RU2808079C2 - Biomarker - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method of detecting the effect of zearalenone is described, the said method including the following: determining in the test sample the expression level of at least one microRNA selected from the group consisting of ssc-miR-1, ssc-miR-181c, ssc-miR-206, ssc-miR-503, ssc-miR-542-3p, ssc-miR-135, ssc-miR-129a-3p, ssc-miR-142-3p, ssc-miR-432-5p, ssc-miR-455-5p, ssc-miR- 182, ssc-miR-493-3p, ssc-miR-455-3p, ssc-miR-183, and ssc-miR-140-3p, or at least one expression level ratio between two of said microRNAs, the said test being the sample is a blood sample; and comparing the said expression level or at least one ratio of expression levels with a reference value. Also a data processing system containing a processor configured to implement the specified method is described. The following is proposed: a sequencing device capable of determining the level of at least one microRNA selected from the group consisting of ssc-miR-1, ssc-miR-181c, ssc-miR-206, ssc-miR-503, ssc-miR-542-3p, ssc-miR-135, ssc-miR-135a-5p, ssc-miR-129a-3p, ssc-miR-142-3p, ssc-miR-432-5p, ssc-miR-455-5p, ssc- miR-182, ssc-miR-493-3p, ssc-miR-455-3p, ssc-miR-183, and ssc-miR-140-3p containing data processing system. A machine-readable medium that stores on it a computer program containing instructions for operating the said sequencing system or device is proposed. A kit for implementing this method is presented.

EFFECT: group of inventions relates to a method of detecting exposure to zearalenone (ZEN), including determining the expression level of at least one microRNA in a test sample, as well as to a new way to use at least one microRNA to detect exposure to zearalenone (ZEN) in a test sample.

14 cl, 8 dwg, 1 tbl, 5 ex

Description

TECHNICAL FIELD

[0001] The present invention relates to a novel method for detecting exposure to zearalenone (ZEN), including determining the expression level of at least one microRNA in a test sample, as well as a novel use of at least one microRNA to detect exposure to zearalenone (ZEN) in a test sample.

DESCRIPTION

[0002] Mycotoxins are secondary metabolites of molds that lead to concentration-dependent effects adverse to animal health. In Northern and Central Europe, mycotoxins produced by field fungi such as Fusarium graminearum or F. culmorum are most often found in animal feed (Streit et al. 2012. Toxins, 4, 788-809). Zearalenone (ZEN) is considered one of the most important Fusarium mycotoxins, especially in pig production. ZEN often contaminates corn, but can also be found in a variety of other foods such as wheat, barley or oats. In a recent study, 88% of feed samples tested were positive for ZEN, with median and maximum concentrations of 20 µg/kg and 11,192 µg/kg, respectively (Kovalsky et al. 2016. Toxins, 8, E363). Pigs are particularly susceptible to the effects of ZEN due to the species-specific metabolism of this mycotoxin (Fink-Gremmels and Malekinejad. 2007. Animal Feed Science and Technology, 137, 326-341). ZEN exhibits low acute toxicity but acts as a full and partial agonist of estrogen receptors α and β, respectively (Zinedine et al. 2007. Food and Chemical Toxicology, 45, 1-18). As a consequence, exposure to ZEN in pigs leads to reproductive and fertility disorders. For example, immature pigs begin to develop mammary glands when fed feed containing 1-5 ppm ZEN. Vulvovaginitis, vulvar edema and rectal prolapse have also been observed (Osweiler, G. 1999. Mycotoxins. In: STRAW, V., DALLAIRE, S., MENGELING, W. & TAYLOR, D. (eds.) 8th Diseases of swine. Ames , Iowa, USA: Iowa State University Press; Osweiler, G. D. 2000. Mycotoxins: Contemporary issues of food animal health and productivity. Veterinary Clinics of North America: Food Animal Practice, 16, 511-530). In mature sows, the effects of ZEN depend on the concentration in the feed, duration of exposure and gestational phase and range from anestrus to a decrease in the number of live embryos, an increase in the number of stillborn piglets and abortions (Kordic et al. 1992. Journal of environmental pathology, toxicology and oncology: official organ of the International Society for Environmental Toxicology and Cancer, 11, 53-55). Newborn piglets from exposed sows have been shown to have enlarged external genitalia and a higher incidence of piglet hip dysplasia and tremors (Osweiler, G. 1999. Mycotoxins. In: STRAW, W., DALLAIRE, S., MENGELING, W. & TAYLOR , D. (eds.) 8th Diseases of swine (Ames, Iowa, USA: Iowa State University Press). In addition, ZEN activates various receptors, especially the pregnane X receptor, which is involved in the regulation of cytochrome P450 isoforms in vitro, thereby potentially influencing the phase I metabolism of various endo- and xenobiotics (Ayed-Boussema et al. 2011. Environmental Toxicology and Pharmacology, 31, 79-87).

[0003] Until now, detection of ZEN mycotoxicosis has primarily been based on observation of clinical signs and feed analysis. However, ZEN, like many other mycotoxins, exhibits effects adverse to livestock health before clinical symptoms become apparent. The analysis of feed samples comes with certain challenges: Representative feed samples must be taken to account for the heterogeneous distribution of mycotoxins in the lot, as well as the timing of sampling, since feed lots may have changed between sampling and the onset of mycotoxin effects. In addition, co-contamination of feed samples with mycotoxin, which may lead to additive, synergistic or antagonistic effects in animals, must be considered (Grenier and Oswald. 2011. World Mycotoxin Journal, 4, 285-313). Moreover, the presence of modified forms of ZEN is not regularly monitored, but is added to the total ZEN load of an individual (Binder et al. 2017. Toxins, 9, 56). In contrast to feed analysis, detection of specific biomarkers in pig biological matrices such as urine, feces, bile or tissue would allow detection of mycotoxicosis at the individual level. Two types of biomarkers are used in mycotoxin research: exposure-based biomarkers and mechanism-based biomarkers (Baldwin et al. 2011. World Mycotoxin Journal, 4, 257-270). While the former refers to the measurement of the mycotoxin itself and/or its metabolites, the latter refers to a specific biological response, such as an increase/decrease in protein or cellular metabolite levels, that may be associated with mycotoxin consumption. Regarding ZEN, efforts have been made in recent years to identify an exposure-based biomarker in pigs. Under controlled conditions, numerous studies have examined the effects of increasing toxin concentrations on the levels of ZEN and/or its metabolites in blood, urine, and bile, as well as in feces, cerebrospinal fluid, liver, or spinal fat. Generally, the highest concentrations of ZEN and its metabolites were extracted from bile and urine with the best correlation coefficients, i.e. ratio of ZEN ingested to ZEN extracted, for bile and urine as matrices than for other matrices such as blood ( et al. 2003. Archives of Animal Nutrition, 57, 311-334; Goyarts et al. 2007. Food Additives and Contaminants, 24, 369-380; Brezina et al. 2014. Archives of animal nutrition, 68, 425-447). Accordingly, a recent field study was unable to correlate ZEN levels in sow feed with plasma levels because plasma toxin concentrations were below the limit of quantitation in most samples (Van Limbergen et al. 2017. Veterinary Record, 181, 539) . Exposure-based biomarkers still cannot be recommended for predicting ZEN intake in the field, since comparable ZEN concentrations in bile, blood, or urine can be obtained from a wide range of ZEN exposure levels ( and Winkler. 2015. Food and Chemical Toxicology, 84, 225-249). More often than not, ZEN residues do not indicate the severity of the adverse health effects caused by ZEN, as evidenced, for example, by the lack of correlation between total bile ZEN levels and uterine weight after ZEN exposure ( and Winkler. 2015. Food and Chemical Toxicology, 84, 225-249). Regarding the mechanism-based biomarkers of ZEN, changes in some biological parameters such as hematological and biochemical parameters have been found (Goyarts et al. 2007. Food Additives and Contaminants, 24, 369-380; et al. 2016. Research in Veterinary Science, 109, 169-180) or mRNA expression (Dai et al. 2016. Animal Reproduction Science, 168, 126-137; Reddy et al. 2017. Asian-Australasian Journal of Animal Sciences, 31, 595 -606; Zhou et al. 2018. Asian-Australasian Journal of Animal Sciences, 31, 32), when exposed to ZEN in vivo. However, the reproducibility and specificity of the studied parameters is limited to the extent that it has not yet been possible to establish a clear dose-response relationship between the increase/decrease of these biological parameters in the blood, urine or bile and the use of ZEN. Not et al. (He et al. 2018. Endocrinology, 159, 2993-3006) describe an increase in the expression of microRNA (miRNA) miR-7 in pituitary tissue of ovariectomized pigs after intraperitoneal injection of 112.5 mg ZEN per day. However, He et al. used an experimental design that does not reflect natural exposure to ZEN from contaminated feed because the amount of 112.5 mg ZEN is much higher than the actual exposure level. In addition, administration by injection does not reflect natural feed intake. Additionally, ovariectomized pigs are not good models for ZEN exposure in healthy, non-ovariectomized pigs because ZEN exposure is generally correlated with reproductive and fertility impairment. Additionally, it would be desirable to identify a biomarker that would be suitable for detecting ZEN exposure in a more accessible sample than pituitary tissue, such as serum.

[0004] The present invention has been made in view of the prior art described above. In general, we have found that mechanism-based biomarkers would be more suitable for use in the detection of mycotoxicosis than exposure-based biomarkers due to simpler detection methods and direct correlation with mycotoxin exposure in an individual, but to date So far, no suitable exposure-based biomarker or mechanism-based biomarker has been identified.

[0005] The present invention relates to a method for detecting exposure to zearalenone (ZEN), the method comprising determining the expression level of at least one microRNA described herein in a test sample; and comparing the expression level with the expression level of said at least one microRNA in a control sample. MicroRNAs (also called microRNAs) are short RNA molecules typically ranging from approximately 19 to approximately 24 nucleotides in length. MicroRNAs are negative regulators of gene expression that can block the translation of mRNA into proteins or degrade mRNA molecules. By providing a method for detecting exposure to ZEN, comprising determining in a test sample the expression level of at least one microRNA described herein; and comparing the expression level with a reference value, we unexpectedly identified a reliable method for detecting ZEN exposure using an exposure-based biomarker that is suitable for use in the art.

[0006] The term "expression level" of a polynucleotide, such as, for example, an mRNA or microRNA molecule, refers to the relative or absolute amount of said polynucleotide. Expression levels of polynucleotides can be determined using appropriate strategies known to one skilled in the art, including, but not limited to, bioarray experiments, quantitative real-time PCR, or so-called next generation sequencing (NGS) technologies such as those offered by, for example, Illumina, Pacific Biosciences, Oxford Nanopore Technologies etc.

[0007] Zearalenone (ZEN) is a non-steroidal macrocyclic lactone with the following structural formula that can be synthesized through the polyketide metabolic pathway:

Its name according to IUPAC nomenclature is: (2E,11S)-15,17-dihydroxy-11-methyl-12-oxabicyclo[12.4.0]octadeca-1(18),2,14,16-tetraene-7 ,13-dione. Various ZEN derivatives also occur in nature and can be formed by enzymatic or chemical modifications of ZEN. Examples include, but are not limited to, ZEN glycosidic conjugates or sulfate-containing conjugates formed by fungal, plant or mammalian metabolism, and ZEN metabolites formed in humans or animals, among others. By ZEN derivatives below we mean ZEN conjugates or ZEN metabolites that occur naturally or are synthesized by chemical or biochemical synthesis, but in particular α-zearalenol (α-ZEL; (2E,7R,11S)-7,15,17- trihydroxy-11-methyl-12-oxabicyclo[12.4.0]-octadeca-1(18),2,14,16-tetraen-13-one), β-zearalenol (β-ZEL; (2E,78,118)-7 ,15,17-trihydroxy-11-methyl-12-oxabicyclo[12.4.0]octadeca-1(18),2,14,16-tetraen-13-one), α-zearalanol (α-ZAL; (7R, 11S)-7,15,17-trihydroxy-11-methyl-12-oxabicyclo[12.4.0]octadeca-1(18), 14,16-trien-13-one), β-zearalanol(β-ZAL; ( 78,118)-7,15,17-trihydroxy-11-methyl-12-oxabicyclo[12.4.0]octadeca-1(14),15,17-trien-13-one), zearalenone 14-sulfate (Z14S; [( 2E, 11S)-15-hydroxy-11-methyl-7,13-dioxo-12-oxabicyclo[12.4.0]octadeca-1(18),2,14,16-tetraen-17-yl]hydrosulfate), zearalenone -14-glycoside (Z14G; (2E,11S)-15-hydroxy-11-methyl-17-[(3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)-tetrahydropyran- 2-yl]oxy-12-oxabicyclo[12.4.0]octadeca 1(18)2,14,16-tetraene-7,13-dione), as well as zearalanone (ZAN; (118)-15,17-dihydroxy-11-methyl-12-oxabicyclo-[12.4.0]octadeca-1(18),14,16-triene-7,13-dione). ZEN, as well as ZEN derivatives, particularly α-ZEL, β-ZEL, Z14S, α-ZAL, β-ZAL, Z14G and ZAN, can also be found in processed foods and animal feeds such as bread or beer , due to their high chemical and physical stability.

[0008] As used herein, the term "zearalenone exposure" or "ZEN effect" refers to the effect of zearalenone on a biological system or the (physiological) effect of zearalenone on a biological system. The biological system can be any biological system such as a cell, cell culture, tissue or animal. In some embodiments, “zearalenone effect” may further refer to the effect on a biological system of a zearalenone derivative or analogue, or to the (physiological) effect of a zearalenone derivative or analogue on a biological system. The zearalenone derivative is preferably the derivative described above in the present application. It should be understood that the zearalenone derivative or analogue preferably has the same or similar effect as zearalenone on a biological system. Preferably, "zearalenone effect" refers to the effect of zearalenone.

[0009] As used herein, "test sample" preferably refers to a sample for which it is desired to determine whether or not it has come into contact with ZEN (or a ZEN analogue or derivative), or whether the subject from which it was obtained has been exposed to ZEN ( or an analogue or derivative of ZEN) or not. The methods of the present invention may thus include step (b) of obtaining a test sample.

