Primers set and crrna, reagent system and assay thereof for determination of fungal nucleic acid

Abstract

The present disclosure provides a recombinase polymerase amplification (RPA) primers set including a forward primer having 70% to 100% sequence identity to SEQ ID NO: 1; a reverse primer having 70% to 100% sequence identity to SEQ ID NO: 2; and a crRNA sequence having 70% to 100% sequence identity to SEQ ID NO: 3 and capable of 5 hybridizing to the region amplified by the forward primer and the reverse primer of the present invention to aid in the determination of fungal nucleic acid in a sample with high specificity and sensitivity. Also provided is an assay protocol based on recombinase polymerase amplification (RPA), Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) adaptive immune systems for rapid determination of 0 fungal nucleic acid in the sample.

Description

PRIMERS SET AND CRRNA, REAGENT SYSTEM AND ASSAY THEREOF FOR DETERMINATION OF FUNGAL NUCLEIC ACIDFIELD OF THE INVENTION

[0001] The present disclosure pertains to an oligonucleotide RPA primers and crRNA sequence for determination of fungi in a sample. In particular, the present disclosure provides recombinase polymerase amplification (RPA) primers set and crRNA sequence, a reagent system comprising the same and an assay for determination of fungal nucleic acid in a sample.BACKGROUND OF THE INVENTION

[0002] The background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0003] The present-day tools and assay protocols for determination of fungi present in a sample in resource-limited countries is challenging because of the following reasons - i) requirement of specialized equipment and trained personnel for the collection of samples in specific manner, ii) requirement of expensive equipment and trained mycologists to establish the assay protocols and iii) even with access to the equipment and personnel, the present-day tools and assay modalities lack sensitivity and specificity and are time-consuming such as culture methodologies which require very long duration of around 3-14 days and often give false positive or incorrection results. Determination of fungal, bacterial, and viral species in a sample is challenging and incorrect determination pose sever challenges in the clinical, industrial and basic research set ups.

[0004] Nucleic acids have been often used as a signature of biological information. The ability to rapidly detect nucleic acids with high sensitivity and single-base specificity has been considered to serve as a generalizable scientific tool and revolutionize assay protocols for determination and differentiation of various fungal species in a given sample. Although attempts have been made to develop various tools and techniques for detecting nucleic acids for determination of fungal species they inevitably suffer from trade-offs among sensitivity, specificity, simplicity, and speed. For example, for detecting and analyzing a small quantity of nucleic acids, nucleic acid amplification technologies are used, the polymerase chain reaction (PCR) being the most widely used method. However, the PCR technique has certainlimitations such as requirement of multiple reiterative thermal cycles among different temperatures for different stages, e.g., denaturing, extension, and re-annealing respectively, wherein each stage must be sufficiently long, making the entire PCR reaction very time consuming over a period of around 4-8 hours. Further, during the reiterative thermal cycling, a target sequence is extended and amplified efficiently only at the extension stage, thus, PCR is limited in the size of the sequence to be amplified and the efficient amplification range is generally below 2000 bp. Besides, relying on precise cycling of a reaction system between different temperatures, PCR requires expensive equipment such as a thermocycler (Do we need to include about AGE for detection). In addition, the repeated denaturing stage exposes the reaction system to a temperature as high as 90° C. or above. Such a high temperature can damage some components of the reaction system and thereby negatively impact the length and quality of the amplified products.

[0005] Hence, though PCR approaches are sensitive but are expensive and rely on complex instrumentation, limiting usability to highly trained operators in laboratory settings. Other approaches, such as recent ones combining isothermal nucleic acid amplification with portable platforms can provide high specificity, but have somewhat limited applications due to low sensitivity. As nucleic acid signatures become increasingly relevant for determination of fungal species in samples for a variety of applications, tools and technologies that provide high specificity and sensitivity in a quicker, simple manner without having need for very expensive specialized equipment will be of great importance in both clinical, industrial and basic research settings.

[0006] Accordingly, there remains an unmet need to provide means and assay protocol that can determine fungal species in samples with high specificity and sensitivity in a simpler and quicker manner as well as obviate need of highly specialized equipment or personnel.OBJECTS OF THE INVENTION

[0007] It is an object of the present disclosure to provide a recombinase polymerase amplification (RPA) primers set including a forward primer (Why special mention for forward primers) for determination of fungal nucleic acid in a sample.

[0008] It is an object of the present disclosure to provide a crRNA sequence capable of hybridizing to the region amplified by the forward primer and the reverse primer of the present invention to aid in the determination of fungal nucleic acid in a sample.

[0009] It is another object of the present disclosure to provide a reagent system comprising a recombinase polymerase amplification (RPA) primers set and crRNA sequence.

[0010] It is another object of the present disclosure to provide a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) based assay to determine fungal nucleic acid and thereby fungal organisms in samples.SUMMARY

[0011] The present disclosure provides tools and techniques based on recombinase polymerase amplification (RPA), Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) adaptive immune systems for determination of fungal nucleic acid in a sample.

[0012] In an aspect, the present disclosure provides a recombinase polymerase amplification (RPA) primers set including a forward primer (Why special mention for forward primers) for determination of fungal nucleic acid in a sample. The forward primer having 70% to 100% sequence identity to SEQ ID NO: 1, and the reverse primer has 70% to 100% sequence identity to SEQ ID NO: 2.

[0013] In one aspect, the present disclosure provides a crRNA sequence capable of hybridizing to the region amplified by the forward primer and the reverse primer of the present invention to aid in the determination of fungal nucleic acid in a sample. The crRNA sequence has 70% to 100% sequence identity to SEQ ID NO: 3.

