ or 3′ end) of the linking sequence and the second nucleotide sequence is coupled to a second end (e.g., 5′ or 3′ end) of the linking sequence. In one example, the first nucleotide sequence can be directly coupled to a first end of the linking sequence and the second nucleotide can be directly coupled to the second end of the linking sequence.
As used herein, a “forward inner primer (FIP)” can be a combination of an F1c primer and an F2 primer.
As used herein, an “F1c,” “F2,” “backward inner primer (BIP),” “B1c,” “B2,” “forward outer primer (F3),” “backward outer primer (B3),” “forward loop primer (LF),” “backward loop primer (LB),” refer to various primers used in an RT-LAMP reaction. These terms are well known in the art and their accepted meaning is intended herein.
In this disclosure, “comprises,” “comprising,” “containing” and “having” and the like can have the meaning ascribed to them in U.S. Patent law and can mean “includes,” “including,” and the like, and are generally interpreted to be open ended terms. The terms “consisting of” or “consists of” are closed terms, and include only the components, structures, steps, or the like specifically listed in conjunction with such terms, as well as that which is in accordance with U.S. Patent law. “Consisting essentially of” or “consists essentially of” have the meaning generally ascribed to them by U.S. Patent law. In particular, such terms are generally closed terms, with the exception of allowing inclusion of additional items, materials, components, steps, or elements, that do not materially affect the basic and novel characteristics or function of the item(s) used in connection therewith. For example, trace elements present in a composition, but not affecting the compositions nature or characteristics would be permissible if present under the “consisting essentially of” language, even though not expressly recited in a list of items following such terminology. When using an open ended term, like “comprising” or “including,” in the written description it is understood that direct support should be afforded also to “consisting essentially of” language as well as “consisting of” language as if stated explicitly and vice versa.
The terms “first,” “second,” “third,” “fourth,” and the like in the description and in the claims, if any, are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It is to be understood that any terms so used are interchangeable under appropriate circumstances such that the embodiments described herein are, for example, capable of operation in sequences other than those illustrated or otherwise described herein. Similarly, if a method is described herein as comprising a series of steps, the order of such steps as presented herein is not necessarily the only order in which such steps may be performed, and certain of the stated steps may possibly be omitted and/or certain other steps not described herein may possibly be added to the method.
As used herein, comparative terms such as “increased,” “decreased,” “better,” “worse,” “higher,” “lower,” “enhanced,” “maximized,” “minimized,” and the like refer to a property of a device, component, composition, or activity that is measurably different from other devices, components, compositions or activities that are in a surrounding or adjacent area, that are similarly situated, that are in a single device or composition or in multiple comparable devices or compositions, that are in a group or class, that are in multiple groups or classes, or as compared to the known state of the art.
The term “coupled,” as used herein, is defined as directly or indirectly connected in a chemical, mechanical, electrical or nonelectrical manner. Objects described herein as being “adjacent to” each other may be in physical contact with each other, in close proximity to each other, or in the same general region or area as each other, as appropriate for the context in which the phrase is used. “Directly coupled” refers to objects, components, or structures that are in physical contact with one another and attached.
Occurrences of the phrase “in one embodiment,” or “in one aspect,” herein do not necessarily all refer to the same embodiment or aspect.
As used herein, the term “substantially” refers to the complete or nearly complete extent or degree of an action, characteristic, property, state, structure, item, or result. For example, an object that is “substantially” enclosed would mean that the object is either completely enclosed or nearly completely enclosed. The exact allowable degree of deviation from absolute completeness may in some cases depend on the specific context. However, generally speaking the nearness of completion will be so as to have the same overall result as if absolute and total completion were obtained. The use of “substantially” is equally applicable when used in a negative connotation to refer to the complete or near complete lack of an action, characteristic, property, state, structure, item, or result. For example, a composition that is “substantially free of” particles would either completely lack particles, or so nearly completely lack particles that the effect would be the same as if it completely lacked particles. In other words, a composition that is “substantially free of” an ingredient or element may still actually contain such item as long as there is no measurable effect thereof.
As used herein, the term “about” is used to provide flexibility to a numerical range endpoint by providing that a given value may be “a little above” or “a little below” the endpoint. Unless otherwise stated, use of the term “about” in accordance with a specific number or numerical range should also be understood to provide support for such numerical terms or range without the term “about”. For example, for the sake of convenience and brevity, a numerical range of “about 50 angstroms to about 80 angstroms” should also be understood to provide support for the range of “50 angstroms to 80 angstroms.” Furthermore, it is to be understood that in this specification support for actual numerical values is provided even when the term “about” is used therewith. For example, the recitation of “about” 30 should be construed as not only providing support for values a little above and a little below 30, but also for the actual numerical value of 30 as well.
As used herein, a plurality of items, structural elements, compositional elements, and/or materials may be presented in a common list for convenience. However, these lists should be construed as though each member of the list is individually identified as a separate and unique member. Thus, no individual member of such list should be construed as a de facto equivalent of any other member of the same list solely based on their presentation in a common group without indications to the contrary.
Concentrations, amounts, levels and other numerical data may be expressed or presented herein in a range format. It is to be understood that such a range format is used merely for convenience and brevity and thus should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges or decimal units encompassed within that range as if each numerical value and sub-range is explicitly recited. As an illustration, a numerical range of “about 1 to about 5” should be interpreted to include not only the explicitly recited values of about 1 to about 5, but also include individual values and sub-ranges within the indicated range. Thus, included in this numerical range are individual values such as 2, 3, and 4 and sub-ranges such as from 1-3, from 2-4, and from 3-5, etc., as well as 1, 2, 3, 4, and 5, individually. This same principle applies to ranges reciting only one numerical value as a minimum or a maximum. Furthermore, such an interpretation should apply regardless of the breadth of the range or the characteristics being described.
Reference throughout this specification to “an example” means that a particular feature, structure, or characteristic described in connection with the example is included in at least one embodiment. Thus, appearances of the phrases “in an example” in various places throughout this specification are not necessarily all referring to the same embodiment.
An initial overview of invention embodiments is provided below and specific embodiments are then described in further detail. This initial summary is intended to aid readers in understanding the technological concepts more quickly, but is not intended to identify key or essential features thereof, nor is it intended to limit the scope of the claimed subject matter.
Selecting primer sets for loop-mediated isothermal amplification (LAMP) and reverse transcription LAMP (RT-LAMP) can be difficult because of the various constraints involved. First, the primer should have adequate stability to allow the LAMP reaction to proceed in a timely manner. Second, when used as a diagnostic test for a specific pathogen, the primer should target a unique sequence have minimal overlap with other potential pathogens, commensals, or background genome. Third, the limit of detection of a target pathogen should be low enough to allow detection of the target pathogen at low concentrations. Fourth, the false positive and false negative rates should be controlled to allow a reliability and a significant degree of confidence in the test results. Fifth, when conducting LAMP reactions on a solid-reaction medium (e.g. paper) slight defects which may not be an issue in liquid LAMP may pose an issue. Finally, in some cases, the reaction speed in a solid-based medium can be more than twice as slow as the reaction speed in a liquid-based medium.
A generalized approach to primer selection can rely on selected properties of the primers. For example, the guanine and cytosine (GC) content of a primer can provide a rough and ready way to approximate the stability of a primer. However, the GC content of a primer and related tools, can be misleading. As such, finding a specific primer sequence in a genome of tens of thousands of nucleotides can involve an extreme amount of experimentation. The amount of experimentation can be significantly controlled by using a process that uses a selected combination of primer parameters (e.g., nucleotide region length, length of primers, distance between primers, end stabilities, melting temperatures, minimum primer dimerization energy, distance between loop primers and inner primers, and screening based on reaction speed, limit of detection, and reducing false positives).
The nucleotide sequences resulting from such a process can have performance properties (e.g., low false positives, fast reaction speed, and low limit of detection). One of the primer sets identified based on this process is the RegX3.1 primer set, as identified herein. In one embodiment, the RegX3.1 primer set can include ten primers as follows: an F1c primer, an F2 primer, a B1c primer, a B2 primer, an F3 primer, a B3 primer, an LF primer, an LB primer, an FIP primer, and a BIP primer that can be associated with 10 distinct nucleotide sequences.
For example, in one disclosure embodiment, an isolated complementary DNA (cDNA) of a nucleic acid molecule can include a nucleotide sequence that is at least 85% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or a combination thereof.
In another disclosure embodiment, a primer set for reverse transcription loop-mediated isothermal amplification (RT-LAMP) analysis can include: a forward inner primer (FIP) sequence that is at least 85% identical to a combination of SEQ ID NO: 1 and SEQ ID NO: 2; a backward inner primer (BIP) sequence that is at least 85% identical to a combination of SEQ ID NO: 3 and SEQ ID NO: 4; a forward outer primer (F3) sequence that is at least 85% identical to SEQ ID NO: 5; a backward outer primer (B3) sequence that is at least 85% identical to SEQ ID NO: 6; a forward loop primer (LF) sequence that is at least 85% identical to SEQ ID NO: 7; and a backward loop primer (LB) sequence that is at least 85% identical to SEQ ID NO: 8. In some embodiments, the combination of SEQ ID NO: 1 and SEQ ID NO: 2 can be SEQ ID NO: 9. In other embodiments, the combination of SEQ ID NO: 3 and SEQ ID NO: 4 can be SEQ ID NO:10.
In yet another disclosure embodiment, a method of detecting a target pathogen from a Sarbecovirus in a subject can include providing a primer set. In one aspect, the primer set can include: a forward inner primer (FIP) sequence that is at least 85% identical to a combination of SEQ ID NO: 1 and SEQ ID NO: 2; a backward inner primer (BIP) sequence that is at least 85% identical to a combination of seq ID NO: 3 and SEQ ID NO: 4; a forward outer primer (F3) sequence that is at least 85% identical to SEQ ID NO: 5; a backward outer primer (B3) sequence that is at least 85% identical to SEQ ID NO: 6; a forward loop primer (LF) sequence that is at least 85% identical to SEQ ID NO: 7; and a backward loop primer (LB) sequence that is at least 85% identical to SEQ ID NO: 8. In some embodiments, the combination of SEQ ID NO: 1 and SEQ ID NO: 2 can be SEQ ID NO: 9. In other embodiments, the combination of SEQ ID NO: 3 and SEQ ID NO: 4 can be SEQ ID NO:10.