[0010] The reference value may correspond to the expression level of the at least one microRNA according to the present invention, which was determined in one or more control samples. Depending on the type of sample, such a control sample could come into contact with (preferably a known amount of) ZEN or a ZEN analogue or derivative, or the control sample could not come into contact with ZEN or a ZEN analogue or derivative. Such a control sample could be obtained from a subject who was exposed to (preferably a known amount) ZEN or a ZEN analog or derivative, or could be obtained from a subject who was not exposed to ZEN or a ZEN analog or derivative. In this application, contact of a sample with ZEN or a ZEN analogue or derivative that is not bioavailable, for example due to binding to another substance, which prevents ZEN or a ZEN analogue or derivative from interacting with a biological system, or exposure of the subject to ZEN or a ZEN analogue or derivative, that are not bioavailable is equivalent to no contact of ZEN or a ZEN analog or derivative with the sample or no exposure of the subject to ZEN or a ZEN analog or derivative. The reference value may also be an average or average value that has been determined from several control samples. The at least one microRNA according to the present invention may be selected from the group consisting of ssc-miR-1, ssc-miR-125a, ssc-miR-125b, ssc-miR-127, ssc-miR-129a, ssc-miR- 133a-5p, ssc-miR-135, ssc-miR-136, ssc-miR-140-3р, ssc-miR-142-3р, ssc-miR-143-5p, ssc-miR-146b, ssc-miR- 181c, ssc-miR-182, ssc-miR-183, ssc-miR-187, ssc-miR-195, ssc-miR-204, ssc-miR-206, ssc-miR-22-3p, ssc-miR- 22-5p, ssc-miR-335, ssc-miR-34a, ssc-miR-369, ssc-miR-378, ssc-miR-424-5p, ssc-miR-432-5p, ssc-miR-450a, ssc-miR-450b-5p, ssc-miR-450c-5p, ssc-miR-455-3р, ssc-miR-455-5p, ssc-miR-486, ssc-miR-493-3р, ssc-miR- 493-5p, ssc-miR-497, ssc-miR-503, ssc-miR-542-3р, ssc-miR-708-3р, ssc-miR-758, ssc-miR-7135-3р, hsa-miR- 135b-5p, hsa-miR-301a-5p, put-miR-300. Preferably, at least one microRNA according to the present invention may be selected from the group consisting of ssc-miR-1, ssc-miR-181c, ssc-miR-206, ssc-miR-503, ssc-miR-542-3p, ssc-miR-135, ssc-miR-129a-3p, ssc-miR-142-3р, ssc-miR-432-5p, ssc-miR-455-5p, ssc-miR-182, ssc-miR-493- 3р, ssc-miR-455-3р, ssc-miR-183, ssc-miR-140-3р, ssc-miR-7135-3р, ssc-miR-204, ssc-miR-143-5p, ssc-miR- 187, ssc-miR-335, ssc-miR-424-5p, ssc-miR-450a, ssc-miR-450b-5p, ssc-miR-450c-5p, ssc-miR-497. The at least one miRNA according to the present invention may also be a combination of two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or even more or all miRNAs selected from the above group.

[0011] Preferably, at least one microRNA according to the present invention is selected from the group consisting of ssc-miR-1, ssc-miR-181c, ssc-miR-206, ssc-miR-503, ssc-miR-542-3p , ssc-miR-135, ssc-miR-129a-3p, ssc-miR-142-3р, ssc-miR-432-5p, ssc-miR-455-5p, ssc-miR-182, ssc-miR-493 -3р, ssc-miR-455-3р, ssc-miR-183 and ssc-miR-140-3р. The at least one miRNA according to the present invention may also be a combination of two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or all fifteen miRNAs selected from the above group. Thus, the effect of zearalenone can be determined with particularly high reliability. Preferably, at least one microRNA according to the present invention is selected from the group consisting of ssc-miR-542-3p, ssc-miR-1, ssc-miR-493-3p, ssc-miR-135, ssc-miR-432- 5p and ssc-miR-455-5р. The at least one miRNA according to the present invention may also be a combination of two, three, four, five or all six miRNAs selected from the above group. Preferred combinations of two microRNAs selected from the above group comprise a pair selected from the group consisting of ssc-miR-542-3p and ssc-miR-1, ssc-miR-542-3p and ssc-miR-493-3p, ssc -miR-135 and ssc-miR-432-5р, and ssc-miR-455-5p and ssc-miR-493-3р. Preferred combinations may also contain two, three or four of the above pairs. In this way, the accuracy of the results of the method can be increased.

[0012] Preferably, at least one microRNA according to the present invention is selected from the group consisting of ssc-miR-1, ssc-miR-181c, ssc-miR-206, ssc-miR-503, ssc-miR-542-3p , ssc-miR-135, ssc-miR-7135-3р, ssc-miR-204, ssc-miR-143-5p, ssc-miR-187, ssc-miR-335, ssc-miR-424-5p, ssc -miR-450a, ssc-miR-450b-5p, ssc-miR-450c-5p and ssc-miR-497. The at least one miRNA according to the present invention may also be a combination of two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen or all sixteen miRNAs selected from the above group. Preferably, at least one microRNA according to the present invention is ssc-miR-542-3p and/or ssc-miR-1.

[0013] In this application, unless otherwise noted, miRNAs are designated by miRBase sequence database accession numbers as of June 21, 2014 (Kozomara A, Griffiths-Jones S. Nucleic Acids Res. 2014 42:D68-D73; Kozomara A , Griffiths-Jones S. Nucleic Acids Res. 2011 39:D152-D157;Griffiths-Jones S, Saini HK, van Dongen S, Enright AJ. Nucleic Acids Res. 2008 36:D154-D158;Griffiths-Jones S, Grocock RJ , van Dongen S, Bateman A, Enright AJ Nucleic Acids Res 2006 34:D140-D144; Griffiths-Jones S Nucleic Acids Res 2004 32:D109-D111). The following table briefly describes some of the microRNAs of the present invention.

[0014] The methods of the present invention may include comparing the level of at least one microRNA detected in a test sample with a reference value. If at least one miRNA includes more than one miRNA, a comparison of the level between these miRNAs may include a comparison of the ratio between the two miRNAs. For example, if at least one miRNA includes two, three, four or five miRNAs selected from the group consisting of or containing at least some, preferably all of ssc-miR-542-3p, ssc-miR-1, ssc- miR-493-3p, ssc-miR-135, ssc-miR-432-5p and ssc-miR-455-5p, the level comparison may refer to the comparison of the ratio between ssc-miR-542-3p and ssc-miR-1, ssc-miR-542-3р and ssc-miR-493-3р, ssc-miR-135 and ssc-miR-432-5p and/or ssc-miR-455-5p and ssc-miR-493-3р. Rate comparison can also refer to comparison of the ratio between any pairs of miRNAs shown in Figure 7 .

[0015] Comparison of the level of the at least one microRNA may be based on comparison of individual values of one or more microRNAs. For example, a deviation in miRNA expression levels between a test sample and a reference value may indicate an effect of zearalenone (e.g., exposure to zearalenone), particularly if the reference value corresponds to the expression level of said miRNA in a control sample that was not exposed to or that received zearalenone. in a subject who was not exposed to zearalenone. Deviation in expression levels may be indicative of the effects of zearalenone. The deviation may be expressed on a normal scale or preferably on a logarithmic scale such as a log2 scale. The deviation is preferably statistically significant. Several methods are known to those skilled in the art to test the statistical significance of results, and one skilled in the art can select and apply the most appropriate method for analyzing statistical significance. Statistical significance may be, for example, expressed as a p-value or FDR adjusted p-value, and the significance level may be ≤0.07, preferably ≤0.05, or more preferably ≤0.001. The deviation may be an increase or a decrease. The deviation indicative of ZEN exposure may, for example, be at least about 1.3 times, more preferably at least about 1.5 times, and most preferably at least about 2 times the reference value corresponding to the sample, not exposed to zearalenone or obtained from a subject not exposed to zearalenone.

[0016] Alternatively, if the reference value corresponds to the expression level that was determined in a control sample exposed to ZEN or a ZEN analog or ZEN derivative, or was obtained from a subject that was exposed to ZEN or a ZEN analog or ZEN derivative, no deviation, slight deviation, or (atypically) low deviation between the values of the test sample and the reference value may be indicative of zearalenone exposure, such as zearalenone exposure.

[0017] The term "detection" or "detection" as used herein in the context of detecting the effects of zearalenone can generally refer to quantitative or qualitative detection. For example, "detection" may refer to qualitative detection, that is, whether the effect of zearalenone is present based on the comparison step. "Detection" can also refer to quantitative detection, i.e. to the degree of effect of zearalenone based on the comparison stage.

[0018] As used herein, the term “sample” refers to a biological sample. The sample may contain a cell. The sample may be obtained from the subject. Biological samples include, but are not limited to, blood, serum, peripheral blood mononuclear cells (PBMC), urine, feces, semen, or tissue such as uterine tissue. To ensure rapid detection of ZEN exposure in a subject and thus achieve maximum applicability in the field, the detection method is preferably applied to a sample that can be obtained with minimal effort. Therefore, the sample is preferably a blood sample, in particular a blood serum sample. By providing a method in which the sample is a blood serum sample, the potential exposure of a subject to ZEN can be assessed in the most convenient manner. A blood serum sample can be obtained, for example, by puncture of the cranial vena cava (Vena cava cranialis) or jugular vein (Vena jugalaris). Serum samples can be stored at -80°C for later analysis. Another preferred sample is a uterine sample. The sample may also be a cell culture sample. The cell culture sample may contain intact cells, but may also contain or consist of disrupted cells and/or supernatant of intact or disrupted cells. The cells contained in the cell culture may express estrogen receptors α and/or β, preferably mammalian estrogen receptors α and/or β such as human or porcine. Preferred cell lines for the cell culture sample may be MCF-7, T47D, SUM159, HeLa or Ishikawa In addition, primary cells, such as, for example, porcine granulosa cells, may be the preferred cells for the cell culture sample.

[0019] If the sample is a uterine tissue sample, the at least one miRNA is preferably selected from the group consisting of ssc-miR-1, ssc-miR-181c, ssc-miR-206, ssc-miR-503, ssc-miR -542-3р, ssc-miR-135, ssc-miR-7135-3р, ssc-miR-204, ssc-miR-143-5p, ssc-miR-187, ssc-miR-335, ssc-miR-424 -5p, ssc-miR-450a, ssc-miR-450b-5p, ssc-miR-450c-5p and ssc-miR-497, wherein at least one microRNA is preferably ssc-miR-542-3p and/or ssc-miR-1.

[0020] If the sample is a blood sample, such as a serum sample, the at least one miRNA may be selected from the group consisting of ssc-miR-1, ssc-miR-181c, ssc-miR-206, ssc-miR- 503, ssc-miR-542-3р, ssc-miR-135, ssc-miR-129a-3p, ssc-miR-142-3р, ssc-miR-432-5p, ssc-miR-455-5p, ssc- miR-182, ssc-miR-493-3p, ssc-miR-455-3p, ssc-miR-183 and ssc-miR-140-3p, preferably selected from the group consisting of ssc-miR-542-3p, ssc -miR-1, ssc-miR-493-3p, ssc-miR-135, ssc-miR-432-5p and ssc-miR-455-5p.

[0021] Comparison of the level of the at least one miRNA can also be based on comparison of individual values of several miRNAs. In this case, the relative (or less preferably absolute) deviations of the expression level in the test sample from the reference value can be combined to obtain an even more meaningful result than comparing the level of only one microRNA. Alternatively, the deviation values can be multiplied or divided by each other, depending on the expected direction of deviation in case of exposure to zearalenone.

[0022] Comparing the level of the at least one microRNA may also refer to comparing the ratio of two microRNAs. The ratio can be determined by dividing the level of one miRNA by the level of the second miRNA, as known to those skilled in the art. The present inventors have found that determining the deviation of miRNA ratios, as described above, allows surprisingly good discrimination between samples that were exposed to zearalenone (ZEN) and samples that were not exposed to zearalenone (ZEN). If individual measured miRNAs are analyzed by qPCR, a possible way to calculate the ratio is to determine the ΔCt (eg, Ct miRNA - Ct UniSp4) for each miRNA to calculate the ratio. As an illustrative example, the ratio between miR-135a and miR-1a can be calculated using ΔCt miRNA 135a - ΔCt miRNA 1a.

[0023] Exceptionally good detection of the effect of ZEN can be achieved when at least one ratio of expression levels between two miRNAs is selected from the group consisting of ratios between miR-542-3p and miR-1, miR542-3p and miR493-3p, miR -135a-5p and miR-432-5p, and miR-455-5p and miR-493-3p. A ratio may be considered preferable to a method described herein if an operating characteristic (ROC) analysis (Grand and Sabin. 2010. Current Opinion in HIV and AIDS, 5, 473-479) to distinguish exposure to ZEN compared to no exposure to ZEN results in to an area under the curve (AUC) value that is greater than or equal to 0.7. Alternatively or additionally, the fold change in miRNA ratio in the ZEN-containing sample is preferably at least about 1.3-fold, more preferably at least about 1.5-fold, most preferably at least about 2-fold compared to the sample not exposed to ZEN or obtained from a subject not exposed to ZEN. The multiple change can be either positive or negative, i.e. positively or negatively regulated compared to the control sample. The level of at least one miRNA further preferably comprises at least one expression level ratio between two miRNAs selected from the group consisting of miR-542-3p and miR-1, miR-542-3p and miR-493-3p, miR -135a-5p and miR-432-5p, and miR-455-5p and miR-493-3p. In this way, the effects of ZEN can be detected with particularly good reliability and reproducibility.

[0024] Comparison of the level of the at least one microRNA with a reference value, in particular if several microRNAs are involved, can also be carried out using complex methods, such as computerized methods, in particular methods involving machine learning. As an illustrative example, an artificial neural network system can be used to compare expression level(s) to a reference value. A neural network can be trained by providing the value of one or more reference values, which can correspond to miRNA levels from one or more samples. Preferably, both ZEN exposure reference values and ZEN non-exposure reference values can be used to train the neural network. The trained neural network can then be used to detect the effects of zearalenone. For this purpose, the miRNA level(s) values of the corresponding miRNA of the test sample may be used as input to a neural network, and the neural network may provide exposure detection of zearalenone, such as qualitative information, quantitative information, estimate, probability, or any other type of information, suitable for detecting or characterizing the effects of zearalenone. Methods for constructing, training and using a neural network are well known to one skilled in the art. Other (computerized) approaches may also be suitable for use in the present invention and are also known to one skilled in the art, such as, for example, Bayesian networks or decision trees.

[0025] Generally, the result of comparison step (d) may be any type of information indicating whether the sample came into contact with ZEN (or an analogue or derivative of ZEN) or whether the subject from whom the sample was obtained was exposed to ZEN (or analogue or derivative of ZEN). The information may be qualitative information, quantitative information, estimate, probability, or any other type of useful information. The method of the present invention may thus include the additional step(s) of providing information regarding the effects of zearalenone. The information may be provided by any suitable means known to one of ordinary skill in the art.

