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    • 专利摘要:
    Method for the rapid detection of Candida auris and the diagnosis of the infection caused by this pathogen. The present invention relates to a biosensor comprising a porous support with an indicator inside the pores, and an oligonucleotide that specifically recognizes Candida auris, where the oligonucleotide is anchored to the surface of the porous support in such a position that controls, in the absence/presence of C.auris DNA, the indicator's exit to the outside. Likewise, the invention also relates to the use of said biosensor for the detection and/or quantification of C. auris, and for the diagnosis of diseases caused by C. auris. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2763043A1
    申请号:ES202030357
    申请日:2020-04-27
    公开日:2020-05-26
    发明作者:Felipe Sara Santiago；Blasco Luis Pla；Mas María Angeles Tormo；Gimeno Elena Aznar；Gaitán Alba Cecilia Ruiz；Gómez Eulogio Valentín；García Javier Pemán；Mañez Ramón Martínez
    申请人:Fundacion Para La Investigacion Del Hospital Univ La Fe；Universidad Politecnica de Valencia；Centro de Investigacion Biomedica en Red de Enfermedades Hepaticas y Digestivas CIBEREHD；Universitat de Valencia；Consorcio Centro de Investigacion Biomedica en Red MP；
    IPC主号:
    专利说明:

    [0002] Method for rapid detection of Candida auris and diagnosis of infection caused by this pathogen
    [0004] The present invention falls within the field of rapid detection of pathogenic fungi, specifically Candida auris, and diagnosis of infections caused by C. auris by using materials capable of specifically recognizing the DNA of the pathogenic species.
    [0006] BACKGROUND OF THE INVENTION
    [0008] C. auris is an emerging species that is highly resistant to currently available antifungal drugs that can cause fungemia and deep infections associated with high mortality, especially in patients admitted to medical intensive care units (ICU) or surgical (Reanimation). The first reported isolation of C. auris was in a patient in Japan with otitis externa in 2009. Since then, disseminated infections and yeast infections caused by this species have been described in several countries, such as India, South Korea, South Africa, United Kingdom, United United, Colombia, Venezuela, Kuwait and, recently, Spain, causing a worldwide health problem (Ruiz-Gaitán. Rev Iberoam Micol. 2017, 34, 23-27). None of the outbreaks of invasive candidiasis caused by C. auris described has been of the magnitude of the one detected at the Hospital Universitario y Politécnico La Fe since April 2016: 52 candidemias and 178 patients colonized or with mild infections in the last thirteen months. The magnitude of this outbreak has created a unique opportunity to deepen knowledge, has favored the development of a new detection system for this new and multi-resistant fungal species, very different from the known Candida species.
    [0010] The identification of C. auris in biological samples represents an important diagnostic challenge due to the difficulty in correctly identifying it with the biochemical techniques commonly used in microbiology laboratories. Many of these techniques mistakenly identify it as Candida haemulonii, Candida famata, Saccharomyces cerevisiae or Rhodotorula glutinis. To correctly identify an isolate of C. auris molecular methods are needed outside the scope of most laboratories and clinical amplification and sequencing of ITS (the Internal acronym ! Transcribed Spacer), D1 / D2 regions of ribosomal DNA, AFLP (from the English acronym Amplified Fragment Length Po! Ymorphism), and from the genes RPB1 and RPB2 or, recently, by MALDI-TOF MS mass spectrometry (from the acronym in English Matrix Assisted Laser Desorption lonization Time Of Flight Mass Spectrometry). Due to these important methodological difficulties for the correct identification of C. auris, which also require a lot of time, specialized personnel and equipment, it is very likely that the infections caused by this species have been underdiagnosed or misidentified in recent years; therefore, the development of rapid and reliable diagnostic methods is necessary.
    [0012] Methods developed to improve the identification of C. auris are described, for example, in WO2019208691, US2019338241 and WO2018213641. WO2019208691 discloses a set of primers for the detection and diagnosis of C. auris in the clinical setting based on nucleic acid amplification by the polymerase chain reaction. US2019338241 discloses a method for the detection of C. auris based on a culture medium that supports or inhibits the growth of C. auris relative to other fungal species. Finally, WO2018213641 discloses methods also based on nucleic acid amplification by polymerase chain reaction to detect various Candida species , including C. auris. Despite the advantages provided by these types of methods, they still have drawbacks such as the need for specialized personnel and equipment to carry out the extraction and amplification of nucleic acids, or the extensive time necessary for the cultivation of fungi in the case of the based method. in a selective culture medium, which, despite selectivity, may result in false negatives or results that are difficult to interpret.
    [0014] Thus, in view of the state of the art, it is necessary to develop alternative methods to those already described for the detection and / or diagnosis of C. auris that do not present the disadvantages and disadvantages of the previous devices / methods, especially the need of specialized personnel and equipment, the long time necessary for crops and the lack of robustness in the result obtained.
    [0016] DESCRIPTION OF THE INVENTION
    [0018] The present invention describes a biosensor for the detection of Candida auris DNA. and / or diagnosis of the infection caused by C. auris that solves the problems of the state-of-the-art methods, that is, it allows the detection of C. auris quickly, easily and with a high level of sensitivity and selectivity. The biosensor developed by the authors of the present invention is based on a porous material that comprises a single-stranded DNA indicator and oligonucleotides that specifically recognize a specific region of the C. auris DNA , so that when the C. auris DNA is present in the medium, the oligonucleotides recognize this region, bind to it, and the release of the indicator occurs, which can be detected (see Figure 1). Thus, the detection of the indicator is indicative of the presence of C. auris in the analyzed sample.
    [0020] Based on this biosensor, the inventors have also developed a method for detecting C. auris DNA and a method for diagnosing diseases caused by said pathogenic fungus.
    [0022] Biosensor of the invention
    [0024] In a first aspect, the present invention refers to a biosensor, hereinafter "the biosensor of the invention", comprising
    [0025] - a porous support comprising an indicator inside the pores, and - one or more types of oligonucleotides that specifically recognize the DNA of Candida auris,
    [0026] where
    [0027] - a plurality of pores comprise access to the outside of the porous support, and - the oligonucleotides are anchored to the surface of the porous support in such a position that
    [0028] (i) in the absence of C. auris DNA , the oligonucleotides block the access of the pores to the exterior, preventing the indicator from leaving, and
    [0029] (ii) in the presence of C. auris DNA , the oligonucleotides recognize and bind to the DNA of said fungus, unblocking the access of the pores to the exterior and allowing the indicator to exit.