With the above-described background in mind, the present disclosure is drawn to cDNA, primer sets, and methods for reverse transcription loop-mediated isothermal amplification (RT-LAMP) analysis. The present disclosure is also drawn to detecting a target pathogen from a Sarbecovirus subgenus in a subject. The present disclosure is also drawn to various primer sets for reverse transcription loop-mediated isothermal amplification (RT-LAMP) analysis.
In one disclosure embodiment, an isolated complementary DNA (cDNA) of a nucleic acid molecule can have a specific nucleotide sequence. In one aspect, the nucleotide sequence can be at least 85% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, the like, or a combination thereof. In another aspect, the nucleotide sequence can be at least 90% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, the like, or a combination thereof. In another aspect, the nucleotide sequence can be at least 95% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, the like, or a combination thereof. In another aspect, the nucleotide sequence can be 100% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, the like, or a combination thereof.
In one example, the nucleotide sequence can be identical to SEQ ID NO: 9. In one aspect, SEQ ID NO: 9 can be a combination of SEQ ID NO: 1 and SEQ ID NO: 2. In one example, SEQ ID NO: 9 can be a combination of SEQ ID NO: 1 and SEQ ID NO: 2 when SEQ ID NO: 9 is 100% identical to a concatenation of SEQ ID NO: 1 and SEQ ID NO: 2 (e.g., SEQ ID: 1 is joined to SEQ ID NO: 2 without any intervening sequences between SEQ ID: 1 and SEQ ID: 2).
In one aspect, the nucleotide sequence can be at least 85% identical to SEQ ID NO: 9. In another aspect, the nucleotide sequence can be at least 90% identical to SEQ ID NO: 9. In yet another aspect, the nucleotide sequence can be at least 95% identical to SEQ ID NO: 9. In yet another aspect, the nucleotide sequence can be 100% identical to SEQ ID NO: 9.
In another aspect, the nucleotide sequence can include SEQ ID NO: 1 joined to SEQ ID NO: 2 by a linking sequence selected from Table 11. In this example, the linking sequence can be an intervening sequence between SEQ ID NO: 1 and SEQ ID NO: 2 without any other sequences between SEQ ID NO: 1 and SEQ ID NO: 2. In this example, when the linking sequence between SEQ ID NO: 1 and SEQ ID NO: 2 is removed, then the resulting sequence can be 85% identical, 90% identical, 95% identical, or 100% identical to SEQ ID NO: 9.
In one example, the nucleotide sequence can be identical to SEQ ID NO: 10. In one aspect, SEQ ID NO: 10 can be a combination of SEQ ID NO: 3 and SEQ ID NO: 4. In one example, SEQ ID NO: 10 can be a combination of SEQ ID NO: 3 and SEQ ID NO: 3 when SEQ ID NO: 10 is 100% identical to a concatenation of SEQ ID NO: 3 and SEQ ID NO: 3 (e.g., SEQ ID: 3 is joined to SEQ ID NO: 4 without any intervening sequences between SEQ ID: 3 and SEQ ID: 4).
In one aspect, the nucleotide sequence can be at least 85% identical to SEQ ID NO: 10. In another aspect, the nucleotide sequence can be at least 90% identical to SEQ ID NO: 10. In yet another aspect, the nucleotide sequence can be at least 95% identical to SEQ ID NO: 10. In yet another aspect, the nucleotide sequence can be 100% identical to SEQ ID NO: 10.
In another aspect, the nucleotide sequence can include SEQ ID NO: 3 joined to SEQ ID NO: 4 by a linking sequence selected from Table 11. In this example, the linking sequence can be an intervening sequence between SEQ ID NO: 3 and SEQ ID NO: 4 without any other sequences between SEQ ID NO: 3 and SEQ ID NO: 4. In this example, when the linking sequence between SEQ ID NO: 3 and SEQ ID NO: 4 is removed, then the resulting sequence can be 85% identical, 90% identical, 95% identical, or 100% identical to SEQ ID NO: 10.
In some cases, the thermodynamic parameters and other properties of the nucleotide sequence can impact the stability and performance of the RT-LAMP reaction. As depicted in Table 1, the thermodynamic parameters of the F3, B3, FIP, BIP, LF, LB, F2, F1c, B2, and B1c primers can fall within a selected range.
The 10 primers included in the orf7ab.1 (e.g., RegX3.1) primer set have relatively low guanine/cytosine (GC) content (30%-50%), with the average being about 39% GC for this primer set. Typically, a GC content between 45% and 65% can be achieved for many primer sets. As the GC content decreases below the range of 45% to 65%, decreasing stability is expected. However, that is not the case with the orf7ab.1 primer set because the end stabilities (the free energy change upon the binding of the last 6 base pairs on either the 5′ end of the 3′ end of the primer) are more negative than −2.5 kcal/mol for the orf7ab.1 primer set (with the exception of the 5′ end of LB and the 3′ ends of F2 and B2). This increased end stability relative to the −4.0-threshold combined with the lower GC content relative to a random sample of nucleotides may provide increased stability and performance for the orf7ab.1 primer set.
In one aspect, the guanine and cytosine (GC) content of the nucleotide sequence can be less than or equal to a selected percentage. The selected percentage of GC content can be based on an end stability of the nucleotide sequence. In one example, the GC content of the nucleotide sequence can be 50% or less (e.g., less than or equal to 50% of the nucleotide sequence are comprised of guanine (G) or cytosine (C), with the remaining nucleotides of the nucleotide sequence being comprised of adenine (A) or thymine (T)). In one example, the GC content of the nucleotide sequence can be 45% or less. In another example, the GC content of the nucleotide sequence can be 40% or less. In yet another example, the GC content of the nucleotide sequence can be 35% or less.
In another aspect, at least one end stability of the nucleotide sequence (e.g., the 5′ end, the 3′ end, or both the 5′ end and the 3′ end of the nucleotide sequence) can have a stability that is less than or equal to a selected stability number. The selected stability number can be based on one or more of: the selected percentage of GC content, the selected temperature range, the like, or combinations thereof. In one example, the at least one end stability of the nucleotide sequence can be less than −2.5 kcal/mol (i.e., more negative). In another example, the at least one end stability of the nucleotide sequence can be less than −5.0 kcal/mol. In another example, the at least one end stability of the nucleotide sequence can be less than −6.0 kcal/mol. In another example, the at least one end stability of the nucleotide sequence can be less than −7.0 kcal/mol. In one aspect, both the 5′ end and the 3′ end of the nucleotide sequence can be less than at least one of −2.5 kcal/mol, −4.0 kcal/mol, −5.0 kcal/mol, −6.0 kcal/mol, −7.0 kcal/mol, the like, or combinations thereof.
In another aspect, the nucleotide sequence can have a melting temperature within a selected temperature range. The selected temperature range can be based on one or more of: the temperature range for activation of a reverse transcriptase, a temperature range for a DNA polymerase, the like, or a combination thereof. In one example, the nucleotide sequence can have a melting temperature of from about 40° C. to about 62° C. In another example, the nucleotide sequence can have a melting temperature of from about 50° C. to about 62° C. In one example, the nucleotide sequence can have a melting temperature of from about 55° C. to about 62° C.
In yet another aspect, the nucleotide sequence can have a selected minimum primer dimerization energy. The selected minimum primer dimerization energy can be based on one or more of: the selected percentage of GC content, the selected stability number, the selected temperature range, the like, or combinations thereof. In one example, the minimum primer dimerization energy can be less than −0.5 kcal/mol. In another example, the minimum primer dimerization energy can be less than −1.0 kcal/mol. In another example, the minimum primer dimerization energy can be less than −2.5 kcal/mol. In yet another example, the minimum primer dimerization energy can be less than −5.0 kcal/mol.
In yet another aspect, the nucleotide sequence can have a cross-contamination homology that can be less than a cross-contamination percentage. In one example, the nucleotide sequence can be less 50% identical to nucleotide sequences associated with non-target agents (commensal microorganisms, other pathogens, and human genome). In one example, the nucleotide sequence can be less 40% identical to nucleotide sequences associated with non-target agents (commensal microorganisms, other pathogens, and human genome). In one example, the nucleotide sequence can be less 30% identical to nucleotide sequences associated with non-target agents (commensal microorganisms, other pathogens, and human genome). In one example, the nucleotide sequence can be less 20% identical to nucleotide sequences associated with non-target agents (commensal microorganisms, other pathogens, and human genome). In one example, the nucleotide sequence can be less 10% identical to nucleotide sequences associated with non-target agents (commensal microorganisms, other pathogens, and human genome).
In some disclosure embodiments, a primer set for RT-LAMP analysis can include: a forward inner primer (FIP) sequence that is at least 85% identical to a combination of SEQ ID NO: 1 and SEQ ID NO: 2; a backward inner primer (BIP) sequence that is at least 85% identical to a combination of seq ID NO: 3 and SEQ ID NO: 4; a forward outer primer (F3) sequence that is at least 85% identical to SEQ ID NO: 5; a backward outer primer (B3) sequence that is at least 85% identical to SEQ ID NO: 6; a forward loop primer (LF) sequence that is at least 85% identical to SEQ ID NO: 7; a backward loop primer (LB) sequence that is at least 85% identical to SEQ ID NO: 8, the like, or combinations thereof. In some embodiments, the combination of SEQ ID NO: 1 and SEQ ID NO: 2 can be SEQ ID NO: 9. In other embodiments, the combination of SEQ ID NO: 3 and SEQ ID NO: 4 can be SEQ ID NO:10.