[0014] In an aspect, the present disclosure provides a reagent system for determination of fungal nucleic acid in a sample, the system comprising: a recombinase polymerase amplification (RPA) primers set including a forward primer having 70% to 100% sequence identity to SEQ ID NO: 1, and the reverse primer having 70% to 100% sequence identity to SEQ ID NO: 2 and a crRNA sequence having 70% to 100% sequence identity to SEQ ID NO: 3. The reagent system additionally comprises a probe having 70% to 100% sequence identity (sequence identity % not applicable here) to 5’-L / TTATT / Q-3’, wherein L is label, and Q is quencher.

[0015] In further aspect, the present disclosure provides a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) based assay to determine fungal nucleic acid and thereby fungal organisms in samples. The method to determine fungal species based on fungal nucleic acid in a sample comprises steps of: adding a first forward primer that includes SEQ ID NO: 1 to a first mixture, wherein the first mixture comprises a nucleic acid isolated from the sample; adding a first reverse primer that includes SEQ ID NO: 2 to the first mixture; and subjecting the first mixture to conditions that allow nucleic acid amplification; and determining the presence of the fungus on the basis of a nucleic acid amplified.

[0016] In another aspect, the present disclosure provides an assay for determining fungus in a sample, the method comprising: adding crRNA of SEQ ID NO: 3 to a reaction mixture that contains the reactants and / or the end products of the method of comprising addition of a first forward primer having SEQ ID NO: 1 to a first mixture, wherein the first mixture comprises a nucleic acid isolated from the sample; adding a first reverse primer having SEQ ID NO: 2 to the first mixture; and subjecting the first mixture to conditions to allow nucleic acid amplification and determining the presence of the fungus on the basis of a nucleic acid amplified. The method further comprises adding an oligonucleotide probe to the first mixture; wherein the oligonucleotide probe comprises ssDNA reporter with a 5' end labelled and a 3' end attached to a quencher. The probe is represented as 5’-L / TTATT / Q-3’, wherein L is label, and Q is quencher.

[0017] In yet another aspect, the present disclosure provides a kit to determine fungus in a sample, the kit comprising without limitation: a forward primer having SEQ ID NO: 1; a reverse primer having SEQ ID NO:2; a probe comprising a ssDNA reporter with a 5' end labelled and a 3' end attached to a quencher, said probe being represented as 5’-L / TTATT / Q-3’, wherein L is label, and Q is quencher; and a crRNA having SEQ ID NO:3, wherein the crRNA is complexed with LbCasl2a protein.

[0018] Various objects, features, aspects and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments.BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 is an exemplary schematic representation of a protocol of an an assay for determining fungus in a sample. Abbreviations include: RPA - Recombinase Polymerase Amplification, CRISPR - Clustered Regularly Interspaced Short Palindromic Repeats, Casl2a - CRISPR associated protein, UV - Ultraviolet, RT - Room Temperature.

[0020] Figure 2 is an Agarose gel electrophoresis image showing the specificity of the RPA primers.

[0021] Figure 3 is a graph showing specificity of real-time assay for determination of fungal nucleic acids.

[0022] Figure 4 is a graph showing specificity of endpoint / visual of assay for determination of fungal nucleic acids.

[0023] Figure 5 is graph showing real-time fluorescence kinetics of amplified Aspergillus flavus DNA gene fragments from 105-1 copies per pl.

[0024] Figure 6 is a graph showing real-time fluorescence kinetics of amplified Fusarium solani DNA gene fragments from 105-1 copies per pl.

[0025] Figure 7 is a graph showing real-time fluorescence kinetics of amplified Curvularia sp. DNA gene fragments from 105-1 copies per pl.

[0026] Figure 8 is a graph showing a graph showing real-time fluorescence kinetics of amplified Candida albicans DNA gene fragments from 105-1 copies per pl.

[0027] Figure 9 are graphs showing real-time fluorescence kinetics of the assay for determination of fungal nucleic acids in corneal swab samples, wherein S1-S36: clinical sample 1-36.

[0028] Figure 10 are graphs showing real-time fluorescence kinetics of the assay for determination of fungal nucleic acids in comeal swab samples, wherein S37-S75: clinical sample 37-S75.

[0029] Figure 11 shows endpoint fluorescence / visual of two independent assays (two independent assays, what does it mean?) for determination of fungus in 75 clinical swab samples.

[0030] Figure 12 shows endpoint fluorescence / visual of two independent assays for determination of fungus in 75 clinical swab samples and non-template control reaction (NTC).

[0031] Figure 13 shows endpoint fluorescence in assay for determination of fungus in 75 clinical swab samples.DETAILED DESCRIPTION OF THE INVENTION

[0032] The following is a detailed description of embodiments of the present disclosure. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.

[0033] Unless the context requires otherwise, throughout the specification which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be constmed in an open, inclusive sense that is as “including, but not limited to.”

[0034] Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, stmcture or characteristic described in connection with theembodiment is included in at least one embodiment. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0035] As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.

[0036] In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable.

[0037] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.

[0038] All methods described herein can be performed in suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0039] The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.

[0040] Various terms are used herein. To the extent a term used in a claim is not defined below, it should be given the broadest definition persons in the pertinent art have given that term as reflected in printed publications and issued patents at the time of filing.

[0041] A nucleic acid may be identified by the IUPAC letter code which is as follows: A -Adenine base; C- Cytosine base; G - guanine base; T or U - thymine or uracil base. M - A or C; R - A or G; W - A or T; S - C or G; Y - C or T; K - G or T; V - A or C or G; H - A or C or T; D - A or G or T; B - C or G or T; N or X - A or C or G or T. Note that T or U may be used interchangeably depending on whether the nucleic acid is DNA or RNA. A sequence having less than 60% 70%, 80%, 90%, 95%, 99% or 100% identity to the identifying sequence may still be encompassed by the invention if it is able of binding to its complimentary sequence and / or priming nucleic acid amplification of a desired target sequence. If a sequence is represented in degenerate form; for example, through the use of codes other than A, C, G, T, or U; the concept of a nucleic acid including the sequence also encompasses a mixture of nucleic acids of different sequences that still meet the conditions imposed by the degenerate sequence.

