Application of diagnostic marker for diarrhea of lambs in lactation period

Abstract

The invention belongs to the technical field of markers, and particularly relates to application of a diagnostic marker for diarrhea of lambs in a lactation period. The invention provides application of a diagnostic marker for diagnosing diarrhea of lambs in the lactation period in preparation of a reagent or a kit for diagnosing and predicting diarrhea of lambs in the lactation period. The diagnostic marker is selected from at least one of the following metabolic pathways: a glycerophospholipid metabolic pathway, a fatty acid metabolic pathway and an amino acid metabolic pathway. The diagnostic marker provided by the invention can comprehensively analyze the health condition of the lambs based on detection data of three dimensions of serum inflammatory factors, intestinal permeability markers and fecal metabonomics, can accurately predict potential diarrhea risks at the stage that the lambs do not present clinical symptoms of diarrhea, creates favorable opportunities for timely intervention, and has a good application prospect. The occurrence and diffusion of diarrhea diseases are effectively prevented.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of markers, and particularly relates to application of a diagnostic marker for diarrhea of lactating lambs. BACKGROUND

[0002] The average incidence of diarrhea of lactating lambs is 47.30% and the mortality rate is 22.20% throughout the year, which has become a bottleneck restricting the economic benefits of large-scale sheep farms. Early and accurate diagnosis of diarrhea is the key to effective intervention and reduction of economic losses. At present, the diagnosis methods for intestinal pathogenic bacteria causing diarrhea of lambs mainly include fecal sample shape scoring, pathogen isolation, microscopic examination, PCR examination or ELISA examination. These methods are either highly subjective or have a long detection period, and cannot reflect the overall pathophysiological state of lambs, which cannot meet the needs of early warning and accurate diagnosis.

[0003] The development of metabolomics technology provides a new perspective for early diagnosis of diseases. Metabolomics can directly reflect the specific physiological or pathological state of the body by systematically analyzing the overall changes of small molecule metabolites in the body, thereby providing specific and sensitive biomarkers for disease diagnosis. Diarrhea of lambs can cause damage to the intestinal barrier and be accompanied by inflammation, but there is still a lack of efficient and reliable means for early and accurate diagnosis of the disease. The existing methods mainly rely on clinical symptom observation and pathogenic detection, and it is difficult to achieve early warning and comprehensive evaluation of the cause. SUMMARY

[0004] To solve the problem that the existing methods in the prior art mainly rely on clinical symptom observation and pathogenic detection, and it is difficult to achieve early warning and comprehensive evaluation of the cause, the application aims to provide an application of a molecular marker that can comprehensively reflect the intestinal barrier function, inflammation state and metabolic response, so as to improve the diagnostic efficiency of diarrhea of lactating lambs. The application provides an application of a diagnostic marker for diarrhea of lactating lambs.

[0005] To achieve the above-mentioned purpose, the application adopts the following technical solutions.

[0006] The application provides an application of a diagnostic marker for diagnosing diarrhea of lactating lambs in preparation of a reagent or kit for diagnosing and predicting diarrhea of lactating lambs, wherein the diagnostic marker is selected from at least one metabolic marker in the following metabolic pathways: a glycerophospholipid metabolism pathway, a fatty acid metabolism pathway and an amino acid metabolism pathway.

[0007] The metabolic markers selected from the glycerophospholipid metabolism pathway are at least one of 1-stearoyl-LPC [Lyso-Pc (18:0)], 1-oleoyl-LPC [Lpc (18:1)], 1-nonadecanoyl-LPC [Pc (19:0 / 0:0)], ether phosphatidylethanolamine [Pe (O-13:1 / 7:0)], and 1-stearoyl-LPE [Pe (18:0 / 0:0)].

[0008] The metabolic marker selected from the fatty acid metabolism pathway is 7-dodecenoylcarnitine.

[0009] The metabolic marker selected from the amino acid metabolism pathway is succinic acid and / or creatinine.