The forward inner primer (FIP) and the backward inner primer (BIP) can be generated by combining two primers (e.g., the F1c and the F2 primers for the FIP primer, or the B1c and the B2 primers for the BIP primer). The F1c, F2, B1c, and B2 sequences can have linker sequences (L) such that the FIP primer can be F1c-L-F2 and the BIP primer can be B1c-L-B2. Table 11 contains a list of the F1c, F2, B1c, and B2 sub-primers that were used when generating the FIP and BIP primers.
In one example, the FIP sequence can be at least 90% identical to a combination of SEQ ID NO: 1 and SEQ ID NO: 2. In another example, the FIP sequence can be at least 95% identical to a combination of SEQ ID NO: 1 and SEQ ID NO: 2. In yet example, the FIP sequence can be 100% identical to a combination of SEQ ID NO: 1 and SEQ ID NO: 2, which is equivalent to SEQ ID NO: 9.
In one aspect, the FIP sequence can include a linking sequence joining SEQ ID NO: 1 and SEQ ID NO: 2. Regardless of the percentage homology between the FIP sequence and the combination of SEQ ID NO: 1 and SEQ ID NO: 2, the linking sequence between SEQ ID NO: 1 and SEQ ID NO: 2 can be a linking sequence that is selected from Table 11. In one example, the linking sequence joining SEQ ID NO: 1 and SEQ ID NO: 2 can be 85%, 90%, 95%, 100%, the like, or a combination thereof, identical to the linking sequence that is selected from Table 11.
In one example, the BIP sequence can be at least 90% identical to a combination of SEQ ID NO: 3 and SEQ ID NO: 4. In another example, the BIP sequence can be at least 95% identical to a combination of SEQ ID NO: 3 and SEQ ID NO: 4. In yet example, the BIP sequence can be 100% identical to a combination of SEQ ID NO: 3 and SEQ ID NO: 4, which is equivalent to SEQ ID NO: 10.
In one aspect, the BIP sequence can include a linking sequence joining SEQ ID NO: 3 and SEQ ID NO: 4. Regardless of the percentage homology between the BIP sequence and the combination of SEQ ID NO: 3 and SEQ ID NO: 4, the linking sequence between SEQ ID NO: 3 and SEQ ID NO: 4 can be a linking sequence that is selected from Table 11. In one example, the linking sequence joining SEQ ID NO: 3 and SEQ ID NO: 4 can be 85%, 90%, 95%, 100%, the like, or a combination thereof, identical to the linking sequence that is selected from Table 11.
The homology percentage between F3 and SEQ ID NO: 5 can vary within a selected percentage range. In one example, the F3 sequence can be at least 90% identical to SEQ ID NO: 5. In another aspect, the F3 sequence can be at least 95% identical to SEQ ID NO: 5. In another aspect, the F3 sequence can be 100% identical to SEQ ID NO: 5.
The homology percentage between B3 and SEQ ID NO: 6 can vary within a selected percentage range. In another aspect, the B3 sequence can be at least 90% identical to SEQ ID NO: 6. In another aspect, the B3 sequence can be at least 95% identical to SEQ ID NO: 6. In another aspect, the B3 sequence can be 100% identical to SEQ ID NO: 6.
The homology percentage between LF and SEQ ID NO: 7 can vary within a selected percentage range. In one aspect, the LF sequence can be at least 90% identical to SEQ ID NO: 7. In another aspect, the LF sequence can be at least 95% identical to SEQ ID NO: 7. In another aspect, the LF sequence can be 100% identical to SEQ ID NO: 7.
The homology percentage between LB and SEQ ID NO: 8 can vary within a selected percentage range. In another aspect, the LB sequence can be at least 90% identical to SEQ ID NO: 8. In another aspect, the LB sequence can be at least 95% identical to SEQ ID NO: 8. In another aspect, the LB sequence can be 100% identical to SEQ ID NO: 8.
In another aspect, the GC content of the FIP, the BIP, the F3, the B3, the LF, the LB, the like, or a combination thereof can be one or more of: 50% or less, 45% or less, 40% or less, 35% or less, the like, or a combination thereof.
In yet another aspect, an end stability of the FIP, the BIP, the F3, the B3, the LF, the LB, the like, or a combination thereof can be less than one or more of: −2.5 kcal/mol, −4.0 kcal/mol, −5.0 kcal/mol, −6.0 kcal/mol, −7.0 kcal/mol, the like, or a combination thereof.
In another aspect, the FIP, the BIP, the F3, the B3, the LF, the LB, the like, or a combination thereof can have a melting temperature in a temperature range of from: about 40° C. to about 62° C.; or about 50° C. to about 62° C.; or about 55° C. to about 62° C.
In yet another aspect, the FIP, the BIP, the F3, the B3, the LF, the LB, the like, or a combination thereof can have a minimum primer dimerization energy of less than one or more of: −0.5 kcal/mol, −1.0 kcal/mol, −2.0 kcal/mol, −4.0 kcal/mol, −5.0 kcal/mol, the like, or combinations thereof.
In another aspect, the FIP, the BIP, the F3, the B3, the LF, the LB, the like, or a combination thereof can be less identical to nucleotide sequences associated with non-target agents (commensal microorganisms, other pathogens, and human genome) than a selected percentage. In one example, the selected percentage can be less than or equal to one or more of: 50%, 40%, 30%, 20%, 10%, the like, or combinations thereof.
In another disclosure embodiment, a method of detecting a target pathogen in a subject can include providing a primer set. In one aspect, the primer set can include: a forward inner primer (FIP) sequence that is at least 85% identical to a combination of SEQ ID NO: 1 and SEQ ID NO: 2; a backward inner primer (BIP) sequence that is at least 85% identical to a combination of seq ID NO: 3 and SEQ ID NO: 4; a forward outer primer (F3) sequence that is at least 85% identical to SEQ ID NO: 5; a backward outer primer (B3) sequence that is at least 85% identical to SEQ ID NO: 6; a forward loop primer (LF) sequence that is at least 85% identical to SEQ ID NO: 7; a backward loop primer (LB) sequence that is at least 85% identical to SEQ ID NO: 8; the like, or combinations thereof.
The target pathogen can comprise various pathogen types. In one aspect, the pathogen target can be one or more of a viral pathogen, a bacterial pathogen, a fungal pathogen, a protozoa pathogen, the like, or combinations thereof. The pathogen target can be detected when the nucleic acid from the pathogen target can be released from a cell wall, a cell membrane, a protein coat, or the like.
More specifically, in one aspect, the pathogen target can be a viral target. In some aspects, the viral target can be H1N1, H2N2, H3N2, H1N1pdm09, severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Middle East respiratory syndrome (MERS), influenza, the like, or combinations thereof.
In one example, the target pathogen can be a human coronavirus selected from: Severe Acute Respiratory Syndrome (SARS)-CoV (SARS-CoV), Severe Acute Respiratory Syndrome (SARS)-CoV 2 (SARS-CoV-2), Middle East Respiratory Syndrome (MERS)-CoV (MERS-CoV), SARS-CoV hCoV-HKU1, hCoV-0C43, hCoV-NL63, and hCoV-229E. In one example, the subject can be a human subject. In yet another example, the target pathogen can be Severe Acute Respiratory Syndrome (SARS)-CoV 2 (SARS-CoV-2).
When the pathogen target includes RNA, the RNA can be reverse transcribed. Therefore, in another aspect, the LAMP detection can be reverse transcription RT-LAMP. In this example, cDNA can be generated from a target RNA with a reverse transcriptase enzyme. The cDNA can be amplified to a detectable amount. When the pathogen target can be detected directly from DNA, then LAMP can be used to amplify the DNA to a detectable amount without reverse transcribing the RNA to DNA.
In another disclosure embodiment, a primer set for RT-LAMP analysis can include: (a) an FIP sequence that is at least 85% identical to a combination of SEQ ID NO: 11 and SEQ ID NO: 12; (b) a BIP sequence that is at least 85% identical to a combination of seq ID NO: 13 and SEQ ID NO: 14; (c) an F3 sequence that is at least 85% identical to SEQ ID NO: 15; (d) a B3 sequence that is at least 85% identical to SEQ ID NO: 16; (e) an LF sequence that is at least 85% identical to SEQ ID NO: 17; and (f) an LB sequence that is at least 85% identical to SEQ ID NO: 18. In one aspect, the FIP sequence can be 100% identical to a combination of SEQ ID NO: 11 and SEQ ID NO: 12, which can be equivalent to SEQ ID NO: 19. In another aspect, the FIP sequence can include a linking sequence joining SEQ ID NO: 11 and SEQ ID NO: 12, wherein the linking sequence is selected from Table 11. In another aspect, the BIP sequence can be 100% identical to a combination of SEQ ID NO: 13 and SEQ ID NO: 14, which can be equivalent to SEQ ID NO: 20. In another aspect, the BIP sequence can include a linking sequence joining SEQ ID NO: 13 and SEQ ID NO: 14, wherein the linking sequence is selected from Table 11.
In another disclosure embodiment, a primer set for RT-LAMP analysis can include: (a) an FIP sequence that is at least 85% identical to a combination of SEQ ID NO: 21 and SEQ ID NO: 22; (b) a BIP sequence that is at least 85% identical to a combination of seq ID NO: 23 and SEQ ID NO: 24; (c) an F3 sequence that is at least 85% identical to SEQ ID NO: 25; (d) a B3 sequence that is at least 85% identical to SEQ ID NO: 26; (e) an LF sequence that is at least 85% identical to SEQ ID NO: 27; and (f) an LB sequence that is at least 85% identical to SEQ ID NO: 28. In one aspect, the FIP sequence can be 100% identical to a combination of SEQ ID NO: 21 and SEQ ID NO: 22, which can be equivalent to SEQ ID NO: 29. In another aspect, the FIP sequence can include a linking sequence joining SEQ ID NO: 21 and SEQ ID NO: 22, wherein the linking sequence is selected from Table 11. In another aspect, the BIP sequence can be 100% identical to a combination of SEQ ID NO: 23 and SEQ ID NO: 24, which can be equivalent to SEQ ID NO: 30. In another aspect, the BIP sequence can include a linking sequence joining SEQ ID NO: 23 and SEQ ID NO: 24, wherein the linking sequence is selected from Table 11.