[0042] An oligonucleotide includes any DNA or RNA reagent of two or more nucleotides, whether from a natural source, artificially synthesized, or produced through the use of recombinant DNA technology. A nucleotide is an individual deoxyribonucleotide or ribonucleotide base such as A, C, G, T, or U. An oligonucleotide is often engineered to be capable of binding a nucleic acid sequence. While oligonucleotides are often linear, they may, depending on their sequence and conditions, assume a two- or three-dimensional structure. Oligonucleotides may be chemically synthesized by any of a number of methods including sequential synthesis, solid phase synthesis, or any other synthesis method now known or yet to be disclosed. Alternatively, oligonucleotides may be produced by recombinant DNA based methods. One skilled in the art would understand the length of oligonucleotide necessary to perform a particular task.

[0043] Oligonucleotides may be directly labelled, used as primers in RPA, crRNA in CRISPR sequencing reactions, or bound directly to a solid substrate as in oligonucleotide arrays.

[0044] The present disclosure provides tools and techniques based on recombinase polymerase amplification (RPA), Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) adaptive immune systems for determination of fungal nucleic acid in a sample.

[0045] In one embodiment, the present disclosure provides a recombinase polymerase amplification (RPA) primers set including a forward primer (?) for determination of fungal nucleic acid in a sample.

[0046] The forward primer having 70% to 100% sequence identity to SEQ ID NO: 1, and the reverse primer has 70% to 100% sequence identity to SEQ ID NO: 2.

[0047] In certain embodiment, the forward primer comprises less than 50 nucleotides.

[0048] In certain embodiment, the reverse primer comprises less than 50 nucleotides.

[0049] These primers may also comprise sequences that facilitate cloning of the amplification product into a cloning vector such as restriction enzyme sites.

[0050] In one embodiment, the present disclosure provides a crRNA sequence capable of hybridizing to the region amplified by the forward primer and the reverse primer of the present invention to aid in the determination of fungal nucleic acid in a sample. The crRNA sequence has 70% to 100% sequence identity to SEQ ID NO: 3.

[0051] The crRNA sequence may be complexed with a protein. For example, the crRNA sequence may be complexed with LbCasl2a protein in order to determination of amplification of sequence of interest, specific to fungi. This sequence can be designed within the RPA amplicons based upon the protospacer adjacent motif (PAM) recognized by Casl2a.

[0052] The RPA primers and crRNA sequence of the present invention are capable of detecting nearly 90% of the known species of human pathogenic fungi in a sample.

[0053] In an aspect, the present disclosure provides a reagent system for determination of fungal nucleic acid in a sample, the system comprising: a recombinase polymerase amplification (RPA) primers set including a forward primer having 70% to 100% sequence identity to SEQ ID NO: 1, and the reverse primer having 70% to 100% sequence identity to SEQ ID NO: 2 and a crRNA sequence having 70% to 100% sequence identity to SEQ ID NO: 3. The reagent system additionally comprises a probe having 70% to 100% identity to 5’-L / TTATT / Q-3’, wherein L is label, and Q is quencher.

[0054] In certain embodiment, the probe comprises less than 50 nucleotides.

[0055] In certain embodiment, the probe optionally may be labelled with a fluorescent label and a quencher molecule in order to help quantify the amount of the specific nucleic acid present in the sample.

[0056] In an embodiment, the probe comprises ssDNA reporter with a 5' end labelled with a label, preferably fluorescent label and a 3' end attached to a quencher. The probe is represented as 5’-L / TTATT / Q-3’, wherein L is label, and Q is quencher.

[0057] An oligonucleotide probe may include any label. A label may be any substance capable of aiding a machine, detector, sensor, device, or enhanced or unenhanced human eye from differentiating a labelled composition from an unlabelled composition. Examples of labels include but are not limited to: a radioactive isotope or chelate thereof, dye (fluorescent or nonfluorescent) stain, enzyme, or nonradioactive metal. Specific examples include but are not limited to: fluorescein, biotin, digoxigenin, alkaline phosphatase, biotin, streptavidin, 3H, 14C, 32P, 35S, or any other compound capable of emitting radiation, rhodamine, 4-(4'- dimethylamino- phenylazo)benzoic acid ("Dabsyl"); 4-(4'-dimethylamino-phenylazo)sulfonic acid (sulfonyl chloride) ("Dabsyl"); 5-((2-aminoethyl)-amino)-naphtalene-l -sulfonic acid ("EDANS"); Psoralene derivatives, haptens, cyanines, acridines, fluorescent rhodol derivatives, cholesterol derivatives; ethylenediaminetetraaceticacid ("EDTA") and derivatives thereof or any other compound that may be differentially detected.

[0058] Examples of fluorescent labels that may be used in quantitative PCR include but need not be limited to: HEX, TET, 6-FAM, JOE, Cy3, Cy5, ROX, TAMRA, and Texas Red.

[0059] An oligonucleotide probe used in quantitative PCR may also comprise a quencher. Examples of quenchers that may be used in quantitative PCR include, but need not be limited to TAMRA (which may be used with any of a number of fluorescent labels such as HEX, TET, or 6-FAM), BHQ1, BHQ2, or DABCYL.

[0060] When a nucleic acid such as a primer, oligonucleotide, oligonucleotide probe or any nucleic acid sequence includes a particular sequence, the sequence may be a part of a longer nucleic acid or may be the entirety of the sequence. The nucleic acid that includes the sequence may contain nucleotides 5' of the sequence, 3' of the sequence, or both. The concept of a nucleic acid including a particular sequence further encompasses nucleic acids that contain less than the full sequence that are still capable of specifically hybridizing to the target sequence under any conditions to which a mixture comprising a nucleic acid may be subjected.