    [0031] The biosensor of the invention has advantages that are ideal for the methods and uses described in the invention. One of the main advantages is that the oligonucleotides specifically recognize C. auris DNA , which together with the great capacity of signal amplification due to the presence of rhodamine B in the pores, allows a detection level of up to a concentration between 0.3 and 0.5 pg / pL of DNA. This combination of characteristics allows the biosensor of the invention to have great sensitivity and reliability to detect C. suris DNA . Furthermore, the biosensor of the invention, the methods and uses of the invention do not require specialized personnel or equipment, allowing, in the simplest case, detection by direct visual observation of the medium. Furthermore, the detection and diagnostic methods of the invention can be carried out in 1 hour, reducing the time necessary to determine the presence of C. suris DNA in a sample, comparing with the commonly used techniques.
    [0033] In the present invention the term "biosensor" refers to a device that is used to detect C. suris DNA and / or diagnose a C. suris infection based on the specific molecular interaction between C. suris DNA and the oligonucleotides that coat the sensor. The interaction gives rise to an intermediate complex that leads to signal transduction. A biosensor can be an analytical device that converts a biological response to an electrical signal, an optical signal, or other transduction scheme.
    [0035] The biosensor of the invention comprises: a porous support and one or more types of oligonucleotides anchored to the surface of the porous support.
    [0037] In the present invention the term "porous support" refers to that substrate that comprises pores and that has a surface to which molecules can be attached indirectly through a binding element. The porous support used in the present invention may comprise any substrate material that comprises pores along its surface with an access to the outside of the porous support, and that provides physical support for capturing elements or probes that are attached to said surface. . The pores are classified by the Union of Pure snd Applied Chemistry (IUPAC) by the pore size, measuring said size by its internal diameter assuming that it is cylindrical, or by the distance between the internal and opposite walls of a pore with a different configuration. Thus, following the IUPAC criteria, if the diameter of a pore (or the distance between the inner or opposite walls of a pore) is approximately 2 nm or less than 2 nm, then the pore is called micropore, if it is greater than 2 nm but less than 50 nm is called mesopore (or nanopore), and if it is approximately 50nm or greater than 50nm, then it is a macropore. Taking this classification into account, a material is considered to be microporous when the average pore size of said material is approximately 2 nm or less than 2 nm, which is mesoporous (or nanoporous) when the average pore size of said material material is greater than 2 nm but less than 50 nm, and it is macroporous when the average pore size of said material is approximately 50 nm or greater than 50 nm. In the present invention, the average pore size of the porous support is between 2 and 100 nm. Thus, in another preferred embodiment of the biosensor of the invention, the porous support is microporous, mesoporous or macroporous. Preferably, the porous support comprises pores with an average size of between about 2 and about 100 nm, more preferably with sizes between 2 and 50 nm, even more preferably with a size of 3, 4, 5, 6, 7, 8, 9 , or 10 nm. In the examples of the present invention the average pore size used is 5 nm. Methods for determining pore size are widely known in the state of the art. Examples of these methods include, without limitation, adsorption-desorption experiments, mercury porosimetry, SAS (■ small-angle scattering), NMR ( nuclear magnetic resonance) and STM-AFM ( scanning tunneling and atomic force microscopies) among others.
    [0039] In addition to the pore size, another parameter that characterizes the porous support is its porosity. Porosity is defined as the measure of empty spaces in a material, and it is a fraction of the volume of voids over the total volume of the material that goes from 0 to 1 or, if expressed as a percentage, from 0 to 100%. The porosity of a system can be measured by various methods. The simplest is the direct method, in which the total volume of the system is measured and the sample is subsequently compacted to remove all the pore space. Then the difference in these volumes gives us the total porosity of the system. To measure accessible porosity, the most widely used method is the so-called gas expansion, but there are others such as polarization light microscopy, or scanning electron microscopy. Regarding indirect methods of porosity measurement, these techniques consist of introducing fluids (liquids such as water or mercury, or gases such as nitrogen or helium) in the empty spaces of the material, and determining the volume of Fluid introduced, indicating the existing volume of voids. In the present invention, the porous support can comprise any porosity. However, it preferably comprises a porosity of between 109 and 1012 cm-2, more preferably, between 1011 and 1012 cm-2.
    [0041] Another characteristic of the porous support of the biosensor of the invention is that it comprises a plurality of pores with access to the outside of the porous support. In the state of the art, this type of pores are called open pores. As explained at the beginning of the present description, said access to the exterior of the porous support is the one that is blocked by the oligonucleotide in the absence of C. suris DNA and that prevents the indicator that is stored inside the pores from leaving the exterior. .
    [0043] The porous support of the biosensor of the invention can be of any material that is porous as defined in the preceding paragraphs. Examples of porous materials that can be used in the context of the present invention as a porous support include, but are not limited to, nanoporous anodic alumina, nanoporous silica, titanium oxide, and graphene. However, in a preferred embodiment of the biosensor of the invention, the porous support is silicon dioxide or aluminum oxide.
    [0045] The porous support of the biosensor of the invention comprises an indicator inside the pores. In the present invention the term "indicator" refers to any compound that can be visualized and / or quantified. Examples of indicators include, but are not limited to, dyes, fluorophores, substances with redox activity, plasmonic resonance, or biologically active as cytotoxic agents, proteins, small biomolecules, enzymes, or nucleic acid fragments. In a preferred embodiment of the biosensor of the invention, the indicator is rhodamine B.
    [0047] Another component of the biosensor of the invention are the oligonucleotides that specifically recognize C. suris DNA . Thus, in the present invention the term "oligonucleotides" refers to short-sequence single-stranded DNA or RNA molecules, both natural and artificial, that can fold into a variety of structures and where nucleotides can be modified, for example, by methylation in 5'.