In another disclosure embodiment, a primer set for RT-LAMP analysis can include: (a) an FIP sequence that is at least 85% identical to a combination of SEQ ID NO: 31 and SEQ ID NO: 32; (b) a BIP sequence that is at least 85% identical to a combination of seq ID NO: 33 and SEQ ID NO: 34; (c) an F3 sequence that is at least 85% identical to SEQ ID NO: 35; (d) a B3 sequence that is at least 85% identical to SEQ ID NO: 36; (e) an LF sequence that is at least 85% identical to SEQ ID NO: 37; and (f) an LB sequence that is at least 85% identical to SEQ ID NO: 38. In one aspect, the FIP sequence can be 100% identical to a combination of SEQ ID NO: 31 and SEQ ID NO: 32, which can be equivalent to SEQ ID NO: 39. In another aspect, the FIP sequence can include a linking sequence joining SEQ ID NO: 31 and SEQ ID NO: 32, wherein the linking sequence is selected from Table 11. In another aspect, the BIP sequence can be 100% identical to a combination of SEQ ID NO: 33 and SEQ ID NO: 34, which can be equivalent to SEQ ID NO: 40. In another aspect, the BIP sequence can include a linking sequence joining SEQ ID NO: 33 and SEQ ID NO: 34, wherein the linking sequence is selected from Table 11.
In another disclosure embodiment, a primer set for RT-LAMP analysis can include: (a) an FIP sequence that is at least 85% identical to a combination of SEQ ID NO: 41 and SEQ ID NO: 42; (b) a BIP sequence that is at least 85% identical to a combination of seq ID NO: 43 and SEQ ID NO: 44; (c) an F3 sequence that is at least 85% identical to SEQ ID NO: 45; (d) a B3 sequence that is at least 85% identical to SEQ ID NO: 46; (e) an LF sequence that is at least 85% identical to SEQ ID NO: 47; and (f) an LB sequence that is at least 85% identical to SEQ ID NO: 48. In one aspect, the FIP sequence can be 100% identical to a combination of SEQ ID NO: 41 and SEQ ID NO: 42, which can be equivalent to SEQ ID NO: 49. In another aspect, the FIP sequence can include a linking sequence joining SEQ ID NO: 41 and SEQ ID NO: 42, wherein the linking sequence is selected from Table 11. In another aspect, the BIP sequence can be 100% identical to a combination of SEQ ID NO: 43 and SEQ ID NO: 44, which can be equivalent to SEQ ID NO: 50. In another aspect, the BIP sequence can include a linking sequence joining SEQ ID NO: 43 and SEQ ID NO: 44, wherein the linking sequence is selected from Table 11
In another disclosure embodiment, a primer set for RT-LAMP analysis can include: (a) an FIP sequence that is at least 85% identical to a combination of SEQ ID NO: 51 and SEQ ID NO: 52; (b) a BIP sequence that is at least 85% identical to a combination of seq ID NO: 53 and SEQ ID NO: 54; (c) an F3 sequence that is at least 85% identical to SEQ ID NO: 55; (d) a B3 sequence that is at least 85% identical to SEQ ID NO: 56; (e) an LF sequence that is at least 85% identical to SEQ ID NO: 57; and (f) an LB sequence that is at least 85% identical to SEQ ID NO: 58. In one aspect, the FIP sequence can be 100% identical to a combination of SEQ ID NO: 51 and SEQ ID NO: 52, which can be equivalent to SEQ ID NO: 59. In another aspect, the FIP sequence can include a linking sequence joining SEQ ID NO: 51 and SEQ ID NO: 52, wherein the linking sequence is selected from Table 11. In another aspect, the BIP sequence can be 100% identical to a combination of SEQ ID NO: 53 and SEQ ID NO: 54, which can be equivalent to SEQ ID NO: 60. In another aspect, the BIP sequence can include a linking sequence joining SEQ ID NO: 53 and SEQ ID NO: 54, wherein the linking sequence is selected from Table 11.
In another disclosure embodiment, a primer set for RT-LAMP analysis can include: (a) an FIP sequence that is at least 85% identical to a combination of SEQ ID NO: 61 and SEQ ID NO: 62; (b) a BIP sequence that is at least 85% identical to a combination of seq ID NO: 63 and SEQ ID NO: 64; (c) an F3 sequence that is at least 85% identical to SEQ ID NO: 65; (d) a B3 sequence that is at least 85% identical to SEQ ID NO: 66; (e) an LF sequence that is at least 85% identical to SEQ ID NO: 67; and (f) an LB sequence that is at least 85% identical to SEQ ID NO: 68. In one aspect, the FIP sequence can be 100% identical to a combination of SEQ ID NO: 61 and SEQ ID NO: 62, which can be equivalent to SEQ ID NO: 69. In another aspect, the FIP sequence can include a linking sequence joining SEQ ID NO: 61 and SEQ ID NO: 62, wherein the linking sequence is selected from Table 11. In another aspect, the BIP sequence can be 100% identical to a combination of SEQ ID NO: 63 and SEQ ID NO: 64, which can be equivalent to SEQ ID NO: 70. In another aspect, the BIP sequence can include a linking sequence joining SEQ ID NO: 63 and SEQ ID NO: 64, wherein the linking sequence is selected from Table 11.
The primer sets that follow comprise: (1) an F1c primer, (2) an F2 primer, (3) a B1c primer, (4) a B2 primer, (5) an F3 primer, (6) a B3 primer, (7) an LF primer, (8) an LB primer, (9) an FIP primer, and (10) a BIP primer in that order for each primer set.
As used herein, the terms “REGX3.1” and “orf7ab.1” are used interchangeably and refer to the same primer set.
The following examples are provided to promote a clearer understanding of certain embodiments of the present invention, and are in no way meant as a limitation thereon.
As illustrated in
The illustration in
An in silico study was performed to characterize inclusivity and cross-reactivity of the LAMP assay primers. One assay included three primer sets: (a) targeting the E-gene (the envelope small membrane protein), (b) the RdRp gene (also known as the nsp12 gene which encodes viral polymerase), and (c) ORF lab region (encoding multiple non-structural proteins of clinical significance). Each primer set contained 6 primers. For both inclusivity and cross-reactivity studies, the BLASTn tool was used to align each primer sequence with the appropriate reference genomes.
The inclusivity study, as depicted in Table 2 shows the proportion of SARS-CoV-2 genomes that were detected by each primer set. Inclusivity was calculated by aligning each primer against 5332 SARS-CoV genome sequences downloaded from NCBI (txid2697049) on 12 Jun. 2020. A primer set was considered inclusive if all six primers in the set had 100% match for the target genome. The test employed 3 primer sets in which each set contained 6 individual primers. In addition, a positive SARS-CoV-2 test uses 2 of the 3 primer sets to show a positive reaction. Thus, the demonstrated 92-94% inclusivity across individual genes was an acceptable level for the test's individual gene components.
Due to the large number of mutations SARS-CoV-2 has undergone, the primer sets exhibited mismatches of varying lengths for 5.7-7.6% of the tested strains. While the presence of a single mismatch within a target genome suggests a lack of inclusivity for that particular strain, this conclusion is not definitive. For example, previous work on MERS-CoV has demonstrated that a single nucleotide mismatch in one of the primers may not have an impact on the limit of detection of LAMP assays. Additionally, the LAMP reaction used 6 primers per set and two of them (e.g., the loop primers) were not used for amplification but rather contribute to the increase of the rate of the reaction. Successful amplification was possible even with mismatches in the loop primers. Therefore, the inclusivity percentages in Table 2 represent a worst-case assumption.
1n-silico inclusivity studies were then conducted to verify detection of SARS-CoV-2 with orflab.II primer set. RT-LAMP primers for orflab.II were aligned against publicly available SARS-CoV-2 whole genomes from the NCBI Nucleotide database as of Aug. 5, 2020. The orflab.II primer set had 100% sequence identity with 98.72% of the 8,844 sequences available; and 99.79% of the sequences contained 1 mismatch or less when aligned with the orflab.II primer set. The alignments which contained 2 or more mismatches (19 sequences) with the orflab.II primer set had multiple mismatches within an individual primer. Although the frequency of this occurrence was less than 0.5%, these types of mismatches had been shown to affect RT-LAMP reactions and could lead to false negatives.
Whole SARS-CoV-2 genomes were identified by filtering all SARS-CoV-2 genomes (as identified by the taxonomy ID #2697049) by: (i) genomic sequence type, (ii) inclusion of the phrase “whole genome” in the sequence name, and (iii) sequences between the lengths of 28,000 and 30,000 base pairs. This was performed by using the following Entrez query with the Entrez esearch utility to obtain the accession numbers: “txid2697049[Organism:noexp] AND (viruses[filter] AND biomol_genomic[PROP] AND (28000[SLEN]: 30000[SLEN])) AND (complete genome[All Fields]).” The Entrez efetch utility was used to download the complete FASTA sequences for each accession number. Primers were aligned to each sequence using the msa.sh (i.e., MultiStateAligner) function of BBMap v38.86. The CIGAR string contained in the resulting SAM file for each primer was used to determine the number of matches between the aligned primer and the subject sequence. Percent sequence identity was calculated using the number of matches divided by the alignment length (which was equal to the primer length for all cases). Inclusivity was determined by calculating the portion of SARS-CoV-2 whole genome sequences that had 100% sequence identity with all of the aligned primers. For a more flexible analysis, the number of mismatches was calculated for each primer alignment. For each sequence, if the sum of mismatches across all primers was less than a predetermined mismatch threshold, then the particular sequence was used for sequence inclusivity. For this analysis, the constituent primers of FIP (e.g., F1c and F2) and BIP (B1c/B2) were used in lieu of the FIP and BIP primers.
To predict cross-reactivity for each LAMP primer set, sequence similarity was calculated for each primer against a list of relevant off-target background genomes. The alignments were subsequently filtered for a ≥80% sequence match, as depicted in Table 3.