[0061] In another embodiment, the present disclosure provides a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) based assay to determine fungal nucleic acid and thereby fungal organisms in samples. The method to determine fungal species based on fungal nucleic acid in a sample comprises steps of: adding a first forward primer that includes SEQ ID NO: 1 to a first mixture, wherein the first mixture comprises a nucleic acid isolated from the sample; adding a first reverse primer that includes SEQ ID NO: 2 to the first mixture; and subjecting the first mixture to conditions that allow nucleic acid amplification; and determining the presence of the fungus on the basis of a nucleic acid amplified.

[0062] In one embodiment, the present disclosure provides an assay for determining fungus in a sample, the method comprising: adding crRNA of SEQ ID NO: 3 to a reaction mixture that contains the reactants and / or the end products of the method of comprising addition of a first forward primer having SEQ ID NO: 1 to a first mixture, wherein the first mixture comprises a nucleic acid isolated from the sample; adding a first reverse primer having SEQ ID NO: 2 to the first mixture; and subjecting the first mixture to conditions to allow nucleic acid amplification and determining the presence of the fungus on the basis of a nucleic acid amplified. The method further comprises adding an oligonucleotide probe to the first mixture; wherein the oligonucleotide probe is represented as 5’-L / TTATT / Q-3’, wherein L is label, and Q is quencher.

[0063] Quantitative PCR platforms may use a probe labelled with a fluorescent label and a quencher molecule in order to help quantify the amount of the specific nucleic acid present in the sample. Qualitative examination of the sample with naked eye may also be done by examining the assay mixture under blue / ultraviolet light.

[0064] In some embodiments of the invention, the amplification is performed with a forward primer and a reverse primer intended to amplify the entirety of the 18S rRNA gene of a sample. This is intended to produce an amplification product that may be used as either a standard (after the copy number of the amplification product is determined) or as a library that includes the 18S rRNA from all fungal species in a sample. These primers may also comprise sequences that faciliofate cloning of the amplification product into a cloning vector such as restriction enzyme sites.

[0065] In general, nucleic acid amplification is a process by which copies of a nucleic acid may be made from a source nucleic acid. In some nucleic amplification methods, the copies are generated exponentially. Examples of nucleic acid amplification include but are not limited to: the Recombinase Polymerase Amplification (RPA), polymerase chain reaction (PCR), ligase chain reaction (LCR), self-sustained sequence replication (3 SR), nucleic acid sequence based amplification (NASBA), strand displacement amplification (SDA), amplification with Q replicase, whole genome amplification with enzymes such as cp29, whole genome PCR, in vitro transcription with Klenow or any other RNA polymerase, or any other method by which copies of a desired sequence are generated.

[0066] Recombinase Polymerase Amplification (RPA) is a particular method of amplifying DNA, generally involving the making of a reaction mixture by mixing a nucleic acid sample, two or more primers, three core enzymes - a recombinase, a single stranded DNA-binding protein and a strand displacing polymerase, and deoxyribose nucleosidetriphosphates (dNTP's). In general, the reaction mixture is subjected to temperature of 39°C for 30 minutes.

[0067] In the present invention a crRNA sequence complexed with LbCasl2a protein in order to detect amplification of sequence of interest, specific to fungi. This sequence was designed within the RPA amplicons based upon the protospacer adjacent motif (PAM) recognized by Casl2a. This mixture is incubated with RPA amplicon and an oligonucleotide probe with a quenched fluorescent ssDNA reporter at 37°C for 30 minutes. The quencher hides from detection the majority of the fluorescence that may be emitted by the fluorescent label when the oligonucleotide probe is in solution. For fluorescence detection, an ssDNA reporter with a 5' end-labelled FAM group and a 3' end attached to a Black quencher represented as / 5’-FAM / TTATT / BHQ-37 is used. Once the Casl2a protein is activated by binding to its complementary sequence, it cleaves the sequence followed by cleaving any ssDNA sequence exposed to it. This transcleavage activity also cleaves the quencher and the fluorescent reporter. This cleavage removes the quencher from the probe, rendering the fluorescent molecule detectable. Therefore, the quantity or intensity of the fluorescence may be correlated with the amount of product formed in the reaction. The resultant fluorescence may be detected using quantitative PCR or using naked eye by examining the reaction mixture under blue LED light or UV light, a positive result denoted by presence of green coloured fluorescence.

[0068] One skilled in the art would be capable of calculating the amount of target nucleic acid (either DNA or RNA) present in a reaction mixture comprising a sample from the quantity of the change in fluorescence on quantitative PCR.

[0069] The invention encompasses methods of identifying fungi through the use of DNA sequencing, such as Sanger sequencing, next generation sequencing, pyrosequencing, SOLID sequencing, massively parallel sequencing, pooled, and barcoded DNA sequencing or any other sequencing method now known or yet to be disclosed.

[0070] In Sanger Sequencing, a single-stranded DNA template, a primer, a DNA polymerase, nucleotides and a label such as a radioactive label conjugated with the nucleotide base or a fluorescent label conjugated to the primer, and one chain terminator base comprising a dideoxynucleotide (ddATP, ddGTP, ddCTP, or ddTTP, are added to each of four reactions (one reaction for each of the chain terminator bases). The sequence may be determined by electrophoresis of the resulting strands. In dye terminator sequencing, each of the chain termination bases is labelled with a fluorescent label of a different wavelength which allows the sequencing to be performed in a single reaction.

[0071] In pyrosequencing, the addition of a base to a single stranded template to be sequenced by a polymerase results in the release of a pyrophosphate upon nucleotide incorporation. An ATP sulfurylase enzyme converts pyrophosphate into ATP which in turn catalyzes the conversion of luciferin to oxyluciferin which results in the generation of visible light that is then detected by a camera.

[0072] In SOLID sequencing, the molecule to be sequenced is fragmented and used to prepare a population of clonal magnetic beads (in which each bead is conjugated to a plurality of copies of a single fragment) with an adaptor sequence and alternatively a barcode sequence The beads are bound to a glass surface. Sequencing is then performed through 2- base encoding.