    [0049] An oligonucleotide sequence can specifically bind to its target with an affinity in the micro-picomolar range. The oligonucleotides of the present invention have two functions: on the one hand, to specifically recognize a region of the C. suris DNA , and on the other hand, to block the pores of the porous material, preventing the exit of the indicator. Thus, in a preferred embodiment of the biosensor of the invention, the nucleotide sequences used are: SEQ ID NO: 2 (5'-TTTTGGGGGGTACGCAAGGCGAATCTACCCGGGGGGTTTT-3 ') or the nucleotide sequence SEQ ID NO: 3 (5'-TTTTGGGGGTGTGTGTGTGTGTGT ). Said sequence SEQ ID NO: 2 specifically recognizes the region 892198 to 892217 of chromosome 2 of the C. suris DNA that encodes an uncharacterized putative C. suris gene . The sequence SEQ ID NO: 3 specifically recognizes the region 1056775 to 1056787 of chromosome 3 of the C. suris DNA that encodes an uncharacterized putative C. suris gene . These sequences are specific for C. suris and do not exist in other pathogenic organisms, guaranteeing the specificity of the biosensor for C. suris.
    [0051] In the biosensor of the invention, the oligonucleotide sequence is attached to the surface of the porous support at a location such that, in the absence of C. suris DNA , the pores of the porous material are blocked, preventing leakage of the indicator. Said union can take place directly to the surface of the support or through a linker or linker, which facilitates its union through electrostatic, supramolecular or covalent interactions.
    [0053] However, in a preferred embodiment of the biosensor of the invention, the oligonucleotides are anchored to the surface of the porous support through a polar organic group.
    [0055] In another preferred embodiment, the oligonucleotides are attached to the surface of the porous support through a polar organic group derivatized with a binding oligonucleotide, wherein the nucleotide sequence of said binding oligonucleotide is partially complementary to the oligonucleotide that recognizes the DNA of C. suris.
    [0057] In the present invention the term "polar organic group" refers to compounds that contain carbon and that have different regions of positive and negative charge as a result of the union with atoms such as nitrogen, oxygen or sulfur. In the present invention the terms "neutral organic group" and "cationic organic group" refer to compounds in which the sum of the charges of the different regions is neutral or positive, respectively.
    [0058] Thus, in a preferred embodiment of the biosensor of the invention, the complementary DNA sequence is attached to the outer surface of the support by a cationic organic group. In a more particular embodiment, the cationic organic group is selected from among amines (NH3 +), guanidinium (HN = (NHR) NH 2 ), phosphonium (PH4 +) or quaternary ammonium (NR4 +) groups. R is selected from linear or branched C 1 -C 6 alkyl and C 3 -C 6 cycloalkyl.
    [0060] In another preferred embodiment of the biosensor of the invention, the neutral organic group is selected from the group consisting of carboxylic acid (-COOH), alcohol (-OH), aldehyde (-CHO), alkenyl C 2 -C 30 alkynyl C 2 -C 30 , amine (-NH 2 or -NR'R "), amide (-C (O) NR'R”), azide (-N 3 ), ketone (-C = 0), ester (-COOR ' ), ether (R'-OR ”), halogen, imine (RR'C = NR"), isocyanate (-NCO), isothiocyanate (-N = C = S), nitrile (-C = N), nitro (- NO 2) and thiol (-SH), each representing R 'and R ", independently, a hydrogen, a C 2 -C 30, alkenyl C 2 -C 30 alkynyl or C 2 -C 30, All of these groups can be linear or branched or substituted or unsubstituted. In a more preferred embodiment, the neutral organic group is isocyanate (-N = C = S).
    [0062] In a more particular embodiment of the invention, the polar organic group is derivatized with a binding oligonucleotide comprising the sequence SEQ ID NO: 1 (NH2- (CH2) 6-5'-AAA AAA CCC CCC-3 '). As can be seen, the binding oligonucleotide has a nucleotide sequence such that it partially hybridizes with the oligonucleotide comprising sequence SEQ ID NO: 2 or 1 to sequence SEQ ID NO: 3.
    [0064] In the present invention, the term "derivatized" refers to the conversion of an original compound into a chemically similar substance by chemical changes.
    [0066] As indicated in previous paragraphs, in the biosensor of the invention the oligonucleotide is anchored to the surface of the porous support in a position such that (i) in the absence of C. suris, the oligonucleotides block the access of the pores to the exterior preventing the indicator from leaving, and
    [0067] (ii) in the presence of C. suris, the oligonucleotides recognize and bind to the DNA of said fungus, unblocking the access of the pores to the exterior and allowing the indicator to exit.
    [0068] Therefore, the oligonucleotide anchor covers the entire outer surface of the porous material, including the edge of the pores so that it can function as a "molecular gate" and, depending on the presence or absence of the C. suris DNA in the medium, the oligonucleotide binds or hybridizes to the C. suris DNA that either allows the indicator to exit (the access of the pore with the exterior is not blocked) or prevents its exit (the access of the pore to the exterior is blocked) .
    [0070] Uses of the biosensor of the invention
    [0072] Throughout the preceding paragraphs, the operation of the biosensor of the invention and its utility in the detection of C. suris DNA have been demonstrated .
    [0074] Therefore, in another aspect, the present invention relates to the use of the biosensor of the invention for the detection of C. suris DNA in a sample, hereafter the "use of the biosensor of the invention".
    [0076] C. suris can be present in a wide variety of environments, from soil, water and air, utensils and surfaces, to humans and animals. Thus, the detection and / or quantification of C. suris by means of the biosensor of the invention can be carried out on any sample that is susceptible to being contaminated by C. suris.
    [0078] In the present invention, "sample" is understood to be a small part or quantity of a thing that is considered representative of the whole and that is taken or separated from it to be studied, analyzed or experimented with. In the present invention, said study, analysis or experimentation refers to the presence / absence of C. suris DNA . Examples of samples suitable for the detection method of the invention include, without limitation, clinical samples and environmental samples. The term "sample" also includes samples that have been manipulated in any way after obtaining them, for example, by treatment with reagents, solubilization or enrichment of certain components. In a preferred embodiment, the isolated samples are processed to obtain a liquid solution in which the components that make up the sample include C. suris DNA . In a preferred embodiment of the use of the invention, the sample is an environmental sample or a clinical sample.
    [0079] In the present invention, an “environmental sample” is understood to be a sample that comes from the environment or the environment and that is susceptible to being or being contaminated by C. suris, such as industrial or household wastewater, laboratory solutions such as buffer solutions, culture liquids, reaction solutions, washes and the like.