[0026] As used herein, “subject” refers to a vertebrate, preferably a mammal. As used herein, the term "mammal" refers to any animal classified as a mammal, including, but not limited to, humans, domestic, farm and zoo animals, sporting animals or pets such as sheep, dogs, horses, cats, cows, rats, pigs, primates such as cynomolgus monkeys, to name just a few illustrative examples. The preferred subject is of the genus Sus, preferably the species Sus scrofa. Another preferred subject is a human.

[0027] The effect of ZEN, in particular the effects of ZEN, manifests itself in the form of adverse health effects such as reproductive and fertility disorders, especially in mammals and particularly significant in Sus subjects. To provide the most rapid detection of ZEN exposure in these subjects, the present invention also provides the method described above, where the subject is preferably a mammal, more preferably from the genus Sus.

[0028] The methods of the present invention can be used for diagnosis, in particular, diagnosis of exposure to zearalenone in a subject. Such methods may be designed to determine the health status of a subject in order to decide whether measures need to be taken to maintain or improve the subject's health.

[0029] The methods of the present invention may also be non-diagnostic. As an illustrative example, the non-diagnostic method may be a feed sampling method. Feed sampling is usually not very accurate due to the heterogeneous distribution of mycotoxins in the feed and the typical sample sizes used for this analysis. Due to the ingestion of a relatively large amount of feed by a subject, such as a pig, determining the subject's exposure to ZEN may provide a more reliable test result for feed contamination with zearalenone than testing the feed itself. Thus, the methods of the present invention may be methods for detecting zearalenone in feed or food. The methods of the present invention may include, prior to obtaining a test sample from a subject, the step of (a) feeding the subject a food or food suspected of containing zearalenone.

[0030] To avoid the undesirable effects of long-term exposure to zearalenone, it may be necessary to treat ZEN-contaminated feed or food with a ZEN neutralizing agent or employ a ZEN neutralizing method if exposure to zearalenone is detected in a subject. Therefore, the present invention further provides the method described above, characterized in that the method is for selecting food or feed to be brought into contact with a zearalenone neutralizing agent or for applying a zearalenone neutralizing method. By applying the described method of selecting a food or feed to be brought into contact with a ZEN neutralizing agent or using a ZEN neutralizing method, the adverse health effects can be at least mitigated or completely avoided. The methods of the present invention may thus include the step of (a) feeding a subject a food believed to contain zearalenone prior to obtaining a test sample from the subject. The methods of the present invention may also include the step (f) of selecting a food to contact with a zearalenone neutralizing agent or employing a zearalenone neutralizing method if a deviation in the expression levels of at least one microRNA between the test sample and the control sample indicates exposure to zearalenone. The methods of the present invention may thus include providing a zearalenone neutralizing agent or using a method for neutralizing zearalenone. The methods of the present invention may also include contacting the feed or food selected by the method with a zearalenone neutralizing agent or applying a zearalenone neutralizing method to the feed.

[0031] A zearalenone neutralizing agent or method may refer to an agent or method that degrades zearalenone, for example, by changing its molecular structure, which may be accomplished by chemical, enzymatic, or physical interaction with a zearalenone neutralizing agent. This treatment may result in a less toxic molecule. The zearalenone neutralizing agent or method may also reduce the bioavailability of zearalenone, such that zearalenone becomes unavailable to the subject, preferably to the subject's digestive system. This can be achieved, for example, by using an agent that binds or adsorbs zearalenone. In general, the zearalenone neutralizing agent may be any suitable type of agent, which may be selected from the group consisting of one or more polypeptides, one or more microorganisms, and one or more zearalenone binding agents.

[0032] The zearalenone neutralizing agent may also be an α/β-hydrolase capable of transforming zearalenone or derivatives or analogs thereof. Various α/β-hydrolases are known to those skilled in the art, which are described, among others, by Lenfant et al. (2013) 'ESTHER, the database of the α/β-hydrolase fold superfamily of proteins: tools to explore diversity of functions' Nucleic Acids Research, Volume 41, Issue D1, D423-D429 and Mindrebo et al. (2016) 'Unveiling the functional diversity of the Alpha-Beta hydrolase fold in plants' Curr Opin Struct Biol. 233-246. Briefly, all α/β-hydrolases have a specific fold property called the α/β-fold (alpha/beta fold), the α/β-fold being common to a number of hydrolytic enzymes of very different phylogenetic origins and catalytic functions. The core of each enzyme is an α/β structure (rather than a cylinder) containing 8 β-strands (b1-b8) connected by α-helices (aA-aF) (Ollis et al. (1992) 'The alpha/beta hydrolase fold' Protein Eng. 5(3): 197-211). Therefore, the α/β-hydrolase described herein may contain an α/β fold. The α/β-hydrolase described herein preferably includes a core α/β-hydrolase domain consisting of 8 β-strands (b1-b8) located on a central β-sheet and additionally includes 6 intersecting α-helices (aA-aF ).

[0033] Members of the various classes of α/β-hydrolases, as well as their structural characteristics, are described, among others, in Kourist et al. (2010) 'The alpha/beta-hydrolase fold 3DM database (ABHDB) as a tool for protein engineering.' Chembiochem. 11(12): 1635-43).

[0034] α/β-hydrolases are often described as enzymes responsible for the hydrolysis of ester and peptide bonds. However, α/β-hydrolases are also involved in the cleavage of carbon-carbon bonds, decarboxylation reactions, and cofactor-independent dioxygenation of heteroaromatic rings. Thus, α/β-hydrolases may include catalytic members (enzymes) in this superfamily. Non-limiting examples are hydrolases (acetylcholinesterase, carboxylesterase, dienelactone hydrolase, lipase, cutinase, thioesterase, serine carboxypeptidase, proline iminopeptidase, prolin oligopeptidase, epoxide hydrolase) together with enzymes that require activation of _the reaction by HCN, _H2O2 or _O2 instead of _H2O for the reaction mechanism ( haloalkane dehalogenase, haloperoxidase, hydroxynitrile lyase). Non-catalytic members may include neuroligins, glutactin, neurotactin, thyroglobulin C-terminal domain, yolk proteins, CCG1-interacting factor-B, and dipeptidyl aminopeptidase VI.

[0035] The ESTHER database collects and annotates published information related to the sequences of genes and proteins of a given superfamily. Thus, one skilled in the art can also obtain α/β-hydrolases from ESTHER (http://bioweb.supagro.inra.fr/ESTHER/general?what=index), a database of the folded α/β-hydrolase protein superfamily .

[0036] As used herein, an α/β-hydrolase may contain the sequence of SEQ ID NO: 45 or a sequence that has 60% or more identity with the sequence of SEQ ID NO: 45. Thus, any α/β-hydrolase containing this sequence , is covered by the term α/β-hydrolase.

[0037] Additionally or alternatively, the α/β-hydrolase of the present invention may also contain the sequence of SEQ ID NO: 46 or a sequence that has 60% or more identity with the sequence of SEQ ID NO: 46. Thus, any α/ A β-hydrolase containing this sequence is covered by the term α/β-hydrolase.

[0038] Additionally or alternatively, the α/β-hydrolase of the present invention may also contain the sequence of SEQ ID NO: 47, 48 or 49 or a sequence that has 60% or more identity with the sequence of SEQ ID NO: 47, 48 or 49. Thus, any α/β-hydrolase containing this sequence is encompassed by the term α/β-hydrolase.

[0039] Additionally or alternatively, the α/β-hydrolase of the present invention may also contain the sequence of SEQ ID NO: 50 or a sequence that has 60% or more identity with the sequence of SEQ ID NO: 50. Thus, any α/ A β-hydrolase containing this sequence is covered by the term α/β-hydrolase.

[0040] For example, an α/β hydrolase may contain a sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% sequence identity to SEQ ID NO: 45. Additionally or alternatively, the α/β-hydrolase may contain a sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95 %, 97%, 98%, 99% identity to SEQ ID NO: 46. Additionally or alternatively, the α/β-hydrolase may contain a sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% identity to SEQ ID NO: 47, 48 and/or 49. Additionally or alternatively, the α/β hydrolase may contain the sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% identity with the sequence of SEQ ID NO: 50.

[0042] The term “identical” or “percentage identity” in the context of two or more polypeptide sequences, such as SEQ ID NO: 45-50, refers to two or more sequences or subsequences that are the same or that have a specified percentage of the same amino acids ( e.g., are at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identical when compared and aligned for maximum matching in the comparison window or designated area, as determined using a sequence comparison algorithm, known in the art, or by manual alignment and visual inspection. Sequences having, for example, 80% to 95% or more sequence identity are considered substantially identical. Those skilled in the art will know how to determine the percent identity between sequences using, for example, algorithms such as those based on the computer program CLUSTALW (Thompson Nucl. Acids Res. 2 (1994), 4673-4680) or FASTDB (Brutlag et al. App. Biosci. 6 (1990), 237-245), as is known in the art.

[0043] Also available to those skilled in the art are the BLAST and BLAST 2.6 algorithms (Altschul Nucl. Acids Res. 25 (1977), 3389-3402). The BLASTP program for amino acid sequences uses a default segment length (W) of 6, an expected threshold of 10, and a comparison of both strands. In addition, the BLOSUM62 scoring matrix can be used (Henikoff Proc. Natl. Acad. Sci., USA, 89, (1989), 10915; Henikoff and Henikoff (1992) 'Amino acid substitution matrices from protein blocks.' Proc Natl Acad Sci USA 1992 Nov 15;89(22):10915-9).

[0044] For example, BLAST2.6, which stands for Basic Local Alignment Search Tool (Altschul, Nucl. Acids Res. 25 (1997), 3389-3402; Altschul, J. Mol. Evol. 36 (1993), 290-300; Altschul, J Mol Biol 215 (1990), 403-410) can be used to search for local sequence alignments.

[0045] The zearalenone neutralizing agent may also be ozone, as, for example, described in Qi L et al. Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 2016 Nov;33(11):1700-1710. The zearalenone neutralizing agent may also be a microorganism, such as, for example, Clonostachys rosea, as described in Kosawang C et al., Fungal Biol. 2014 Apr; 118(4):364-73, or Lactobacillus pentosus, as described in Arunrussamee Sangsila et al., Food Control, Volume 62, April 2016, Pages 187-192, or a yeast strain such as Trichosporon mycotoxinivorans MTV, as described in Schatzmayr et al. Mycot. Res. 2003 19: 124-128. Additional zearalenone neutralizing agents are, for example, described in Zinedine A et al., Food Chem Toxicol. 2007 Jan;45(l):1-18 or Ji C et al., Animal Nutrition Volume 2, Issue 3, 2016, Pages 127-133.

[0046] As described above, a zearalenone neutralizing agent can also reduce the bioavailability of zearalenone, for example, by binding and/or adsorption. Examples of such agents may include cholestyramine, divinylbenzene styrene, bentonites, montmorillonite-rich clays, zeolites, organoclays, activated carbon, yeast or products derived from yeast cell walls.

[0047] Methods of neutralizing zearalenone may include, but are not limited to, methods of contacting a zearalenone neutralizing agent with a feed. The methods may also include heat treatment or ultrasonic treatment.

[0048] Evaluating the productivity and/or effectiveness of mycotoxin neutralizing agents or mycotoxin neutralizing methods poses a significant challenge due to the lack of suitable methods for determining whether the mycotoxin neutralizing agent or method used achieves the desired effect. Thus, the methods of the present invention can also serve to evaluate the ability of a method or (test) compound to neutralize ZEN. In other words, the method of the present invention can be used to evaluate the ability of a method or compound to neutralize ZEN. Such a compound may be selected from the group consisting of one or more polypeptides, one or more microorganisms, including live, dead, lyophilized, autolyzed and/or dormant microorganisms, and one or more zearalenone binding agents, including organic and/or inorganic zearalenone -binding agent(s). Using the methods of the present invention, an assessment of the productivity and/or effectiveness of a ZEN neutralizing agent or method can be obtained in a particularly fast and efficient manner.

[0049] In cases where the sample was obtained from a subject, the method of the present invention may thus include, prior to obtaining the test sample from the subject, step (a3) of feeding the subject a food that has come into contact with zearalenone and the test compound. In such a case, the method may further include the steps of contacting the food with zearalenone, contacting the food with a test compound, and/or contacting the zearalenone with a test compound. Typically, such steps can be carried out in any order. Accordingly, the method before step (a3) may include the steps of: (a1) contacting the food with zearalenone, and (a2) bringing the food that has come into contact with the zearalenone into contact with the test compound; or (a1') contacting the food with the test compound, and (a2') contacting the food that has come into contact with the test compound with zearalenone; or (a1'') contacting the zearalenone with the test compound, and (a2'') contacting the zearalenone contacted with the test compound in contact with the feed.

[0050] In some cases, which may represent the case where the test sample is a cell culture, the sample could directly come into contact with zearalenone and the test compound. The control sample used in such a method may be a negative control sample or a positive control sample. A negative control sample used in this context refers to a sample that has come into contact with zearalenone and optionally a compound that does not neutralize zearalenone. A positive control sample used in this context refers to a sample that came into contact with zearalenone and a compound that neutralizes zearalenone, or a sample that did not come into contact with zearalenone. In such a method, the absence of deviation or slight deviation in expression levels between the test sample and the positive control sample may indicate the ability of the test compound to neutralize zearalenone. Alternatively or additionally, the presence of a (preferably significant) deviation in expression levels between the test sample and the negative control sample may indicate the ability of the test compound to neutralize zearalenone. In such methods, the sample may be a sample that was not obtained from the subject.

[0051] To avoid time-consuming, time-consuming and costly animal testing, research experiments are preferably performed on cultured cells. Therefore, the test sample, positive control sample and/or negative control sample may be a cell culture. As an illustrative example, the cell culture may contain MCF-7, T47D, SUM159, HeLa or Ishikawa. In addition, primary cells, such as, for example, porcine granulosa cells, can be maintained in cell culture. By providing the method described herein, wherein the test sample, control sample, such as a positive control sample or negative control sample, is a cell culture, valuable data can be obtained, for example, about the ability of the method or compound to neutralize ZEN, in a particularly effective manner.

