Diagnosis and treatment of constipation

Abstract

The present invention relates to a method and product for diagnosing and treating constipation, and specifically provides the use of detecting DPA in diagnosing, preventing or treating constipation. The present invention can detect and warn patients with intractable constipation and guide patients to make correct drug selections.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to the diagnosis and treatment of constipation. Background Art

[0002] Chronic constipation is a common chronic disease, with a prevalence of about 4-10% in my country and an average prevalence of 16% worldwide. Systemic treatment can often improve symptoms, but a large number of patients still suffer from long-term recurrences, and there is almost no treatment except surgery. The cause of this type of intractable chronic constipation is unknown, which is extremely harmful to the patient's physical and mental health. Exploring its cause and pathogenic mechanism is the key to preventing and treating this type of constipation.

[0003] At present, the clinical diagnosis of chronic constipation is still based on the Rome IV criteria. This standard divides chronic constipation into four types: functional constipation, opioid constipation, constipation-type irritable bowel syndrome, and functional defecation disorder. The common manifestations are: difficulty in defecation, emptying hard stools, feeling of incomplete defecation, feeling of anorectal obstruction during defecation, and the need for manual assistance. Conventional treatments for chronic constipation can often effectively improve symptoms, such as: 1. General treatment: defecation physiology education, changing dietary habits, etc.; 2. Drug treatment: laxatives, bulking agents, osmotic laxatives, etc.; 3. Psychological therapy: cognitive therapy, eliminating tension, etc. The course of the disease is often more than 6 months. Conventional treatment methods (such as high-fiber diet, exercise, drugs, etc.), pelvic floor biofeedback therapy (biofeedback-aided pelvic floor retraining) and surgical treatment (such as rectal anastomosis) can effectively improve constipation symptoms. However, nearly 1 / 3 of patients still have poor treatment effects and recurrent episodes, which manifests as intractable chronic constipation. Intractable chronic constipation is the most serious functional slow-transmission constipation. The cause of its formation is still unclear, and there is an urgent need to seek new research ideas. Previous studies have shown that intractable constipation is caused by infection with PIB bacteria (a new species of Shigella), and the detection of PIB in fecal bacteria can effectively detect and warn patients with intractable constipation. However, direct detection of live bacteria from feces has disadvantages such as complex sample processing, high environmental requirements, and low positive rate. It is very necessary to develop new and simple detection methods to detect PIB infection. Summary of the invention

[0004] The first aspect of the present invention provides the use of a reagent in preparing a detection kit for diagnosing constipation, wherein the reagent comprises (1) a reagent for detecting DPA, and the reagent optionally further comprises (2) a reagent for detecting PIB.

[0005] In one or more embodiments, the constipation is refractory constipation, preferably refractory constipation caused by PIB.

[0006] In one or more embodiments, the reagent for detecting DPA is a reagent for detecting DPA using one or more detection methods selected from the group consisting of chromatography (e.g., gas chromatography, HPLC), mass spectrometry, gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, colorimetry, enzyme-linked immunosorbent assay, ultra-high performance liquid chromatography, capillary gas chromatography, and the like.

[0007] In one or more embodiments, the reagent for detecting DPA includes a solvent for DPA, such as dichloromethane and / or chloroform.

[0008] In one or more embodiments, the reagent for detecting DPA includes a reagent for extracting DPA from feces. In one or more embodiments, the reagent for detecting DPA includes one or more of methanol, dichloromethane, and water. Preferably, the reagent for detecting DPA includes methanol, dichloromethane, and water mixed in a certain proportion.

[0009] In one or more embodiments, the reagent for detecting DPA includes a substance specific for DPA or a derivative thereof.

[0010] In one or more embodiments, the DPA derivative is an in vivo metabolite of DPA, including one or more selected from the group consisting of 17-oxo-DPA, 17S-HDPA, 13-oxo-DPA, 7,12,13R-triHDPA, 7,13R-diHDPA, 7,13R,20-triHDPA, 7,8,13R-triHDPA, 13R-HDPA, 13-F3-IsoP-DPA, 7-F3-IsoP-DPA, 10-F3-IsoP-DPA, 20-F3-IsoP-DPA, 14-F3-IsoP-DPA, 17-F3-IsoP-DPA, 18-F3-IsoP-DPA, 19-F3-IsoP-DPA, 20-F3-IsoP-DPA, 21-F3-IsoP-DPA, 22-F3-IsoP-DPA, 23-F3-IsoP-DPA, 24-F3-IsoP-DPA, 25-F3-IsoP-DPA, 26-F3-IsoP-DPA, 27-F3-IsoP-DPA, 28-F3-IsoP-DPA, 29-F3-IsoP-DPA, 30-F3-IsoP-DPA, 31-F3-IsoP-DPA, 32-F3-IsoP-DPA, 33-F3-IsoP-DPA, 34-F3-IsoP-DPA, 35-F3-IsoP-DPA, 36-F3-IsoP-DPA, 37-F3-IsoP-DPA, 38-F3-IsoP-DPA, 39-F3-IsoP-DPA, oP-DPA, 14S-H(p)-DPA, 13,14S-epoxy-DPA, 7S,14S-dihydroxy-8E,10E,12Z,16Z,19Z-n-3DPA, 13,14-dihydroxy-7Z,9,11,16Z,19Z-n-3DPA, 14,21-dihydroxy-7Z,10Z,12E,16Z,19Z-n-3DPA, 17S-H(p)-DPA, 16S,17S-epoxy-DPA, 10,17S-diHDPA, 16,17S-diH-DPA, 10R,17S-dihydroxy-7Z,11E,13E,15Z,19Zn-3 DPA, 16,17R-dihydroxy-7Z,10,13,14,19Zn-3 DPA, 7,17S-diH(p)-DPA, 7,8,17S-trihydroxy-9,11,13,15E,19Z-n-3DPA, 7,16,17-trihydroxy-8,10,12,14E,19Z-n-3DPA, 7S,17S-trihydroxy-8E,10Z,13Z,15E,19Z-n-3DPA, 7,17-diHDPA, 10,17S-diHDPA, 11-HDPA, 17-HDPA, 8,14-diHDPA, 10-HDPA, 11-HDPA, 13-HDPA, 14-HDPA, 16-HDPA, 17-HDPA, 20-HDPA, 16,20-diHDPA (HDPA is the abbreviation of hydroxy-DPA and H(p)-DPA is the abbreviation of hydroxy(peroxy)-DPA).

[0011] In one or more embodiments, the reagent for detecting DPA carries a detectable label, such as a detectable label selected from the group consisting of a radioisotope, a fluorophore, a chemiluminescent moiety, an enzyme, an enzyme substrate, an enzyme cofactor, an enzyme inhibitor, a fuel, a metal ion, or a ligand (e.g., biotin or a hapten).

[0012] In one or more embodiments, the reagent for detecting DPA is used for qualitatively or quantitatively detecting DPA in a subject or a sample thereof. In one or more embodiments, the subject is a mammal suffering from or suspected of suffering from constipation or at risk of suffering from constipation.

[0013] In one or more embodiments, the sample is feces, preferably mammalian feces.

[0014] In one or more embodiments, compared to the control level, the DPA content in the subject sample is increased, indicating that the subject suffers from or is suspected of suffering from constipation, or has a risk of suffering from constipation. Further, compared to the control level, the DPA content in the subject sample is increased, and the subject sample contains PIB or the PIB content is increased, indicating that the subject suffers from or is suspected of suffering from constipation, or has a risk of suffering from constipation.

[0015] In some embodiments, the control level is the amount of DPA in the stool of a subject who does not suffer from constipation.

[0016] In some embodiments, the control level is less than or equal to 0.07±0.01 ng / mg, preferably less than or equal to 0.1±0.01 ng / mg.

[0017] In one or more embodiments, the detection kit further comprises one or more substances selected from the group consisting of: a container, a buffer, an adjuvant, a solvent, a negative control substance, a positive control substance, and instructions for use.

[0018] In one or more embodiments, the reagents for detecting DPA include a DPA standard.

[0019] In one or more embodiments, the reagents for detecting PIB include primers or probes that specifically amplify marker nucleic acid sequences of PIB, antibodies or ligands that specifically bind to surface proteins or secretions of PIB. In one or more embodiments, the marker nucleic acid sequence is SNP.

[0020] The present invention also provides a method for extracting DPA from feces, the method comprising: treating a feces sample obtained from a subject with an extraction solution, wherein the extraction solution comprises alcohol, dichloromethane or chloroform and water.

[0021] In one or more embodiments, the alcohol comprises methanol, ethanol, or propanol.

[0022] In one or more embodiments, the extract comprises methanol, dichloromethane, and water.

[0023] In one or more embodiments, the volume ratio of alcohol, dichloromethane or chloroform to water is 1-10:1-5:1-10, such as 2-6:1-4:2-6, preferably 3:2:3.

[0024] In another aspect of the present application, a product for diagnosing constipation, predicting the risk of occurrence or progression of constipation in a subject, or evaluating the prognosis of constipation, or providing a treatment plan is provided, such as a detection kit or device, which comprises a reagent for detecting DPA in a subject's sample, and optionally also comprises a reagent for detecting PIB in a subject's sample.

[0025] In one or more embodiments, the sample comprises feces.

[0026] In one or more embodiments, the constipation is refractory constipation, preferably refractory constipation caused by PIB.

[0027] In one or more embodiments, the reagent for detecting DPA is a reagent for detecting DPA using one or more detection methods selected from the following group: chromatography (e.g., gas chromatography, HPLC), mass spectrometry, gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, colorimetry, enzyme-linked immunosorbent assay, ultra-high performance liquid chromatography, capillary gas chromatography, and the like.

[0028] In one or more embodiments, the reagent for detecting DPA includes a reagent for extracting DPA from feces. In one or more embodiments, the reagent for detecting DPA includes one or more of methanol, dichloromethane, and water. Preferably, the reagent for detecting DPA includes methanol, dichloromethane, and water mixed in a certain proportion.

[0029] In one or more embodiments, the reagent for detecting DPA includes a substance specific to DPA or its derivatives. In one or more embodiments, the DPA derivative is an in vivo metabolite of DPA.

[0030] In one or more embodiments, the reagent for detecting DPA carries a detectable label, such as a detectable label selected from the group consisting of a radioisotope, a fluorophore, a chemiluminescent moiety, an enzyme, an enzyme substrate, an enzyme cofactor, an enzyme inhibitor, a fuel, a metal ion, or a ligand (e.g., biotin or a hapten).

[0031] In one or more embodiments, the reagents for detecting PIB include primers or probes that specifically amplify marker nucleic acid sequences of PIB, antibodies or ligands that specifically bind to surface proteins or secretions of PIB. In one or more embodiments, the marker nucleic acid sequence is SNP.