    [0081] In the present invention, “clinical sample” is understood as that sample isolated from a subject. The term "clinical specimen" encompasses both blood and other biologically derived liquid specimens, solid tissue specimens, such as a biopsy specimen or tissue cultures or cells derived from them and their progeny, such as cells in cell culture, supernatants cell phones, Cell phones, serum, plasma, biological fluids and tissue samples. Examples of clinical samples suitable for the use of the invention include, but are not limited to, cellular tissues, fecal material, and body fluids, such as urine, saliva, serum and pleural fluid, peritoneal fluid, synovial fluid, and cerebrospinal fluid. In a preferred embodiment in the use of the invention, the biological sample is selected from the group consisting of blood, serum, sputum, pleural fluid, peritoneal fluid, synovial fluid, and cerebrospinal fluid.
    [0083] In the present invention, the clinical sample is isolated from a subject. The term "subject" as used in the present description refers to any animal, preferably a mammal, and includes, but is not limited to, domestic and farm animals, primates, and humans. In a preferred embodiment, the subject is a human of any sex, age, or race.
    [0085] In the present invention, "detect" or "detection" is understood when reporting or identifying C. suris DNA in a sample.
    [0087] The biosensor of the invention is also useful in diagnosing C. suris infection . Thus, in another aspect, the present invention relates to the use of the biosensor of the invention to diagnose in vitro, hereinafter "the diagnostic use of the biosensor of the invention", an infection caused by C. suris in a subject.
    [0089] The term "subject" has been defined in previous paragraphs and is applicable to the present inventive aspect. Thus, in a preferred embodiment of the diagnostic use of the biosensor of the invention, the subject is human.
    [0091] In the present invention, "diagnose" is understood to be the procedure by which a certain disease, nosological entity, syndrome, or any health-disease condition is identified, through the analysis of a series of clinical parameters or characteristic symptoms of said disease, and that distinguish it from other diseases with similar clinical symptoms. In the present invention, the disease to be identified is a yeast infection, that is, an infection caused by C. suris, and the clinical parameter is the presence of C. suris DNA in a sample isolated from the subject.
    [0093] C. suris is a multiresistant pathogenic ascomycete fungus that grows like yeast, forms smooth, shiny, whitish viscous colonies in growth media. Microscopically the cells are ellipsoid in shape. Its growth develops well at 42 ° C, but it has variable growth at higher temperatures. The strong association of this organism with intensive care settings, especially patients with central venous catheters and long-term urinary catheters, suggests a potential of the fungus for biofilm formation. Its multiple resistance to antifungals and the ability to colonize intensive care environments, makes it easier for this fungus to cause a large number of nosoconial infections, where many evolve to fatal systemic infections. Examples of infections caused by C. suris are ear and wound infections, superficial infections (such as skin and mucosal infections), deep infections (peritonitis, meningitis, endocarditis, osteomyelitis, etc.) and systemic infections such as candidemia ( presence of candida in blood). However in a preferred embodiment of the diagnostic use of the biosensor of the invention, the infection caused by C. suris is selected from the group consisting of: candidemia, oropharyngeal candidiasis, vulvovaginal candidiasis, oral candidiasis, cutaneous candidiasis, deep candidiasis (such as peritonitis , meningitis, endocarditis, osteomyelitis, arthritis, etc.), candidiasic esophagitis, onychomycosis and sepsis.
    [0095] Methods of the invention
    [0097] Analogously to the uses of the biosensor of the invention described in previous paragraphs, methods aimed at detecting C. suris in a sample as well as those aimed at diagnosing infections caused by C. suris in a subject are also contemplated in the present invention. .
    [0098] Therefore, another aspect of the invention relates to an in vitro method for detecting C. auris, hereinafter the "detection method of the invention" in a sample comprising:
    [0099] a) contacting the biosensor of the invention with a sample, and
    [0100] b) detect or measure the indicator in the medium,
    [0101] where the presence of the indicator in the medium is indicative of the presence of the C. auris DNA in the sample.
    [0103] The terms "detect", "C. auris ” and“ sample ”have been defined or explained in previous paragraphs, and said definitions and the particular embodiments thereof are applicable to the present inventive aspect. Thus, in a particular embodiment of the detection method of the invention, the sample is selected from the group consisting of an environmental sample or a clinical sample, preferably, the clinical sample is selected from the group consisting of blood, serum, sputum, fluid pleural, peritoneal, synovial, or cerebrospinal.
    [0105] The first step of the detection method of the invention [step a)] comprises contacting the biosensor of the invention with a sample. As explained in previous paragraphs, said sample could have been previously treated before being contacted with the biosensor. Techniques for handling or preparing samples to be subjected to the analysis of their components, in particular for the analysis of the detection of microorganisms, are widely known in the state of the art. In a preferred embodiment of the detection and / or quantification method of the invention, prior to step a), the method comprises mixing the sample with a buffer solution.
    [0107] The term "buffer solution" in the present invention refers to solutions in relatively high concentrations of an acid and its conjugate base, that is, hydrolytically active salts. They have the property of keeping the pH of a solution stable against the addition of relatively small amounts of strong acids or bases. In the detection and / or quantification method of the present invention, the buffer solution allows the pH of the medium to be kept at a narrow threshold so that changes in the solution that may affect the result of the method can be avoided. In a preferred embodiment the "buffer solution" is tris (hydroxymethyl) aminomethane (TRIS) buffer solution. In a preferred embodiment, the pH of the medium is 6.5 to 8.5, preferably p H from 7 to 8, even more preferably pH 7.5.
    [0109] In a second step [step b)], the detection and / or quantification method of the invention comprises detecting or measuring the indicator in the medium. The means and techniques necessary to detect or quantify the indicator in the medium are determined by the indicator used, and such means and techniques are known in the state of the art to a person skilled in the art who will know which means and techniques to use. Examples of methods or techniques include, but are not limited to, visual observation, fluorescence spectroscopy, and ultraviolet-visible spectrophotometry. In a preferred embodiment of the detection method of the invention, detection and quantification is performed by measuring the fluorescence intensity of the medium, more preferably, measuring the fluorescence intensity with a wavelength of 550 to 625 nm from the means, medium.
    [0111] Once the indicator is detected in the middle, the biosensor allows us to conclude that
    [0112] - the presence of the indicator in the medium is indicative of the presence of C. suris DNA in the sample.