[0010] The application provides a use of a diagnostic marker for diagnosing diarrhea of lactating lambs in the preparation of a reagent or kit for diagnosing and predicting diarrhea of lactating lambs, the diagnostic marker being at least one metabolic marker in the glycerophospholipid metabolism pathway, the fatty acid metabolism pathway, and the amino acid metabolism pathway. Based on the detection data of fecal metabolomics, by constructing a diagnostic model based on metabolites, the application overcomes the limitations of long detection period of clinical symptoms observation and etiology detection, inability to achieve early warning and comprehensive assessment of causes, and difficulty in comprehensive assessment of the severity of intestinal barrier damage and systemic stress response, and can accurately predict the potential risk of diarrhea at the stage when the lamb has not yet shown clinical symptoms of diarrhea, creating a favorable opportunity for timely intervention and effectively preventing the occurrence and spread of diarrhea diseases. The diagnostic marker provided by the application can comprehensively reflect the intestinal barrier function and metabolic response to improve the diagnostic efficiency of diarrhea of lactating lambs.

[0011] Further, the reagent or kit comprises a preparation for detecting the diagnostic marker.

[0012] Further, the preparation for detecting the diagnostic marker comprises a chromatographic separation reagent, an isotopic internal standard solution, a protein extraction agent, and a mass spectrometry tuning solution.

[0013] Further, the chromatographic separation reagent is a liquid chromatographic separation reagent.

[0014] Further, the protein extraction agent comprises RIPA lysis buffer and / or trichloroacetic acid precipitant.

[0015] Further, the isotopic internal standard solution is a stable isotope-labeled amino acid homolog.

[0016] Further, the mass spectrometry tuning solution is a mixed tuning solution containing polytyrosine, caffeine, and methanesulfonic acid len.

[0017] Further, the reagent or kit is used for detecting the fecal sample of the lactating lamb.

[0018] In the embodiments provided by the present application, the fecal sample is taken from healthy and diarrhea lambs, and the diarrhea risk of the lamb can be predicted in advance before the lamb shows diarrhea symptoms.

[0019] The present application also provides an application of a lamb diarrhea early judgment method based on metabolomics and inflammation indicators, which comprises the following steps: Step 1, collecting blood and fecal samples of the lactating lamb.

[0020] Step 2, determining the inflammation factors in the serum by using the enzyme-linked immunosorbent assay (ELISA) method.

[0021] Step 3, determining the intestinal permeability indicators in the serum by using the enzyme-linked immunosorbent assay (ELISA) method.

[0022] Step 4, detecting the fecal microbial flora metabolites of the lactating lamb.

[0023] Step 5, performing PAC and PLS-DA analysis on the fecal microbial flora metabolites of the lactating lamb.

[0024] Step 6, performing enrichment analysis on the differential fecal microbial flora metabolites of the lactating lamb.

[0025] Step 7, performing inter-group difference analysis on the differential fecal microbial flora metabolites of the lactating lamb.

[0026] Step 8, performing ROC analysis on the differential fecal microbial flora metabolites of the lactating lamb.

[0027] Further, the prediction criteria are the increase of metabolites Lyso-Pc (18:0), Lpc (18:1), Pc (19:0 / 0:0), Pe (O-13:1 / 7:0), Pe (18:0 / 0:0), 7-Dodecenoylcarnitine, Succinic Acid and Creatinine.

[0028] In an embodiment of the present application, it is found that the lactating lamb enters the diarrhea high-incidence period after 7 days old. In the test process, it is found that 8-15 days old is the diarrhea high-incidence time of the lamb. According to the diarrhea condition of 8-15 days old, the lactating lamb is divided into a Health group and a Diarrhea group, and the serum inflammation factors, intestinal permeability indicators and fecal microbial flora metabolomics analysis of the two groups of lambs are compared, and the present application discloses the influence of the body metabolism and intestinal metabolites of the lactating lamb on the occurrence of diarrhea, so as to provide a prevention strategy and basis for diarrhea.

[0029] In the application, the diarrhea of the lambs in the Health group and the Diarrhea group occurs successively at 8-15 days old, and the serum inflammatory factor results show that the serum IL-1beta, TNF-alpha and IL-6 of the lambs in the Diarrhea group are significantly higher than those in the Health group, which shows that the lambs in the Diarrhea group have inflammatory reactions.

[0030] In the application, the D-LA and DAO contents in the serum of the lambs in the Diarrhea group are significantly higher than those in the Health group, which shows that the intestinal mucosa of the lambs in the Diarrhea group is damaged and the intestinal flora is disordered.