[0032] In one or more embodiments, the product is a device, comprising a memory, a processor, and a computer program stored in the memory and operable to run on the processor, wherein when the processor executes the program, the following steps are implemented:

[0033] a) detecting DPA and optionally PIB in a subject sample, and

[0034] b) diagnosing constipation, predicting the risk of occurrence or progression of constipation in a subject, or evaluating the prognosis of constipation, or providing a treatment plan based on the result of step a).

[0035] In one or more embodiments, step a) comprises: a1) detecting DPA in a sample from a subject, and a2) determining whether the abundance of DPA detected in step a1) is higher than a control level.

[0036] In one or more embodiments, step a) includes: a1) detecting DPA and PIB in the subject sample, and a2) determining whether the abundance of DPA detected in step a1) is higher than the control level, and whether PIB is present or its content is higher than the control.

[0037] In one or more embodiments, an increase in the level of DPA in a subject's sample compared to a control level indicates that the subject suffers from or is suspected of suffering from constipation, or is at risk of suffering from constipation, or has a poor prognosis for constipation, or indicates that a change in treatment regimen is needed.

[0038] In one or more embodiments, an increased level of DPA in a subject's sample compared to a control level and the presence of PIB or an increased level of PIB in the subject's sample indicate that the subject suffers from or is suspected of suffering from constipation, or is at risk of suffering from constipation, or has a poor prognosis for constipation, or indicates that a change in treatment regimen is needed.

[0039] In some embodiments, the control level is the DPA content in a sample of a subject not suffering from constipation. In some embodiments, the control level is less than or equal to 2.26±0.43 ng / mg, such as less than or equal to 0.07±0.01 ng / mg, preferably less than or equal to 0.05±0.01 ng / mg, more preferably less than or equal to 0.02±0.01 ng / mg.

[0040] In some embodiments, the characteristics involved in the reagents used in the products of the present application and the performance of the products are as defined or described herein.

[0041] Another aspect of the present invention provides the use of antibiotics or their derivatives in the preparation of drugs for treating constipation, wherein the antibiotics include but are not limited to quinolone antibiotics, carbapenem antibiotics, mitoflavin, bleomycin sulfate, and sulfonamide enhancers.

[0042] In one or more embodiments, the derivative is a hydrate.

[0043] In one or more embodiments, the constipation is refractory constipation, preferably refractory constipation caused by PIB.

[0044] In one or more embodiments, the quinolones include but are not limited to levofloxacin, balofloxacin, besifloxacin hydrochloride, enrofloxacin, moxifloxacin, pazufloxacin mesylate, gatifloxacin, sparfloxacin, sarafloxacin hydrochloride, norfloxacin, ciprofloxacin, levofloxacin, enoxacin, lomefloxacin, sitafloxacin, difloxacin hydrochloride. Preferred are norfloxacin, sitafloxacin hydrate, sarafloxacin hydrochloride, besifloxacin hydrochloride, levofloxacin hemihydrate, lomefloxacin hydrochloride, sparfloxacin, lomefloxacin, levofloxacin, and gatifloxacin.

[0045] In one or more embodiments, the carbapenems include but are not limited to meropenem, doripenem, biapenem, ertapenem sodium; preferably doripenem.

[0046] Another aspect of the present invention provides use of a bacteriophage in preparing a drug for treating constipation, wherein the bacteriophage is a T4 bacteriophage.

[0047] In one or more embodiments, the constipation is refractory constipation, preferably refractory constipation caused by PIB.

[0048] In one or more embodiments, the bacteriophage is a Myoviridae T4 phage.

[0049] In one or more embodiments, the bacteriophage can be applied to enema, gavage, or injection. The preferred administration route is oral administration, enema, or injection. The preferred administration amount is 1×10 9 phages / times / person.

[0050] On the other hand, the present invention also provides a pharmaceutical composition for treating constipation, comprising a pharmaceutically acceptable excipient and: (1) an antibiotic or a derivative thereof, and / or (2) a bacteriophage, wherein the antibiotic includes but is not limited to quinolone antibiotics, carbapenem antibiotics, mitoflavin, bleomycin sulfate, and sulfonamide enhancers, and the bacteriophage is T4 phage.

[0051] In one or more embodiments, the derivative is a hydrate.

[0052] In one or more embodiments, the constipation is refractory constipation, preferably refractory constipation caused by PIB.

[0053] In one or more embodiments, the quinolones include but are not limited to levofloxacin, balofloxacin, besifloxacin hydrochloride, enrofloxacin, moxifloxacin, pazufloxacin mesylate, gatifloxacin, sparfloxacin, sarafloxacin hydrochloride, norfloxacin, ciprofloxacin, levofloxacin, enoxacin, lomefloxacin, sitafloxacin, difloxacin hydrochloride. Preferred are norfloxacin, sitafloxacin hydrate, sarafloxacin hydrochloride, besifloxacin hydrochloride, levofloxacin hemihydrate, lomefloxacin hydrochloride, sparfloxacin, lomefloxacin, levofloxacin, and gatifloxacin.

[0054] In one or more embodiments, the carbapenems include but are not limited to meropenem, doripenem, biapenem, ertapenem sodium; preferably doripenem.

[0055] In one or more embodiments, the bacteriophage is a Myoviridae T4 phage. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 : The chemical substance secreted by PIB is docosapentaenoic acid (DPA). Figure 1 , A and B: Inhibitory activity of polar and non-polar phase substances on intestinal contraction after extraction of PIB culture supernatant with organic solvent (n=3). Figure 1 , C: Elution peak pattern of non-polar substances by HPLC (C18 column) chromatography analysis, the arrow indicates the additional peak appearing in the PIB supernatant. Figure 1 , D: Additional peaks of PIB were collected and their activity in inhibiting intestinal contraction was determined. Figure 1 , E: The active peak molecular weight was determined by LC-MASS. Figure 1 , F: molecular weight of each fragment of the active peak. Figure 1 , G: Inhibitory effect of pure DPA on intestinal contraction. Figure 1 , H: Dose-effect of pure DPA in inhibiting intestinal contraction. **p<0.01.

[0057] Figure 2 : Colonic administration was used to determine the activity of DPA on colonic excretion function.

[0058] Figure 3 : DPA content in feces of constipated mice.

[0059] Figure 4 : The DPA content in feces of patients with intractable chronic constipation is higher. Figure 4 , A and B: HPLC analysis of feces of people with intractable chronic constipation (A) and normal healthy people (B), the arrows point to the characteristic peak of DPA. Figure 4 , C: Abundance of characteristic peaks in HPLC experiment of 0.03125ng DPA standard. Figure 4, D: Comparison of fecal DPA content between people with intractable chronic constipation and normal healthy people. **p<0.01.

[0060] Figure 5 : Determination of PIB bacteria content in feces of PIB-constipated mice after norfloxacin treatment.

[0061] Figure 6 : Phage restriction enzyme map.

[0062] Figure 7 : PCR test results of T4 g23.

[0063] Figure 8 : In vitro bactericidal effect of bacteriophages.

[0064] Fig. 9 : In vivo therapeutic effect of phage cocktail therapy on PIB constipation model. DETAILED DESCRIPTION

[0065] Previous studies have shown that PIB bacteria (CGMCC 20603) is a pathogenic bacterium of intractable chronic constipation, but its pathogenic mechanism is unclear. The inventors speculate that PIB may secrete a substance that can directly inhibit the rhythmic contraction of the intestine. This substance may be a protein or an organic substance. The present invention separates and identifies the substance, and systematically studies the pathogenic substances of PIB bacteria and their correlation with the disease.

[0066] Specifically, the present invention provides an application of a DPA detection reagent in the preparation of a medicine box for preventing or treating intractable chronic constipation. The application directly determines the specific metabolite of PIB in feces, docosapentaenoic acid (DPA), so as to determine whether PIB infection and intractable constipation occur.

[0067] DPA and its detection

[0068] Previous studies have shown that PIB bacteria are pathogenic bacteria of intractable chronic constipation, and the DPA secreted by them can directly inhibit rhythmic intestinal contractions. The inventors also found that when DPA abundance and PIB were analyzed together, the correlation between both and the disease was very high. DPA is a long-chain unsaturated fatty acid that is often found in deep-sea fish, seals, seaweed and other organisms, and is an important component of deep-sea fish oil. Existing studies have shown that DPA and its similar long-chain unsaturated fatty acids DHA and EPA, as nutritional health foods, have obvious beneficial effects on improving cardiovascular diseases, neurological diseases, etc., but there are no reports on the effects of these fatty acids on the intestines.

[0069] The present invention is the first to discover the relationship between DPA in feces and constipation. The abundance of DPA can be detected by chromatography (such as HPLC), mass spectrometry, gas chromatography, gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, colorimetry, enzyme-linked immunosorbent assay, ultra-high performance liquid chromatography, capillary gas chromatography, etc.

[0070] As used herein, the terms "detection substance", "detection reagent" or "reagent for detecting DPA" are used interchangeably and include any substance used to detect DPA or its derivatives.

[0071] For example, the reagent for detecting DPA is a reagent for detecting DPA or its derivatives using one or more detection methods selected from the group consisting of chromatography (e.g., gas chromatography, HPLC), mass spectrometry, gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, colorimetry, enzyme-linked immunosorbent assay, ultra-high performance liquid chromatography, capillary gas chromatography, etc. Such a reagent may be a solvent for DPA, such as dichloromethane and / or chloroform.

[0072] Alternatively, the reagent for detecting DPA may also include a reagent for extracting DPA from feces. In one or more embodiments, the reagent for detecting DPA includes one or more of methanol, dichloromethane, and water. Preferably, the reagent for detecting DPA includes methanol, dichloromethane, and water mixed in a certain proportion.

[0073] For another example, the reagent for detecting DPA includes a substance that is specific to DPA or its derivatives. The types and methods of use of these substances are within the scope of conventional knowledge in the art. In order to facilitate detection, the detection reagent of the present invention may also carry a detectable label, and the detectable label includes but is not limited to: a radioisotope, a fluorophore, a chemiluminescent moiety, an enzyme, an enzyme substrate, an enzyme cofactor, an enzyme inhibitor, a dye, a metal ion, a ligand (e.g., biotin or a hapten), etc.