[0052] The present invention also relates to the use of at least one microRNA according to the present invention to detect the effect of zearalenone in a test sample. The at least one miRNA may be selected from the group consisting of ssc-miR-1, ssc-miR-125a, ssc-miR-125b, ssc-miR-127, ssc-miR-129a, ssc-miR-133a-5p , ssc-miR-135, ssc-miR-136, ssc-miR-140-3р, ssc-miR-142-3р, ssc-miR-143-5p, ssc-miR-146b, ssc-miR-181c, ssc -miR-182, ssc-miR-183, ssc-miR-187, ssc-miR-195, ssc-miR-204, ssc-miR-206, ssc-miR-22-3р, ssc-miR-22-5p , ssc-miR-335, ssc-miR-34a, ssc-miR-369, ssc-miR-378, ssc-miR-424-5p, ssc-miR-432-5p, ssc-miR-450a, ssc-miR -450b-5p, ssc-miR-450c-5p, ssc-miR-455-3р, ssc-miR-455-5p, ssc-miR-486, ssc-miR-493-3р, ssc-miR-493-5p , ssc-miR-497, ssc-miR-503, ssc-miR-542-3р, ssc-miR-708-3р, ssc-miR-758, ssc-miR-7135-3р, hsa-miR-135b-5p , hsa-miR-301a-5p, put-miR-300. Preferably, at least one microRNA according to the present invention may be selected from the group consisting of ssc-miR-1, ssc-miR-181c, ssc-miR-206, ssc-miR-503, ssc-miR-542-3p, ssc-miR-135, ssc-miR-129a-3p, ssc-miR-142-3р, ssc-miR-432-5p, ssc-miR-455-5p, ssc-miR-182, ssc-miR-493- 3р, ssc-miR-455-3р, ssc-miR-183, ssc-miR-140-3р, ssc-miR-7135-3р, ssc-miR-204, ssc-miR-143-5p, ssc-miR- 187, ssc-miR-335, ssc-miR-424-5p, ssc-miR-450a, ssc-miR-450b-5p, ssc-miR-450c-5p, ssc-miR-497. Application can be carried out in any method according to the present invention. Such use may be for the purpose of detecting a subject's exposure to zearalenone, assessing the ability of a test compound or method to neutralize zearalenone, or selecting a food to be treated with a zearalenone neutralizing agent or method. By using at least one microRNA, as described herein, to detect the effect of zearalenone in a test sample, the effect of ZEN can be detected with particularly high reliability and specificity.

[0053] To ensure a rapid response, such as when applying a counteracting agent in the event of exposure to ZEN, rapid availability of test results is necessary. This can be achieved, for example, if determining the expression level of at least one microRNA in a test sample and comparing the expression level with a reference value is carried out using an integrated system. Thus, the methods of the present invention may be computer-assisted methods that can be implemented by an apparatus for performing such a method.

[0054] Thus, the present invention also relates to a data processing system comprising a processor configured to implement a method including the steps of comparing the expression level of at least one microRNA in accordance with the invention determined in a test sample obtained from a subject with a reference meaning. The expression level of at least one miRNA must be entered. Information may be transmitted to a data processing system through a device that is capable of detecting such information, for example, through some transmission medium such as an electrical wire or cable, through a fiber optic cable, or through electromagnetic radiation. Such a device may be integrated into or connected to a data processing system. Information regarding the expression level of at least one miRNA may also be provided to the data processing system via input via a user interface. If the reference value corresponds to the expression level of the specified miRNA in a control sample that was obtained from a subject not exposed to zearalenone, a (significant) deviation in miRNA expression levels between the test sample and the reference value may indicate an effect of zearalenone (such as exposure to zearalenone). In such a case, the method may include detecting the subject's exposure to zearalenone if an abnormality is determined. Alternatively, if the reference value corresponds to the expression level that was determined to correspond to a control sample that was obtained from a subject exposed to zearalenone, the absence of deviation between the values of the test sample and the reference value may not indicate an effect of zearalenone, such as exposure to zearalenone. In such a case, the method may include detecting the subject's exposure to zearalenone if no abnormality or significant abnormality is detected. "No deviation" in this context may also include minor deviation or (atypically) low deviation. The at least one microRNA may be selected from the group consisting of ssc-miR-1, ssc-miR-125a, ssc-miR-125b, ssc-miR-127, ssc-miR-129a, ssc-miR-133a-5p , ssc-miR-135, ssc-miR-136, ssc-miR-140-3р, ssc-miR-142-3р, ssc-miR-143-5p, ssc-miR-146b, ssc-miR-181c, ssc -miR-182, ssc-miR-183, ssc-miR-187, ssc-miR-195, ssc-miR-204, ssc-miR-206, ssc-miR-22-3р, ssc-miR-22-5p , ssc-miR-335, ssc-miR-34a, ssc-miR-369, ssc-miR-378, ssc-miR-424-5p, ssc-miR-432-5p, ssc-miR-450a, ssc-miR -450b-5p, ssc-miR-450c-5p, ssc-miR-455-3р, ssc-miR-455-5p, ssc-miR-486, ssc-miR-493-3р, ssc-miR-493-5p , ssc-miR-497, ssc-miR-503, ssc-miR-542-3р, ssc-miR-708-3р, ssc-miR-758, ssc-miR-7135-3р, hsa-miR-135b-5p , hsa-miR-301a-5p, put-miR-300. Preferably, at least one microRNA according to the present invention may be selected from the group consisting of ssc-miR-1, ssc-miR-181c, ssc-miR-206, ssc-miR-503, ssc-miR-542-3p, ssc-miR-135, ssc-miR-129a-3p, ssc-miR-142-3р, ssc-miR-432-5p, ssc-miR-455-5p, ssc-miR-182, ssc-miR-493- 3р, ssc-miR-455-3p, ssc-miR-183, ssc-miR-140-3р, ssc-miR-7135-3р, ssc-miR-204, ssc-miR-143-5p, ssc-miR- 187, ssc-miR-335, ssc-miR-424-5p, ssc-miR-450a, ssc-miR-450b-5p, ssc-miR-450c-5p, ssc-miR-497.

[0055] Ideally, the data processing system is coupled or integrated into a device that is capable of determining the expression level of at least one microRNA according to the present invention in a test sample. The device may provide the data processing system with information regarding the expression level of the at least one microRNA. Such a device may, for example, be a sequencing device, such as a quantitative real-time sequencing device. Sequencing devices are known to one skilled in the art and are, for example, commercially available from Illumina, Pacific Biosciences, Oxford Nanopore Technologies, etc. Particularly suitable are portable sequencing devices such as MinION sequencing (Oxford Nanopore Technologies). A device capable of detecting the expression level of at least one microRNA in accordance with the present invention may also be a microarray reader.

[0056] Accordingly, the present invention also relates to a sequencing device capable of determining the level of at least one microRNA according to the present invention, comprising a data processing system according to the present invention. The at least one microRNA may be selected from the group consisting of ssc-miR-1, ssc-miR-125a, ssc-miR-125b, ssc-miR-127, ssc-miR-129a, ssc-miR-133a-5p , ssc-miR-135, ssc-miR-136, ssc-miR-140-3р, ssc-miR-142-3р, ssc-miR-143-5p, ssc-miR-146b, ssc-miR-181c, ssc -miR-182, ssc-miR-183, ssc-miR-187, ssc-miR-195, ssc-miR-204, ssc-miR-206, ssc-miR-22-3р, ssc-miR-22-5p , ssc-miR-335, ssc-miR-34a, ssc-miR-369, ssc-miR-378, ssc-miR-424-5p, ssc-miR-432-5p, ssc-miR-450a, ssc-miR -450b-5p, ssc-miR-450c-5p, ssc-miR-455-3р, ssc-miR-455-5p, ssc-miR-486, ssc-miR-493-3р, ssc-miR-493-5p , ssc-miR-497, ssc-miR-503, ssc-miR-542-3р, ssc-miR-708-3р, ssc-miR-758, ssc-miR-7135-3р, hsa-miR-135b-5p , hsa-miR-301a-5p, put-miR-300. Preferably, at least one microRNA according to the present invention may be selected from the group consisting of ssc-miR-1, ssc-miR-181c, ssc-miR-206, ssc-miR-503, ssc-miR-542-3p, ssc-miR-135, ssc-miR-129a-3p, ssc-miR-142-3р, ssc-miR-432-5p, ssc-miR-455-5p, ssc-miR-182, ssc-miR-493- 3р, ssc-miR-455-3р, ssc-miR-183, ssc-miR-140-3р, ssc-miR-7135-3р, ssc-miR-204, ssc-miR-143-5p, ssc-miR- 187, ssc-miR-335, ssc-miR-424-5p, ssc-miR-450a, ssc-miR-450b-5p, ssc-miR-450c-5p, ssc-miR-497.

[0057] The present invention also provides a computer program containing instructions for causing a data processing system described herein or a sequencing device described herein to perform the steps of (a) comparing the expression level of at least one microRNA in accordance with the invention , determined in a test sample obtained from a subject, with a reference value; and (b) identifying the subject's exposure to zearalenone if an abnormality is determined, wherein the reference value corresponds to the expression level that was determined in a control sample obtained from a subject who was not exposed to zearalenone ; or identifying the subject's exposure to zearalenone if no abnormality is determined, wherein the reference value corresponds to the expression level that was determined in a control sample obtained from a subject who was exposed to zearalenone. When a computer program is downloaded and executed by a data processing system, the data processing system becomes a device for implementing the methods described in this application.

[0058] A computer program may be a computer program product accessible from a computer usable or computer readable medium that provides program code for use by or in connection with a computer or any instruction execution system. The present invention, therefore, also relates to a computer-readable medium storing on it a computer program described in this application. A computer-usable or machine-readable medium can be any device that can contain, store, transmit, distribute, or move a program for use by or in connection with a system, device, or instruction execution apparatus. The medium may be an electronic, magnetic, optical, electromagnetic, infrared, semiconductor, or paper system (or apparatus or device) or propagation medium. The medium may be any suitable tangible medium, such as a floppy disk, CD, DVD, BD, hard disk, memory stick, memory stick, or any other computer-readable storage medium.

[0059] To achieve particularly cost-effective and rapid detection of ZEN exposure, a kit is provided for implementing a method for detecting ZEN exposure in the sense of the present invention, containing a specific binding agent for at least one microRNA according to the present invention contained on or conjugated to a solid support. The at least one microRNA may be selected from the group consisting of ssc-miR-1, ssc-miR-125a, ssc-miR-125b, ssc-miR-127, ssc-miR-129a, ssc-miR-133a-5p , ssc-miR-135, ssc-miR-136, ssc-miR-140-3р, ssc-miR-142-3р, ssc-miR-143-5p, ssc-miR-146b, ssc-miR-181c, ssc -miR-182, ssc-miR-183, ssc-miR-187, ssc-miR-195, ssc-miR-204, ssc-miR-206, ssc-miR-22-3р, ssc-miR-22-5p , ssc-miR-335, ssc-miR-34a, ssc-miR-369, ssc-miR-378, ssc-miR-424-5p, ssc-miR-432-5p, ssc-miR-450a, ssc-miR -450b-5p, ssc-miR-450c-5p, ssc-miR-455-3р, ssc-miR-455-5p, ssc-miR-486, ssc-miR-493-3р, ssc-miR-493-5p , ssc-miR-497, ssc-miR-503, ssc-miR-542-3р, ssc-miR-708-3р, ssc-miR-758, ssc-miR-7135-3р, hsa-miR-135b-5p , hsa-miR-301a-5p, put-miR-300. Preferably, at least one microRNA according to the present invention may be selected from the group consisting of ssc-miR-1, ssc-miR-181c, ssc-miR-206, ssc-miR-503, ssc-miR-542-3p, ssc-miR-135, ssc-miR-129a-3p, ssc-miR-142-3р, ssc-miR-432-5p, ssc-miR-455-5p, ssc-miR-182, ssc-miR-493- 3р, ssc-miR-455-3р, ssc-miR-183, ssc-miR-140-3р, ssc-miR-7135-3р, ssc-miR-204, ssc-miR-143-5p, ssc-miR- 187, ssc-miR-335, ssc-miR-424-5p, ssc-miR-450a, ssc-miR-450b-5p, ssc-miR-450c-5p, ssc-miR-497. The kit may contain an unlabeled specific binding partner and a labeled specific partner of at least one miRNA. Preferably, the unlabeled specific binding partner is conjugated to a solid support. A set may contain specific binding partners for no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 microRNAs. In some applications, the kit may contain specific binding partners of no more than 24, 48, 96 or 384 miRNAs. A set may contain specific binding partners for no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 microRNAs according to the present invention. The kit may only contain specific binding partners for miRNAs in accordance with the methods of the present invention.

[0060] Such a kit may be designed for a lateral flow test. A schematic illustration of a side flow device is shown in FIG. 8. The kit may contain a lateral flow device (LFD) that contains a support (solid support) that has at least two zones, a conjugate pad and a test zone. The conjugate pad contains a labeled binding agent for at least one miRNA according to the present invention, which can be mobilized in a solvent (eg, a solvent that contains the target molecule) and transported therein to the test site. The test area contains an unlabeled binding agent that is also capable of binding the target molecule. Thus, the targets (at least one microRNA), which are also bound by the labeled agent from the conjugate cushion, are immobilized in the test area. The optional additional control zone may contain another immobilized coupling agent capable of binding labeled coupling agent from the conjugate cushion that is not retained in the test zone. Examples of lateral flow devices are described in Koczula and Gallotta. Essays Biochem. 2016 Jun 30;60(1):111-20 or Krska and Molinelli. Anal Bioanal Chem. 2009 Jan; 393(l):67-71.

[0061] In addition, immunochemical detection can be performed using an ELISA, such as a sandwich ELISA. Thus, the kit may contain a (preferably unlabeled) specific binding agent for the target microRNA, which may be immobilized on a solid support or immobilized on a solid support. The test sample can be added to the substrate. In addition, a labeled binding agent for the target miRNA can be added. The tag associated with the miRNA can then be used to quantify the miRNA.

[0062] The specific binding agent may be, for example, a nucleic acid, an antibody, an antigen-binding fragment of an antibody, or a protein-like molecule having antibody-like properties. In cases where both a labeled and an unlabeled specific binding partner are used in the kit, it is preferable that both binding partners can simultaneously bind to the target microRNA. This can be achieved, for example, if one specific binding partner can bind (eg, hybridize) to one part of the target miRNA, while another specific binding partner can bind (eg, hybridize) to another part of the target miRNA.

[0063] The kit may further comprise a microRNA detection agent that is coupled to a specific binding agent, such as an unlabeled specific binding agent described herein, which is preferably conjugated to a solid support. Such an agent may contain a probe conjugated to a detectable label. For example, such an agent may be a labeled specific binding agent, as described herein. Typically, such a "detectable label" may be any suitable chemical substance or enzyme that directly or indirectly generates a detectable compound or signal in a chemical, physical, optical or enzymatic reaction. For example, a fluorescent or radioactive label may be used to generate fluorescence or x-ray emission as a detectable signal. The dye can be used to generate an optical signal. Alkaline phosphatase, horseradish peroxidase and β-galactosidase are examples of enzyme tags (and at the same time optical tags) that catalyze the formation of chromogenic reaction products. Preferably, the label does not adversely affect the characteristics of the binding partner to which the label is conjugated. A preferred detectable label is an optically detectable label such as a chromophore or a fluorescent label.