[0073] In massively parallel sequencing, randomly fragmented targeted DNA is attached to a surface. The fragments are extended and bridge amplified to create a flow cell with clusters, each with a plurality of copies of a single fragment sequence. The templates are sequenced by synthesizing the fragments in parallel. Bases are indicated by the release of a fluorescent dye correlating to the addition of the particular base to the fragment.

[0074] An oligonucleotide may be added to a mixture by any of a number of methods including manual methods, mechanical methods, or any combination thereof. An oligonucleotide may be added to a mixture by adding the mixture to an oligonucleotide that is conjugated to a substrate such as in a microarray. One may also add the oligonucleotide to a mixture in which the target allele to which the nucleic acid has specificity is absent.

[0075] In certain embodiment of the present invention, an oligonucleotide is bound to a substrate such as a microarray. Examples of microarrays include constructs in which a plurality of single stranded oligonucleotide probes are affixed to a substrate such as silicon glass. Oligonucleotides with a sequence complementary to an allele are capable of specifically binding to that allele to the exclusion of alleles that differ from the specific allele by one or more nucleotides. Labelled sample DNA may be hybridized to the oligonucleotides and detection of the label is correlated with binding of the sample and consequently the presence of the target nucleic sample.

[0076] Any oligonucleotide bound to a substrate may be covalently bound to the substrate or it may be bound by some non-covalent interaction including electrostatic, hydrophobic, hydrogen bonding, Van Der Waals, magnetic, or any other interaction by which the oligonucleotide may be attached to a substrate while maintaining its ability to recognize the allele to which it has specificity. A substrate may be any solid or semisolid material onto which a probe may be affixed, attached or printed, either singly or in the presence of one ormore additional probes or samples. Examples of substrate materials include but are not limited to polyvinyl, polystyrene, polypropylene, polyester or any other plastic, glass, silicon dioxide or other silanes, hydrogels, gold, platinum, microbeads, micelles and other lipid formations, nitrocellulose, or nylon membranes. The substrate may take any form, including a spherical bead or flat surface. For example, the probe may be bound to a substrate in the case of an array. The sample may be bound to a substrate in the case of a Southern Blot.

[0077] The present invention encompasses methods of determining the presence of fungus in a sample. A sample may be derived from anywhere that a fungus or any part of a fungus including fungal spores, buds, or hyphae may be found including soil, air, water, solid surfaces (whether natural or artificial,) culture media, foodstuffs, and any interfaces between or combinations of these elements. A sample may be derived from a subject, such as a plant or animal, including humans. Samples derived from animals include but are not limited to biopsy necropsy, or other in vivo or ex vivo collection of eye, prostate, breast, skin, muscle, fascia, brain, endometrium, lung, head and neck, pancreas, small intestine, blood, liver, testes, ovaries, colon, skin, stomach, esophagus, spleen, lymph node, bone marrow, kidney, placenta, or fetus. Samples derived from subjects may also take the form of a fluid sample such as peripheral blood, aqueous humor, vitreous humor, lymph fluid, ascites, serous fluid, pleural effusion, sputum, bronchial wash, bronchioalveolar lavage fluid (BALF), cerebrospinal fluid, semen, amniotic fluid, lacrimal fluid, stool, urine, hair, or any other source of material that may be collected from a living or dead animal. Samples collected from a plant may be collected from part of a plant or from an entire plant. Samples may be collected by any method now known or yet to be disclosed, including swiping, scraping or swabbing an area or orifice, removal of a piece of tissue as in a biopsy, or any method known to collect bodily fluids. Samples may be suspected of containing a fungus if they are derived from a subject displaying symptoms of a fungal infection or from an environmental sample from an area in which a fungus is thought to be present.

[0078] Examples of fungi that may be detected with the primers, crRNA sequence, reagent system, and the assay of the present invention include but need not be limited to Acremonium falciforme, Acremonium kiliense, Acrophialophora fusispora, Altemaria alternata, Alternaria chlamydospora, Aspergillus nidulans, Aspergillus flavipes, Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Aspergillus tamarii, Aspergillus terreus, Beauveria bassiana, Bipolaris australiensis, Bipolaris hawaiiensis, Candida albicans, Candida krusei, Candida parapsilosis, Candida tropicalis, Chaetomium globosum, Cladophialophora carrion, Colletorichum truncatum, Curvularia spicifera, Cylindrocarpondestructans, Epicoccum nigrum, Exserohilum rostratum, Exophiala dermatitidis, Fonsecaea pedrosoi, Fusarium chlamydosporum, Lasiodiplodia theobromae, Fusarium dimerum, Fusarium incarnatum, Fusarium lichenicola, Fusarium oxysporum, Fusarium sacchari, Fusarium solani, Nigrospora sphaerica, Pseudallescheria boydii, Rhizopus arrhizus, Sarcinomyces phaeomuriformis, Scedosporium apiospermum, Scedosporium prolificans and Trichosporon asahii.

[0079] In yet another embodiment, the present disclosure provides a kit to determine fungus in a sample, the kit comprising without limitation: (Do we need to mention RPA enzymes and dNTPs in it) a forward primer having SEQ ID NO: 1; a reverse primer having SEQ ID NO:2; a probe comprising ssDNA reporter with a 5' end labelled with a label, preferably fluorescent label and a 3' end attached to a quencher., said probe being represented as 5’-L / TTATT / Q-3’, wherein L is label, and Q is quencher; and a crRNA having SEQ ID NO: 3, wherein the crRNA is complexed with LbCasl2a protein.

[0080] The kit may be any assemblage or collection of components that comprises the primers set and crRNA sequence of the present invention and that facilitates to carry out the assay of the present invention. The kit that facilitates the assay of the present invention can include specific nucleic acids such as oligonucleotides, labelling reagents, enzymes including recombinase, single-stranded DNA binding protein and strand displacing polymerase, reverse transcriptase, or any other enzymes and / or reagents that facilitate detection of the target nucleic acids. Specific nucleic acids may include nucleic acids, polynucleotides, oligonucleotides (DNA, or RNA), or any combination of molecules that includes one or more of the above, or any other molecular entity capable of specific binding to a nucleic acid marker. In one embodiment of the present invention, the specific nucleic acid comprises one or more oligonucleotides capable of hybridizing to the marker.