[0074] Herein, DPA derivatives refer to the in vivo metabolites of DPA, including: 17-oxo-DPA, 17S-HDPA, 13-oxo-DPA, 7,12,13R-triHDPA, 7,13R-diHDPA, 7,13R,20-triHDPA, 7,8,13R-triHDPA, 13R-HDPA, 13-F3-IsoP-DPA, 7-F3-IsoP-DPA, 10-F3-IsoP-DPA, 20-F3-IsoP-DPA, 14-F3-IsoP-DPA, 17-F3-IsoP-DPA, 14S-H(p)-DPA, 13,1 4S-epoxy-DPA, 7S,14S-dihydroxy-8E,10E,12Z,16Z,19Z-n-3DPA, 13,14-dihydroxy-7Z,9,11,16Z,19Z-n-3DPA, 14,21-dihydroxy-7Z,10Z, 12E,16Z,19Z-n-3DPA, 17S-H(p)-DPA, 16S,17S-epoxy-DPA, 10,17S-diHDPA, 16,17S-diH-DPA, 10R,17S-dihydroxy-7Z,11E,13E,15Z,19Zn-3 DPA, 16,17R-dihydroxy-7Z,10,13,14,19Zn-3 DPA, 7,17S-diH(p)-DPA, 7,8,17S-trihydroxy-9,11,13,15E,19Z-n-3DPA, 7,16,17-trihydroxy-8,10,12,14E,19Z-n-3DPA, 7S,17S-dihydroxydocosae-8E,10Z,13Z ,15E,19Z-n-3DPA, 7,17-diHDPA, 10,17S-diHDPA, 11-HDPA, 17-HDPA, 8,14-diHDPA, 10-HDPA, 11-HDPA, 13-HDPA, 14-HDPA, 16-HDPA, 17-HDPA, 20-HDPA, 16,20-diHDPA. Wherein, HDPA is the abbreviation of hydroxy-DPA, and H(p)-DPA is the abbreviation of hydroxy(peroxy)-DPA. Generally, the reagent for detecting DPA may also include a reagent for extracting the above derivatives from feces.

[0075] Typically, before DPA detection, the sample (e.g., feces) may be pretreated to remove substances that may affect the detection. Preferably, the sample is a fecal sample, and the fecal sample may be derived from fresh human feces, frozen feces, or feces diluted with liquid. Therefore, the present invention also provides a method for extracting DPA from feces, the method comprising: treating a fecal sample obtained from a subject with a pretreatment liquid extract, the extract comprising alcohol, dichloromethane or chloroform and water. The volume ratio of alcohol, dichloromethane or chloroform to water is 1 to 10: 1 to 5: 1 to 10, for example 2 to 6: 1 to 4: 2 to 6, preferably 3: 2: 3. The alcohol includes methanol, ethanol or propanol. In an exemplary embodiment, the extract comprises methanol, dichloromethane and water in a volume ratio of 3: 2: 3.

[0076] Methods for detecting DPA are common knowledge in the art. Exemplary DPA detection methods include HPLC chromatography, liquid chromatography-mass spectrometry, gas chromatography-mass spectrometry, enzyme-linked immunosorbent assay, colorimetry, and the like.

[0077] HPLC chromatography: After the pre-treated sample is dissolved, it is injected and eluted. The chromatographic column is usually a C18 column, and the mobile phase is generally acetonitrile / methanol / ammonium formate / isopropanol. After step-by-step elution, the abundance of the DPA characteristic peak is analyzed and compared with the abundance of the known concentration standard for quantification.

[0078] Liquid chromatography-mass spectrometry: 50 mg of feces was collected, and an organic extraction reagent (methanol / dichloromethane / water = 420 uL: 280 uL: 420 uL) was added. The organic phase was collected, dried, dissolved, and the sample was prepared. The molecular weight and structure were then determined by mass spectrometry, and the results were compared with the known concentration of DPA standard.

[0079] Gas chromatography-mass spectrometry: Collect 50 mg of feces, methylate with potassium hydroxide and methanol, extract unsaturated fatty acid methyl esters with chloroform or other organic solvents, and dry to prepare samples. Load the samples into gas chromatography or gas chromatography-mass spectrometry to analyze the peak characteristics of DPA methyl ester.

[0080] Enzyme-linked immunosorbent assay: Use anti-DPA or long-chain unsaturated fatty acid antibody as the primary antibody to coat the ELISA plate, add the sample to be tested (feces after pretreatment), incubate, wash the plate, add the enzyme-linked secondary antibody, wash the plate, and develop color.

[0081] Colorimetric method: Mix potassium iodide solution with the stool sample, and after the addition reaction is completed, add 1% starch solution for color development.

[0082] The reagents involved in the above-mentioned DPA detection method are included in the detection kit of the present invention.

[0083] Testing products

[0084] Also provided herein is a product for detecting refractory chronic constipation in a patient, for example, a kit, which comprises a reagent for detecting DPA and optionally further comprises a reagent for detecting PIB.

[0085] According to the needs of the detection method used, appropriate DPA detection reagents can be selected and made into products starting from the detection method used, such as kits. Those skilled in the art can adjust and change the detection method and the reagents contained in the product according to actual conditions and needs.

[0086] The kit may also include one or more substances selected from the following group: containers, instructions for use, positive controls, negative controls, buffers, solvents or adjuvants, such as solutions for suspending or fixing cells, detectable labels or markers, and reagents for removing impurities that affect the test results from the sample, such as proteins, nucleic acids, etc.

[0087] In one example, the present invention provides a detection kit suitable for detecting DPA and / or PIB in a biological sample by chromatography. The detection kit may include: reagents required for chromatographic analysis to detect DPA (methanol, acetonitrile, etc.) and one or more reagents for detecting PIB; and optional containers containing the above reagents and instructions for use.

[0088] The reagent for detecting PIB includes a primer or probe for a marker nucleic acid sequence of specific amplification PIB, an antibody or ligand that specifically binds to a surface protein or secretion of PIB. In one or more embodiments, the marker nucleic acid sequence is a SNP. For examples of the primer or probe, see CN202011023676.4, which is incorporated herein by reference in its entirety. For example, the product of amplifying the genome of Shigella bacteria (CGMCC 20603) by the oligonucleotide primers comprises: (1) nucleotides located at positions in the genome of the bacteria corresponding to one or more positions selected from the following positions of the genome of the bacterial strain with accession number CGMCC 20603: PIB2013.g.331, PIB2324.g.562, PIB2324.g.601, PIB2629.g.1126, PIB3156.g.2396, (2) nucleotides selected from the following one or more positions: the 21st nucleotide of the genomic amplification fragment using SEQ ID NOs: 9 and 10 as primers; the 21st nucleotide of the genomic amplification fragment using SEQ ID NOs: 11 and 12 as primers; the 21st nucleotide of the genomic amplification fragment using SEQ ID NOs: 13 and 14 as primers; the 20th nucleotide of the genomic amplification fragment using SEQ ID NOs: 15 and 16 as primers; NO:17 and 18 as primers for the 21st nucleotide of the genomic amplification fragment; (3) any one or more sequences selected from SEQ ID NO:2-6 or fragments thereof having the nucleotides described in (2) or variants thereof having at least 98% sequence identity therewith, or (4) the 16S rDNA sequence shown in SEQ ID NO:1 or a sequence having at least 98% sequence identity therewith. In one or more embodiments, the amplification product of the oligonucleotide primers comprises any sequence described in SEQ ID NO:1-6. In one or more embodiments, the oligonucleotide primers are selected from any one or more of SEQ ID NO:7-18.

[0089] The detection kit in this article may also be accompanied by an instruction manual for the kit, which records how to use the kit for detection, how to diagnose intractable chronic constipation, and how to choose a treatment plan.

[0090] diagnosis

[0091] The inventors found that there is a high concentration of DPA in the feces of patients with intractable constipation, and the DPA content in the feces of positive patients is 2.26±0.43ng / mg (0.07±0.01ng / mg in the normal population). Therefore, the detection of DPA in feces can diagnose intractable constipation, with a positive rate of 44.12%. In addition, the simultaneous determination of DPA and PIB can increase the positive detection rate to 75%.

[0092] The present invention provides the use of (1) a reagent for detecting DPA and (2) a reagent for detecting PIB in preparing a kit for diagnosing intractable constipation. The reagent for detecting DPA and the reagent for detecting PIB are as described in any embodiment of the present invention.

[0093] The present invention also provides a method for diagnosing intractable constipation, comprising detecting DPA in the feces of a subject, and optionally further comprising detecting PIB in the feces of the subject. Preferably, if the DPA content in the feces of the subject is greater than or equal to 0.07±0.01 ng / mg (e.g., greater than or equal to 0.1 ng / mg, greater than or equal to 0.5 ng / mg, greater than or equal to 1.0 ng / mg, greater than or equal to 1.5 ng / mg, greater than or equal to 2.0 ng / mg, greater than or equal to 2.26±0.43 ng / mg), the subject is a potential patient with intractable constipation.

[0094] Antibiotics or bacteriophages

[0095] The present invention also provides the use of antibiotics or bacteriophages in the preparation of drugs for treating intractable constipation. The inventors found that when PIB-sensitive antibiotics or bacteriophages are used to treat intractable constipation, DPA in feces is significantly reduced.

[0096] The invention obtains PIB-sensitive antibiotics by screening the existing anti-biological drug library, and provides a drug formula and a treatment method for clearing PIB and treating intractable constipation.

[0097] As used herein, the antibiotics include, but are not limited to, quinolone antibiotics, carbapenem antibiotics, bleomycin sulfate, mitomycin, and sulfonamide potentiators. The quinolones may include levofloxacin, balofloxacin, besifloxacin hydrochloride, enrofloxacin, moxifloxacin, pazufloxacin mesylate, gatifloxacin, sparfloxacin, sarafloxacin hydrochloride, norfloxacin, ciprofloxacin, levofloxacin, enoxacin, lomefloxacin, sitafloxacin, difloxacin hydrochloride, etc. The carbapenems may include meropenem, doripenem, biapenem, and ertapenem sodium.

[0098] As used herein, among the quinolones, norfloxacin is the most sensitive, and the order is: norfloxacin>sitafloxacin hydrate>sarafloxacin hydrochloride>besifloxacin hydrochloride>levofloxacin hemihydrate>lomefloxacin hydrochloride>sparfloxacin>lomefloxacin>levofloxacin>gatifloxacin>ciprofloxacin hydrochloride monohydrate>moxifloxacin>difloxacin hydrochloride>danofloxacin mesylate>tosufloxacin p-toluenesulfonate hydrate>enoxacin sesquihydrate>balofloxacin. As used herein, among the carbapenems, PIB is most sensitive to doripenem.

[0099] PIB is a pathogenic bacterium that causes intractable chronic constipation. Eliminating PIB from the body is an ideal method for treating the disease. Bacteriophages are bacterial viruses that can specifically recognize specific bacteria and invade the bacteria to produce bacterial lytic enzymes and lyse the bacteria. Bacteriophages can eliminate specific bacteria with high efficiency and specificity. They are very safe and efficient antibacterial viruses and have been widely used in many countries. Clinical trials have been conducted in the United States and will soon be put on the market. This article screened PIB-specific phages and conducted biological identification to screen out phages that are sensitive to PIB.

[0100] As used herein, the bacteriophage is a T4 phage, such as a Myoviridae T4 phage.