[0031] The fecal microbial flora of the healthy and diarrhea lambs is analyzed by metabolomics, the differential metabolites Lyso-Pc (18:0), Lpc (18:1), Pc (19:0 / 0:0), Pe (O-13:1 / 7:0) and Pe (18:0 / 0:0) involved in glycerophospholipid metabolism, 7-Dodecenoylcarnitine involved in fatty acid metabolism, Succinic Acid and Creatinine involved in amino acid metabolism are all significantly up-regulated in the Diarrhea group, which means that glycerophospholipid metabolism, fatty acid metabolism and amino acid metabolism are disordered in the Diarrhea group. The fecal metabolites are closely related to serum inflammatory factors, intestinal permeability and diarrhea. Eight metabolites (Lyso-Pc (18:0), Lpc (18:1), Pc (19:0 / 0:0), Pe (O-13:1 / 7:0), Pe (18:0 / 0:0), 7-Dodecenoylcarnitine, Succinic Acid and Creatinine) can achieve a high prediction effect of diarrhea, and the AUC values are all greater than 0.75.

[0032] Compared with the prior art, the application has the following beneficial effects: The application provides application of a diagnostic marker for diagnosing diarrhea of lactating lambs in preparation of a reagent or kit for diagnosing and predicting diarrhea of lactating lambs, the diagnostic marker being selected from at least one metabolic marker in a metabolic pathway, a glycerophospholipid metabolic pathway, a fatty acid metabolic pathway and an amino acid metabolic pathway. The metabolic marker selected from the glycerophospholipid metabolic pathway is selected from at least one of 1-stearoyl-LPC [Lyso-Pc (18:0)], 1-oleoyl-LPC [Lpc (18:1)], 1-nonadecanoyl-LPC [Pc (19:0 / 0:0)], ether phosphatidylethanolamine [Pe (O-13:1 / 7:0)] and 1-stearoyl-LPE [Pe (18:0 / 0:0)]; the metabolic marker selected from the fatty acid metabolic pathway is 7-dodecenoyl carnitine; and the metabolic marker selected from the amino acid metabolic pathway is succinic acid and / or creatinine. Based on detection data of fecal metabolomics, the application overcomes the limitations of long period of clinical symptom observation and pathogenic detection, inability to realize early warning and comprehensive assessment of causes, and difficulty in comprehensively assessing the severity of intestinal barrier damage and systemic stress response, and can accurately predict the potential diarrhea risk of lambs at the stage when the lambs have not yet shown clinical symptoms of diarrhea, thereby creating a favorable opportunity for timely intervention and effectively preventing the occurrence and spread of diarrhea diseases. The diagnostic marker provided by the application can comprehensively reflect the intestinal barrier function, inflammatory state and metabolic response, thereby improving the diagnostic efficiency of diarrhea of lactating lambs. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 FIG. 1 is a graph of the influence of serum inflammatory factors in the Health group and the Diarrhea group in the application; wherein: FIG. 1A is IL-1β; FIG. 1B is TNF-α; FIG. 1C is IL-6.

[0034] Figure 2 FIG. 2 is a graph of serum intestinal permeability D-lactic acid and diamine oxidase in the Health group and the Diarrhea group in the application; wherein: FIG. 2A is DAO; FIG. 2B is D-LA.

[0035] Figure 3 FIG. 3 is a PCA and PLS-DA graph of rectal fecal microbial metabolome in the application.

[0036] Figure 4 FIG. 4 is a differential metabolite enrichment analysis graph in the application.

[0037] Figure 5 FIG. 5 is a column chart of differences among four metabolite groups in the application; wherein: FIG. 5A is Lpc (18:1); B is (Lyso-Pc (18:0); C is Pe (18:0 / 0:0); D is Pe (O-13:1 / 7:0).

[0038] Figure 6 Figure B is a bar chart of the difference between the four metabolites in the application; wherein: A is Pc (19:0 / 0:0); B is 7-Dodecenoylcarnitine; C is Succinic Acid; D is Creatinine.

[0039] Figure 7 Figure B is a bar chart of the difference between the four metabolites in the application; wherein: DETAILED DESCRIPTION

[0040] The application will be described in detail below with reference to the accompanying drawings and specific examples, but should not be understood as limiting the application. If not specifically stated, the technical means used in the following examples are conventional means familiar to those skilled in the art, and the materials, reagents, etc. used in the following examples, if not specifically stated, can be obtained from commercial channels.