[0064] The present invention is further characterized by the following points:

[0065] Clause 1. A method for detecting the effects of zearalenone, comprising: (c) determining in a test sample the expression level of at least one microRNA selected from the group consisting of ssc-miR-1, ssc-miR-181c, ssc-miR- 206, ssc-miR-503, ssc-miR-542-3р, ssc-miR-135, ssc-miR-129a-3p, ssc-miR-142-3р, ssc-miR-432-5p, ssc-miR- 455-5p, ssc-miR-182, ssc-miR-493-3р, ssc-miR-455-3р, ssc-miR-183, ssc-miR-140-3р, ssc-miR-7135-3р, ssc- miR-204, ssc-miR-143-5p, ssc-miR-187, ssc-miR-335, ssc-miR-424-5p, ssc-miR-450a, ssc-miR-450b-5p, ssc-miR- 450c-5p, ssc-miR-497; and (d) comparison of the expression level with a reference value.

[0066] Clause 2. The method according to claim 1, characterized in that the reference value corresponds to the expression level of said at least one microRNA in the control sample.

[0067] Claim 3. The method according to claim 1 or 2, characterized in that the effect of zearalenone is the effect of zearalenone.

[0068] Claim 4. The method of any of the preceding claims, characterized in that the test sample is obtained from a subject.

[0069] Claim 5. The method of any of the preceding claims, characterized in that a deviation of the expression level from the reference value indicates exposure to zearalenone.

[0070] Claim 6. The method according to any of the preceding claims, characterized in that the deviation includes an increase and/or decrease in the expression level of at least one of the microRNAs of claim 1.

[0071] Claim 7. The method according to any of the preceding claims, characterized in that the deviation includes an increase and/or decrease in at least one ratio of the expression levels of at least two of the microRNAs of claim 1.

[0072] Clause 8. The method of any of the preceding clauses, characterized in that the deviation is statistically significant.

[0073] Claim 9. The method of any of the preceding claims, wherein the control sample was obtained from a subject who was not exposed to zearalenone, or is the average of several samples that were obtained from one or more subjects who were not exposed. effects of zearalenone.

[0074] Clause 10. The method of any of the preceding claims, wherein the sample is a blood sample or a tissue sample.

[0075] Clause 11. The method according to any of the preceding claims, characterized in that the sample is a blood serum sample.

[0076] Clause 12. The method according to any of the preceding claims, characterized in that at least one microRNA is selected from the group consisting of ssc-miR-1, ssc-miR-181c, ssc-miR-206, ssc-miR- 503, ssc-miR-542-3р, ssc-miR-135, ssc-miR-129a-3p, ssc-miR-142-3р, ssc-miR-432-5p, ssc-miR-455-5p, ssc- miR-182, ssc-miR-493-3р, ssc-miR-455-3р, ssc-miR-183 and ssc-miR-140-3р.

[0077] Clause 13. The method according to claim 11 or 12, characterized in that at least one microRNA includes two microRNAs selected from the group consisting of ssc-miR-542-3p and ssc-miR-1, ssc-miR -542-3р and ssc-miR-493-3р, ssc-miR-135 and ssc-miR-432-5p, and ssc-miR-455-5p and ssc-miR-493-3р.

[0078] Clause 14. The method of any one of clauses 11-13, wherein the expression level of at least one miRNA includes at least one expression level ratio between two microRNAs selected from the group consisting of ssc-miR-542- 3p and ssc-miR-1, ssc-miR-542-3p and ssc-miR-493-3p, ssc-miR-135 and ssc-miR-432-5p, and ssc-miR-455-5p and ssc-miR -493-3r.

[0079] Clause 15. The method of claim 14, wherein at least one ratio of expression levels between two microRNAs is selected from the group consisting of a ratio between ssc-miR-542-3p and ssc-miR-1, ssc- miR-542-3р and ssc-miR-493-3р, ssc-miR-135 and ssc-miR-432-5p, and ssc-miR-455-5p and ssc-miR-493-3р.

[0080] Item 16. The method of any one of items 1-10, wherein the sample is a sample of uterine tissue.

[0081] Clause 17. The method according to any of the preceding claims, characterized in that at least one microRNA is selected from the group consisting of ssc-miR-1, ssc-miR-181c, ssc-miR-206, ssc-miR- 503, ssc-miR-542-3р, ssc-miR-135, ssc-miR-7135-3р, ssc-miR-204, ssc-miR-143-5p, ssc-miR-187, ssc-miR-335, ssc-miR-424-5p, ssc-miR-450a, ssc-miR-450b-5p, ssc-miR-450c-5p and ssc-miR-497.

[0082] Claim 18. The method according to claim 16 or 17, characterized in that at least one microRNA contains miR-542-3p and miR-1.

[0083] Clause 19. The method of claim 18, wherein the expression level of at least one miRNA includes a ratio of expression levels between miR-542-3p and miR-1.

[0084] Clause 20. The method according to any one of claims. 4-19, characterized in that the subject is a mammal.

[0085] Clause 21. The method according to any one of claims. 4-20, characterized in that the subject is a subject of the genus Sus.

[0086] Item 22. The method according to item 21, characterized in that the subject belongs to the species Sus scrofa.

[0087] Clause 23. The method of any of the preceding claims, further comprising (e) providing information regarding the effects of zearalenone.

[0088] Clause 24. The method of any of the preceding claims, further comprising (b) providing a test sample.

[0089] Clause 25. The method according to any of the preceding claims, characterized in that the method is intended for diagnosis.

[0090] Clause 26. The method of any of the preceding claims, wherein the method is for diagnosing exposure to zearalenone.

[0091] Clause 27. The method according to any one of claims. 1-24, characterized in that the method is not intended for diagnostics.

[0092] Item 28. The method according to any of items 4-24 and 27, characterized in that the method is designed to detect zearalenone in feed or food.

[0093] Clause 29. The method of claim 28, wherein the method comprises, before obtaining a test sample from the subject, (a) feeding the subject a food purported to contain zearalenone.

[0094] Item 30. The method of any one of items 4-24 and 27-29, wherein the method is for selecting a feed to be brought into contact with a zearalenone neutralizing agent or using a zearalenone neutralizing method.

[0095] Clause 31. The method of claim 29 or 30, comprising (a) feeding a subject a food believed to contain zearalenone until a test sample is obtained from the subject; and/or (f) selecting a feed to contact with a zearalenone neutralizing agent or using a zearalenone neutralizing method if a deviation in the expression levels of at least one microRNA between the test sample and the control sample indicates exposure to zearalenone.

[0096] Clause 32. The method of claim 30 or 31, wherein the zearalenone neutralizing agent is selected from the group consisting of one or more polypeptides, one or more microorganisms, and one or more zearalenone-binding agents.

[0097] Clause 33. The method of any one of clauses 30-32, further comprising providing a zearalenone neutralizing agent.

[0098] Clause 34. The method of any one of clauses 30-33, further comprising contacting the feed or food selected using the method with a zearalenone neutralizing agent or applying the zearalenone neutralizing method to the feed.

[0099] Item 35. The method of any one of items 4-22 and 25-27, wherein the method is designed to evaluate the ability of the method or test compound to neutralize zearalenone.

[0100] Clause 36. The method of claim 35, wherein the test compound is selected from the group consisting of one or more polypeptides, one or more microorganisms, and one or more zearalenone-binding agents.

[0101] Clause 37. The method of claim 35 or 36, wherein the method comprises, before obtaining a test sample from a subject, (a3) feeding the subject a food that has come into contact with zearalenone and the test compound.

[0102] Item 38. The method of item 37, wherein the method before (a3) includes the steps of: (a1) bringing the feed into contact with zearalenone, and (a2) bringing the feed that has come into contact with the zearalenone into contact with test connection; or (a1') contacting the food with the test compound, and (a2') contacting the food that has come into contact with the test compound with zearalenone; or (a1'') contacting the zearalenone with the test compound, and (a2'') contacting the zearalenone contacted with the test compound in contact with the feed.

[0103] Item 39. The method of any one of items 1-3, wherein the method is designed to evaluate the ability of the method or test compound to neutralize zearalenone.

[0104] Clause 40. The method of claim 39, wherein the test compound is selected from the group consisting of one or more polypeptides, one or more microorganisms, and one or more zearalenone binding agents.

[0105] Clause 41. The method of claim 39 or 40, wherein the test sample comes into contact with zearalenone and the test compound.

[0106] Clause 42. The method of any one of clauses 39-41, wherein the control sample is a negative control sample or a positive control sample.

[0107] Clause 43. The method of claim 42, wherein the absence of a deviation in expression levels between the test sample and the positive control sample or the presence of a deviation in expression levels between the test sample and the negative control sample indicates the ability of the test compound to neutralize zearalenone.

[0108] Clause 44. The method of any one of clauses 40-43, wherein the test sample, control sample, positive control sample, or negative control sample is a cell culture.

[0109] Clause 45. The method according to claims. 30-44, characterized in that neutralization is carried out by degradation.

[0110] Clause 46. The method according to claims. 30-44, characterized in that neutralization is carried out by binding.

[0111] Clause 47. Use of at least one microRNA selected from the group consisting of miR-1, miR-181c, miR-206, miR-503, miR-542-3p, miR-135, miR-135a-5p , miR-129a-3p, miR-142-3p, miR-432-5p, miR-455-5p, miR-182, miR-493-3p, miR-455-3p, miR-183, miR-140-3p , miR-7135-3p, miR-204, miR-143-5p, miR-187, miR-335, miR-424-5p, miR-450a, miR-450b-5p, miR-450c-5p, miR-497 , to detect the effects of zearalenone in the test sample.

[0112] Clause 48. The use of claim 47, wherein said use is to detect the effects of zearalenone on a subject.

[0113] Clause 49. A data processing system comprising a processor configured to implement a method comprising the steps of (a) comparing the expression level of at least one microRNA selected from the group consisting of miR-1, miR-181c, miR-206 , miR-503, miR-542-3p, miR-135, miR-135a-5p, miR-129a-3p, miR-142-3p, miR-432-5p, miR-455-5p, miR-182, miR -493-3р, miR-455-3р, miR-183, miR-140-3р, miR-7135-3p, miR-204, miR-143-5p, miR-187, miR-335, miR-424-5p , miR-450a, miR-450b-5p, miR-450c-5p and miR-497 determined in a test sample obtained from a subject with a reference value; and (b) identifying the subject's exposure to zearalenone if abnormal is determined, wherein the reference value corresponds to the expression level that was determined in a control sample obtained from a subject who was not exposed to zearalenone; or identifying the subject's exposure to zearalenone if no abnormality is determined, wherein the reference value corresponds to the expression level that was determined in a control sample obtained from a subject who was exposed to zearalenone.

[0114] Clause 50. A sequencing device capable of determining the level of at least one microRNA selected from the group consisting of miR-1, miR-181c, miR-206, miR-503, miR-542-3p, miR-135 , miR-135a-5p, miR-129a-3p, miR-142-3p, miR-432-5p, miR-455-5p, miR-182, miR-493-3p, miR-455-3p, miR-183 , miR-140-3р, miR-7135-3р, miR-204, miR-143-5p, miR-187, miR-335, miR-424-5p, miR-450a, miR-450b-5p, miR-450c -5p and miR-497, containing the data processing system according to claim 49.

[0115] Clause 51. A computer program containing instructions for the data processing system of claim 49 or the sequencing device of claim 50 to perform the steps of (b) comparing the expression level of at least one microRNA selected from the group consisting of miR-1 , miR-181с, miR-206, miR-503, miR-542-3р, miR-135, miR-135a-5p, miR-129a-3p, miR-142-3р, miR-432-5p, miR-455 -5p, miR-182, miR-493-3p, miR-455-3p, miR-183, miR-140-3p, miR-7135-3p, miR-204, miR-143-5p, miR-187, miR -335, miR-424-5p, miR-450a, miR-450b-5p, miR-450c-5p and miR-497 determined in a subject sample with a reference value; and (c) identifying the subject's exposure to zearalenone if abnormal is determined, wherein the reference value corresponds to the expression level that was determined in a control sample obtained from a subject who was not exposed to zearalenone; or identifying the subject's exposure to zearalenone if no abnormality is determined, wherein the reference value corresponds to the expression level that was determined in a control sample obtained from a subject who was exposed to zearalenone.

[0116] Clause 52. A computer-readable medium storing on it the computer program of claim 51.

[0117] Item 53. A kit for carrying out the method of any one of items 1-38, containing a specific binding agent for at least one microRNA selected from the group consisting of miR-1, miR-181c, miR-206, miR-503 , miR-542-3р, miR-135, miR-135a-5p, miR-129a-3p, miR-142-3р, miR-432-5p, miR-455-5p, miR-182, miR-493-3p , miR-455-3р, miR-183, miR-140-3р, miR-7135-3р, miR-204, miR-143-5p, miR-187, miR-335, miR-424-5p, miR-450a , miR-450b-5p, miR-450c-5p and miR-497, located on or conjugated to a solid support.

[0118] Item 54. The kit of item 53, wherein the specific binding agent is a nucleic acid, an antibody, an antigen-binding fragment of an antibody, or a protein-like molecule having antibody-like properties.

[0119] Item 55. The kit of claim 53 or 54, further comprising an agent for detecting microRNA that is bound to a specific binding agent.

[0120] Item 56. The kit according to item 55, characterized in that the means for detecting microRNA that is associated with a specific binding agent contains a probe conjugated to a detectable label.

[0121] Clause 57. The kit of claim 56, wherein the detectable mark is an optically detectable mark.

[0122] It should be noted that in this application, the singular and definite articles include references to the plural and vice versa, unless the context clearly dictates otherwise. Thus, for example, a reference to a “microRNA” or a “method” includes one or more such microRNAs or methods, respectively, and a reference to a “method” includes equivalent steps and methods that may be modified or substituted as known to those of ordinary skill in the art. field of technology. Likewise, for example, reference to “microRNA” in the plural includes “microRNA” in the singular.

[0123] Unless otherwise indicated, the term “at least” preceding a series of elements should be understood to refer to each element in the series. Those skilled in the art will be able to understand or determine, using no more than routine experimentation, many equivalents to the specific embodiments described herein. Such equivalents are intended to be within the scope of the present invention.

[0124] The term "more" includes a specific number. For example, "more than 20" means ≥20.