[0101] As used herein, the phage is a PIB-specific phage, which can be obtained by screening the following steps: the phage stock solution obtained above is identified by a double-layer plate method, and the double-layer plate method includes two layers of LB solid culture medium with different concentrations, the lower layer is a 1.5% agar LB culture medium, which is mainly used to support nutrition; the upper layer is a 0.5% agar LB culture medium, which is conducive to the growth and spread of host bacteria and phage. This method is also more convenient for the observation of plaques. The specific operation is: first prepare a 1.5% lower agar culture medium, mix 100μl of logarithmic phase bacterial solution and 100μl of phage stock solution evenly, let it stand at room temperature for about 10min, quickly add it to the 0.5% upper culture medium preheated to 48°C, quickly mix it and pour it into the lower culture medium, evenly spread it on the lower plate, let it stand at room temperature for 20min, and place it at 37°C to observe whether plaques are formed. Pick the plaques on the plate, add them to LB medium, then add PIB bacterial solution at a ratio of 5%, and culture at 37℃, 200rpm shaking for 6-8h. After filtration and centrifugation, use the double-layer plate method to coat the plate, and repeat the above operation two to three times until a round transparent plaque with uniform size and complete edges is observed. Pick the plaque with a sterilized gun tip, and add 5% bacterial solution to LB medium at the same time, culture and amplify overnight at 37℃, 200rpm, shaker, 12000rpm, 5min, 4℃ centrifuge, filter with a 0.22μm filter membrane, add to 10% SM buffer, and store in a -80℃ refrigerator.

[0102] As used herein, the phage therapy regimen and efficacy analysis may be to prepare cocktails of three phages, phage1, 2, and 3, by co-shaking culturing of phages and PIB. After thorough mixing, each mouse in the Phage treatment group is intragastrically administered with 100 μl of phage cocktail each time.

[0103] Pharmaceutical composition

[0104] The antibiotics and / or bacteriophages described herein can be used for scientific research or for preparing therapeutic drugs for corresponding cancers. Herein, "pharmaceutical composition" refers to a composition for administration to an individual and encompasses a composition of antibiotics and / or bacteriophages for treatment. The pharmaceutical composition of the present invention may also contain a pharmaceutically acceptable excipient.

[0105] As used herein, a "pharmaceutically acceptable" ingredient is a substance that is suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and reaction), ie, at a reasonable benefit / risk ratio.

[0106] The pharmaceutical composition of the present invention contains a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable adjuvant. The term "pharmaceutically acceptable adjuvant" refers to an adjuvant used for the administration of a therapeutic agent, including various excipients and diluents. Such adjuvants include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Usually, the pharmaceutical preparation matches the mode of administration, and the dosage form of the pharmaceutical composition of the present invention is an injection, an oral preparation (tablet, capsule, oral liquid), a transdermal agent, and a sustained-release agent. For example, it is prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutically acceptable adjuvant of the present invention includes adjuvants suitable for delivery of antibiotics and / or bacteriophages. The pharmaceutical composition is preferably manufactured under sterile conditions. Compositions containing these adjuvants can be prepared by well-known conventional methods.

[0107] The composition of the present invention can be administered topically or systemically. In certain embodiments, the composition provided by the present invention can be administered orally or parenterally, such as intravenously, intra-arterially, intrathecally, subdermally or intramuscularly. Parenteral formulations include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous vehicles include water, alcoholic solutions / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral carriers include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution or fixed oil. Intravenous carriers include liquid and nutritional supplements, electrolyte supplements (such as those based on Ringer's dextrose), etc. Preservatives and other additives may also be present, such as antimicrobial agents, antioxidants, chelating agents and inert gases, etc. Antibiotics described herein can be administered orally, intramuscularly or intravenously. The bacteriophage described herein can be administered orally, by enema, intragastric gavage, intramuscularly or intravenously.

[0108] The pharmaceutical compositions herein can be administered to a subject at an appropriate dose. The active ingredients described herein may vary depending on the mode of administration and the severity of the disease to be treated. The dosage regimen may be determined by the attending physician and clinical factors. As is well known in the medical field, the dosage for any one patient depends on a variety of factors, including the patient's size, body surface area, age, specific compound to be administered, sex, time and route of administration, overall health, and other drugs administered simultaneously. For example, depending on the urgency of the treatment condition, several divided doses may be administered daily, or the dose may be reduced proportionally. Exemplarily, the dosage of the bacteriophage described herein is 1x10 9 phages / times / person.

[0109] As used herein, the term "subject" or "patient" is a mammal, such as a mouse, rabbit, human, etc.

[0110] As used herein, the term "control level" refers to the level of DPA or PIB used as a reference, including but not limited to: the level of DPA or PIB in a non-constipated subject, the level of DPA or PIB measured in a non-constipated normal biological sample of the same subject, a statistically determined population standard level, or a standardized level. In one or more embodiments, the control level is determined by the following steps: (1) before treatment, detecting the level of DPA or PIB in a biological sample from a patient, (2) the level of DPA or PIB in the biological sample of the patient after treatment, (3) correlating the response of the patient after treatment with the level of DPA or PIB determined in step (2), and then comparing the biological level of DPA or PIB in (1) to determine the control level.

[0111] The compounds of the present invention can be obtained from commercial sources, or can be synthesized according to disclosed methods or with reference to disclosed methods. Other aspects of the present invention are obvious to those skilled in the art due to the disclosure of this article. The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Specific examples, materials, quantities and operating procedures will be more broadly interpreted according to the scope and spirit of the present invention as described herein. The experimental methods for which specific conditions are not indicated in the following examples are generally based on conventional conditions such as the conditions described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.

[0112] Exemplary embodiments

[0113] Sample source: PIB is an intestinal bacterium that has the potential to infect multiple tissues. DPA is a characteristic metabolite of PIB and therefore may be present in multiple tissues. Feces is the main source of DPA produced by PIB, and the sample source is preferably feces. Fecal samples can come from fresh human feces, frozen feces, or feces diluted with liquid.

[0114] Sample processing: Fecal samples are preferably selected for pretreatment, which is characterized by mixing a certain amount of feces with an organic extraction reagent in a certain proportion to extract organic matter from the organic phase.

[0115] HPLC purification and separation of DPA: The treated sample is separated and purified by HPLC, which is characterized by eluting with a carbon column (preferably a C18 column) and acetonitrile / methanol / ammonium formate / isopropanol mobile phase and collecting active peaks to prepare the sample.

[0116] LC-MASS (liquid chromatography-mass spectrometry) confirmed the molecular structure: the active peak was loaded for LC-MASS analysis to determine the substance peak with a molecular weight of 330 Daltons, the molecular weight of its fragments was determined again, and then the structure of the substance was extracted.

[0117] Preparation of fecal DPA detection kit: The kit contains at least the following components: fecal collection device, organic solvent, positive standard, negative control, DPA standard. The specific uses are as follows:

[0118] Stool collection devices: equipment and containers for patients to collect stool themselves

[0119] Organic solvents: used for sample extraction

[0120] Positive standard: Positive fecal extract

[0121] Standard product: DPA pure product

[0122] Negative control: negative fecal extract

[0123] Purpose of the PIB-DPA kit: The present invention is the first to discover the relationship between fecal DPA and constipation. Currently, gas chromatography or HPLC chromatography is often used to determine the content of DPA in food and fish oil to evaluate the nutritional components of food. DPA in food is mainly absorbed in the small intestine and does not reach the colon, so it will not affect the fecal DPA content and interfere with diagnosis. The fecal DPA detection method described in the present invention can be used to diagnose and screen for intractable constipation or PIB bacteria infection constipation.

[0124] The present invention will be further described below by way of specific examples. It should be understood that these examples are merely illustrative and are not intended to limit the scope of the present invention. The methods and reagents used in the examples are, unless otherwise stated, conventional methods and reagents in the art.

[0125] Example 1: It was found that the material basis for PIB bacteria to inhibit intestinal contraction is docosapentaenoic acid (DPA)

[0126] Materials and Methods

[0127] Animals: C57BL / 6 mice (half male and half female, 6-8 weeks old, weighing 20-22 grams)

[0128] PIB bacteria: CGMCC 20603, stored by Zhu Minsheng's laboratory (50% glycerol, -80°C). Main reagents: Docosapentaenoic acid (DPA) purchased from Aladdin, proteinase K purchased from Sigma.

[0129] Colon contraction tension measurement:

[0130] Extraction of organic matter secreted by PIB: Inoculate the frozen PIB bacteria in LB, culture at 37°C overnight (12-16 hours), and collect the culture supernatant; at the same time, inoculate DH5a Escherichia coli in LB, culture at 37°C overnight (12-16 hours), and collect the culture supernatant to obtain the control supernatant. Divide 15ml of supernatant into 10 tubes with extraction solution and drain with SpeedVac instrument (Thermofisher). Dissolve it fully with pre-cooled methanol solution (410ul methanol + 210ul water), then add 280ul dichloromethane and 210ul water. After shaking and mixing, collect the non-polar phase by centrifugation and drain for later use.

[0131] HPLC purification of organic matter: The instrument is Agilent 1200HPLC. The chromatographic column is a C18 column (150 mm × 4.6 mm) from Yiliteng. The elution mobile phase is A: methanol / acetonitrile / 10 mM ammonium formate (1:1:1, v / v / v); B: acetonitrile / isopropanol (1:1, v / v). 20 tubes of eluent are collected and dried.

[0132] LCMS / MS: On-machine analysis: A hybrid quadrupole time-of-flight mass spectrometer (brand model: AB Sciex TripleTOF 4600instruments) was used, combined with a C18 column (Phenomenex Accucore C18, 150mm×2.1mm, 2.6μm) for separation, and the mobile phases were: Phase A: acetonitrile / methanol / 10mM ammonium formate (1:1:1, v / v / v); Phase B: acetonitrile / isopropanol (1:1, v / v), and the gradient elution order was: 0-1min, 20% B; 1-4min, 60% B; 4-10min, 70% B; 10-15min, 95% B; 15-20min, 95% B. The mass spectrometry analysis used DuoSpray Ion Source combined with negative ion mode, and the parameters were set as follows: auxiliary gas pressure, 55 psi; nebulizer gas pressure, 55 psi; air curtain gas, 35 psi; ion source temperature, 600 °C; voltage, 4500 V; potential energy, 80 eV, collision energy, 10 eV. Peakview software was used for data processing.

[0133] result

[0134] 1. PIB secretions are present in the organic phase

[0135] First, we need to rule out whether the active substance secreted by PIB is a protein. The culture supernatant of PIB was treated with proteinase K (37°C, 60 minutes). After treatment, the supernatant still had the activity of inhibiting intestinal contraction, so the active product could not be a protein. When the culture supernatant of PIB was treated with dichloromethane / methanol / water extract, the organic phase material was taken for activity measurement. The results showed that the organic phase material in the culture supernatant of PIB had obvious activity of inhibiting intestinal contraction, while the organic phase material in the culture supernatant of the control bacteria (DH5a) was inactive. Figure 1 As shown in A and B.