[0081] The kit may optionally contain an indication of a result that indicates a particular outcome. For example, an indication may be a DNA sequence signifies the identification of a particular fungal phylum, class, order, family, genus species, subspecies, strain or any other delineation of a group of fungi. An indication may be a standard curve configured to quantify the amount of fungus present in a sample. An indication may include a specific colour fluorescence as an indication of the presence of fungus in the sample. An indication may include a set of standards that is included in the kit that has been premeasured with regard to copy number. An indication may be a positive control included with the kit or a set of primersand / or other reagents that may be used to generate the positive control. In the case of a quantitative PCR reaction, the indication may be a particular Ct level or a range of Ct levels. An indication may also include but need not be limited to: a level of fluorescence or radioactive decay, a value derived from a standard curve or from a control, or any combination of these and other outputs. The indication may be printed on a writing that may be included in the kit or it may be posted on the Internet or embedded in a software package.

[0082] The present disclosure provides primers set, crRNA sequence, reagent system, kit and assay protocol that can determine fungal species in samples with high specificity and sensitivity in a simpler and rapid manner as well as obviate need of highly specialized equipment or personnel.

[0083] In the above description, and for the purposes of explanation, various specific details are set forth in order to provide a thorough understanding of the various aspects of the invention. It will be understood, however, by those skilled in the relevant arts, that the present invention may be practiced without these specific details. It is intended that the inventions not be limited only to the specific structure, material or acts that are described in the preferred embodiments, but in addition, include any and all structures, materials or acts that perform the claimed function as described in alternative embodiments or forms of the invention, or that are well known presently or later-developed, equivalent structures, material or acts for performing the same function.

[0084] Thus, while the foregoing description discloses various embodiments of the disclosure, other and further embodiments of the invention may be devised without departing from the basic scope of the disclosure. The invention is not limited to the described embodiments, versions or examples, which are included to enable a person having ordinary skill in the art to make and use the invention when combined with information and knowledge available to the person having ordinary skill in the art.EXAMPLESThe above disclosure generally describes the present disclosure, which is further exemplified by the following examples. These specific examples are described solely for purposes of illustration, and are not intended to limit the scope of this disclosure. Although specific targets, terms, and values have been employed herein, such targets, terms, and values will likewise be understood as exemplary and non-limiting to the scope of this disclosure.Example 1ASSAY FOR DETERMINATION OF FUNGUS THOUGH FUNGAE NUCEEIC ACID(a) Construction of recombinase polymerase amplification (RPA) primers set and crRNA

[0085] Small-subunit (18S) rRNA gene sequences from 49 relevant fungal species were accessed via the GenBank database and were aligned by using CLUSTAL Omega as per Sievers F, Wilm A, Dineen D, et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol. 2011; 7:539. Conserved sequences were identified to generate target recombinase polymerase amplification (RPA) primers and CRISPR guide RNA (gRNA) sequences. The specificity of the designed RPA primers and gRNA sequences were tested using the PRIMER-Blast Program (Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction | BMC Bioinformatics | Full Text. Accessed August 17, 2022. https: / / bmcbioinformatics.biomedcentral.com / articles / 10.1186 / 1471-2105-13-134). The RPA primers were constructed as per manufacturer’s instructions and the gRNA sequences were designed within the RPA amplicons based upon the protospacer adjacent motif (PAM) recognized by Casl2a. After screening and optimization, the RPA primer set was arrived at containing forward primer having nucleic acid sequence ascribed as SEQ ID NO: 1 and reverse primer having nucleic acid sequence ascribed as SEQ ID NO: 2. The crRNA sequences arrived had a nucleic acid sequence ascribed as SEQ ID NO: 3.(b) Optimization of the protocol for assay for determination of fungal nucleic acid

[0086] The assay combines recombinase polymerase amplification (RPA) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) adaptive immune systems technologies. The RPA reaction was performed as per the manufacturer’s instructions (TwistAmp Basic, TwistDx, Cambridge, United Kingdom). The 50pl reaction mixture containing 0.48pM forward and reverse primers, 29.5pl primer free rehydration buffer, lOpl template DNA, and 14mM magnesium acetate (MgOAc) was incubated at 39°C for 30 minutes. LbCasl2a trans-cleavage assays were performed similarly to those previously described. 16, 18 Briefly, a total of 50nM LbCasl2a (New England Biolabs Inc., Ipswich, MA USA) was pre-incubated with 500nM gRNA in l x NEBuffer 2.1 for 10 minutes at 25 °C. After formation of the RNA-protein complex, IpM of a quenched fluorescent ssDNA reporter and 13 pl of the RPA amplicon were added and incubated at 37°C for 30 minutes or the denoted time in figures. For fluorescence detection, an ssDNA reporter with a 5' end-labelled FAM group and a 3' end attached to a Black quencher / 5’-FAM / TTATT / BHQ-37 was used. Real-time and endpoint fluorescence was the raw fluorescence determined by the Real-Time PCR Detection System. Visual detection was accomplished through imaging the tubes in the LED blue light illuminator and the Bio-Rad ChemiDoc MP Imaging System (Bio-Rad., Hercules, CA USA) with its built-in UV channel.(c) Samples