    [0114] In the present invention, the term "the presence of the indicator in the medium is indicative of the presence of C. suris DNA in the sample" refers to the fact that the biosensor oligonucleotide of the invention has hybridized, bound, or recognized to C. suris DNA , unlocking the pores of the porous support and releasing the indicator into the medium.
    [0116] Due to the characteristics of the biosensor of the invention, it can also be used in the diagnosis of C. suris infections . Therefore, in another aspect, the present invention relates to an in vitro method for diagnosing a C. suris infection , hereafter the "diagnostic method of the invention", in a subject comprising:
    [0117] a) contacting a biosensor of the invention with a biological sample from a subject, and
    [0118] b) detect the indicator in the middle,
    [0119] where
    [0120] - the presence of the indicator in the medium is indicative that the subject suffers from an infection caused by C. suris.
    [0122] The terms "detect", "C. suris ”, “ sample ”,“ diseases caused by a C. suris infection " have been defined and explained in previous inventive aspects, and said definitions and the particular embodiments thereof are applicable to the diagnostic method of the invention.
    [0124] Thus, in a preferred embodiment of the diagnostic method of the invention, the sample is selected from the group consisting of an environmental sample or a clinical sample, more preferably, the clinical sample is selected from the group consisting of blood, serum, sputum, pleural, peritoneal, synovial, or cerebrospinal fluid.
    [0126] In another preferred embodiment of the diagnostic method of the invention, the infection caused by C. suris is selected from the group consisting of: candidemia, oropharyngeal candidiasis, vulvovaginal candidiasis, oral candidiasis, cutaneous candidiasis, deep candidiasis (such as peritonitis, meningitis , endocarditis, osteomyelitis, arthritis, etc.), candidiasis esophagitis, onychomycosis and sepsis.
    [0128] In another preferred embodiment of the diagnostic method of the invention, the subject is a human.
    [0130] In another preferred embodiment of the diagnostic method of the invention, prior to step a), the method comprises mixing the sample with a buffer solution. In another more preferred embodiment, the "buffer solution" is tris (hydroxymethyl) aminomethane (TRIS) buffer solution. In another preferred embodiment, the pH of the medium is 6.5 to 8.5, preferably p H d e 7 to 8, even more preferably pH 7.5.
    [0132] As used in the present description, the term "the presence of the indicator in the medium is indicative that the subject suffers from a disease caused by C. suris" refers to the fact that due to the interaction of the biosensor oligonucleotide of the invention with C. suris cells , the indicator is released by the pores into the external environment, revealing the presence in said medium of C. suris cells , and by association the disease caused by the pathogen C. suris.
    [0134] Kit of the invention and its uses
    [0136] The implementation of the uses and methods of the invention includes the use of the biosensor of the invention, which may be part of a kit.
    [0137] Therefore, in another aspect, the present invention relates to a kit, hereinafter the "kit of the invention", comprising the biosensor of the invention. In addition to the biosensor, the kit may comprise other components useful in practicing the present invention, such as buffers, delivery vehicles, material carriers, positive and / or negative control components, etc. Optionally, such controls comprise the biosensor of the invention with modifications for use as controls, eg, oligonucleotides that do not recognize C. suris DNA , containers with solutions. In addition to the components mentioned, the kits may also include instructions for practicing the object of the invention. These instructions may be present on the mentioned kits in a variety of ways, one or more of which may be present on the kit. One way these instructions can be present is as information printed on a suitable medium or substrate, eg. eg, a sheet or sheets of paper on which the information is printed, on the kit packaging, on a package insert, etc. Another medium would be a computer-readable medium, for example, a CD, a USB, etc., on which the information was recorded. Another means that may be present is a website address that can be used over the Internet to access information at a remote site. Any convenient means can be present in the kits.
    [0139] In another aspect, the invention relates to the use of the kit of the invention for the detection of C. suris, hereafter the "detection kit of the invention", in a sample.
    [0141] In another aspect, the invention relates to the use of the kit of the invention to diagnose an infection caused by C. suris in a subject in vitro .
    [0143] The terms "detection", "diagnosis", "sample", "subject" and "infection caused by C. suris" have been defined in other aspects of the present invention and are applicable to the kit of the invention and its uses. Likewise, the preferred embodiments of said terms are also applicable to the kit of the invention and its uses. Therefore, in a preferred embodiment, the sample is selected from the group consisting of blood, serum, sputum, pleural, peritoneal, synovial, or cerebrospinal fluid. In another preferred embodiment, the infection caused by C. suris is selected from the group consisting of: candidemia, oropharyngeal candidiasis, vulvovaginal candidiasis, oral candidiasis, cutaneous candidiasis, deep candidiasis (such as peritonitis, meningitis, endocarditis, osteomyelitis, arthritis, etc.), candidiasic esophagitis, onychomycosis and sepsis.
    [0145] In another preferred embodiment of using the kit of the invention to diagnose an infection caused by C. auris in a subject in vitro , the subject is human.
    [0147] Preparation method of the biosensor of the invention
    [0149] Another aspect of the present invention is a method of preparing the biosensor of the invention comprising:
    [0150] a) Insertion of the indicator into the porous support,
    [0151] b) Functionalization of the material from step a) by attaching a neutral or cationic organic group to the surface, and
    [0152] c) Addition of the aptamer recognized by C. auris.
    [0154] In a preferred embodiment of the method of preparing the biosensor of the invention, between step b) and c), the neutral or cationic organic group attached to the surface of the material of step a) is derivatized.