[0136] 2. The active substance of PIB is docosapentaenoic acid

[0137] To determine the chemical structure of the active substance in the PIB supernatant, the organic phase mixture was analyzed by liquid chromatography-mass spectrometry. When the samples were subjected to liquid chromatography, a specific peak appeared in the PIB culture supernatant sample at the end of the elution period, while the control sample did not ( Figure 1 C). The peak samples were collected and their biological activity was determined. The results showed that this peak had significant biological activity, while other elution peaks had no obvious intestinal contraction inhibitory activity ( Figure 1D), indicating that the active peak is a characteristic active peak of PIB culture supernatant. The active peak was further analyzed by mass spectrometry, and the molecular weight of the active substance in the peak was 329.25 Daltons; the fragments were analyzed and it was found that the substance contained two fragments with molecular weights of 285.26 Daltons and 59.01 Daltons, respectively. The above data were entered into the Preview database, and it was found that docosapentaenoic acid was the only candidate molecule.

[0138] Conclusion: This study first found that the enteroinhibitory active substance secreted by PIB is DPA.

[0139] Example 2: Docosapentaenoic acid can inhibit intestinal contraction and intestinal transit rate

[0140] DPA is a long-chain unsaturated fatty acid that is often found in deep-sea fish, seals, seaweed and other organisms, and is an important component of deep-sea fish oil. Existing studies have shown that DPA and its similar long-chain unsaturated fatty acids DHA and EPA, as nutritional health foods, have obvious beneficial effects on improving cardiovascular diseases, neurological diseases, etc., but there are no reports on the effects of these fatty acids on the intestine. The above results suggest that the material basis for PIB to inhibit intestinal contraction may be DPA, but it is unclear whether DPA has a direct effect of inhibiting intestinal contraction. This embodiment intends to determine whether DPA has the function of inhibiting intestinal contraction through in vivo studies.

[0141] Materials and Methods

[0142] DPA: purchased from Aladdin

[0143] DPA administration and colon motility measurement: DPA was introduced into the colon through a catheter, and glass microspheres were added at the same time, and the discharge time of the microspheres was observed.

[0144] result:

[0145] Commercial DPA pure product has in vivo activity of directly inhibiting colon contraction: To confirm whether DPA has intestinal function in vivo, DPA was infused into the colon and then the colon transit time was measured. The results showed that after intestinal infusion of DPA, the intestinal transit time was extended by about 150% ( Figure 2 ). The above results confirmed that DPA has the function of inhibiting intestinal contraction and intestinal transit and is a pathogenic molecule of PIB.

[0146] Conclusion: This study first found that DPA can significantly inhibit intestinal contraction and reduce intestinal motility, thereby leading to constipation.

[0147] Example 3: PIB constipation animal phenotype is correlated with DPA content in feces

[0148] The results of Examples 1 and 2 indicate that DPA is a pathogenic molecule of PIB, but it is unclear whether there is a correlation between the DPA content in feces and the constipation phenotype. To this end, the present invention analyzed the DPA content in feces of PIB constipation mice and its correlation with PIB infection by HPLC.

[0149] Materials and Methods

[0150] PIB bacteria: preserved by Zhu Minsheng Laboratory, Nanjing University

[0151] Animals: C57BL / 6 mice (half male and half female, 6-8 weeks old, weighing 20-22 g)

[0152] LC-MASS analysis of fecal DPA: 50 mg of mouse feces was collected, organic matter extraction reagent (methanol / dichloromethane / water) was added, the organic phase was collected, dried, dissolved, and prepared for HPLC loading, and then the molecular weight and structure were determined by mass spectrometry.

[0153] Experimental results:

[0154] In this example, feces of 5 PIB constipation mice, 5 C57BL / 6 control mice, and 5 phage-treated constipation mice were collected. The content of DPA was analyzed by LC-MASS. The results showed that the signal intensity of DPA in the feces of PIB constipation mice was significantly higher than that of the control group and the phage-treated group (p<0.01) ( Figure 3 ). The DPA concentration was converted into absolute concentration. The results showed that the DPA content in the feces of PIB constipated mice was about 7.51ug / mg, while the DPA content in the feces of control mice was about 0.009ug / mg, and there was a very significant difference between the two groups.

[0155] in conclusion

[0156] The DPA content in the feces of PIB-constipated mice was increased, and there was a significant correlation between DPA and constipation.

[0157] Example 4: Significant increase in DPA content in feces of patients with intractable constipation

[0158] According to previous studies, about 56% of patients with intractable constipation have positive PIB bacteria in their feces, but the content of DPA and its correlation with the disease are still unclear. Clarifying this issue is an important basis for evaluating the diagnostic value of DPA. The purpose of this example is to determine the DPA content in human feces by HPLC and compare the difference between the constipation group and the normal group.

[0159] Materials and Methods

[0160] Population data: Cases of intractable constipation were obtained from Nanjing Jinling Hospital. All patients were diagnosed with constipation according to the Rome III criteria, all patients had slow-transmission constipation, and irritable bowel syndrome constipation was excluded. All patients were willing to undergo surgical treatment and had undergone surgical treatment. The normal population was selected from healthy volunteers who were in good health and had no complaints of constipation.

[0161] Fecal DPA determination: 50 mg of fresh feces were collected, 6 mg of dry feces were taken after drying, extracted with an alkane / alcohol organic solvent, the organic phase was collected, dried, redissolved with an organic solvent, and loaded on HPLC. The HPLC analysis steps were the same as those described in Example 1.

[0162] Fecal PIB determination: see CN202011023676.

[0163] result

[0164] This example analyzes the content of DPA in the feces of 68 patients with intractable constipation. When the sample contains DPA molecules, an obvious DPA characteristic peak can appear at a peak time of about 10.1-10.4 min (verified by LC-MASS), and the peak height is positively correlated with the DPA concentration. When the sample does not contain DPA, no characteristic peak appears ( Figure 4 A, 4B). Among the 68 cases, 30 were DPA positive, accounting for 44.12%, and 38 were DPA negative, accounting for 55.88%. At the same time, the PCR method was used to determine the PIB bacteria in the feces of 68 cases, and it was found that 38 were PIB positive (55.88%) and 30 were PIB negative (44.12%). DPA-positive patients are not consistent with PIB-positive patients. 21 of the 38 PIB+ feces were DPA negative, and 13 of the 30 PIB- feces were DPA positive. There were 17 samples that were both PIB+ and DPA+, accounting for 25% of the total samples. The same method was used to determine the feces of 30 normal people, and the results showed that all samples were PIB negative or DPA negative. The results show that testing PIB or DPA can detect 44-55% of patients with intractable constipation. If the two indicators are tested at the same time, the positive detection rate can be increased to 75%.

[0165] DPA cannot be detected in the feces of some PIB-positive patients. The possible reason is that DPA is not evenly distributed in the feces. When the sampling volume is small, the DPA-positive area may be lost. The inventor took small samples from different parts of the same fecal sample to measure DPA, and indeed observed that some samples had high DPA content, while some samples had low content or could not be detected. In addition, PIB could not be detected in some DPA-positive feces. The PCR method for detecting PIB requires that the bacteria in the feces are alive. If the sampling is not good or the transportation and storage are not good, the PIB bacteria will die and the PCR method will not be able to detect it. Therefore, the detection accuracy can be improved by simultaneously detecting the two indicators of PIB and DPA.

[0166] If the signal intensity of DPA measured above is converted into absolute concentration, the results show that the DPA content in the feces of DPA-positive patients is about 2.26±0.43ng / mg; while the DPA content in the feces of normal people or DPA-negative people is 0.07±0.01ng / mg.

[0167] in conclusion

[0168] There is a high concentration of DPA in the feces of patients with intractable constipation, with a positive rate of 44.12%. Determining the concentration of DPA in feces can diagnose intractable constipation. Simultaneous determination of DPA and PIB can increase the positive detection rate to 75%.

[0169] Example 5: Screening of PIB-sensitive antibiotics

[0170] Previous studies have found that PIB infection is an important cause of intractable constipation, but how to eliminate PIB in the body is still unclear. The purpose of this embodiment is to obtain PIB-sensitive antibiotics by screening existing antibiotic libraries, and to provide a drug formulation and treatment method for eliminating PIB and treating intractable constipation.

[0171] Materials and Methods

[0172] Antibiotic library: purchased from Selleck, containing a total of 308 antibiotics.

[0173] Screening method: Dilute each compound in the drug library 200 times with LB culture medium, and the final concentration is 3-80μg / mL. Take 10μl of antibiotics and add them to the PIB bacterial solution wells (96-well plate, 100ul bacterial solution per well). After culturing at 37℃ for 16 hours, measure OD600nm. The blank control is LB culture medium.

[0174] result

[0175] The screening results showed that most antibiotics were insensitive to PIB, and the more sensitive ones included: quinolones (levofloxacin, balofloxacin, besifloxacin hydrochloride, enrofloxacin, moxifloxacin, pazufloxacin mesylate, gatifloxacin, sparfloxacin, sarafloxacin hydrochloride, norfloxacin, ciprofloxacin, levofloxacin, enoxacin, lomefloxacin, sitafloxacin, difloxacin hydrochloride), carbapenems (meropenem, doripenem, biapenem, ertapenem sodium), mitomycin, bleomycin sulfate, and sulfonamide enhancers.

[0176] In order to further evaluate the antibacterial potency of sensitive drugs, the present invention dilutes these drugs in concentration gradients to determine their dosage effects. At the same time, DH5a Escherichia coli is used as a control bacterium to compare the sensitivity differences of the target antibiotics to different intestinal bacteria. The results show that among quinolones, norfloxacin is the most sensitive, and the order of arrangement is: norfloxacin>sitafloxacin hydrate>sarafloxacin hydrochloride>besifloxacin hydrochloride>levofloxacin hemihydrate>lomefloxacin hydrochloride>sparfloxacin>lomefloxacin>levofloxacin>gatifloxacin>ciprofloxacin hydrochloride monohydrate>moxifloxacin>difloxacin hydrochloride>danofloxacin mesylate>tosufloxacin p-toluenesulfonate hydrate>enoxacin sesquihydrate>balofloxacin. Among them, balofloxacin, besifloxacin hydrochloride, norfloxacin, lomefloxacin hydrochloride, enoxacin sesquihydrate, tosufloxacin p-toluenesulfonate hydrate, and lomefloxacin have weak bactericidal effects on DH5a Escherichia coli and have certain specificity for PIB bacteria. Among carbapenems, doripenem is the most sensitive to PIB. Bleomycin sulfate has almost no bactericidal effect on DH5a Escherichia coli. Specific data are shown in Table 1.

[0177] Table 1 MIC and MBC determination of antibiotics against PIB (mg / L) PIB and DH5α

[0178]

[0179]

[0180] Note: "None" means that the test bacteria are not sensitive to the drug at the gradient dilution concentration of the drug in this example.

[0181] in conclusion

[0182] Quinolones, carbapenems, and bleomycin sulfate are sensitive to PIB.

[0183] Example 6: Evaluation of the effect of norfloxacin antibiotic in clearing PIB in vivo

[0184] Example 5 Several antibiotics sensitive to PIB were screened, but it is still unclear whether these antibiotics can eliminate PIB in the body. The purpose of this example is to evaluate the ability of sensitive antibiotics to eliminate PIB in animals.