[0087] Seventy-five comeal swabs / scrapings from 75 consecutive patients with presumed microbial keratitis specifically fungal keratitis were collected as previously described by “Lalitha P, Prajna NV, Sikha M, et al. Evaluation of Metagenomic Deep Sequencing as a Diagnostic Test for Infectious Keratitis. Ophthalmology. 2021; 128(3):473-475” and “Leek A. Taking a comeal scrape and making a diagnosis. Community Eye Health. 2009; 22(71):42-43”. Informed consent was obtained from all participants, and the trial conformed to the Declaration of Helsinki. Ethical approval was obtained from the Institutional Review Board. For the assay according to the present invention and PCR, the affected cornea was swabbed with a sterile polyester tipped applicator (Puritan Medical Products, Guilford, ME, USA) and transferred to a 1.5 ml Eppendorf tube. DNA extraction was performed using the QIAmp DNA Blood Mini kit (Qiagen, Hilden, Germany) and stored at -20°C until further use. The RPA reaction was performed as described above with lOpl of the eluted DNA at 39°C for 30 minutes. LbCasl2a trans-cleavage assays were performed as described above. Both real-time and endpoint fluorescence detection was performed for all clinical samples. For the analysis of clinical samples, the result of the present invention assay was considered positive if it was equal or greater than a cut-off threshold equal to the mean signal of the negative control samples plus three times its standard deviation. Figure 1 illustrates the workflow of the assay of the present invention for clinical specimens. PCR was performed as previously described and the amplified product was visualized on an UV transilluminator using 2% AGE incorporating 0.5 pg / ml ethidium bromide. Previously described panfungal primers [Forward primer sequence, 5'- GTG AAA TTG TTG AAA GGG AA-3' (SEQ ID NO: 4); and reverse primer sequence, 5'-GAC TCC TTG GTC CGT GTT -3’ (SEQ ID NO: 5) specific for the 28S rRNA gene were used in the study.(d) Conventional microbiologic investigations

[0088] A standard scraping procedure for comeal debridement was performed for patients with clinically suspected microbial (fungal) keratitis as previously described (Prajna NV, Krishnan T, Mascarenhas J, et al. The mycotic ulcer treatment trial: a randomized trial comparing natamycin vs voriconazole. JAMA Ophthalmol. 2013; 131(4): 422-429). Briefly, two scrapings were smeared directly on separate glass slides for Gram staining and potassiumhydroxide (KOH) wet mount, and three further scrapings were taken and directly inoculated onto sheep’s-blood agar, chocolate agar, and potato dextrose agar or Sabouraud’s agar for bacterial and fungal cultures. Fungal smears were considered positive when fungal elements were seen under low-power magnification and reduced light. Fungal cultures were considered positive with growth on any two media or moderate to heavy growth on one medium.(e) Determination of the specificity and analytical sensitivity of the assay

[0089] To determine the specificity and sensitivity of the RPA primers in accordance with the present invention, the DNA extracted from cultures of the following organisms isolated from patients with fungal keratitis (FK) was used: Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Fusarium oxysporum, Candida albicans, Lasiodiplodia theobromae, Alternaria alternata, Bipolaris, Curvularia, Exserohilum, Pseudomonas aeruginosa, Streptococcus pneumoniae, Staphylococcus aureus and Escherichia coli. To determine the sensitivity of the RPA primers, the RPA reactions were performed as described above and the amplified products were investigated using 2% agarose gel electrophoresis (AGE) (Figure 2). Real-time and endpoint fluorescence detection was used to determine the specificity of the assay using 10 ng DNA of the above listed isolates. The analytical limit of detection (LoD) was evaluated by testing the calibration standards of the following fungal species: Aspergillus flavus, Candida albicans, Curvularia and Fusarium oxysporum, prepared by serial dilutions at the following concentrations: 165 fM (105 copies), 16 fM (104 copies), 1.6 fM (103 copies), 165 aM (102 copies), 16 aM (10 copies) and 1.6 aM (1 copy).

[0090] The specificity of the assay determined using DNA isolated from patient isolates demonstrated detection of Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Fusarium oxysporum, Candida albicans, Lasiodiplodia theobromae, Alternaria alternata, Bipolaris, Curvularia, and Exserohilum and no detection of Pseudomonas aeruginosa, Streptococcus pneumoniae, Staphylococcus aureus, Escherichia coli and human DNA, confirming high specificity (Figures 3,4). The analytical sensitivity of the assay of the present invention was determined and the LoD was 13.8 genomic copies for Aspergillus flavus (Figure 5), 16.6 for Fusarium solani (Figure 6), 13.9 for Curvularia lunata (Figure 7), and 13.3 for Candida albicans (Figure 8).

[0091] The results of the assay in correlation with microscopy, culture and PCR for patients with suspected microbial keratitis are shown in Table 1.Table 1: Results of microscopic examination, culture, PCR, assay of the present invention in Microbial Keratitis samples

[0092] The real-time fluorescence and visual results of the present invention assay are presented in Figures 9-13. Of the 75 scrapes, 72 (96 %) were positive for fungus by standard mycologic methods of which 55 (73%) were positive by both microscopy and culture and 17 (22.7%) were positive only by microscopy. Between the assay of the present invention and culture, 56 concordant (52 positive and 4 negative) and 19 discordant (16 present invention assay positive but culture negative, 3 assay of the present invention negative but culture positive) results were observed. All the 16 scrapes positive for the assay but negative for culture were found to be microscopy-positive for fungus. Of the 55 scrapes positive on fungal culture, 36 harbored Fusarium,' 8 had Aspergillus; Cylindrocarpon and

[0093] Scedosporium were identified in 2 cultures each, Lasiodiplodia, Curvularia and Exserohilum were identified in 1 culture each while 4 culture isolates were not speciated. Among the 4 specimens negative by both assay of the present invention and fungal culture, 3 showed bacterial growth (Pseudomonas in 2 and Streptococcus in 1) and one had no growth.