[0125] Throughout this application and claims or claims, unless the context otherwise requires, the word “comprise” and variations such as “comprises” and “comprising” will be understood to include the specified integer (or step) or group integers (or stages). It does not exclude any other integer (or stage) or group of integers (or stages). As used herein, the term “comprising” may be replaced by the term “comprising,” “consisting of,” “including,” “having,” or “carrying.”

[0126] As used herein, the term “about” or “approximately” means within 20%, preferably within 10%, and more preferably within 5% of a target value or range. It does, however, also include a specific number, for example approximately 20 includes 20.

[0127] As used herein, the term “consisting of” excludes any element, step or ingredient not specified in the claims. As used herein, the term "consisting essentially of" does not exclude materials or steps that do not substantially affect the essential and novel features of the claims.

[0128] In each case in this application, any of the terms “comprising,” “consisting essentially of,” and “consisting of” may be replaced by any of the other two terms. The possibility of replacing terms with each other should not be understood that these terms are necessarily synonymous.

[0129] It should be understood that the present invention is not limited to the specific methodology, protocols, materials, reagents and substances, etc. described in this application, and as such may vary. The terminology used in this application is intended to describe specific embodiments only and is not intended to limit the scope of the present invention, which is defined only by the claims.

[0130] All publications cited throughout this application (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.) are incorporated herein by reference in their entirety. Nothing in this application should be construed as an admission that the present invention is not dated earlier by reason of the prior invention. To the extent that material incorporated by reference is inconsistent with or inconsistent with this application, this application supersedes any such material.

[0131] To further explain all aspects of the present invention, the invention is described below by way of figures and examples. In him:

[0132] In FIG. Figure 1 shows the average vulvar surface areas of animals (n=6) from the four treatment groups over the course of the experiment.

[0133] In FIG. Figure 2 shows the number of differentially expressed miRNAs in uterine samples from ZEN-exposed piglets (with adjusted p-values ≤0.07).

[0134] In FIG. 3A and B show the fold change values of S. scrofa -specific miRNAs in uterine tissue that were significantly affected (ie, with an adjusted FDR p value of ≤0.07) in at least one ZEN group by ZEN exposure. Fold change values can be either positive or negative, i.e. positively or negatively regulated compared to the control sample.

[0135] In FIG. Figures 3C-3E show fold change values for S. scrofa -specific miRNAs in uterine tissue that were not significantly affected (i.e., adjusted FDR p value >0.07). Fold change values can be either positive or negative, i.e. positively or negatively regulated compared to the control sample.

[0136] In FIG. Figure 4 shows log2-transformed fold changes and expression levels (measured as marks per million, TPM) of putative and predicted miRNAs significantly increased or decreased (adjusted FDR p value ≤0.07) by ZEN exposure in uterine tissue.

[0137] In FIG. Figure 5 shows the results of targeted qPCR serum analysis on days 6 and 28, comparing the control group and the high ZEN group. Only miRNAs with quantifiable levels in at least four individuals in each treatment group were considered.

[0138] In FIG. Figure 6 shows miRNA fold change values from the qPCR-based serum target assay. Fold change values can be either positive or negative, i.e. positively or negatively regulated compared to the control sample.

[0139] In FIG. Figure 7 shows the fold change values of miRNA ratios from the qPCR-based serum target assay for all three ZEN groups, as well as the combined value of the three ZEN groups compared to control (i.e., ZEN vs. no ZEN). Ratios were considered suitable when the area under the curve (AUC) was ≥0.7 for at least one sampling day. Fold change values can be either positive or negative, i.e. positively or negatively regulated compared to the control sample.

[0140] In FIG. 8 illustrates a schematic diagram of a side flow device.

EXAMPLES

[0141] Example 1: Animals and Study Design

[0142] Female weaned piglets (sow: Landrace x Large White, boar: Pietrain; age 30 +/- 2 days) were obtained from a local producer and individually marked. Piglets were housed in pens with slatted floors under controlled environmental conditions. After a seven-day acclimation period, twenty-four female piglets were selected based on body weight and vulval size and allocated to one of four groups (6 animals per group). Piglets received either blank feed (negative control; Ctrl) or feed contaminated with ZEN in three increasing concentrations (low ZEN: 0.17 mg/kg feed; medium ZEN: 1.5 mg/kg feed; high ZEN: 4.6 mg/kg food). During the 28-day experimental period, each treatment group was given free access to water and the assigned food. Pigs were observed daily and weighed weekly.

[0143] The diet was manufactured by BIOMIN AN (Herzogenburg, Austria) and formulated to meet the energy and amino acid requirements of piglets. Four different batches were prepared, one control batch and three batches artificially contaminated with ZEN (Fermentek LTD, Israel; purity >99.2%). Uniform distribution of ZEN in the diet was achieved by preparing maltodextrin premixes that contained an appropriate amount of ZEN and which were added to the main feed at 0.9% (w/w). The final concentrations of ZEN were tested by HPLC-MS (Romer Labs GmbH) and confirmed.

[0144] Blood was collected weekly (days 0, 6, 14, 21 and 28) from individual animals by puncture of the cranial vena cava vena cava cranialis (Primavette® EDTA, Kabe Labortechnik GmbH, Numbrecht-Elsenroth, Germany). After centrifugation (1500 x g, 20 min), serum samples were stored at -80°C for subsequent analysis. The length and width of the vulva were measured at regular intervals using calipers to determine the effects of ZEN in nutrition. Vulval area was calculated as vulval length x vulval width as previously reported (Jiang et al. 2011. Journal of animal science, 89, 3008-3015). During the experimental period, eight measurements were taken for the piglet.

[0145] After 28 days of exposure to the experimental diet, pigs were anesthetized with an intramuscular injection of ketamine (0.1 ml/kg body weight (bw) Narketan®, Vetoquinol GmbH, Ravensburg, Germany) and azaperone (0.03 ml/kg bw). t.; Stresnil®, Janssen-Cilag, Neuss, Germany). The animals were subsequently sacrificed by intracardiac injection of T61® (Intervet, Unterschlei*Bheim, Germany; 0.1 ml/kg bw). The entire reproductive tract was collected, weighed, and examined for any clinical signs. Small pieces of the uterine body and midjejunum were dissected, placed in 1 ml RNAlater and stored overnight at 4°C. The next day, all tissue samples were transferred to -80°C for long-term storage until processing for transcriptomic analysis.

[0146] Results: Exposure to ZEN increased vulvar surface area in a dose- and time-dependent manner (see FIG. 1). Specifically, piglets from the medium and high ZEN groups showed significant increases in vulvar surface area after thirteen and six days of exposure, respectively. At day 26, the increase in vulval surface compared to the control group was +90% for the medium ZEN group and +230% for the high ZEN group. Likewise, the weight of the entire reproductive tract (including vagina, uterus, fallopian tubes, ovaries) was significantly increased in piglets in the high ZEN group. Reproductive tract weight was expressed relative to individual body weight ([reproductive tract (grams)/body weight (kg)]*100), and the estimated mean (±SD) was as follows: 51.8 ± 20.6, 55.8 ± 17.2, 121.4 ± 43.4 and 353.4 ± 110.6 in the control, low ZEN, medium ZEN and high ZEN groups, respectively. Significant differences were observed between the high ZEN group and the control group (p-values <0.001). Regardless of concentration, exposure to ZEN did not affect the body weight of piglets.

[0147] Example 2: microRNA analysis in tissues

[0148] MicroRNA Extraction and Library Preparation: A total of 18 animals were used for microRNA tissue profiling studies: 6 from the control group, 4 from the low ZEN group, 4 from the medium ZEN group, and 4 from the high ZEN group. Approximately 30 mg of tissue from the uterus or jejunum was disrupted using a bead mill, and total RNA, including RNA of approximately 18 nucleotides or more, was extracted and purified using the miRNeasy Mini Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. RNA concentration was first assessed on a Nanodrop (Termo Scientific NanoDrop 2000, USA), and again using an Agilent 2100 Bioanalyzer (Agilent Technologies, Germany) during RIN measurement to determine RNA quality. A total of 1 μg of total RNA was used to construct small RNA sequencing libraries using the NEBNext Multiplex SmallRNA Library kit for Illumina (New England Biolabs). The adapter-ligated total RNA was reverse transcribed into cDNA, which was used as a template for PCR amplification (15 cycles) using the SR Illumina primer and index primers 1–18. Barcoded DNA libraries were purified using the QIAQuick PCR purification protocol (Qiagen, Hilden, Germany) and quantified using a high-sensitivity DNA 1000 chip (Agilent Technologies, Germany). Purified samples were pooled at equimolar (50 nM) concentration. Size selection was performed using gel extraction to select insert sizes from 18 to 50 nucleotides. The final multiplexed (n=18) libraries were sequenced on a NextSeq 500 sequencing instrument according to the manufacturer's instructions. The raw data were demultiplexed and FASTQ files for each sample were generated using bcl2fastq software (Illumina inc.). FASTQ data were checked using the online FastQC tool (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/).

[0149] Bioinformatics analysis of RNA sequencing (RNA-Seq): For total read counts, adapter sequences were trimmed using Cutadapt and quality filtered (Q-Score >30) using the FastQC tool. Trimmed and quality-filtered reads were aligned to miRNA reference sequences in miRBase release 21 (www.mirbase.org; Kozomora and Griffiths-Jones. 2014. Nucleid Acids Research, 42, D68-D73) using bowtie2. Reads were subsequently mapped against the Sus scrofa Sscrofal0.2 genome assembly as well as Ensembl's small noncoding RNA reference databases. Reads were normalized to the total number of reads in the library to generate tags per million (TPM) values. Clustvis was used to analyze unsupervised clustering. EdgeR (Robinson et al. 2010. Bioinformatics, 26, 139-140) was used to analyze differential expression based on TPM values. The resulting p-values were adjusted for multiple testing using the Benjamini-Hochberg false discovery rate (FDR). Adjusted p-values ≤0.07 were considered significant.

[0150] RNA Sequence Validation: Real-time polymerase chain reaction, also called quantitative polymerase chain reaction (qPCR), was used to verify some of the results obtained for the RNA sequence from the uterus. Starting from the same total RNA samples used for sequencing analysis, cDNA was synthesized using the Universal cDNA Synthesis Kit II (Exiqon, Denmark) in a reverse transcription (RT) reaction using 50 ng of total RNA. Reaction conditions were used according to the manufacturer's recommendations. To control the effectiveness of RT and the presence of impurities with inhibitory activity, synthetic spike-in RNA (cel-miR-39-3p) was added to the RT reaction. PCR amplification was carried out in a 96-well plate format in a Roche LC480 II instrument (Roche, Germany) and using EXiLENT SYBR Green mastermix (Exiqon, Denmark) with the following settings: 95 °C for 10 min, 45 cycles of 95 °C in for 10 s and 60 °C for 60 s, followed by melting curve analysis. The second derivative method was used to calculate the cycle of quantitation values (Cq-values). Cq values were normalized to the mean Cq value in each sample (global mean normalization) by subtracting the individual miRNA Cq value from the mean Cq value calculated for that sample.

[0151] Results: Small RNA sequencing identified the presence of 224 miRNAs (with >1 tag per million) known in swine species (ssc) in uterine samples, out of a total of 461 mature miRNAs currently known in porcine miRBase. Of these, 16 (medium ZEN) and 74 (high ZEN) miRNAs were significantly affected following ZEN exposure. In FIG. Figure 2 provides an overview of the number of up- and down-regulated miRNAs in different treatment groups. In the group with average ZEN, multiple changes in species-specific microRNAs were achieved, which were significantly influenced, from -3.4 (ssc-miRNA-187) to +3.9 (ssc-miRNA-542-3p) compared to the control group (FIG .3A and 3B). In the high ZEN group, the effects were more pronounced, with fold changes reaching from -4.1 (ssc-miRNA-204) to +12.6 (ssc-miRNA-542-3p). A total of 14 microRNAs (ssc-miR-1, ssc-miR-143-5p, ssc-miR-181c, ssc-miR-187, ssc-miR-206, ssc-miR-335, ssc-miR-424 -5p, ssc-miR-450a, ssc-miR-450b-5p, ssc-miR-450c-5p, ssc-miR-497, ssc-miR-503, ssc-miR-542-3р, ssc-miR-7135 -3p) had an effect in the medium ZEN and high ZEN groups. Exposure to ZEN resulted in significant divergence in the expression of 13 putative or predicted miRNAs (FIG. 4). Of these, put-miR-300 was significantly affected at medium ZEN and high ZEN. To validate next generation RNA sequencing results, ssc-miR-542-3p, ssc-miR-450c-5p, ssc-miR-503, ssc-miR-424-5p, ssc-miR-181c were successfully quantified in uterine samples with using qPCR for all groups.

[0152] Example 3: MicroRNA Analysis in Serum Samples

[0153] MicroRNA Extraction and qPCR Analysis: In contrast to small RNA-seq analysis, all animals from the feeding study were used for serum miRNA profile studies (6 per group; 24 animals total). RNA was isolated from serum samples using the miRNeasy Mini Kit (Qiagen, Germany). Serum samples were thawed on ice and centrifuged at 12,000 g for 5 minutes to remove any cellular debris. For each sample, 200 μl of serum was mixed with 1000 μl of Qiazol and 1 μl of synthetic spike-in (Exiqon, Denmark). After a 10-minute incubation at room temperature, 200 μl of chloroform was added to the lysates, followed by centrifugation and cooling at 12,000 g for 15 minutes at 4 °C. A volume of 650 μL of the upper aqueous phase was mixed with 7 μL of glycogen (50 mg/mL) to enhance precipitation. Samples were transferred to a miRNeasy mini column and RNA was precipitated using 750 μL ethanol followed by a wash with RPE buffer and RWT. RNA was eluted in 30 μl of nuclease-free water and stored at −80°C until further analysis. Starting from total RNA samples, cDNA was synthesized using the Universal cDNA Synthesis Kit II (Exiqon, Denmark). Reaction conditions were selected according to the manufacturer's recommendations. A total of 2 μl of purified total RNA per 10 μl reverse transcription (RT) reaction was used. To control the effectiveness of RT and the presence of impurities with inhibitory activity, synthetic spike-in RNA (cel-miR-39-3p) was added to the RT reaction. PCR amplification was carried out in a 384-well plate format using special Pick&Mix plates (Exiqon, Denmark) in a Roche LC480 II device (Roche, Germany) and EXiLENT SYBR Green mastermix (Exiqon, Denmark) with the following settings: 95 °C for 10 min, 45 cycles of 95 °C for 10 s and 60 °C for 60 s, followed by melting curve analysis. To calculate the cycle of quantitation values (Cq-values), the second derivative method was used, as known to one skilled in the art. Cq values were normalized to the mean Cq value in each sample (global mean normalization) by subtracting the individual miRNA Cq value from the mean Cq value calculated for that sample. The miRNAs selected for targeted analysis, their responses to ZEN treatment in uterine samples and their relevance are shown in Table 1.