[0185] Materials and Methods

[0186] Animals: C57BL / 6 animals were purchased from Nanjing Jicui Yaokang Biotechnology Co., Ltd., and the PIB infection model was established in the laboratory.

[0187] Drug: Norfloxacin, purchased from CSPC Pharmaceutical Group Ouyi Pharmaceutical Co., Ltd.

[0188] Administration: Oral administration of norfloxacin (50 mg / kg), once a day for 7 consecutive days.

[0189] Effect observation: Culture animal feces, harvest bacterial fluid, and use PCR to detect PIB bacteria.

[0190] result

[0191] After one week of norfloxacin treatment of PIB constipation animals, the PIB bacteria in the animal feces began to turn negative. One month after stopping the drug, PIB was still not detected in the feces. Figure 5 ).

[0192] in conclusion

[0193] Norfloxacin can effectively eliminate PIB bacteria in the body and can be used to treat constipation.

[0194] Example 7. Screening and identification of PIB-specific phage

[0195] PIB is a pathogenic bacterium that causes intractable chronic constipation. Eliminating PIB in the body is an ideal method for treating the disease. Bacteriophages are bacterial viruses that can specifically identify specific bacteria and invade the bacteria to produce bacterial lytic enzymes and lyse bacteria. Bacteriophages can efficiently and specifically eliminate specific bacteria and are very safe and efficient antibacterial viruses. They have been widely used in many countries, and clinical trials have been carried out in the United States and will soon be put on the market. This embodiment will screen PIB-specific phages and conduct biological identification to lay the foundation for drug development.

[0196] Materials and Methods

[0197] Strains: PIB bacteria and K12 Escherichia coli provided by Zhu Minsheng's laboratory

[0198] Culture medium: LB liquid medium, 1.5% solid LB medium

[0199] Reagents: restriction endonucleases EcoRV, NcoI, SspI, DNase I, RNaseA, sodium dodecyl sulfate (SDS), chloroform, isopropanol, agarose, agar powder, Trypton, Yeast extract

[0200] Instruments and equipment: ultra-low temperature refrigerator, 4℃ refrigerator, electrophoresis instrument, constant temperature shaker, biological safety cabinet, low-temperature centrifuge, sterilizer, spectrophotometer, Beckman Conlter ultracentrifuge

[0201] Preparation of host bacteria: Take the PIB strain stored in a -80°C refrigerator, culture it in a shaking incubator at 37°C and 200 rpm, and use a spectrophotometer to detect that the OD600 value of the bacterial solution is between 0.6 and 0.9, reaching the logarithmic phase.

[0202] Sewage sample collection and treatment: Untreated domestic sewage samples were collected in Pukou District, Nanjing. The sewage samples were centrifuged at 12000rpm, 4℃, for 10min to remove precipitated impurities, and the supernatant was taken and filtered through a 0.22μm filter membrane to remove bacterial impurities and stored in a 4℃ refrigerator for future use.

[0203] Isolation and screening of bacteriophages: The treated sewage sample was added to an equal volume of 2xLB liquid culture medium, and the host bacteria was added at a 5% inoculation rate and inoculated into sterile LB liquid culture medium. The culture was cultured overnight at 37°C and 200 rpm. The next day, the culture was centrifuged at 12000 rpm and 4°C for 10 min. The supernatant was taken and filtered through a 0.22 μm filter membrane to obtain the bacteriophage stock solution.

[0204] Screening of PIB-specific phages: The phage stock solution obtained above was identified by the double-layer plate method. The double-layer plate method includes two layers of LB solid culture medium with different concentrations, the lower layer is 1.5% agar LB culture medium, which is mainly used to support nutrition; the upper layer is 0.5% agar LB culture medium, which is conducive to the growth and spread of host bacteria and phages. This method is also more convenient for the observation of plaques. The specific operation is: first prepare 1.5% lower agar culture medium, mix 100μl logarithmic phase bacterial solution and 100μl phage stock solution evenly, let it stand at room temperature for about 10min, quickly add it to the 0.5% upper culture medium preheated to 48℃, quickly mix it and pour it into the lower culture medium, evenly spread it on the lower plate, let it stand at room temperature for 20min, and place it at 37℃ to observe whether plaques are formed.

[0205] Purification, amplification and storage of PIB-specific phage: Pick the plaques on the plate, add them to LB medium, then add PIB bacterial solution at a ratio of 5%, and culture at 37°C, 200rpm shaking for 6-8h. After filtration and centrifugation, spread the plate using the double-layer plate method, and repeat the above operation two to three times until a round transparent plaque with uniform size and complete edges is observed. Pick the plaque with a sterilized gun tip, and add 5% bacterial solution to LB medium at the same time, culture and amplify overnight at 37°C, 200rpm, shaker, 12000rpm, 5min, 4°C centrifuge, filter with a 0.22μm filter membrane, add to 10% SM buffer, and store in a -80°C refrigerator.

[0206] Preparation of phage concentrate and phage DNA: Mix the stored phage with the PIB strain and add them to LB medium, culture overnight at 37°C in a shaking incubator, centrifuge at 12,000 rpm for 5 min at 4°C, take the supernatant and filter it through a 0.22 μm filter to obtain the phage stock solution, freeze-ultrapentrifuge the phage stock solution at 60,000 rpm at 4°C for 1 h, remove the supernatant, and dissolve the precipitate with SM buffer to obtain the phage concentrate. Extraction of phage DNA: 1) Add 5 μl 1 mg / ml DNaseI and 2.5 μl 12.5 mg / ml RNaseA to 500 μl phage concentrate, incubate at 37°C in a water bath for 30 min; 2) Add 25 μl 10% SDS and 5 μl 10 mg / ml protein K, incubate at 37°C in a water bath for 30 min; 3) Add 250 μl phenol and 250 μl chloroform (trichloromethane), centrifuge at 12,000 rpm for 10 min at RT, and take 500 μl of the original supernatant of the lysate (aspirate as little as possible and avoid aspirating the middle protein layer); 4) Add an equal volume of 500 μl chloroform, centrifuge at 12,000 rpm for 10 min at RT; 5) Add 45 μl 3M NaAc, pH 5.2, 500 μl isopropanol, -20°C, 30 min, 12000 rpm, 10 min; 6) Wash twice with 70% ethanol; 7) Dissolve in 10-20 μl H2O; 8) Determine the concentration; 9) Store at -20°C.

[0207] Preparation of restriction enzyme digestion map of phage DNA: take 10μl phage DNA, add restriction endonuclease, for example, EcoRV, NcoI, SSPI, ddH2O, 10x buffer, digest in a water bath at 37℃ for 6h, when the reaction is finished, add 2μl 10xloading buffer to terminate the digestion reaction, prepare 1% agarose gel, 100v, 1h, UV irradiation, and obtain the restriction enzyme digestion map of phage DNA, which can be used to distinguish different types of phages.

[0208] Genomic analysis of phage DNA: The genomic DNA samples of phage phage3 and phage4 were sent to Shanghai TechPu Bio-Sequencing Service Co., Ltd. for whole genome sequencing. The whole genome of these two phages was sequenced by Illumina second-generation sequencing, and the CARD database (arpcard.mcmaster.ca) was used for drug resistance analysis. The core of the database is ARO (Antibiotic Resistance Ontology), which contains antibiotic resistance genes, resistance mechanisms, etc. At the same time, virulence gene comparison was also performed, and the database was VFDB (www.mgc.ac.cn / VFs / ) to determine whether the phage contains virulence genes and antibiotic resistance genes.

[0209] Experimental Results

[0210] 1. Screening and obtaining four specific PIB phages:

[0211] By co-culturing the phage stock solution with PIB and then repeatedly screening and purifying it by the double-layer plate method, a large number of translucent, nearly circular plaques can be observed on the surface of the plate, i.e., the presence of plaques. The purified plaques are picked and co-cultured with PIB again. By removing the host strain and using ultra-high-speed centrifugation, the enriched phage concentrate is obtained. The phage DNA is extracted and prepared by the phenol-chloroform method. Then, by the restriction fragment polymorphism method, the whole genome of the phage is digested with restriction endonucleases EcoRV, NcoI, and SSPI to obtain a variety of different types of phages ( Figure 6 ).from Figure 6 It can be seen that when the SSPI restriction endonuclease was used for digestion, phage1, phage2 and phage3 were distinguished as different types of phages. Then, EcoRV and NcoI were used for digestion again, and it was found that phage2, phage3 and phage4 were also different types of phages. Therefore, four different types of PIB-specific phages were screened and isolated.

[0212] 2. Genetic characteristics of the four phages

[0213] Since the structure of bacteriophage is relatively simple, usually a single nucleic acid (DNA / RNA) wrapped in a protein shell, and there is a lack of universal marker genes for virus research, some highly conserved structural or functional genes are currently selected as marker genes for a certain type of virus to study the phylogenetic status of the virus. Common genes include the g23 gene of T4 bacteriophage, the g20, psbA and psbD genes of cyanobacterial phages, etc. This example selects the conserved capsid protein gene g23 of the commonly used T4 phage, and through PCR experiments, Figure 3The experimental results also showed the identification of the T4g23 gene, and the results also showed that the screened phages all belonged to the T4 phage of the Myoviridae family, which is a relatively common phage.

[0214] The results of whole genome sequencing showed that Phage3 / 4 were both double-stranded DNA phages. According to the genome, both were T4 phages of the Myoviridae family. According to the lytic characteristics, both strains were virulent phages. The genome of Phage3 was 64058 bp long, with a GC content of 43.26%. The genome of Phage4 was 238877 bp long, with a GC content of 44.42%.

[0215] in conclusion

[0216] Four PIB-specific phages were successfully screened and obtained, all of which were T4 phages, and no harmful genes or drug-resistant genes were detected.

[0217] Example 8: Therapeutic effect of PIB-specific bacteriophage on PIB constipation

[0218] The phages screened in Example 7 have good bactericidal effects in vitro, but it is unclear whether they have bactericidal effects in vivo. The purpose of this example is to evaluate the efficacy of the three PIB-specific phages screened in the present invention in PIB constipation animals.

[0219] Materials and Methods

[0220] Bacteria: PIB bacteria were provided by Zhu Minsheng's laboratory.

[0221] PIB-specific phages: PIB-specific phages phage1, 2, 3, and 4 are as described in Example 7 and are stored and provided by Zhu Minsheng's laboratory.

[0222] Experimental animals: C57BL / 6 mice, purchased from Jicui Yaokang, 8-week-old male mice, were raised in an SPF environment with an ambient temperature of 18-24°C and a 12-h light / 12-h dark cycle.