[0094] Being a nucleic acid determination modality, the performance of the assay of the present invention was compared to PCR. A sample was considered positive for FK if it demonstrated positive results by culture or microscopy. This definition of positive results was used as the gold standard to calculate and compare the test performances (sensitivity, specificity, PPV and NPV) between the present invention assay, PCR and culture (Table 2).Table 2: Performance of PCR, the present invention assay and culture in comparison to reference standard in microbial keratitis samples

[0095] The sensitivity, specificity, PPV and NPV of the present invention assay were 94.4%, 100%, 100 % and 42.9% respectively. The sensitivity, specificity, PPV and NPV ofPCR were 87.5%, 100%, 100% and 25% respectively. There was no significant difference between the sensitivities and specificities of PCR and the present invention assay (p=0.16). The sensitivity, specificity, PPV and NPV of culture were 76.4%, 100%, 100 % and 15% respectively. The sensitivity of the present invention assay was significantly higher than the sensitivity of culture (p=0.002).ADVANTAGES OF THE INVENTION

[0096] The present disclosure provides primers set, crRNA sequence, reagent system and kit capable of determining fungal nucleic acid and thereby fungal species present in the sample with high specificity and sensitivity.

[0097] The present disclosure assay protocol can determine fungal species in samples not only with high specificity and sensitivity but in a simpler and rapid manner.

[0098] The present disclosure assay protocol obviates need of highly specialized equipment or personnel.SEQUENCE LISTINGS

[0099] SEQ ID NO: 1:5 ’ -CGGCACCTTACGAGAAATCAAAGTTTTTGG-3 ’

[0100] SEQ ID NO:2:5 -ACCACCACCTGAAAAATCAAGAAAGAGCTCTC- 3 ’

[0101] SEQ ID NO:3 5 ’ -UAAUUUCUACUAAGUGUAGAUACUCAACACGGGGAAACUCACCAG- 3 ’

[0102] SEQ ID NO:4:5'- GTG AAA TTG TTG AAA GGG AA-3'

[0103] SEQ ID NO:5:5'-GAC TCC TTG GTC CGT GTT -3’

Claims

aim:

1. A recombinase polymerase amplification (RPA) composition for determining fungal nucleic acid in a sample comprising: forward primer having 70% to 100% sequence identity to SEQ ID NO: 1; reverse primer having 70% to 100% sequence identity to SEQ ID NO: 2; and crRNA sequence having 70% to 100% sequence identity to SEQ ID NO: 3.

2. The RPA composition as claimed in claim 1 further comprises a probe comprising a ssDNA reporter with a 5' end labelled and a 3' end attached to a quencher, said probe being represented as 5’-L / TTATT / Q-3’, wherein L is label, and Q is quencher.

3. The RPA composition as claimed in claim 2, wherein the probe is an ssDNA reporter with a 5' end-labelled FAM group and a 3' end attached to a Black quencher represented by / 5’-FAM / TTATT / BHQ-374. The RPA composition as claimed in claim 2, wherein the fungus is selected from a group consisting of Acremonium falciforme, Acremonium kiliense, Acrophialophora fusispora, Alternaria alternata, Alternaria chlamydospora, Aspergillus nidulans, Aspergillus flavipes, Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Aspergillus tamarii, Aspergillus terreus, Beauveria bassiana, Bipolaris australiensis, Bipolaris hawaiiensis, Candida albicans, Candida krusei, Candida parapsilosis, Candida tropicalis, Chaetomium globosum, Cladophialophora carrion, Colletorichum truncatum, Curvularia spicifera, Cylindrocarpon destructans, Epicoccum nigrum, Exserohilum rostratum, Exophiala dermatitidis, Fonsecaea pedrosoi, Fusarium chlamydosporum, Lasiodiplodia theobromae, Fusarium dimerum, Fusarium incamatum, Fusarium lichenicola, Fusarium oxysporum, Fusarium sacchari, Fusarium solani, Nigrospora sphaerica, Pseudallescheria boydii, Rhizopus arrhizus, Sarcinomyces phaeomuriformis, Scedosporium apiospermum, Scedosporium prolificans and Trichosporon asahii.

5. A Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based assay to determine fungal nucleic acid in a sample comprises steps of: a. adding the forward primer as claimed in claim 1 to a first mixture, wherein the first mixture comprising the fungal nucleic acid; b. adding the reverse primer as claimed in claim 1 to the first mixture;c. subjecting the first mixture to conditions that allow nucleic acid amplification; and d. determining the presence of the fungus on the basis of a nucleic acid amplified.

6. An assay for determining the presence of fungal nucleic acid in a sample comprises steps of: a. adding crRNA of SEQ ID NO: 3 to a reaction mixture the forward primer having SEQ ID NO: 1 to a first mixture comprising the fungal nucleic acid, wherein the crRNA is complexed with LbCasl2 a protein; b. adding the reverse primer having SEQ ID NO: 2 to the first mixture; c. subjecting the first mixture to conditions to allow nucleic acid amplification; and d. determining the presence of the fungus on the basis of a nucleic acid amplified.

7. The assay as claimed in claim 5 or 6 further comprises the step of adding an oligonucleotide probe as claimed in claim 2 to the first mixture.

8. The assay as claimed in claim 5 or 6, wherein the fungal nucleic acid is 18S.

9. The assay as claimed in claim 5 or 6, wherein the detection of fungus is effected sequencing of the amplified sequence in step d) by a method selected from a group consisting of Sanger sequencing, next generation sequencing, pyrosequencing, SOLID sequencing, massively parallel sequencing, pooled, and barcoded DNA sequencing.

10. A kit to determine presence of fungus in a sample, the kit comprising: a. a forward primer having SEQ ID NO: 1; b. a reverse primer having SEQ ID NO: 2; c. a probe comprising ssDNA reporter with a 5' end labelled with a label, preferably fluorescent label and a 3' end attached to a quencher., said probe being represented as 5’-L / TTATT / Q-3’, wherein L is label, and Q is quencher; d. a crRNA having SEQ ID NO: 3, wherein the crRNA is complexed with LbCasl2a protein; and e. reagent system comprising dNTPs, buffers, labelling reagents, s recombinase enzyme, single -stranded DNA binding protein and strand displacing polymerase, reverse transcriptase, and enzymes and / or reagents that facilitate detection of the fungal nucleic acid.

Images