Chlamydia pneumoniae
Haemophilus influenzae
Legionella pneumophila
Mycobacterium tuberculosis
Streptococcus pneumoniae
Streptococcus pyogenes
Bordetella pertussis
Mycoplasma pneumoniae
Pneumocystis jirovecii
Pseudomonas aeruginosa
Staphylococcus epidermidis
Streptococcus salivarius
Background genomes tested include those that were reasonably likely to be encountered in the clinical specimen. The primers were compared against the human reference genome (GRCh38.p13), and the nasal microbiome sequencing data (Accession: PRJNA342328) to represent diverse microbial flora in the human respiratory tract.
Results of the cross-reactivity analysis indicated a negligible chance of false-positives on off-target organisms. Columns in the table for each SARS-CoV-2 gene target indicated the number of primers in each set (out of six total) that scored above the 80% threshold. In a few cases (e.g., C. pneumoniae, H. influenzae), one primer in a set of six scored above the threshold. In this case, the risk of non-specific amplification was minimal because amplification cannot occur unless at least two primers bound the target. In the case of MERS, two primers out of six were highly similar to the RdRp gene. However, MERS is not prevalent in the United States, with 2 cases ever reported. Moreover, even if a false-positive for this marker were to occur, the lack of positive amplification on the other two markers would indicate a negative test result to the operator. The highest risk of cross-reactivity with off-target organisms appeared to be with related SARS viruses, especially human SARS-CoV-1, bat, and feline coronaviruses. Because SARS-CoV-1 is not currently extant in human populations, the chance of a false positive on this off-target can be considered negligible. Finally, two primers out of each set of six were similar to the human genome background. However, these primer sets have not exhibited non-specific amplification on human saliva specimens in experiments. These results indicate a low probability of false-positives due to cross-reactivity.
Additional wet lab testing can confirm these computational predictions using commercially-available panels (e.g., ZeptoMetrix Validation panels (#NATRVP-3, NATPPQ-BIO, NATPPA-BIO) with intact, inactivated organisms.
In-silico homology studies were also conducted against several potentially pathogenic microorganisms and viruses that can be found in the human saliva or in the human respiratory tract using BLAST. Organisms were found to be potentially cross-reactive if any primer was >80% identical as determined by percent identity. Consequently, four microorganisms were found to be potentially cross-reactive: SARS-coronavirus, Haemophilus influenzae, Pneumocystis jirovecii, and Pseudomonas aeruginosa. Both P. jirovecii and P. aeruginosa have one primer with >80% homology. As a result, the orflab.II primer set was not expected to be cross-reactive with these pathogens. Two primers were found to be potentially cross-reactive with H. influenzae; however, one of these two primers was a loop primer, which was primarily used to accelerate the RT-LAMP reaction. In the absence of more than one “core” primer (e.g., F3/B3 or FIP/BIP) being reactive, it was not expected that the orflab.II primer set would be cross-reactive with these organisms either. Four primers were found to be potentially cross-reactive with SARS-coronavirus; however, because of the low prevalence of this virus in general populations, there was minimal risk that orflab.II would produce false positives. Comprehensive results of the homology analysis can be found in Table 4A.
Chlamydia
pneumoniae
Haemophilus
influenzae
Legionella
pneumophila
Mycobacterium
tuberculosis
Streptococcus
pneumoniae
Streptococcus
pyogenes
Bordetella
pertussis
Mycoplasma
pneumoniae
Pneumocystis
jirovecii
Candida albicans
Pseudomonas
aeruginosa
Staphylococcus
epidermis
Streptococcus
salivarius
In-silico homology analysis was conducted by performing a BLAST search of each primer against sequences available in the NCBI Nucleotide database for the specific taxon of interest. Parameters that were used in the BLAST search can be found in Table 4B (for the entrez query, “{TaxonID}” is replaced with the TaxonID of the respective microorganism). Sequence identity for each hit in the BLAST analysis was then calculated by using the number of matches for a hit divided by the length of the primer, not the alignment length. Homology was determined by calculating the maximum sequence identity of all hits for a specific primer against an individual organism and is reported in Table 4B. Primers with greater than 80% homology were deemed as potentially cross-reactive.
Interfering substances found in respiratory samples endogenously or exogenously can also be tested to evaluate the extent, if any, of potential assay inhibition. Bio-banked saliva specimens (e.g., frozen samples without preservative) can be spiked with 2× limit of detection (LoD) with inactivated virus to further characterize the potential assay inhibition.
RT-LAMP primer sets were designed using PrimerExplorer v5 and are presented in Table 10. Parameters used to design primers can be found in Table 5A. All other Primer Explorer parameters were kept at their default values. Primer sets were designed using portions of the SARS-CoV-2 reference genome (NCBI accession number: NC 045512). Primer sets for RdRP were designed by first splitting the nsp12 gene sequence into 2 portions. Primer set RdRP.I was designed using the first portion of the nsp12 sequence, while primer sets RdRP.II and RdRP.III were designed using the second portion of the nsp12 sequence. Primer sets for orflab were designed using a portion of the orflab gene sequence. Primer sets for RegX were designed by choosing three random 2,000 nt regions of the reference genome. In-silico analyses were used to the predict sensitivity and specificity of each primer set. Optimal primer sets underwent experimental cross-reactivity studies to ensure specificity to SARS-CoV-2.
Whole SARS-CoV-2 genomes were identified by filtering all publicly available SARS-CoV-2 genomes from the NCBI Nucleotide database as of Feb. 5, 2021 (as identified by the taxon ID 2697049) by genomic sequence type, inclusion of the phrase “whole genome” in the sequence name, and sequences between the lengths of 28,000 and 30,000 base pairs. This identification was accomplished by using the following Entrez query with the Entrez esearch utility to obtain the accession numbers: “txid2697049[Organism:noexp] AND (viruses/filter] AND biomol_genomic[PROP] AND (28000[SLEN]: 30000[SLEN])) AND (complete genome[All Fields]).” The Entrez efetch utility was then used to download the complete FASTA sequences for each accession number. Primers were aligned to each sequence using the msa.sh (which stands for MultiStateAligner not Multiple Sequence Alignment) function of BBMap v38.86. The CIGAR string contained in the resulting SAM file for each primer was used to determine the number of matches between the aligned primer and the subject sequence. Percent sequence identity was calculated using the number of matches divided by the alignment length (which was equal to the primer length for all cases). Inclusivity was then determined by calculating the portion of SARS-CoV-2 whole genome sequences that had 100% sequence identity with all of the aligned primers. For a more relaxed analysis, the number of mismatches was calculated for each primer alignment. For each sequence, if the sum of mismatches across all primers was less than a given mismatch threshold (either 0, 1, or more), this sequence was counted for sequence inclusivity. For this analysis, the constituent primers of FIP and BIP, F1c/F2 and B1c/B2, respectively, were used in lieu of the FIP and BIP primers. The orflab.II and orf7ab.I primers set had 100% sequence identity with 97.52% and 95.12% of the 39,134 sequences available, respectively. When one mismatch was allowed across the entire set, the orflab.II and orf7ab.I primer sets then had 99.63% and 99.29% of the sequences meet this constraint.
We conducted in-silico inclusivity and sequence identity studies to verify the conservation of the RT-LAMP primers with available SARS-CoV-2 sequences and to predict cross-reactivity of our primer sets. In-silico sequence identity analyses were conducted by performing a BLAST search of each primer against sequences available in the NCBI Nucleotide database for the specific taxon of interest. Parameters that were used in the BLAST search can be found in Table 5B. The sequence identity for each hit in the BLAST analysis was then calculated by using the number of matches for a hit divided by the length of the primer, not the alignment length. Overall sequence identity was determined by calculating the maximum sequence identity of all hits for a specific primer against an individual organism and is reported in Table 5C and Table 5D. Primers with greater than 80% sequence identity were deemed as potentially cross-reactive. One primer deemed potentially cross reactive (sequence identity >0.8) was the F2 primer of orflab.II with B. pertussis; all other primers were not predicted to be cross-reactive (Table 5A and Table 5B). Since a single primer is predicted to be cross-reactive, we do not expect that our primer sets are cross-reactive with any of the organisms. We confirmed that these targets were not significantly cross-reactive experimentally using genomic extracts of these targets (Table 5E and Table 5F). One replicate of orf7ab.I was cross-reactive with HRSV Strain A2011 but was not deemed to be a concern since all three replicates did not amplify. The calculated sensitivity was 100% for both orflab.II and orf7ab.I and the calculated specificity was 100% and 99.13% for orflab.II and orf7ab.I, respectively.
The orf7ab.I and orflab.II primer sets were used to test cross-reactivity against several pathogens found in the upper respiratory tract of individuals presenting with symptoms similar to COVID-19. For each pathogen, 5 μL of genomic DNA/RNA at a concentration of 2×103 copies/μL was used as a template to result in a total of 104 copies/reaction. NTC reactions with water were used as negative controls, and heat-inactivated SARS-CoV-2 at a concentration of 2×103 copies/μL to result in a total of 104 copies/reaction was used as a positive control. Positive amplification was determined as any amplification at 30 minutes that was greater than 50% of the average fluorescent intensity value of the positive controls at 30 minutes. Sensitivity and specificity were calculated in the same manner as listed before. The pathogens used and their reactivity with orf7ab.I and orf7ab.II are displayed in Table 5E and Table 5F, respectively.