[0041] Example 1: Collection of samples of lactating diarrhea lambs and healthy lambs The present application evaluates the health of newborn lambs. During the test period, the fecal condition of lambs was observed at 11:00 and 17:00 every day in the lamb stable, and the diarrhea score was evaluated according to the method of Marquardt et al.: fecal bar or granular (A: 0 points); soft feces, can be shaped (B: 1 point); thick, not shaped, fecal water not separated (C: 2 points); liquid, not shaped, fecal water separated (D: 3 points). Diarrhea was defined as Diarrhea group (fecal score ≥ 2) if the diarrhea of lambs lasted for more than 2 days, and the same-age lambs that never had diarrhea were defined as Health group (fecal score < 2). Finally, a total of 12 healthy lambs and 12 diarrhea lambs were selected for subsequent research. Fecal samples were collected in 2 mL sterile enzyme-free cryogenic tubes, frozen in liquid nitrogen and stored at -80°C for standby. At the same time, 5 mL of blood was collected through the jugular vein into a blood collection tube without anticoagulant, centrifuged at 3000 r / min for 15 min, and the supernatant was taken into a 2 mL cryogenic tube and stored at -20°C.

[0042] Among them, the newborn lambs come from sheep intensive breeding farms, which are located in the fine wool sheep breeding base of Gongnaise Sheep Farm in Yili Kazakh Autonomous Prefecture, Xinjiang.

[0043] Example 2: Determination of serum inflammatory factors and intestinal permeability indicators of lactating diarrhea lambs and healthy lambs The levels of tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), interleukin-6 (IL-6), D-lactic acid (D-LA), and diamine oxidase (DAO) in serum were detected by enzyme-linked immunosorbent assay (ELISA).

[0044] Among them, tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), interleukin-6 (IL-6), D-lactic acid (D-LA) and diamine oxidase (DAO) indicators were all tested using an enzyme-linked immunosorbent assay (ELISA) kit (Jingmei Biotechnology Co., Ltd., Jiangsu, China) in strict accordance with the manufacturer's instructions.

[0045] See results Figure 1 .

[0046] Depend on Figure 1 It can be seen that the serum levels of TNF-α, IL-6, and IL-1β in the Diarrhea group were significantly higher than those in the Health group. P <0.05). By Figure 2 It can be seen that the D-LA content in the serum of lambs in the Diarrhea group was significantly higher than that in the Health group. P <0.01), DAO content was significantly higher than that in the Health group ( P <0.05).

[0047] Example 3: Determination of fecal microbiota metabolites in lactating lambs with diarrhea and healthy lambs 20 mg of fecal samples from healthy and diarrheal lambs were weighed into EP tubes, 3 grinding beads were added, and then 400 μL of methanol-water solution (volume ratio 4:1) was added to obtain a mixture. The mixture was then ground at 10℃ and 50 Hz for 6 min on a cryosurfactant, followed by ultrasonic extraction at 5℃ and 40 kHz for 30 min to obtain the fecal extract. The fecal extract was then placed in a -20℃ freezer for 30 min, centrifuged at 13000g for 15 min at 4℃, and the supernatant was transferred to a vial with an inner tube for analysis. The samples were analyzed by LC-MS / MS using an ultra-high performance liquid chromatography (Thermo Fisher, USA) tandem time-of-flight mass spectrometry (UHPLC-Exploris 240) system.

[0048] The chromatographic conditions are as follows: the chromatographic column is ACQUITY UPLC HSS T3 (100 mm x 2.1 mm i.d., 1.8 μm; Waters, Milford, USA); the mobile phase A is 95 % (v / v) water + 5 % (v / v) acetonitrile (containing 0.1 % (v / v) formic acid), the mobile phase B is 47.5 % (v / v) acetonitrile + 47.5 % (v / v) isopropanol + 5 % (v / v) water (containing 0.1 % (v / v) formic acid), the injection amount is 3 μL, and the column temperature is 40 °C.