[0223] Experimental reagents: LB culture medium; Evans Blue indicator preparation: Prepare immediately before use, prepare the indicator according to the ratio of 1.5% Evans Blue + 5% methylcellulose, vortex thoroughly to mix, and sonicate at 37°C for 10 minutes; Maixin Bio UltraSensitive TMS-P (Mouse / Rabbit) Ultrasensitive SP (Mouse / Rabbit) Kit

[0224] Analysis of PIB in vitro bactericidal effect:

[0225] 1. Bacterial preparation: 1) Prepare PIB bacterial solution in advance, and culture overnight on a shaker at 37°C, 200 rpm; 2) Add the bacterial solution to LB at a ratio of bacterial solution: LB = 1:500, and phage stock solution: LB = 1:50, shake evenly, take 60 μl of the liquid in the tube, and measure the OD600 value with a spectrophotometer; 3) Culture on a shaker at the same inclination angle, and culture under the same conditions, 37°C, 200 rpm, and observe the clarity of the liquid in the test tube every 1 hour, and measure the OD600 value of the liquid in the tube.

[0226] 2. Preparation and identification of PIB infection constipation model: 30 C57BL / 6 male mice were divided into two groups, one group of 10 mice as the control group, and the other group of 20 mice as the experimental group. They were treated by gavage once a week with a volume of 100 μl each time. The control group was gavaged with LB, and the experimental group was gavaged with PIB bacterial solution. After 8 gavages, the constipation model of PIB infected mice was established.

[0227] 3. Identification of PIB constipation model: During the gavage process, determine whether the mice are successfully infected with PIB bacteria. Take fresh feces of the mice, add them to LB medium with amp resistance, shake and mash them, and culture them in a shaking incubator at 37°C overnight. The next day, take the bacterial solution for PCR detection.

[0228] 4. Determination of intestinal transit speed: After eight consecutive gavages, a constipation model of PIB-infected mice was established. The defecation frequency and whole intestinal transit time of mice were measured. The mice were fasted one day before gavage. The next day, each mouse was gavaged with 100 μl of Evans Blue indicator gel. The gavage time and the time of excretion of feces with blue indicator were recorded for each mouse. The time difference between the two was calculated, which was the whole intestinal transit time of the mouse.

[0229] 5. Phage therapy regimen and efficacy analysis: Three preserved phages were prepared in advance and cultured with PIB in a shaking incubator to prepare cocktails of the three phages phage1, 2, and 3. After thorough mixing, each mouse in the Phage treatment group was gavaged with 100 μl of phage cocktail each time. After the constipation model of PIB-infected mice was established, the control group was still gavaged with LB once a week. The experimental group was randomly divided into two groups, each with 10 mice. One group was used as the PIB group and was gavaged with LB; the other group was used as the Phage group and was gavaged with phage cocktail instead. Each mouse was gavaged once a week, 100 μl each time. After four consecutive weeks of gavage, the therapeutic effect of phage was determined by measuring the defecation frequency and whole intestinal transit time of the mice per unit time.

[0230] Experimental Results

[0231] 1. Effects of four bacteriophages on killing bacteria in vitro

[0232] In order to further verify whether the screened phages can repeatedly kill PIB, phages were mixed with PIB and added to LB. Figure 8 The five test tubes in A are LB, LB+PIB, LB+PIB+Phage-1, LB+PIB+Phage-2, and PIB+Phage3 from left to right. From the clarity of the test tubes, the clarity of the test tubes with both phage and PIB is similar to that of the test tubes without PIB, and is significantly clearer than the test tubes with PIB. Figure 8 B is the initial measurement of the OD600 value of the liquid in the test tube when phage and PIB are added to the shaker, and then the OD600 value is measured again at regular intervals. It can be seen from the figure that after 100 minutes of co-culture, the OD600 value is significantly lower than that of the test tube with only PIB added, indicating that in the process of co-culture of PIB and phage, the four phages screened have a good in vitro killing effect on PIB.

[0233] 2. Effect of phage therapy on PIB-infected animals

[0234] Fig. 9 The three groups shown from left to right are the control group, PIB constipation group, and phage treatment group. The mice in the PIB constipation group had significant differences in total intestinal transit time and defecation frequency compared with the control group, indicating that the mice in the PIB constipation group still had constipation symptoms, while the phage cocktail treatment group had a significant recovery in constipation symptoms compared with the PIB constipation group.

[0235] in conclusion

[0236] All four phages have the ability to effectively kill PIB; PIB phage has a significant therapeutic effect on constipation symptoms caused by PIB. Sequence Listing <110> Shaanxi Anning Yunsheng Biotechnology Co., Ltd. <120> Diagnosis and treatment of constipation <160> 18 <170> SIPOSequenceListing 1.0 <210> 1 <211> 4392 <212> DNA <213> Unknown <400> 1 attgaacgct ggcggcaggc ctaacacatg caagtcgaac ggtaacagaa atcagcttgc 60 tgatttgctg acgagtggcg gacgggtgag taatgtctgg gaaactgcct gatggagggg 120 gataactact ggaaacggta gctaataccg cataacgtcg caagaccaaa gagggggacc 180 ttcgggcctc ttgccatcgg atgtgcccag atgggattag ctagtaggtg gggtaacggc 240 tcacctaggc gacgatccct agctggtctg agaggatgac cagccacact ggaactgaga 300 cacggtccag actcctacgg gaggcagcag tggggaatat tgcacaatgg gcgcaagcct 360 gatgcagcca tgccgcgtgt atgaagaagg ccttcgggtt gtaaagtact ttcagcgggg 420 aggaagggag taaagttaat acctttgctc attgacgtta cccgcagaag aagcaccggc 480 taactccgtg ccagcagccg cggtaatacg gagggtgcaa gcgttaatcg gaattactgg 540 gcgtaaagcg cacgcaggcg gtttgttaag tcagatgtga aatccccggg ctcaacctgg 600 gaactgcatc tgatactggc aagcttgagt ctcgtagagg ggggtagaat tccaggtgta 660 gcggtgaaat gcgtagagat ctggaggaat accggtggcg aaggcggccc cctggacgaa 720 gactgacgct caggtgcgaa agcgtgggga gcaaacagga ttagataccc tggtagtcca 780 cgccgtaaac gatgtcgact tggaggttgt gcccttgagg cgtggcttcc ggagctaacg 840 cgttaagtcg accgcctggg gagtacggcc gcaaggttaa aactcaaatg aattgacggg 900 ggcccgcaca agcggtggag catgtggttt aattcgatgc aacgcgaaga accttacctg 960 gtcttgacat ccacagaact ttccagagat ggattggtgc cttcgggaac tgtgagacag 1020 gtgctgcatg gctgtcgtca gctcgtgttg tgaaatgttg ggttaagtcc cgcaacgagc 1080 gcaaccctta tcctttgttg ccagcggtcc ggccgggaac tcaaaggaga ctgccagtga 1140 taaactggag gaaggtgggg atgacgtcaa gtcatcatgg cccttacgac cagggctaca 1200 cacgtgctac aatggcgcat acaaagagaa gcgacctcgc gagagcaagc ggacctcata 1260 aagtgcgtcg tagtccggat tggagtctgc aactcgactc catgaagtcg gaatcgctag 1320 taatcgtgga tcagaatgcc acggtgaata cgttcccggg ccttgtacac accgcccgtc 1380 acaccatggg agtgggttgc aaaagaagta ggtagcttaa ccttcgggag ggcgcttacc 1440 actttgtgat tcatgactgg ggtgattgaa cgctggcggc aggcctaaca catgcaagtc 1500 gaacggtaac agaatcagc ttgctgattt gctgacgagt ggcggacggg tgagtaatgt 1560 ctgggaaact gcctgatgga gggggataac tactggaaac ggtagctaat accgcataac 1620 gtcgcaagac caaagagggg gaccttcgg cctcttgcca tcggatgtgc ccagatggga 1680 ttagctagta ggtggggtaa cggctcacct aggcgacgat ccctagctgg tctgagagga 1740 tgaccagcca cactggaact gagacacggt ccagactcct acgggaggca gcagtggga 1800 atattgcaca atgggcgcaa gcctgatgca gccatgccgc gtgtatgaag aaggccttcg 1860 ggttgttaaag tactttcagc ggggagaag ggagtaaagt taataccttt gctcattgac 1920 gttacccgca gaagaagcac cggctactc cgtgccagca gccgcggtaa tacggagggt 1980 gcaagcgtta atcggaatta ctgggcgtaa agcgcacgca ggcggtttgt taagtcagat 2040 gtgaaatccc cgggctcaac ctgggaactg catctgatac tggcaagctt gagtctcgta 2100 gagggggga gaattccagg tgtagcggtg aaatgcgtag agatctggag gaataccggt 2160 ggcgaaggcg gccccctgga cgaagactga cgctcaggtg cgaaagcgtg gggagcaaac 2220 aggattagat accctggtag tccacgccgt aaacgatgtc gacttggagg ttgtgccctt 2280 gaggcgtggc ttccggagct aacgcgttaa gtcgaccgcc tggggagtac ggccgcaagg 2340 ttaaaactca aatgaattga cgggggcccg cacaagcggt ggagcatgtg gtttaattcg 2400 atgcaacgcg aagaacctta cctggtcttg acatccacag aactttccag agatggattg 2460 gtgccttcgg gaactgtgag acaggtgctg catggctgtc gtcagctcgt gttgtgaaat 2520 gttgggttaa gtcccgcaac gagcgcaacc cttatccttt gttgccagcg gtccggccgg 2580 gaactcaaag gagactgcca gtgataaact ggaggaaggt ggggatgacg tcaagtcatc 2640 atggccctta cgaccagggc tacacacgtg ctacaatggc gcatacaaag agaagcgacc 2700 tcgcgagagc aagcggacct cataaagtgc gtcgtagtcc ggattggagt ctgcaactcg 2760 actccatgaa gtcggaatcg ctagtaatcg tggatcagaa tgccacggtg aatacgttcc 2820 cgggccttgt acacaccgcc cgtcacacca tgggagtggg ttgcaaaaga agtaggtagc 2880 ttaaccttcg ggagggcgct taccactttg tgattcatga ctggggtgat tgaacgctgg 2940 cggcaggcct aacacatgca agtcgaacgg taacagaat cagcttgctg atttgctgac 3000 gagtggcgga cgggtgagta atgtctggga aactgcctga tggaggggga taactactgg 3060 aaacggtagc taataccgca taacgtcgca agaccaaga gggggacctt cgggccctt 3120 gccatcggat gtgcccagat gggattagct agtaggtggg gtaacggctc acctaggcga 3180 cgatccctag ctggtctgag aggatgacca gccacactgg aactgagaca cggtccagac 3240 tcctacggga ggcagcagtg gggaatattg cacaatgggc gcaagcctga tgcagccatg 3300 cggcgtgtat gagaaggcc ttcggttgt aaagtacttt cagcggggag gaagggagta 3360 aagttaatac ctttgctcat tgacgttacc cgcagaagaa gcaccggcta actccgtgcc 3420 agcagccgcg gtatacgga gggtgcaagc gttaatcgga attactgggc gtaaagcgca 3480 cgcaggcggt ttgttaagtc agatgtgaa tccccggct CAcctggga actgcatctg 3540 atactggcaa gcttgagtct cgtagagggg ggtagaatttc caggtgtagc ggtgaaatgc 3600 gtagagatct ggaggaatac cggtggcgaa ggcggccccc tggacgaaga ctgacgctca 3660 ggtgcgaaag cgtggggagc aaacaggatt agataccctg gtagtccacg ccgtaaacga 3720 tgtcgacttg gaggttgtgc ccttgaggcg tggcttccgg agctaacgcg ttaagtcgac 3780 cgcctgggga gtacggccgc aaggttaaaa ctcaaatgaa ttgacggggg cccgcacaag 3840 cggtggagca tgtggtttaa ttcgatgcaa cgcgaagaac cttacctggt cttgacatcc 3900 acagaacttt ccagagatgg attggtgcct tcgggaactg tgagacaggt gctgcatggc 3960 tgtcgtcagc tcgtgttgtg aaatgttggg ttaagtcccg caacgagcgc aacccttatc 4020 ctttgttgcc agcggtccgg ccgggaactc aaaggagact gccagtgata aactggagga 4080 aggtggggat gacgtcaagt catcatggcc cttacgacca gggctacaca cgtgctacaa 4140 tggcgcatac aaagagaagc gacctcgcga gagcaagcgg acctcataaa gtgcgtcgta 4200 gtccggattg gagtctgcaa ctcgactcca tgaagtcgga atcgctagta atcgtggatc 4260 agaatgccac ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac accatgggag 4320 tgggttgcaa aagaagtagg tagcttaacc ttcgggaggg cgcttaccac tttgtgattc 4380 atgactgggg tg 4392 <210> 2 <211> 250 <212> DNA <213> Artificial Sequence <400> 2 gcaaatataa catctgcccg aacgttgatt ttatggcgac acaacctaac taccgcggcg 60 cattaacgca ctatctgtgt catccggaga gctttactta caaactgccc gacaatatgg 120 acacgatgga aggggcgctg gtggagcctg ccgcagtcgg gatgcatgcc gcgatgctgg 180 cagatgttaa accgggtaag aagataatta ttctgggagc gggttgtatt ggtttgatga 240 cgttgcaagc 250 <210> 3 <211> 393 <212> DNA <213> Artificial Sequence <400> 3 aaggttgcag ggtggtgggg gttgccggtg gcgcggaaaa atgccgccat gctaccgaga 60 tgttaggctt cgatgtatgc cttgatcacc acgcggatga ttttgccgaa caactggcga 120 aagcgtgccc gaaaggcgtt gatatctatt atgaaaacgt tggcggtaag gtattcgatg 180 cggtgctacc gttacttaat acatctgcgc gcattcccgt ctgcggctta gtgagcagct 240 ataacgctac agagctacca cccggcccgg atcgattacc tctgttgatg gctacggtgc 300 tgaaaaaacg tattcgcctg caaggattta ttatcgctca ggattatggt caccgcatcc 360 atgagtttca gaaggagatg gggcaatggg tga 393 <210> 4 <211> 351 <212> DNA <213> Artificial Sequence <400> 4 aatgccgcca tgctaccgag atgttaggct tcgatgtatg ccttgatcac cacgcggatg 60 attttgccga acaactggcg aaagcgtgcc cgaaaggcgt tgatatctat tatgaaaacg 120 ttggcggtaa ggtattcgat gcggtgctac cgttacttaa tacatctgcg cgcattcccg 180 tctgcggctt agtgagcagc tataacgcta cagagctacc acccggcccg gatcgattac 240 ctctgttgat ggctacggtg ctgaaaaaac gtattcgcct gcaaggattt attatcgctc 300 aggattatgg tcaccgcatc catgagtttc agaaggagat ggggcaatgg g 351 <210> 5 <211> 468 <212> DNA <213> Artificial Sequence <400> 5 gatggtgccg gtatgctggt acttgaagag tacgaacacg cgaaaaaacg cggtgcgaaa 60 atttacgctg aactcgtcgg ctttggtatg agcagcgatg cttatcatat gacgtcaccg 120 ccagaaaatg gcgcaggcgc agctctggca atggcaaatg ctttgcgtga tgcaggcatt 180 gaagcgagtc agattggcta cgttaacgca cacggtactt ctactccggc tggcgataaa 240 gctgaagcgc aggcggtgaa aaccatcttc ggtgaagctg caagccgtgt gttggtaagc 300 tccacgaaat ctatgaccgg tcacctgtta ggtgcggcgg gtgcagtaga atctatctac 360 tccatcctgg cgctgcgcga tcaggctgtt ccgccaacca ttaacctgga taacccggat 420 gaaggttgcg atctggattt cgtaccgtac gaagcgcgtc aggtcagc 468 <210> 6 <211> 426 <212> DNA <213> Artificial Sequence <400> 6 atgcatccgg tgccgccagg tgtggtgggt gatctctatc tcaccggtat tcaactggcg 60 caggggtatc tgggacgacc cgatctgacc gccagtcgct ttattgccga tccttttgcc 120 ccaggtgaac ggatgtaccg taccggagac gttgcccgct ggctggataa cggcgcagtg 180 gagtacctcg ggcgcagtga cgatcagcta aaaattcgcg ggcagcgtat cgaactaggc 240 gaaattgatc gcgtgatgca ggcgctgccg gatgtcgaac aggccgttac ccacgcctgt 300 gtgattaacc aggcggcagc cactggtggt gatgcgcgtc agttggtggg ctatctggtg 360 tcgcaatcgg gcctgccgtt ggataccact gcattgcagg cgcagttgcg cgaaacattg 420 ccaccg 426 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <400> 7 agagtttgat cctggctcag 20 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <400> 8 ggttaccttg ttacgactt 19 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <400> 9 gcaaatataa catctgcccg a 21 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <400> 10 gcttgcaacg tcatcaaacc 20 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <400> 11 aaggttgcag ggtggtgggg g 21 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <400> 12 tcacccattg ccccatctcc 20 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <400> 13 aatgccgcca tgctaccgag a 21 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <400> 14 cccattgccc catctccttc 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <400> 15 gatggtgccg gtatgctggt 20 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <400> 16 gctgacctga cgcgcttcgt a 21 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <400> 17 atgcatccgg tgccgccagg t 21 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <400> 18 cggtggcaat gtttcgcgca 20