    [0156] Particular embodiments of the method of preparing the biosensor of the invention are all those particular embodiments of the biosensor of the invention, such as those previously described in the present description:
    [0157] - the oligonucleotide is anchored to the surface of the porous support through a polar organic group, and / or
    [0158] - the oligonucleotide is anchored to the surface of the porous support through a polar organic group derivatized with a binding oligonucleotide, wherein the nucleotide sequence of said binding oligonucleotide is partially complementary to the oligonucleotide, and / or
    [0159] - the polar organic group is a neutral or cationic organic group, and / or
    [0160] - the neutral organic group is selected from the group consisting of carboxylic acid (-COOH), alcohol (-OH), aldehyde (-CHO), alkene, alkyne, amine (-NH 2 or -NR'R "), amide ( -C (O) NR'R "), azide (-N 3 ), ketone (-C = 0), ester (-COOR '), ether (R'-OR”), halogen, imine (RR'C = NR "), isocyanate (-NCO), isothiocyanate (-N = C = S), nitrile (-C = N), nitro (-NO 2 ) and thiol (-SH), R being a generic label for a side chain , substituent, or chemical group attached to one of the major functional groups described, and / or
    [0161] - the cationic organic group is selected from the group consisting of amines, guanidinium (CHeN 3 +), phosphonium (PH 4 +) or quaternary ammonium (NR 4 +) groups, where R is selected from C 1 -C 30 alkyl linear or branched and C 3 -C 6 cycloalkyl, and / or
    [0162] - the polar organic group is derivatized with a binding oligonucleotide comprising the sequence NH 2 - (CH 2 ) 6 -SEQ ID NO: 1, and / or
    [0163] - the oligonucleotide comprises the nucleotide sequence SEQ ID NO: 2 or the nucleotide sequence SEQ ID NO: 3 and / or
    [0164] - the indicator is rhodamine B, and / or
    [0165] - the porous support is microporous, mesoporous or macroporous, and / or
    [0166] - the average pore size of the porous support is between approximately 2 and 100 nm, more preferably between approximately 2 and 50 nm, and / or
    [0167] - the porous support is silicon dioxide or aluminum oxide.
    [0169] Throughout the description and claims, the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages, and features of the invention will emerge in part from the description and in part from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.
    [0171] BRIEF DESCRIPTION OF THE FIGURES
    [0173] Fig. 1. Diagram of the biosensor of the invention and the succession of events in the presence of C. auris DNA .
    [0175] Fig. 2. FESEM image of the NAA material and the S2-S3 solid (the biosensor of the invention).
    [0177] Fig. 3. Study of releasing rhodamine B from the biosensor of the invention with the oligonucleotide of sequence SEQ ID NO: 2 (A) and from the biosensor of the invention with the oligonucleotide of sequence SEQ ID NO: 3 (B), both in the presence of 100 ng of C. auris genomic DNA (a) and in the absence (b).
    [0179] Fig. 4. Study of the release of rhodamine B from the biosensor of the invention with the oligonucleotide of sequence SEQ ID NO: 2 (A) and from the biosensor of the invention with the oligonucleotide of sequence SEQ ID NO: 3 (B) in the presence of different concentrations of C. suris DNA . The tests were carried out at 60 minutes in PBS at pH 7.5.
    [0181] Fig. 5. Study of the release of rhodamine B from the biosensor of the invention with the oligonucleotide of sequence SEQ ID NO: 2 in the presence of different concentrations of C. suris colony forming units (CFU) . The tests were carried out at 60 minutes in PBS at pH 7.5.
    [0183] Fig. 6. Study of the release of rhodamine B from the biosensor of the invention with the oligonucleotide of sequence SEQ ID NO: 2 and from the biosensor of the invention with the oligonucleotide of sequence SEQ ID NO: 3 in the presence of other fungal species of the genus Candida. The tests were carried out at 60 minutes in PBS at pH 7.5.
    [0185] EXAMPLES
    [0187] Next, the invention will be illustrated by tests carried out by the inventors, which shows the effectiveness of the biosensor of the invention.
    [0189] Materials and Instrumentation
    [0191] For the preparation of the porous materials, a thermostatic magnetic stirrer and usual glass and plastic material were used. All the reagents used for the preparation of the materials were used as they were received from the commercial houses without any extra purification. The materials obtained were characterized by means of field emission scanning electron microscopy (FESEM), X-ray energy dispersion spectroscopy (EDX) and fluorescence spectroscopy. FESEM images and EDX analysis were obtained with an ULTRA 55 microscope (Zeiss). Fluorescence measurements were carried out on a Synergy H1 microplate fluorimeter (BioTek, Winooski, VT, USA).
    [0193] Examples
    [0195] Example 1: Synthesis of solid S1
    [0196] 8 independent plates of mesoporous alumina (2 mm in diameter and 0.1 mm in height) they were immersed in a solution containing 8 mL of 1 mM acetonitrile and rhodamine B, leaving the mixture to stir at room temperature for 24 h. Then 40 pL per plate of (3-aminopropyl) triethoxysilane (1.32 mmol) was added and allowed to react at room temperature for 5.5 h. The solid obtained was characterized by the FESEM and EDX techniques and was identified as S1.
    [0198] Example 2: Synthesis of the biosensor of the invention
    [0199] 2 independent plates of S1 solid were immersed in 485 pL of a pH 7.5 tris (hydroxymethyl) aminomethane (TRIS) buffer solution containing 15 pL of the oligonucleotide SEQ ID NO: 2 (5'- TTT TGG GGG GTA CGC AAG GCG AAT CTA CCC GGG GGG TTT T-3 ') or 15 pL of the oligonucleotide SEQ ID NO: 3 (5'- TTT TGG GGG GTC GCC ATT TTC TTT GTG GCG GGG GGT TTT-3') and allowed to stir at for 1 h at 37 ° C. It was then washed in solid with the buffered solution of TRIS dropwise and the obtained S2 solid was characterized by the FESEM and EDX techniques.
    [0201] Example 3: Characterization of the materials obtained
    [0202] The porous alumina supports consisted of an anodic aluminum oxide film on a 0.1 mm thick aluminum layer with a pore density of 9-1011 cm'2 and pores of 5 nm in diameter and length of 10 pm . Figure 2a shows the images obtained by means of field emission scanning electron microscopy (FESEM), where the porous surface of the alumina plates can be visualized. Likewise, FESEM images of material S2-S3 show the existence of a layer of organic matter on the surface of the material, while the existence of pores in certain areas confirms the conservation of the porous structure throughout the synthesis of the biosensor. of the invention (Figure 2b).
    [0203] The organic matter content corresponding to the different solids was determined by EDX analysis. By means of this technique, the presence of carbon, nitrogen and phosphorous atoms corresponding to the rhodamine B, (3-aminopropyl) triethoxysilane and oligonucleotide molecules of each of the materials was confirmed. The values obtained for C, N and P in reference to the content of aluminum atoms (C / AI, N / AI and P / AI) are presented in Table 1.