Chlamydia pneumoniae
Haemophilus
influenzae
Legionella pneumophila
Mycobacterium
tuberculosis
Streptococcus
pneumoniae
Streptococcus
pyogenes
Bordetella pertussis
Mycoplasma
pneumoniae
Pneumocystis jirovecii
Candida albicans
Pseudomonas
aeruginosa
Staphylococcus
epidermis
Streptococcus salivarius
Chlamydia pneumoniae
Haemophilus
influenzae
Legionella pneumophila
Mycobacterium
tuberculosis
Streptococcus
pneumoniae
Streptococcus
pyogenes
Bordetella pertussis
Mycoplasma
pneumoniae
Pneumocystis jirovecii
Candida albicans
Pseudomonas
aeruginosa
Staphylococcus
epidermis
Streptococcus salivarius
Staphylococcus epidermidis
Mycobacterium Tuberculosis
Haemophilus Influenzae
Legionella pneumophilia
Streptococcus pyogenes (T1)
Streptococcus pneumoniae
Bordetella pertussis
Pseudomonas aeruginosa
Staphylococcus epidermidis
Mycobacterium Tuberculosis
Haemophilus Influenzae
Legionella pneumophilia
Streptococcus pyogenes (T1)
Streptococcus pneumoniae
Bordetella pertussis
Pseudomonas aeruginosa
The following conserved genes of SARS-CoV-2 were targeted to design at least three primer sets per gene: the N gene, the RdRp gene, and the orflab segment using PrimerExplorer V5. Three experiments were performed using heat-inactivated SARS-CoV-2 to select the optimal primer set: (1) using a fluorescent RT-LAMP kit and pooled saliva to determine whether the primers could amplify the target in 18% saliva, which is the maximum concentration of saliva that can be achieved in a liquid format); (2) using a fluorescent RT-LAMP kit and water to determine whether the primers could dimerize (i.e., show amplification in non-template controls (NTC)); and (3) using a colorimetric RT-LAMP kit to determine the limit of detection (LoD) of the primer set.
Primer sets were screened in water using a fluorescent RT-LAMP kit and in-vitro transcribed SARS-CoV-2 RNA for the gene targeted by the primer set to assess performance and ability to dimerize. Water was used to prevent any off-target interactions with the sample background. The assay utilized a no-primer control to ensure that the reaction zones do not change color when heated. Further screening to determine off-target interactions was conducted in 18% saliva using a fluorescent RT-LAMP kit and heat-inactivated SARS-CoV-2 to assess performance in complex samples. After screening the primer sets depicted in Table 6 and based on the results illustrated in
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RT-LAMP primer sets were designed using PrimerExplorer v5. Parameters used to design primers can be found in Table 8. All other Primer Explorer parameters that are not indicated in Table 8 were set to the default values.
Primer sets were designed using portions of the SARS-CoV-2 reference genome (NCBI accession number: NC 045512). Primer sets for RdRP were designed by first splitting the nsp12 gene sequence into 2 portions. Primer set RdRP.I was designed using the first portion of the nsp12 sequence, while primer sets RdRP.II and RdRP.III were designed using the second portion of the nsp12 sequence. Primer sets for orflab were designed using a portion of the orflab gene sequence. Primer sets for RNaseP were designed using the mRNA sequence for the POP7 gene, which encodes for the p20 subunit of RNaseP.
In order to increase the speed of the RT-LAMP reaction, the inclusion of multiple primer sets in the fluorescent RT-LAMP reaction mix was investigated. The investigation was carried out in water using NEB LAMP fluorescent dye as a fluorometric indicator. The inclusion of multiple primer sets did not seem to increase the reaction speed significantly. Rather, the reaction proceeded primarily at the speed of the primer set that had the fastest reaction time when used in isolation.
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A sample list of materials used in a LAMP protocol can be found in Table 9.
Primer Mix
The primer mix was formulated by: (1) Obtaining all 6 diluted primers (100 μM) from the freezer, (2) Mixing 80 μl of FIP, 80 μl of BIP, 20 μl of FB, 20 μl LB, 10 μl of F3 and 10 μl of B3 in a tube; and (3) Adding enough PCR-grade water to reach 500 μl total.
1. Obtain the NEB Bst 2.0 Warmstart kit and the primer mix; 2. While the reagents thaw and after at least 5 minutes of spraying the RNaseAway, wipe the surfaces with a Kimwipe; 3. Label all the PCR tubes needed with the DNA sample and primers that will be used. Make sure to add a negative control which will not have DNA added; 4. Add 5 μl of PCR-grade water (or dye), 12.5 μl of NEB Bst 2.0 Warmstart kit and 2.5 μl of primer mix per reaction. A master mix can be made for however many reactions will be run; 5. If 5 μl of EBT dye are added, it should be in 1500 μM concentration so that the final concentration ends up being 30004; 6. The reactions with no DNA should have an extra 5 μl of PCR-grade water added and not opened again until they have to be loaded on a gel; 7. Once ready, the PCR tubes should be put in the PCR tray previously left in the pass-through chamber and carried out to a different room; 8. Once in the new room, obtain the sample DNA from the −20° C. freezer; 9. Spray your hands with RNaseAway spray and rub your hands around the DNA sample tube so that it is covered in the spray as well; 10. Add 5 μl of the DNA sample where appropriate and close the tubes. Avoid opening 2 DNA tubes at the same time and close the PCR tubes right after adding the DNA; 11. Put the samples in a thermocycler set at 65° C. for 1 hour and 80° C. for 5 minutes (samples may be kept at −20° C. overnight after this operation).
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As shown in the figure, the virus-spiked samples reach a fluorescence of about 5×104 in intensity between 7 to 13 minutes after commencement of the reaction, while the control samples reach a fluorescence of about 5×104 in intensity between 45-60 minutes after commencement of the reaction.
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As shown in the figure, the virus-spiked samples reach a fluorescence of about 5×104 in intensity between 10 to 12 minutes after commencement of the reaction, while the control samples reach a fluorescence of about 5×104 in intensity between 35-45 minutes after commencement of the reaction
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The four virus-spiked samples reach a fluorescence of about 7×104 in intensity 15 minutes after commencement of the reaction for a viral concentration of about 100 k copies of the SARS-CoV-2 virus. One of the four controls spiked after about 40 minutes with the remaining three controls exhibiting no spike in fluorescence above a baseline level.
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A list of primers (F3, B3, FIP, BIP, LF, and LB) with sequences and reverse complements for N.3, N.6, N.10, N.13e, RdRP.1, RdRP.2, RdRP.3, RdRP.4, orflab.1, orflab.2, orflab.3, orflab.4, E.1, E.2, E.3, E.4, E.5, RNaseP.1, RNaseP.2, RNaseP.3, RNaseP.4, RNaseP.5, RegX1.1, RegX1.2, RegX2.1, RegX2.2, RegX2.3, RegX2.4, RegX2.3, RegX2.4, and RegX3.1 can be found in Table 10.
A list of primers (F2, F1c, B2, and Bic) with forward sequences for N.3, N.6, N.10, N.13e, nsp12.1, nsp12.2, nsp12.3, nsp12.4, orflab.1, orflab.2, orflab.3, orflab.4, E.1, E.2, E.3, E.4, E.5, RNaseP.1, RNaseP.2, RNaseP.3, RNaseP.4, RNaseP.5, RegX1.1, RegX1.2, RegX2.1, RegX2.2, RegX2.3, RegX2.4, RegX2.3, RegX2.4, and RegX3.1 can be found in Table 11.
RT-LAMP primers were initially designed using the regions targeted by the CDC SARS-CoV-2 RT-PCR primers and other RT-LAMP primers. Primers were blasted against the target genome using the NCBI's blastn algorithm with the following parameters: word size: 7; expect threshold; 1E11. The regions contained within the resulting alignment of the Forward/Reverse primers (for RT-PCR primers) or F3/B3 primers (for RT-LAMP primers) were exported to FASTA file format. If the region identified by primer alignment was less than 1200 nucleotides, the identified region was padded equally on both sides with nucleotides corresponding to the organism's sequence until the total length of the region was approximately 1200 nucleotides. The RdRP gene was divided into two regions to ensure that the sequences were less than 2,000 nucleotides in length.
Additional regions were identified by separating the SARS-CoV-2 genome (Accession #: NC_045512.2) into portions of 2,000 nucleotides. The regions overlapped by 500 nucleotides. Each of these regions were referred to as Tiled Regions. For example, Tiled region 1 would be the nucleotide sequence from position 0 to position 2000 of the reference genome, tiled region 2 from 1,500 to 3,500, tiled region 3 from 3,000 to 5,000, and so forth.
Each Tiling Region was used as the input into the Primer Explorer v5 algorithm. The algorithm parameters were adjusted to design primers (most notably the length of the primers and distance between primers). Primer sets for targeted regions from the CDC and literature were chosen based on their end stability, namely the 5′ end of the F1c/B1c and the 3′ end of the F3/B3/F2/B2/LF/LB should be less than −4.00 kcal/mol (i.e., more negative). If these restrictions could not be maintained, then the primer sets with closest end stabilities to −4.00 were selected. Selected primer sets were used as inputs to design loop primers in the Primer Explorer v5 algorithm. Loop primers with melting temperatures closest to 65° C. were chosen provided they still maintained the thermodynamic parameters previously described in this disclosure.
Tiled regions were used as input into the Primer Explorer v5 algorithm. Parameters were set to maximize the number of returned primer sets by: (a) reducing the minimum primer dimerization energy, (b) increasing the distance between loop primers and F2, and (c) increasing the maximum number of primer sets returned. Each of the resulting primer sets (which did not include loop primers) was aligned against results from the proprietary FAST-NA algorithm (which determines subsequences with minimal sequence identity to organisms found in the human respiratory tract background, namely human DNA and bacteria/viruses which inhabit the respiratory tract). Primer sets that mostly aligned with these FAST-NA results (less than 5 nucleotides total for all primers outside of the FAST-NA regions) and maintained most of the thermodynamic parameters as previously described were selected for further experimental screening. These primers are indicated by the prefix RegX.
Primer sets selected the preceding were screened experimentally to determine their reaction efficacy and efficiency in order of decreasing priority: (i) number of false positives, (ii) reaction speed, and (iii) limit of detection. Experiments were carried out sequentially in (1) solution (water followed by saliva) using fluorometric RT-LAMP, then (2) colorimetric RT-LAMP in solution, and finally (3) colorimetric RT-LAMP on paper. Screened primer sets were experimentally tested for cross-reactivity against other organisms in the human respiratory tract.
In one example, an isolated complementary DNA (cDNA) of a nucleic acid molecule is provided and can comprise: a nucleotide sequence that is at least 85% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or a combination thereof.