[0154] Results: Targeted analysis of ssc-miR-1, ssc-miR-125a, ssc-miR-125b, ssc-miR-127, ssc-miR-129a, ssc-miR-133a-5p, ssc-miR-135 , ssc-miR-136, ssc-miR-140-3р, ssc-miR-142-3p, ssc-miR-143-5p, ssc-miR-146b, ssc-miR-181c, ssc-miR-182, ssc -miR-183, ssc-miR-187, ssc-miR-195, ssc-miR-204, ssc-miR-206, ssc-miR-22-3р, ssc-miR-22-5p, ssc-miR-335 , ssc-miR-34a, ssc-miR-369, ssc-miR-378, ssc-miR-424-5p, ssc-miR-432-5p, ssc-miR-450a, ssc-miR-450b-5p, ssc -miR-450c-5p, ssc-miR-455-3р, ssc-miR-455-5p, ssc-miR-486, ssc-miR-493-3р, ssc-miR-493-5p, ssc-miR-497 , ssc-miR-503, ssc-miR-542-3р, ssc-miR-708-3р, ssc-miR-758, ssc-miR-7135-3р, hsa-miR-135b-5p, hsa-miR-301a -5p, put-miR-300 was performed in serum samples collected 6 and 28 days after ZEN exposure. For ssc-miR-135, ssc-miR-187, ssc-miR-195, ssc-miR-497, qPCR amplification was performed using primers designed for human orthologous sequences. However, the sequence identity between the pig sequence and the human sequence for these four miRNAs was 100%. After removing several miRNAs from the data set due to too low read counts (ssc-miR-143-5p, ssc-miR-187, ssc-miR-493-5p, ssc-miR-708-3p, ssc-miR-7135-3p , ssc-miR-135b-5p, hsa-miR-301a-5p, put-miR-300), the focus was on comparing quantifiable miRNA levels between the control group and the high ZEN group. Three (day 6) and five (day 28) miRNAs showed up- or down-regulation after ZEN exposure (unadjusted p-value ≤0.1). Up-regulated miRNAs include ssc-miR-140-3p, ssc-miR-455-3p, ssc-miR-455-5p and ssc-miR-542-3p, while down-regulated miRNAs include ssc-miR- 1, ssc-miR-142-3р, ssc-miR-181c and ssc-miR-182. In FIG. Figure 5 provides details of log2-transformed fold changes in serum miRNA levels following ZEN exposure. In addition to the above miRNAs, ZEN had a significant effect on the log2-transformed fold changes (≤-0.8 or ≥0.8) of six miRNAs, namely ssc-miR-129a, ssc-miR-135, ssc-miR-206 , ssc-miR-432-5p, ssc-miR-493-3p and ssc-miR-503. To further explore the global effects of ZEN regardless of concentrations, principal component analyzes (PCA) were performed on the data (fold changes) for all groups and for all miRNAs. This made it possible to detect microRNAs that are not constant across all treatments. Thus, some miRNAs that were already reported to be affected were discovered, as well as new miRNAs such as ssc-miR-183. Based on the data obtained, ssc-miR-1, ssc-miR-129a, hsa-miR-135a-3p, ssc-140-3р, ssc-miR-142-3р, ssc-miR-181c, ssc-miR-182 , ssc-miR-183, ssc-206, ssc-miR-432-5p, ssc-miR-455-3р, ssc-miR-455-5p, ssc-miR-493-3р, ssc-miR-503 and ssc -miR-542-3p were identified as suitable miRNAs for use as mechanism-based biomarkers to determine the effects of ZEN based on significant up- or down-regulation and relevant expression profile that takes into account both relative fold change and absolute expression levels.

[0155] Example 4: Correlation of MicroRNA Levels in Tissue and Serum

[0156] A correlation analysis was performed to compare the miRNA results obtained for uterus (NGS read counts) and serum (qPCR dCq values). To this end, NGS reads were log2-transformed and residuals (difference to mean) were calculated and used for correlation. Two microRNAs showed a tendency to correlate expression levels in tissues and serum: ssc-miR-455-5p (sample size: 17; correlation coefficient r = 0.4045; significance level P = 0.1073; 95% confidence interval for r: from - 0.09449 to 0.7411) and ssc-miR-542-3р (sample size: 17; correlation coefficient r=0.4244; significance level P=0.0895; 95% confidence interval for r: from -0.07066 up to 0.7517).

[0157] Example 5: miRNA ratios and operating characteristic (ROC) analysis

[0158] The relationships between selected miRNAs that were identified as being affected by ZEN in serum samples were determined and subsequently subjected to an ROC (operating characteristic analysis) approach as a validated and common way to evaluate biomarkers (Grand and Sabin. 2010. Current Opinion in HIV and AIDS, 5, 473-479). Thus, fit ratios were determined that allowed for discrimination between pigs not exposed to ZEN (control) and pigs exposed to ZEN (low ZEN, medium ZEN, high ZEN): ssc-miR-135/ssc-miR-432 -5p, ssc-miR-455-5p/ssc-miR-493-3p, ssc-miR-542-3p/ssc-miR-1 and ssc-miR-542-3p/ssc-miR-493-3p (FIG 6).

[0159] In addition to the 15 serum miRNAs identified as miRNAs suitable for use as mechanism-based biomarkers for ZEN exposure described in Example 3, miRNA ssc-miR-135/ssc-miR ratios were found to -432-5p, ssc-miR-455-5p/ssc-miR-493-3p, ssc-miR-542-3p/ssc-miR-1 and ssc-miR-542-3p/ssc-miR-493-3p are particularly robust and well-defined biomarkers for detecting ZEN exposure.

[0160] The invention illustrated herein may be suitably practiced in the absence of any element or elements, limitation or limitations not specifically described herein. Moreover, the terms and expressions used herein are used as terms of description and not limitation, and it is not intended that such terms and expressions be used to exclude any equivalents of the features shown and described, or portions thereof, but it is recognized that various modifications possible within the scope of the claimed invention. Thus, it is to be understood that while the present invention has been specifically disclosed by way of illustrative embodiments and optional features, modifications and variations of the inventions embodied herein may be contemplated by those skilled in the art, and that such modifications and variations are considered included within the scope of the present invention.

[0161] The present invention is described in a broad and general manner in this application. Each of the more specific types and subgroups falling within the general description are also part of the present invention. It includes a general description of the present invention with the condition or negative limitation to the exclusion of any subject from the class, regardless of whether the subject excluded is specifically stated in this application.

[0162] Other embodiments are within the scope of the following claims. In addition, if features or aspects of the present invention are described in terms of Markush groups, those skilled in the art will understand that the present invention is also described in terms of any individual member or subgroup of members of the Markush group.

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LIST OF SEQUENCES

<110> Erber Aktiengesellschaft

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<212> RNA

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acuggacuug gagucagaag gc 22

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<212> RNA

<213> Sus scrofa

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ucuuggagua ggucauuggg u 21

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<212> RNA

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uuuugcgaug uguuccuaau au 22

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<212> RNA

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uuuugcgaug uguuccuaau ac 22

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<212> RNA

<213> Sus scrofa

<220>

<223> microRNA

<400> 31

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<212> RNA

<213> Sus scrofa

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<400> 32

uaugugccuu uggacuacau cg 22

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<212> RNA

<213> Sus scrofa

<220>

<223> microRNA

<400> 33

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<212> RNA

<213> Sus scrofa

<220>

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<400> 34

ugaaggucua cugugucca gg 22

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<213> Sus scrofa

<220>

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<400> 35

uuguacaugg uaggcuuuca uu 22

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cagcagcaca cugugguuug u 21

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Met Val Thr Ser Pro Ala Leu Arg Asp Val His Val Pro His Ala Tyr

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Pro Glu Gln Gln Val Asp Leu Gly Glu Ile Thr Met Asn Tyr Ala Glu

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Ala Gly Asp Pro Asp Arg Pro Ala Val Leu Leu Ile Pro Glu Gln Thr

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Gly Ser Trp Trp Ser Tyr Glu Glu Ala Met Gly Leu Leu Ser Glu His

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Phe His Val Tyr Ala Val Asp Leu Arg Gly Gln Gly Arg Ser Ser Trp

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Phe Ile Ala Leu Val Val Lys Arg Pro Val Val Val Ala Gly Asn Ser

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Ser Gly Gly Val Leu Ala Ala Trp Leu Ser Ala Tyr Ser Met Pro Gly

115 120 125

Gln Leu Arg Gly Val Leu Cys Glu Asp Pro Pro Phe Phe Ala Ser Glu

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Leu Val Pro Ala His Gly His Ser Val Arg Gln Gly Ala Gly Pro Val

145 150 155 160

Phe Glu Leu Phe Arg Thr Tyr Leu Gly Asp Gln Trp Ser Val Gly Asp

165 170 175

Trp Glu Gly Phe Cys Arg Ala Ala Gly Ala Ser Ala Ser Pro Met Ala

180 185 190

Arg Ser Phe Val Ala Asp Gly Ile Pro Gln His Leu Gln Glu Tyr Asp

195 200 205

Pro Glu Trp Ala Arg Val Phe Tyr Glu Gly Thr Val Gly Leu Ser Cys

210 215 220

Pro His Glu Arg Met Leu Gly Gln Val Lys Thr Pro Val Leu Leu Thr

225 230 235 240

His His Met Arg Gly Ile Asp Pro Glu Thr Gly Asn Leu Leu Gly Ala

245 250 255

Leu Ser Asp Glu Gln Ala Leu Arg Ala Arg Arg Leu Met Asp Ser Ala

260 265 270

Gly Val Thr Val Asp Tyr Glu Ser Val Pro Asp Ala Ser His Met Met

275 280 285

His Gln Ser Ala Pro Ala Arg Tyr Val Glu Ile Phe Thr Arg Trp Ala

290 295 300

Ala Ala Leu Ala Pro

305

<210> 46

<211> 308

<212> PROTEIN

<213> Artificial sequence

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<223> Alpha/beta hydrolase domain

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Glu Lys Gln Ala Asp Leu Gly Glu Ile Thr Met Asn Tyr Ala Glu Ala

20 25 30

Gly Glu Pro Asp Met Pro Ala Val Leu Leu Ile Pro Glu Gln Thr Gly

35 40 45

Ser Trp Trp Gly Tyr Glu Glu Ala Met Gly Leu Leu Ala Glu Asn Phe

50 55 60

His Val Tyr Ala Val Asp Leu Arg Gly Gln Gly Arg Ser Ser Trp Ala

65 70 75 80

Pro Lys Arg Tyr Ser Leu Asp Asn Phe Gly Asn Asp Leu Val Arg Phe

85 90 95

Ile Ala Leu Val Val Lys Arg Pro Val Ile Val Ala Gly Asn Ser Ser

100 105 110

Gly Gly Val Leu Ala Ala Trp Leu Ser Ala Tyr Ser Met Pro Gly Gln

115 120 125

Val Arg Gly Ala Leu Cys Glu Asp Ala Pro Phe Phe Ala Ser Glu Leu

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Val Thr Thr Cys Gly His Ser Ile Arg Gln Ala Ala Gly Pro Met Phe

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Glu Leu Phe Arg Thr Tyr Leu Gly Asp Gln Trp Ser Val Gly Asp Trp