    [0204] Table 1: Organic matter content of the different solids
    [0209] Example 4: Behavior of the biosensor of the invention in the presence of C. auris genomic DNA.
    [0210] 2 independent plates of the biosensor of the invention for each oligonucleotide SEQ ID NO: 2 and SEQ ID NO: 3 were immersed in 900 pL of buffer solution at pH 7.5 (TRIS) each. To one of them was added 100 pL of the buffered solution and to the other was added 100 pL of a C. auris genomic DNA solution (1 ng pL "1). In the absence of the target analyte, no release of the dye was observed. as a function of time (Figure 3 Ay-B, curves a) On the other hand, in the presence of C. auris a release of the dye was observed, as shown by curves b in Figure 3A and 3B.
    [0212] Example 5: Behavior of the biosensor of the invention in the presence of different concentrations of C. auris genomic DNA.
    [0213] 7 independent supports of the biosensor of the invention for each oligonucleotide SEQ ID NO: 2 and SEQ ID NO: 3 were individually immersed in 900 pL of buffer solution at pH 7.5 (TRIS) and to one of them was added 100 pL of dilutions Serials of a C. auris genomic DNA solution until final concentrations in the range of 10.2 and 10.6 ng DNA pL_1 are reached. As seen in Figure 4A (for SEQ ID NO: 2) and Figure 4B (for SEQ ID NO: 3), the amount of indicator released after 60 minutes of testing is proportional to the concentration of fungus present in the medium, establishing a detection limit of 0.5 pg / pL.
    [0215] In another test, 6 supports of the biosensor of the invention with the oligonucleotide SEQ ID NO: 2 were immersed in 6 solutions of human blood artificially inoculated with different concentrations of the C. auris fungus (104-0 CFU mL'1) previously cultivated in medium YPD (Difeo®) agar at 370C for 24 hours. The blood solutions were subsequently centrifuged at 3000 xg for 10 min. Finally, each of the supports was immersed in a mixture of 500 pL of each serum in 500 pL of TRIS buffer (pH 7.5). As seen in Figure 5, the amount of indicator released after 60 minutes of test is proportional to the concentration of fungus present in the medium, establishing a detection limit of 6 CFU / mL.
    [0217] Example 6: Behavior of the biosensor of the invention in the presence of other Candida species.
    [0218] Figure 6 shows the rhodamine B released from the biosensor of the invention with SEQ ID NO: 2 (material S2) and the biosensor of the invention with SEQ ID NO: 3 (material S3) in the presence of different species of the genus Candida ( Candida albicans, Candida glabrata, Candida parapsilopsis, Candida tropicalis, Candida pseudohaemulonii, Candida haemulonii, Candida intermedia and Candida lusitaniae). As can be seen, the release of rhodamine B only occurs in the presence of C. auris, demonstrating the high selectivity achieved by the biosensor of the invention.
    [0220] Example 7: Behavior of the biosensor of the invention with the oligonucleotide SEQ ID NO: 2 in the presence of clinical samples from patients infected with C. auris.
    [0221] System validation was performed on real clinical samples. Specifically, the presence of C. auris was analyzed in 22 blood samples from patients with suspected C. auris infection . All the samples were analyzed in parallel by the Microbiology Service of the Hospital Universitari i Politécnic La Fe. As Table 2 shows, the system managed to identify 90% of the infected samples.
    [0223] In addition, validation of the system was also performed on real clinical samples of different geographical origin, in order to demonstrate the system's ability to identify the different globally identified clusters . All the samples were analyzed in parallel by the Microbiology Service of the Hospital Universitari i Politécnic La Fe. Table 3 shows the results. The biosensor of the invention with the oligonucleotide SEQ ID NO: 2 was able to match 93% of the samples analyzed.
    [0225] Table 1: Validation of the diagnostic method of the biosensor of the invention with the oligonucleotide of sequence SEQ ID NO: 2 ( S2 ) in real blood culture samples from patients with suspected C. auris infection.
    [0227] # Sample S2b reference method
    [0228] (blood culture) 3
    [0229] one
    [0230] 2
    [0231] 3
    [0232] 4
    [0233] 5
    [0234] # Sample Reference method
    [0235] (blood culture) 3 S2b
    [0251] a) It is considered positive (+) when a colony is isolated from the blood samples
    [0252] C. suris.
    [0254] b) It is considered positive (+) when the fluorescence intensity at 575 nm (AeXc =
    [0255] 555 nm) is equal to or greater than 3 times the standard deviation of 3 controls
    [0256] negatives.
    [0258] Table 3: Validation of the diagnostic method of the invention with the biosensor of the invention with the oligonucleotide of sequence SEQ ID NO: 2 ( S2 ) in real samples of sera from patients with suspected infection with different isolates of C. auris from various geographical origins.
    [0260] # Isolated Country Reference method
    [0261] (sequencing) 3 S2b
    [0262] 1 Korea
    [0263] 2 Japan
    [0264] 3 India
    [0265] 4 Venezuela
    [0266] 5 Kuwait
    [0267] 6 Oman
    [0268] 7 Colombia -8 Spain -9 Spain
    [0269] 10 Spain
    [0270] 11 Spain
    [0271] 12 Spain
    [0272] 13 Spain
    [0273] 14 Spain
    [0275] a) Samples confirmed by sequencing.
    [0276] b> It is considered positive (+) when the fluorescence intensity at 575 nm (AeXc = 555 nm) is equal to or greater than 3 times the standard deviation of 3 negative controls.
    权利要求:
    Claims (31)
    [1]
    1. A biosensor comprising
    - a porous support comprising an indicator inside the pores, and - one or more types of oligonucleotides that specifically recognize the DNA of Candida auris,
    where
    - a plurality of pores comprise access to the outside of the porous support, and - the oligonucleotides are anchored to the surface of the porous support in such a position that
    (iii) in the absence of C. auris DNA , the oligonucleotides block the access of the pores to the exterior, preventing the indicator from leaving, and
    (iv) in the presence of C. auris DNA , the oligonucleotides recognize and bind to the DNA of said fungus, unblocking the access of the pores to the exterior and allowing the indicator to exit.
    [2]
    2. Biosensor according to claim 1, wherein the oligonucleotides are anchored to the surface of the porous support through a polar organic group.