In one example of an isolated complementary DNA (cDNA) of a nucleic acid molecule, the nucleotide sequence can be at least 85% identical to SEQ ID NO: 9.
In another example of an isolated complementary DNA (cDNA) of a nucleic acid molecule, the nucleotide sequence can comprise SEQ ID NO: 1 joined to SEQ ID NO: 2 by a linking sequence selected from Table 11.
In another example of an isolated complementary DNA (cDNA) of a nucleic acid molecule, the nucleotide sequence can be at least 85% identical to SEQ ID NO: 10.
In another example of an isolated complementary DNA (cDNA) of a nucleic acid molecule, the nucleotide sequence can comprise SEQ ID NO: 3 joined to SEQ ID NO: 4 by a linking sequence selected from Table 11.
In another example of an isolated complementary DNA (cDNA) of a nucleic acid molecule, the guanine and cytosine (GC) content of the nucleotide sequence can be 50% or less.
In another example of an isolated complementary DNA (cDNA) of a nucleic acid molecule, the guanine and cytosine (GC) content of the nucleotide sequence can be 40% or less.
In another example of an isolated complementary DNA (cDNA) of a nucleic acid molecule, an end stability of the nucleotide sequence can be less than −3.5 kcal/mol.
In another example of an isolated complementary DNA (cDNA) of a nucleic acid molecule, the nucleotide sequence can have a melting temperature of from about 40° C. to about 62° C.
In another example of an isolated complementary DNA (cDNA) of a nucleic acid molecule, the nucleotide sequence can have a minimum primer dimerization energy of less than −3 kcal/mol.
In another example of an isolated complementary DNA (cDNA) of a nucleic acid molecule, the nucleotide sequence can be less than 50% identical to nucleotide sequences associated with non-target agents.
In another example of an isolated complementary DNA (cDNA) of a nucleic acid molecule, the nucleotide sequence can be at least 90% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or a combination thereof.
In another example of an isolated complementary DNA (cDNA) of a nucleic acid molecule, the nucleotide sequence can be at least 95% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or a combination thereof.
In another example of an isolated complementary DNA (cDNA) of a nucleic acid molecule, the nucleotide sequence can be 100% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or a combination thereof
In one example there is provided a primer set for reverse transcription loop-mediated isothermal amplification (RT-LAMP) analysis that can comprise: a forward inner primer (FIP) sequence that is at least 85% identical to a combination of SEQ ID NO: 1 and SEQ ID NO: 2; a backward inner primer (BIP) sequence that is at least 85% identical to a combination of seq ID NO: 3 and SEQ ID NO: 4. a forward outer primer (F3) sequence that is at least 85% identical to SEQ ID NO: 5; a backward outer primer (B3) sequence that is at least 85% identical to SEQ ID NO: 6; a forward loop primer (LF) sequence that is at least 85% identical to SEQ ID NO: 7; and a backward loop primer (LB) sequence that is at least 85% identical to SEQ ID NO: 8.
In one example of a primer set for reverse transcription loop-mediated isothermal amplification (RT-LAMP) analysis, the FIP sequence can further comprise a linking sequence joining SEQ ID NO: 1 and SEQ ID NO: 2, wherein the linking sequence is selected from Table 11.
In another example of a primer set for reverse transcription loop-mediated isothermal amplification (RT-LAMP) analysis, the BIP sequence can further comprise a linking sequence joining SEQ ID NO: 3 and SEQ ID NO: 4, wherein the linking sequence is selected from Table 11.
In another example of a primer set for reverse transcription loop-mediated isothermal amplification (RT-LAMP) analysis, the guanine and cytosine (GC) content of the FIP, the BIP, the F3, the B3, the LF, the LB, or a combination thereof can be 50% or less.
In another example of a primer set for reverse transcription loop-mediated isothermal amplification (RT-LAMP) analysis, the guanine and cytosine (GC) content of the FIP, the BIP, the F3, the B3, the LF, the LB, or a combination thereof can be 40% or less.
In another example of a primer set for reverse transcription loop-mediated isothermal amplification (RT-LAMP) analysis, an end stability of the FIP, the BIP, the F3, the B3, the LF, the LB, or a combination thereof can be less than −2.5 kcal/mol.
In another example of a primer set for reverse transcription loop-mediated isothermal amplification (RT-LAMP) analysis, the FIP, the BIP, the F3, the B3, the LF, the LB, or a combination thereof can have a melting temperature of from about 40° C. to about 62° C.
In another example of a primer set for reverse transcription loop-mediated isothermal amplification (RT-LAMP) analysis, the FIP, the BIP, the F3, the B3, the LF, the LB, or a combination thereof can have a minimum primer dimerization energy of less than −3.0 kcal/mol.
In another example of a primer set for reverse transcription loop-mediated isothermal amplification (RT-LAMP) analysis, the FIP, the BIP, the F3, the B3, the LF, the LB, or a combination thereof can be less than 50% identical to nucleotide sequences associated with non-target agents.
In another example of a primer set for reverse transcription loop-mediated isothermal amplification (RT-LAMP) analysis, the FIP sequence can be at least 90% identical to a combination of SEQ ID NO: 1 and SEQ ID NO: 2; the BIP sequence can be at least 90% identical to a combination of seq ID NO: 3 and SEQ ID NO: 4; the F3 sequence can be at least 90% identical to SEQ ID NO: 5; the B3 sequence can be at least 90% identical to SEQ ID NO: 6; the LF sequence can be at least 90% identical to SEQ ID NO: 7; and the LB sequence can be at least 90% identical to SEQ ID NO: 8.
In another example of a primer set for reverse transcription loop-mediated isothermal amplification (RT-LAMP) analysis, the FIP sequence can be at least 95% identical to a combination of SEQ ID NO: 1 and SEQ ID NO: 2; the BIP sequence can be at least 95% identical to a combination of seq ID NO: 3 and SEQ ID NO: 4; the F3 sequence can be at least 95% identical to SEQ ID NO: 5; the B3 sequence can be at least 95% identical to SEQ ID NO: 6; the LF sequence can be at least 95% identical to SEQ ID NO: 7; and the LB sequence can be at least 95% identical to SEQ ID NO: 8.
In another example of a primer set for reverse transcription loop-mediated isothermal amplification (RT-LAMP) analysis, the FIP sequence can be at least 100% identical to a combination of SEQ ID NO: 1 and SEQ ID NO: 2, which is equivalent to SEQ ID NO: 9; the BIP sequence can be at least 100% identical to a combination of seq ID NO: 3 and SEQ ID NO: 4, which is equivalent to SEQ ID NO: 10; the F3 sequence is at least 100% identical to SEQ ID NO: 5; the B3 sequence can be at least 100% identical to SEQ ID NO: 6; the LF sequence can be at least 100% identical to SEQ ID NO: 7; and the LB sequence can be at least 100% identical to SEQ ID NO: 8.
In one example there is provided, a method of detecting a target pathogen from a Coronaviridae family in a sample comprising: providing a primer set comprising: a forward inner primer (FIP) sequence that is at least 85% identical to a combination of SEQ ID NO: 1 and SEQ ID NO: 2; a backward inner primer (BIP) sequence that is at least 85% identical to a combination of seq ID NO: 3 and SEQ ID NO: 4. a forward outer primer (F3) sequence that is at least 85% identical to SEQ ID NO: 5; a backward outer primer (B3) sequence that is at least 85% identical to SEQ ID NO: 6; a forward loop primer (LF) sequence that is at least 85% identical to SEQ ID NO: 7; and a backward loop primer (LB) sequence that is at least 85% identical to SEQ ID NO: 8; and including the primer set in a reverse transcription loop-mediated isothermal amplification (RT-LAMP) procedure containing the sample.
In one example of a method of detecting a target pathogen from a Coronaviridae family in a sample, the target pathogen can be a coronavirus selected from: Severe Acute Respiratory Syndrome (SARS)-CoV (SARS-CoV), Severe Acute Respiratory Syndrome (SARS)-CoV 2 (SARS-CoV-2), Middle East Respiratory Syndrome (MERS)-CoV (MERS-CoV), SARS-CoV hCoV-HKU1, hCoV-0C43, hCoV-NL63, and hCoV-229E.
In another example of a method of detecting a target pathogen from a Coronaviridae family in a sample, the sample can be from a human subject.
In another example of a method of detecting a target pathogen from a Coronaviridae family in a sample, the target pathogen can be Severe Acute Respiratory Syndrome (SARS)-CoV 2 (SARS-CoV-2).
In another example of a method of detecting a target pathogen from a Coronaviridae family in a sample, the method can further comprise observing an output test indicator of the RT-LAMP process indicating the presence or absence of the target pathogen.
In another example of a method of detecting a target pathogen from a Coronaviridae family in a sample, the output test indicator is a color indicator.
In one example there is provided a primer set for reverse transcription loop-mediated isothermal amplification (RT-LAMP) analysis which comprises: a forward inner primer (FIP) sequence that is at least 85% identical to a combination of SEQ ID NO: 11 and SEQ ID NO: 12; a backward inner primer (BIP) sequence that is at least 85% identical to a combination of seq ID NO: 13 and SEQ ID NO: 14. a forward outer primer (F3) sequence that is at least 85% identical to SEQ ID NO: 15; a backward outer primer (B3) sequence that is at least 85% identical to SEQ ID NO: 16; a forward loop primer (LF) sequence that is at least 85% identical to SEQ ID NO: 17; and a backward loop primer (LB) sequence that is at least 85% identical to SEQ ID NO: 18.
In one example of a primer set for reverse transcription loop-mediated isothermal amplification (RT-LAMP) analysis, the primer set can comprise the FIP sequence can be 100% identical to a combination of SEQ ID NO: 11 and SEQ ID NO: 12, which is equivalent to SEQ ID NO: 19.
In another example of a primer set for reverse transcription loop-mediated isothermal amplification (RT-LAMP) analysis, the FIP sequence can further comprise a linking sequence joining SEQ ID NO: 11 and SEQ ID NO: 12, wherein the linking sequence is selected from Table 11.