[0049] The mass spectrometry conditions are as follows: the sample mass spectrometry signal acquisition adopts positive and negative ion scanning modes, the mass scanning range m / z is 70-1050, the ion spray voltage is 3400 V for positive ions and -3000 V for negative ions, the sheath gas is 60 arb, the auxiliary heating gas is 20 arb, the ion source heating temperature is 350 °C, and the 20-40-60 V cyclic collision energy.

[0050] The LC-MS raw data are imported into the metabolomics processing software Progenesis QI v3.0 (Waters Corporation, Milford, USA) for baseline filtering, peak identification, integration, retention time correction, peak alignment, and finally a data matrix with retention time, mass-to-charge ratio and peak intensity is obtained. Then the software is used for feature peak library identification, and the MS and MS / MS mass spectrometry information is matched with the metabolic database, the MS mass error is set to be less than 10 ppm, and the metabolites are identified according to the secondary mass spectrum matching score. The main databases are mainstream public databases such as http: / / www.hmdb.ca / and https: / / metlin.scripps.edu / and the database built by Meiji.

[0051] The data matrix is preprocessed as follows: the data matrix is removed by 80 % rule, that is, at least one group of samples with more than 80 % non-zero values of variables, and then the missing values are filled (the minimum value in the original matrix is filled for the missing values). In order to reduce the error caused by sample preparation and instrument instability, the total normalization method is used for normalizing the response intensity of sample mass spectrometry peaks, and the normalized data matrix is obtained. At the same time, the variables with relative standard deviation (RSD) > 30 % of the QC sample are deleted, and log10 logarithmic processing is performed, and finally the data matrix used for subsequent analysis is obtained.

[0052] The results are shown in Table 1.

[0053] Table 1 Total ion number and identification statistics From Table 1, Ion mode (ion mode of mass spectrometer instrument for detecting substances) mainly includes: pos (positive ion mode) and neg (negative ion mode). All peaks (the number of mass spectrometry peaks extracted by software), in which 4975 in the positive ion mode and 5199 in the negative ion mode; Metabolites in Library (the number of metabolites annotated to the public database such as HMDB and Lipidmaps), in which 728 in the positive ion mode and 714 in the negative ion mode; Metabolites in kegg (the number of metabolites annotated to KEGG database), in which 334 in the positive ion mode and 324 in the negative ion mode.

[0054] Example 4: Differential metabolites and metabolite enrichment pathway analysis of diarrhea and healthy lambs The principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were performed on the pretreated data matrix in R language, and 7 cycles of cross-validation were used to evaluate the stability of the model. Figure 3 The results show that there is a significant difference in the composition of fecal metabolites between the Health group and the Diarrhea group, which indicates that diarrhea in lambs is accompanied by significant fecal metabolic profile disorder, and the metabolite characteristics can be used as potential biomarkers for distinguishing between healthy and diarrhea states, laying a foundation for subsequent analysis of the mechanism of diarrhea and development of early diagnosis indicators. The selection of significant differential metabolites is based on the variable weight value (VIP) and student's t test P value obtained from the OPLS-DA model, and the threshold is set to VIP>1, P <0.05. The differential metabolites are annotated by the metabolic pathway database (https: / / www.kegg.jp / kegg / pathway.html) to obtain the pathways involved in the differential metabolites. The Python package scipy.stats is used for pathway enrichment analysis, and the Fisher's exact test is used to obtain the most relevant biological pathways related to the experimental treatment.

[0055] KEGG pathway topology analysis shows that the differential metabolites found in the present application are significantly enriched in several key metabolic pathways, including glycerophospholipid metabolism, amino sugar and nucleotide sugar metabolism, glycolysis / gluconeogenesis, etc. Figure 4). This result suggests that the diarrhea process may be closely related to cell membrane structural integrity and energy metabolism. Specifically, the glycerophospholipid metabolism pathway was particularly affected, with multiple lipid metabolites in this pathway, such as 1-stearoyl-LPC, 1-oleoyl-LPC, 1-nonadecanoyl-LPC, ether phosphatidylethanolamine, and 1-stearoyl-LPE, significantly upregulated in the Diarrhea group. Figure 5 and Figure 6 ).

[0056] The above results indicate that there are significant metabolic characteristics in the body of diarrhea lambs, which are centered on glycerophospholipid metabolism disorder, accompanied by energy metabolism reprogramming. These disorders collectively point to impaired intestinal cell membrane structural integrity and imbalanced energy homeostasis, which may be an important metabolic basis for the destruction of intestinal barrier function and the occurrence and development of diarrhea.