    [3]
    3. Biosensor according to claim 2, wherein the polar organic group is derivatized with a binding oligonucleotide, wherein the nucleotide sequence of said binding oligonucleotide is partially complementary to the oligonucleotide that recognizes C. auris DNA .
    [4]
    4. Biosensor according to any of claims 1 to 3, wherein the polar organic group is a neutral or cationic organic group.
    [5]
    5. Biosensor according to claim 4, wherein the organic group is selected from neutral the group consisting of carboxylic acid (-COOH), alcohol (-OH), aldehyde (-CHO), alkenyl C 2 -C 30 alkynyl C 2 -C 30 , amine (-NH 2 or -NR'R "), amide (-C (O) NR'R”), azide (-N3), ketone (-C = 0), ester (-COOR ') , ether (R'-OR ”), halogen, imine (RR'C = NR"), isocyanate (-NCO), isothiocyanate (-N = C = S), nitrile (-C = N), nitro (-NO 2) and thiol (-SH), preferably isothiocyanate (-NCO S), each representing R 'and R ", independently, a hydrogen, a C 2 -C 30 alkenyl , C 2 -C 30 , or a C 2 -C 30 alkynyl, all of these groups may be linear or branched or substituted or unsubstituted replaced.
    [6]
    6. Biosensor according to claim 4, wherein the cationic organic group is selected from the group consisting of amines (-NH3 +), guanidinium ([CH6N3] +), phosphonium (-PH4 +) or quaternary ammonium (-NR4 +) groups, wherein each R is independently selected from a linear or branched Ci-C3o alkyl, and a C3-C6 cycloalkyl.
    [7]
    7. Biosensor according to any one of claims 3 to 6, wherein the polar organic group is derivatized with a binding oligonucleotide comprising the sequence NH2- (CH2) 6-SEQ ID NO: 1.
    [8]
    8. Biosensor according to any one of claims 1 to 7, wherein the oligonucleotides comprise the nucleotide sequences SEQ ID NO: 2 or the nucleotide sequence SEQ ID NO: 3.
    [9]
    9. Biosensor according to any one of claims 1 to 8, wherein the indicator is rhodamine B.
    [10]
    10. Biosensor according to any one of claims 1 to 9, wherein the porous support is microporous, mesoporous or macroporous.
    [11]
    11. Biosensor according to claim 10, wherein the average pore size of the porous support is between about 2 and about 100 nm.
    [12]
    12. Biosensor according to any one of claims 1 to 11, wherein the porous support is silicon dioxide or aluminum oxide.
    [13]
    13. Use of a biosensor according to any one of claims 1 to 12 for the detection of C. suris DNA in a sample.
    [14]
    14. Use according to claim 13, wherein the sample is selected from the group consisting of an environmental sample or a clinical sample, preferably, the clinical sample is selected from the group consisting of blood, serum, sputum, pleural fluid, peritoneal, synovial or cerebrospinal.
    [15]
    15. Use of a biosensor according to any one of claims 1 to 12 to diagnose C. auris infection in vitro in a subject.
    [16]
    16. Use according to claim 15, wherein the infection caused by C. auris is selected from the group consisting of: candidemia, oropharyngeal candidiasis, vulvovaginal candidiasis, oral candidiasis, cutaneous candidiasis, deep candidiasis, candidiasis esophagitis, onychomycosis and sepsis.
    [17]
    17. Use according to claim 15 or 16, wherein the subject is human.
    [18]
    18. In vitro method for detecting C. auris DNA in a sample comprising: a) contacting a biosensor according to any one of claims 1 to 12 with a sample, and
    b) detect the indicator in the middle,
    where the presence of the indicator in the medium is indicative of the presence of the C. auris DNA in the sample.
    [19]
    19. In vitro method to diagnose C. auris infection in a subject comprising:
    a) contacting a biosensor according to any one of claims 1 to 12 with a biological sample isolated from a subject, and
    b) detect the indicator in the middle,
    where the presence of the indicator in the medium is indicative that the subject suffers from an infection caused by C. auris.
    [20]
    20. The method according to claim 19, wherein the infection caused by C. auris is selected from the group consisting of: candidemia, oropharyngeal candidiasis, vulvovaginal candidiasis, oral candidiasis, cutaneous candidiasis, deep candidiasis, candidiasis esophagitis, onychomycosis and sepsis.
    [21]
    21. Method according to any one of claims 18 to 20, wherein prior to step a), the method comprises mixing the sample with a buffer solution.
    [22]
    22. Method according to any one of claims 18 to 21, wherein the sample is selected from the group consisting of a food sample or a clinical sample, preferably, the clinical sample is selected from the group consisting of serum, sputum, pleural fluid, peritoneal, synovial, or cerebrospinal blood.
    [23]
    23. Method according to any one of claims 18 to 22, wherein the subject is human.
    [24]
    24. A kit comprising a biosensor according to any one of claims 1 to 12.
    [25]
    25. Use of a kit according to claim 24, for the detection of C. suris DNA in a sample.
    [26]
    26. Use according to claim 25, wherein the sample is selected from the group consisting of blood, serum, sputum, pleural, peritoneal, synovial or cerebrospinal fluid.
    [27]
    27. Use of a kit according to claim 24, to diagnose C. suris infection in vitro in a subject.
    [28]
    28. Use according to claim 27, wherein the infection caused by C. suris is selected from the group consisting of candidemia, oropharyngeal candidiasis, vulvovaginal candidiasis, oral candidiasis, cutaneous candidiasis, deep candidiasis, candidiasis esophagitis, onychomycosis and sepsis.
    [29]
    29. Use according to claim 27 or 28, wherein the subject is human.
    [30]
    30. Method for preparing a biosensor according to any one of claims 1 to 12 comprising
    a) Insertion of the indicator into the porous support,
    b) Functionalization of the material from step a) by attaching a neutral or cationic organic group to the surface, and
    c) Addition of the oligonucleotide that recognizes the C. suris DNA .
    [31]
    31. The method according to claim 30, where after step b) and before step c) the neutral or cationic organic group attached to the surface of the material from step a) is derivatized.
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    ES2763043B2|2021-03-25|
    WO2021219910A1|2021-11-04|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    WO2019048722A1|2017-09-05|2019-03-14|Universitat Politècnica De València|Porous material for the detection of candida albicans, diagnostic method using same and preparation method thereof|
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