165 170 175

Thr Gly Tyr Cys Arg Ala Ala Asp Ala Ser Ser Ser Pro Met Ala Arg

180 185 190

Tyr Phe Val Ala Asp Glu Ile Pro Gln His Met Arg Glu Tyr Asp Pro

195 200 205

Glu Trp Ala Arg Ala Phe Trp Glu Gly Thr Val Ala Leu His Cys Pro

210 215 220

His Glu Gln Leu Leu Thr Gln Val Lys Thr Pro Val Leu Leu Thr His

225 230 235 240

His Met Arg Asp Ile Asp Pro Asp Thr Gly His Leu Val Gly Ala Leu

245 250 255

Ser Asp Glu Gln Ala Ala Arg Ala Arg Leu Leu Met Glu Ser Ala Gly

260 265 270

Val Lys Val Asp Tyr Ala Ser Val Pro Asp Ala Leu His Met Met His

275 280 285

Gln Phe Asp Pro Pro Arg Tyr Val Glu Ile Phe Thr Gln Trp Ala Ala

290 295 300

Thr Leu Ala Ala

305

<210> 47

<211> 309

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Alpha/beta hydrolase domain

<400> 47

Met Val Thr Ser Pro Ala Leu Arg Asp Val His Val Pro His Ala Tyr

1 5 10 15

Pro Glu Gln Gln Val Asp Leu Gly Glu Ile Thr Met Asn Tyr Ala Glu

20 25 30

Ala Gly Asp Pro Gly Arg Pro Ala Val Leu Leu Ile Pro Glu Gln Thr

35 40 45

Gly Ser Trp Trp Ser Tyr Glu Glu Ala Met Gly Leu Leu Ala Glu His

50 55 60

Phe His Val Tyr Ala Val Asp Leu Arg Gly Gln Gly Arg Ser Ser Trp

65 70 75 80

Thr Pro Lys Arg Tyr Ser Leu Asp Asn Phe Gly Asn Asp Leu Val Arg

85 90 95

Phe Ile Ala Leu Val Val Arg Arg Pro Val Val Val Ala Gly Asn Ser

100 105 110

Ser Gly Gly Val Leu Ala Ala Trp Leu Ser Ala Tyr Ser Met Pro Gly

115 120 125

Gln Ile Arg Gly Val Leu Cys Glu Asp Pro Pro Phe Phe Ala Ser Glu

130 135 140

Leu Val Pro Ala His Gly His Ser Val Arg Gln Gly Ala Gly Pro Val

145 150 155 160

Phe Glu Leu Phe Arg Thr Tyr Leu Gly Asp Gln Trp Ser Val Gly Asp

165 170 175

Trp Glu Gly Phe Arg Ser Ala Ala Asp Ala Ser Ala Ser Pro Met Ala

180 185 190

Arg Ser Phe Val Ala Asp Thr Ile Pro Gln His Leu Lys Glu Tyr Asp

195 200 205

Pro Glu Trp Ala Arg Ala Phe Tyr Glu Gly Thr Val Gly Leu Asn Cys

210 215 220

Pro His Glu Arg Met Leu Asn Arg Val Asn Thr Pro Val Leu Leu Thr

225 230 235 240

His His Met Arg Gly Thr Asp Pro Glu Thr Gly Asn Leu Leu Gly Ala

245 250 255

Leu Ser Asp Glu Gln Ala Ala Gln Val Arg Arg Leu Met Glu Ser Ala

260 265 270

Gly Val Lys Val Asp Tyr Glu Ser Val Pro Asp Ala Ser His Met Met

275 280 285

His Gln Ser Asp Pro Ala Arg Tyr Ala Glu Ile Leu Thr Pro Trp Thr

290 295 300

Ala Ala Leu Ala Pro

305

<210> 48

<211> 309

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Alpha/beta hydrolase domain

<400> 48

Met Val Thr Ser Pro Ala Leu Arg Asp Val His Val Pro His Ala Tyr

1 5 10 15

Pro Glu Gln Gln Val Asp Leu Gly Glu Ile Thr Met Asn Tyr Ala Glu

20 25 30

Ala Gly Asp Pro Asp Arg Pro Ala Val Leu Leu Ile Pro Glu Gln Thr

35 40 45

Gly Ser Trp Trp Ser Tyr Glu Glu Ala Met Gly Leu Leu Ala Glu His

50 55 60

Phe His Val Tyr Ala Val Asp Leu Arg Gly Gln Gly Arg Ser Ser Trp

65 70 75 80

Thr Pro Lys Arg Tyr Ser Leu Asp Asn Phe Gly Asn Asp Leu Val Arg

85 90 95

Phe Ile Ala Leu Val Val Lys Arg Pro Val Val Val Ala Gly Asn Ser

100 105 110

Ser Gly Gly Val Leu Ala Ala Trp Leu Ser Ala Tyr Ser Met Pro Gly

115 120 125

Gln Leu Arg Gly Val Leu Cys Glu Asp Pro Pro Phe Phe Ala Ser Glu

130 135 140

Leu Val Pro Ala His Gly His Ser Val Arg Gln Gly Ala Gly Pro Val

145 150 155 160

Phe Glu Leu Phe Arg Thr Tyr Leu Gly Asp Gln Trp Ser Val Ser Asp

165 170 175

Trp Glu Gly Phe Cys Arg Ala Ala Gly Ala Ser Ala Ser Pro Met Ala

180 185 190

Arg Ser Phe Val Ala Asp Gly Ile Pro Gln His Leu Lys Glu Tyr Asp

195 200 205

Pro Glu Trp Ala Arg Ala Phe His Glu Gly Thr Val Gly Leu Asn Cys

210 215 220

Pro His Glu Arg Met Leu Gly Arg Val Asn Thr Pro Val Leu Leu Thr

225 230 235 240

His His Met Arg Gly Thr Asp Pro Glu Thr Gly Asn Leu Leu Gly Ala

245 250 255

Leu Ser Asp Glu Gln Ala Ala Gln Ala Arg Leu Leu Met Glu Ser Ala

260 265 270

Gly Val Arg Val Asp Tyr Glu Ser Val Pro Asp Ala Ser His Met Met

275 280 285

His Gln Ser Asp Pro Ala Arg Tyr Ala Glu Ile Phe Thr Arg Trp Ala

290 295 300

Ala Ala Leu Ala Pro

305

<210> 49

<211> 309

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Alpha/beta hydrolase domain

<400> 49

Met Val Thr Ser Pro Ala Leu Arg Asp Val His Val Pro His Ala Tyr

1 5 10 15

Pro Glu Gln Gln Val Asp Leu Gly Glu Ile Thr Met Asn Tyr Ala Glu

20 25 30

Ala Gly Asp Pro Gly Arg Pro Ala Val Leu Leu Ile Pro Glu Gln Thr

35 40 45

Gly Ser Trp Trp Ser Tyr Glu Glu Ala Met Gly Leu Leu Ala Glu His

50 55 60

Phe His Val Tyr Ala Val Asp Leu Arg Gly Gln Gly Arg Ser Ser Trp

65 70 75 80

Thr Pro Lys Arg Tyr Ser Leu Asp Asn Phe Gly Asn Asp Leu Val Arg

85 90 95

Phe Met Ala Leu Val Val Arg Arg Pro Val Val Val Ala Gly Asn Ser

100 105 110

Ser Gly Gly Val Leu Ala Ala Trp Leu Ser Ala Tyr Ser Met Pro Gly

115 120 125

Gln Ile Arg Gly Val Leu Cys Glu Asp Pro Pro Phe Phe Ala Ser Glu

130 135 140

Leu Val Pro Ala His Gly His Ser Val Arg Gln Gly Ala Gly Pro Val

145 150 155 160

Phe Glu Leu Phe Arg Thr Tyr Leu Gly Asp Gln Trp Ser Val Gly Asp

165 170 175

Trp Glu Gly Phe Arg Ser Ala Ala Gly Ala Ser Ala Ser Pro Met Ala

180 185 190

Arg Ser Phe Val Ala Asp Thr Ile Pro Gln His Leu Lys Glu Tyr Asp

195 200 205

Pro Glu Trp Ala Arg Ala Phe Tyr Glu Gly Thr Val Gly Leu Asn Cys

210 215 220

Pro His Glu Arg Met Leu Asn Arg Val Asn Thr Pro Val Leu Leu Thr

225 230 235 240

His His Met Arg Gly Thr Asp Pro Glu Thr Gly Asn Leu Leu Gly Ala

245 250 255

Leu Ser Asp Glu Gln Ala Ala Gln Ala Arg Arg Leu Met Glu Ser Ala

260 265 270

Gly Val Lys Val Asp Tyr Glu Ser Val Pro Asp Ala Ser His Met Met

275 280 285

His Gln Ser Asp Pro Ala Arg Tyr Ala Glu Ile Leu Thr Pro Trp Ala

290 295 300

Ala Ala Leu Ala Pro

305

<210> 50

<211> 328

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Alpha/beta hydrolase domain

<400> 50

Met Ala Glu Glu Gly Thr Arg Ser Glu Ala Ala Asp Ala Ala Thr Gln

1 5 10 15

Ala Arg Gln Leu Pro Asp Ser Arg Asn Ile Phe Val Ser His Arg Phe

20 25 30

Pro Glu Arg Gln Val Asp Leu Gly Glu Val Val Met Asn Phe Ala Glu

35 40 45

Ala Gly Ser Pro Asp Asn Pro Ala Leu Leu Leu Leu Pro Glu Gln Thr

50 55 60

Gly Ser Trp Trp Ser Tyr Glu Pro Val Met Gly Leu Leu Ala Glu Asn

65 70 75 80

Phe His Val Phe Ala Val Asp Ile Arg Gly Gln Gly Arg Ser Thr Trp

85 90 95

Thr Pro Arg Arg Tyr Ser Leu Asp Asn Phe Gly Asn Asp Leu Val Arg

100 105 110

Phe Ile Ala Leu Val Ile Lys Arg Pro Val Val Val Ala Gly Asn Ser

115 120 125

Ser Gly Gly Leu Leu Ala Ala Trp Leu Ser Ala Tyr Ala Met Pro Gly

130 135 140

Gln Ile Arg Ala Ala Leu Cys Glu Asp Ala Pro Phe Phe Ala Ser Glu

145 150 155 160

Leu Val Pro Ala Tyr Gly His Ser Val Leu Gln Ala Ala Gly Pro Ala

165 170 175

Phe Glu Leu Tyr Arg Asp Phe Leu Gly Asp Gln Trp Ser Ile Gly Asp

180 185 190

Trp Lys Gly Phe Val Glu Ala Ala Lys Ala Ser Pro Ala Lys Ala Met

195 200 205

Gln Leu Phe Pro Thr Pro Asp Glu Ala Pro Gln Asn Leu Lys Glu Tyr

210 215 220

Asp Pro Glu Trp Gly Arg Ala Phe Phe Glu Gly Thr Val Ala Leu His

225 230 235 240

Cys Pro His Asp Arg Met Leu Ser Gln Val Lys Thr Pro Ile Leu Ile

245 250 255

Thr His His Ala Arg Thr Ile Asp Pro Glu Thr Gly Glu Leu Leu Gly

260 265 270

Ala Leu Ser Asp Leu Gln Ala Glu His Ala Gln Asp Ile Ile Arg Ser

275 280 285

Ala Gly Val Arg Val Asp Tyr Gln Ser His Pro Asp Ala Leu His Met

290 295 300

Met His Leu Phe Asp Pro Ala Arg Tyr Ala Glu Ile Leu Thr Ser Trp

305 310 315 320

Ser Ala Thr Leu Pro Ala Asn Asp

325

<---

Claims (25)

1. A method for detecting the effect of zearalenone, including: (c) determining in the test sample the expression level of at least one microRNA selected from the group consisting of ssc-miR-1, ssc-miR-181c, ssc-miR-206, ssc-miR-503, ssc-miR-542 -3p, ssc-miR-135, ssc-miR-129a-3p, ssc-miR-142-3p, ssc-miR-432-5p, ssc-miR-455-5p, ssc-miR-182, ssc-miR -493-3p, ssc-miR-455-3p, ssc-miR-183, and ssc-miR-140-3p, or at least one expression level ratio between two of said microRNAs, wherein said test sample is a blood sample ; And (d) comparing said expression level or said at least one expression level ratio with a reference value. 2. The method according to claim 1, characterized in that said reference value corresponds to the expression level of said at least one microRNA in the control sample. 3. The method according to claim 1, characterized in that said at least one ratio of expression levels between two microRNAs includes at least one ratio of expression levels between two microRNAs selected from the group consisting of ssc-miR-542-3p and ssc -miR-1, ssc-miR-542-3p and ssc-miR-493-3p, ssc-miR-135 and ssc-miR-432-5p, and ssc-miR-455-5p and ssc-miR-493- 3p. 4. Method according to any one of paragraphs. 1-3, wherein said test sample is obtained from a subject, wherein said subject is preferably a mammal, preferably the genus Sus, preferably the species Sus scrofa. 5. Method according to any one of paragraphs. 1-4, additionally including b) provision of a test sample; and/or e) providing information regarding the effect of zearalenone. 6. Method according to any one of paragraphs. 1-5, characterized in that said method is intended for diagnosing, preferably, the effects of zearalenone. 7. Method according to any one of paragraphs. 1-5, wherein said method is for detecting zearalenone in a feed or food, said method preferably being for selecting a feed to be brought into contact with a zearalenone neutralizing agent or for applying a zearalenone neutralizing method. 8. Method according to any one of paragraphs. 4-7, including (a) feeding said subject a food suspected of containing zearalenone prior to obtaining a test sample from said subject; and/or (f) selecting a feed to contact with a zearalenone neutralizing agent or using a zearalenone neutralizing method if a deviation in the expression levels of said at least one microRNA between the test sample and the control sample indicates exposure to zearalenone. 9. Method according to any one of paragraphs. 1-8, characterized in that said method of detecting the effect of zearalenone is carried out to evaluate the ability of the method or test compound to neutralize zearalenone. 10. Use of at least one microRNA selected from the group consisting of ssc-miR-1, ssc-miR-181c, ssc-miR-206, ssc-miR-503, ssc-miR-542-3p, ssc-miR -135, ssc-miR-135a-5p, ssc-miR-129a-3p, ssc-miR-142-3p, ssc-miR-432-5p, ssc-miR-455-5p, ssc-miR-182, ssc -miR-493-3p, ssc-miR-455-3p, ssc-miR-183, and ssc-miR-140-3p, to detect the effect of zearalenone in a test sample, wherein the test sample is a blood sample. 11. A data processing system comprising a processor configured to implement a method including the steps (a) comparing the expression level of at least one microRNA selected from the group consisting of ssc-miR-1, ssc-miR-181c, ssc-miR-206, ssc-miR-503, ssc-miR-542-3p, ssc-miR-135, ssc-miR-135a-5p, ssc-miR-129a-3p, ssc-miR-142-3p, ssc-miR-432-5p, ssc-miR-455-5p, ssc-miR- 182, ssc-miR-493-3p, ssc-miR-455-3p, ssc-miR-183, and ssc-miR-140-3p determined in a test sample obtained from a subject, or at least one expression level ratio between two of said microRNAs with a reference value, wherein said test sample is a blood sample; And (b) identifying exposure to zearalenone in a specified subject if a deviation is determined, where the specified reference value corresponds to the expression level or ratio of expression levels that was determined in a control sample obtained from a subject who was not exposed zearalenone; or identifying exposure to zearalenone in a specified subject if no deviation is determined, wherein said reference value corresponds to the expression level or ratio of expression levels that was determined in a control sample obtained from a subject who was exposed to zearalenone. 12. A sequencing device capable of determining the level of at least one microRNA selected from the group consisting of ssc-miR-1, ssc-miR-181c, ssc-miR-206, ssc-miR-503, ssc-miR-542 -3p, ssc-miR-135, ssc-miR-135a-5p, ssc-miR-129a-3p, ssc-miR-142-3p, ssc-miR-432-5p, ssc-miR-455-5p, ssc -miR-182, ssc-miR-493-3p, ssc-miR-455-3p, ssc-miR-183, and ssc-miR-140-3p, containing the data processing system according to claim 11. 13. A machine-readable medium storing on it a computer program containing instructions for causing the data processing system of claim 11 or the sequencing device of claim 12 to perform the steps of claim 12 (b) comparing the expression level of at least one microRNA selected from the group consisting of ssc-miR-1, ssc-miR-181c, ssc-miR-206, ssc-miR-503, ssc-miR-542-3p, ssc-miR-135, ssc-miR-135a-5p, ssc-miR-129a-3p, ssc-miR-142-3p, ssc-miR-432-5p, ssc-miR-455-5p, ssc-miR- 182, ssc-miR-493-3p, ssc-miR-455-3p, ssc-miR-183, and ssc-miR-140-3p, or at least one ratio of expression levels between two of these microRNAs identified in the sample, obtained from a subject, wherein said sample is a blood sample with a reference value; And (c) identifying exposure to zearalenone in a specified subject if a deviation is determined, where the specified reference value corresponds to the expression level or ratio of expression levels that was determined in a control sample obtained from a subject who was not exposed zearalenone; or identifying exposure to zearalenone in a specified subject if no deviation is determined, wherein said reference value corresponds to the expression level or ratio of expression levels that was determined in a control sample obtained from a subject who was exposed to zearalenone. 14. Kit for implementing the method according to any one of paragraphs. 1-9, containing a specific binding agent for at least one microRNA selected from the group consisting of miR-1, miR-181c, miR-206, miR-503, miR-542-3p, miR-135, miR-135a -5p, miR-129a-3p, miR-142-3p, miR-432-5p, miR-455-5p, miR-182, miR-493-3p, miR-455-3p, miR-183, and miR- 140-3p, wherein said specific binding agent is a nucleic acid molecule that hybridizes to said at least one microRNA conjugated to a solid support.

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