[0057] Example 5: ROC curve analysis of differential metabolites of lactating diarrhea lambs and healthy lambs To evaluate the diagnostic potential of the significantly different metabolites in distinguishing healthy and diarrhea lambs, this example performed a receiver operating characteristic curve (ROC) analysis. Using the R package pROC v1.12.1, the lamb health status (healthy / diarrhea) was used as the state variable, and the standardized peak area of each metabolite was used as the prediction variable, to draw the ROC curve and calculate the area under the curve (AUC) and its 95% confidence interval. AUC>0.7 was considered to have certain diagnostic value.

[0058] Among them, the subjects were 12 healthy and 12 diarrhea lambs, all from the fine wool sheep breeding base of Gongnaies Sheep Farm in Yili Kazakh Autonomous Prefecture, Xinjiang.

[0059] The ROC curve of the significantly different metabolite marker is shown in Figure 7 .

[0060] As Figure 7 can be seen, the AUC values of the eight metabolites Lyso-Pc (18:0), Lpc (18:1), Pc (19:0 / 0:0), Pe (O-13:1 / 7:0), Pe (18:0 / 0:0), 7-Dodecenoylcarnitine, Succinic Acid, and Creatinine are all greater than 0.75, indicating that they have good predictive ability and diagnostic value for lamb diarrhea, and can be used as excellent potential diagnostic biomarkers.

[0061] The above results show that the diagnostic marker provided by the application can accurately diagnose lamb diarrhea with sensitivity and specificity of AUC>0.75, and by revealing the synergistic disorder of glycerophospholipid, fatty acid and amino acid metabolic pathways, the metabolic reprogramming is organically integrated with the increase of proinflammatory factors and intestinal barrier damage, and the occurrence mechanism of lamb diarrhea is revealed from the molecular level: that is, metabolic disorder, intestinal barrier damage and mucosal inflammation activation are interrelated and jointly exacerbate, which constitutes an important pathological basis for the occurrence and development of lamb diarrhea.

[0062] It should be noted that when a numerical range is involved in the present application, both endpoints of each numerical range and any number between the two endpoints can be selected, and in order to prevent repetition, the preferred embodiments are described in the present application.

[0063] Although the preferred embodiments of the present application have been described, those skilled in the art can make further changes and modifications to these embodiments once they know the basic inventive concept, and these changes and modifications all fall within the scope of all changes and modifications of the present application.

Claims

1. Use of a diagnostic marker for diagnosing diarrhoea in a lactating lamb in the manufacture of a reagent or kit for diagnosing and predicting diarrhoea in a lactating lamb, characterised in that, The diagnostic marker is a glycerophospholipid metabolism pathway metabolite, a fatty acid metabolism pathway metabolite, or an amino acid metabolism pathway metabolite; The glycerophospholipid metabolism pathway metabolite is at least one of 1-stearoyl-LPC, 1-oleoyl-LPC, 1-nonadecanoyl-LPC, ether phosphatidylethanolamine, and 1-stearoyl-LPE; The fatty acid metabolism pathway metabolite is 7-dodecenoyl carnitine; The amino acid metabolism pathway metabolite is succinate and / or creatinine.

2. Use according to claim 1, characterized in that, The reagent or kit comprises a preparation for detecting the diagnostic marker.

3. Use according to claim 2, characterized in that, The preparation for detecting the diagnostic marker comprises a chromatographic separation reagent, an isotopic internal standard solution, a protein extraction agent, and a mass spectrometry tuning solution.

4. Use according to claim 3, characterized in that, The chromatographic separation reagent is a liquid chromatographic separation reagent.

5. Use according to claim 3, characterized in that, The protein extraction agent comprises a RIPA lysis buffer and / or a trichloroacetic acid precipitant.

6. The use according to claim 3, characterized in that, The isotopic internal standard solution is a stable isotope-labeled amino acid homolog.

7. The use according to claim 3, characterized in that, The mass spectrometry tuning solution is a mixed tuning solution containing polytyrosine, caffeine, and methanesulfonic acid luminal.

8. The use according to claim 1, characterized in that, The reagent or kit is used for detecting a fecal sample of a lactating lamb.

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