In another example of a primer set for reverse transcription loop-mediated isothermal amplification (RT-LAMP) analysis, the BIP sequence can be 100% identical to a combination of SEQ ID NO: 13 and SEQ ID NO: 14, which is equivalent to SEQ ID NO: 20.
In another example of a primer set for reverse transcription loop-mediated isothermal amplification (RT-LAMP) analysis, the BIP sequence can further comprise a linking sequence joining SEQ ID NO: 13 and SEQ ID NO: 14, wherein the linking sequence is selected from Table 11.
In one example there is provided, a primer set for reverse transcription loop-mediated isothermal amplification (RT-LAMP) analysis that can comprise: a forward inner primer (FIP) sequence that is at least 85% identical to a combination of SEQ ID NO: 21 and SEQ ID NO: 22;
a backward inner primer (BIP) sequence that is at least 85% identical to a combination of seq ID NO: 23 and SEQ ID NO: 24. a forward outer primer (F3) sequence that is at least 85% identical to SEQ ID NO: 25; a backward outer primer (B3) sequence that is at least 85% identical to SEQ ID NO: 26; a forward loop primer (LF) sequence that is at least 85% identical to SEQ ID NO: 27; and a backward loop primer (LB) sequence that is at least 85% identical to SEQ ID NO: 28.
In one example of a primer set for reverse transcription loop-mediated isothermal amplification (RT-LAMP) analysis, the FIP sequence can be 100% identical to a combination of SEQ ID NO: 21 and SEQ ID NO: 22 which is equivalent to SEQ ID NO: 29.
In another example of a primer set for reverse transcription loop-mediated isothermal amplification (RT-LAMP) analysis, the FIP sequence can further comprise a linking sequence joining SEQ ID NO: 21 and SEQ ID NO: 22, wherein the linking sequence is selected from Table 11.
In another example of a primer set for reverse transcription loop-mediated isothermal amplification (RT-LAMP) analysis, the BIP sequence can be 100% identical to a combination of SEQ ID NO: 23 and SEQ ID NO: 24, which is equivalent to SEQ ID NO: 30.
In another example of a primer set for reverse transcription loop-mediated isothermal amplification (RT-LAMP) analysis, the BIP sequence can further comprise a linking sequence joining SEQ ID NO: 23 and SEQ ID NO: 24, wherein the linking sequence is selected from Table 11.
In one example there is provided, a primer set for reverse transcription loop-mediated isothermal amplification (RT-LAMP) analysis that can comprise: a forward inner primer (FIP) sequence that is at least 85% identical to a combination of SEQ ID NO: 31 and SEQ ID NO: 32; a backward inner primer (BIP) sequence that is at least 85% identical to a combination of seq ID NO: 33 and SEQ ID NO: 34. a forward outer primer (F3) sequence that is at least 85% identical to SEQ ID NO: 35; a backward outer primer (B3) sequence that is at least 85% identical to SEQ ID NO: 36; a forward loop primer (LF) sequence that is at least 85% identical to SEQ ID NO: 37; and a backward loop primer (LB) sequence that is at least 85% identical to SEQ ID NO: 38.
In one example of a primer set for reverse transcription loop-mediated isothermal amplification (RT-LAMP) analysis, the FIP sequence can be 100% identical to a combination of SEQ ID NO: 31 and SEQ ID NO: 32, which is equivalent to SEQ ID NO: 39.
In another example of a primer set for reverse transcription loop-mediated isothermal amplification (RT-LAMP) analysis, the FIP sequence can further comprise a linking sequence joining SEQ ID NO: 31 and SEQ ID NO: 32, wherein the linking sequence is selected from Table 11.
In another example of a primer set for reverse transcription loop-mediated isothermal amplification (RT-LAMP) analysis, the BIP sequence can be 100% identical to a combination of SEQ ID NO: 33 and SEQ ID NO: 34 which is equivalent to SEQ ID NO: 40.
In another example of a primer set for reverse transcription loop-mediated isothermal amplification (RT-LAMP) analysis, the BIP sequence can further comprise a linking sequence joining SEQ ID NO: 33 and SEQ ID NO: 34, wherein the linking sequence is selected from Table 11.
In yet another example there is provided, a primer set for reverse transcription loop-mediated isothermal amplification (RT-LAMP) analysis that can comprise: a forward inner primer (FIP) sequence that is at least 85% identical to a combination of SEQ ID NO: 41 and SEQ ID NO: 42; a backward inner primer (BIP) sequence that is at least 85% identical to a combination of seq ID NO: 43 and SEQ ID NO: 44. a forward outer primer (F3) sequence that is at least 85% identical to SEQ ID NO: 45; a backward outer primer (B3) sequence that is at least 85% identical to SEQ ID NO: 46; a forward loop primer (LF) sequence that is at least 85% identical to SEQ ID NO: 47; and a backward loop primer (LB) sequence that is at least 85% identical to SEQ ID NO: 48.
In one example of a primer set for reverse transcription loop-mediated isothermal amplification (RT-LAMP) analysis, the FIP sequence can be 100% identical to a combination of SEQ ID NO: 41 and SEQ ID NO: 42 which is equivalent to SEQ ID NO: 49.
In another example of a primer set for reverse transcription loop-mediated isothermal amplification (RT-LAMP) analysis, the FIP sequence can further comprise a linking sequence joining SEQ ID NO: 41 and SEQ ID NO: 42, wherein the linking sequence is selected from Table 11.
In another example of a primer set for reverse transcription loop-mediated isothermal amplification (RT-LAMP) analysis, the BIP sequence can be 100% identical to a combination of SEQ ID NO: 43 and SEQ ID NO: 44 which is equivalent to SEQ ID NO: 50.
In another example of a primer set for reverse transcription loop-mediated isothermal amplification (RT-LAMP) analysis, the BIP sequence can further comprise a linking sequence joining SEQ ID NO: 43 and SEQ ID NO: 44, wherein the linking sequence is selected from Table 11.
In one example there is provided, a primer set for reverse transcription loop-mediated isothermal amplification (RT-LAMP) analysis which can comprise: a forward inner primer (FIP) sequence that is at least 85% identical to a combination of SEQ ID NO: 51 and SEQ ID NO: 52; a backward inner primer (BIP) sequence that is at least 85% identical to a combination of seq ID NO: 53 and SEQ ID NO: 54. a forward outer primer (F3) sequence that is at least 85% identical to SEQ ID NO: 55; a backward outer primer (B3) sequence that is at least 85% identical to SEQ ID NO: 56; a forward loop primer (LF) sequence that is at least 85% identical to SEQ ID NO: 57; and a backward loop primer (LB) sequence that is at least 85% identical to SEQ ID NO: 58.
In one aspect, the FIP sequence can be 100% identical to a combination of SEQ ID NO: 51 and SEQ ID NO: 52 which is equivalent to SEQ ID NO: 59.
In another example of a primer set for reverse transcription loop-mediated isothermal amplification (RT-LAMP) analysis, the FIP sequence can further comprise a linking sequence joining SEQ ID NO: 51 and SEQ ID NO: 52, wherein the linking sequence is selected from Table 11.
In another example of a primer set for reverse transcription loop-mediated isothermal amplification (RT-LAMP) analysis, the BIP sequence can be 100% identical to a combination of SEQ ID NO: 53 and SEQ ID NO: 54, which is equivalent to SEQ ID NO: 60.
In another example of a primer set for reverse transcription loop-mediated isothermal amplification (RT-LAMP) analysis, the BIP sequence can further comprise a linking sequence joining SEQ ID NO: 53 and SEQ ID NO: 54, wherein the linking sequence is selected from Table 11.
In one example there is provided, a primer set for reverse transcription loop-mediated isothermal amplification (RT-LAMP) analysis which can comprise: a forward inner primer (FIP) sequence that is at least 85% identical to a combination of SEQ ID NO: 61 and SEQ ID NO: 62; a backward inner primer (BIP) sequence that is at least 85% identical to a combination of seq ID NO: 63 and SEQ ID NO: 64. a forward outer primer (F3) sequence that is at least 85% identical to SEQ ID NO: 65; a backward outer primer (B3) sequence that is at least 85% identical to SEQ ID NO: 66; a forward loop primer (LF) sequence that is at least 85% identical to SEQ ID NO: 67; and a backward loop primer (LB) sequence that is at least 85% identical to SEQ ID NO: 68.
In one example of a primer set for reverse transcription loop-mediated isothermal amplification (RT-LAMP) analysis, the FIP sequence can be 100% identical to a combination of SEQ ID NO: 61 and SEQ ID NO: 62 which is equivalent to SEQ ID NO: 69.
In another example of a primer set for reverse transcription loop-mediated isothermal amplification (RT-LAMP) analysis, the FIP sequence can further comprise a linking sequence joining SEQ ID NO: 61 and SEQ ID NO: 62, wherein the linking sequence is selected from Table 11.
In another example of a primer set for reverse transcription loop-mediated isothermal amplification (RT-LAMP) analysis, the BIP sequence can be 100% identical to a combination of SEQ ID NO: 63 and SEQ ID NO: 64, which is equivalent to SEQ ID NO: 70.
In another example of a primer set for reverse transcription loop-mediated isothermal amplification (RT-LAMP) analysis, the BIP sequence can further comprise a linking sequence joining SEQ ID NO: 63 and SEQ ID NO: 64, wherein the linking sequence is selected from Table 11.
It should be understood that the above-described methods are only illustrative of some embodiments of the present invention. Numerous modifications and alternative arrangements may be devised by those skilled in the art without departing from the spirit and scope of the present invention and the appended claims are intended to cover such modifications and arrangements. Thus, while the present invention has been described above with particularity and detail in connection with what is presently deemed to be the most practical and preferred embodiments of the invention, it will be apparent to those of ordinary skill in the art that variations including, may be made without departing from the principles and concepts set forth herein.
This application claims the benefit of U.S. Provisional Patent Application Ser. No. 63/148,527 filed Feb. 11, 2021, the entire contents of which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 63148527 | Feb 2021 | US |