Claims

1. Use of a reagent in preparing a test kit for diagnosing constipation in a subject, the reagent comprising a reagent for detecting DPA in a sample; wherein: The constipation is intractable constipation caused by PIB. The subject is a human or a mouse, The sample is feces.

2. The use according to claim 1, characterized in that The reagents also include reagents for detecting PIB.

3. The use according to claim 2, characterized in that The reagent for detecting DPA is a reagent for detecting DPA in a sample using one or more detection methods selected from the following group: chromatography, mass spectrometry, colorimetry, and enzyme-linked immunosorbent assay.

4. The use according to claim 2, characterized in that The reagent for detecting DPA is a reagent for detecting DPA in a sample using one or more detection methods selected from the following group: gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, ultra-high performance liquid chromatography, and capillary gas chromatography.

5. The use according to claim 3 or 4, characterized in that Also includes one or more of the following characteristics: The reagents for detecting DPA include a solvent for DPA, Reagents for detecting DPA include reagents for extracting DPA from feces; The reagent for detecting DPA includes a substance specific to DPA or its derivatives. The reagents for detecting PIB include primers or probes for specifically amplifying the marker nucleic acid sequence of PIB, antibodies or ligands that specifically bind to the surface protein or secretion of PIB, The detection kit further comprises one or more substances selected from the following group: a container, a buffer, an adjuvant, a solvent, a negative control substance, a positive control substance, and instructions for use.

6. The use according to claim 5, characterized in that The reagent for detecting DPA includes dichloromethane and / or chloroform.

7. The use according to claim 5, characterized in that The reagent for detecting DPA includes one or more of methanol, dichloromethane and water.

8. The use according to any one of claims 6 to 7, characterized in that The reagent for detecting DPA includes a mixture of methanol, dichloromethane and water.

9. The use according to claim 8, characterized in that An increased level of DPA in a sample from a subject compared to a control level indicates that the subject suffers from or is suspected of suffering from, or is at risk of suffering from, constipation, or An increased level of DPA in the subject's sample compared to a control level and the presence of PIB or an increased level of PIB in the subject's sample indicate that the subject suffers from or is suspected of suffering from constipation, or is at risk of suffering from constipation.

10. The use according to claim 9, characterized in that The control level is the amount of DPA in a sample from a subject not suffering from constipation.

11. A product for diagnosing constipation, the product comprising reagents for detecting DPA and PIB in a subject sample, wherein: The constipation is intractable constipation caused by PIB. The subject is a human or a mouse, The sample is feces.

12. The product according to claim 11, characterized in that The product is a detection kit, and the reagent for detecting DPA and PIB is a reagent for detecting DPA and PIB in a sample using one or more detection methods selected from the following group: chromatography, mass spectrometry, colorimetry, and enzyme-linked immunosorbent assay.

13. The product according to claim 11, characterized in that The product is a detection kit, and the reagent for detecting DPA and PIB is a reagent for detecting DPA in a sample using one or more detection methods selected from the following group: gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, ultra-high performance liquid chromatography, and capillary gas chromatography.

14. The product according to claim 12 or 13, characterized in that Also includes one or more of the following characteristics: The reagents for detecting DPA include a solvent for DPA, Reagents for detecting DPA include reagents for extracting DPA from feces; The reagent for detecting DPA includes a substance specific to DPA or its derivatives. The reagents for detecting PIB include primers or probes for specifically amplifying the marker nucleic acid sequence of PIB, antibodies or ligands that specifically bind to the surface protein or secretion of PIB, The detection kit further comprises one or more substances selected from the following group: a container, a buffer, an adjuvant, a solvent, a negative control substance, a positive control substance, and instructions for use.

15. The product according to claim 14, characterized in that The reagent for detecting DPA includes dichloromethane and / or chloroform.

16. The product according to claim 14, characterized in that The reagent for detecting DPA includes one or more of methanol, dichloromethane and water.

17. The product according to any one of claims 15-16, characterized in that The reagent for detecting DPA includes a mixture of methanol, dichloromethane and water.

18. The product according to claim 11, characterized in that The product is a device, comprising a memory, a processor, and a computer program stored in the memory and operable on the processor, wherein when the processor executes the program, the following steps are implemented: a) testing DPA and PIB in the subject sample, and b) diagnosing constipation based on the results of step a).

19. The product according to claim 18, characterized in that The step a) comprises: a1) detecting DPA and PIB in the subject sample, and a2) determining whether the abundance of DPA detected in step a1) is higher than the control level, and whether PIB exists or its content is higher than the control.

20. The product according to claim 19, characterized in that An increased level of DPA in the subject's sample compared to a control level and the presence of PIB or an increased level of PIB in the subject's sample indicate that the subject suffers from or is suspected of suffering from constipation, or is at risk of suffering from constipation.

21. The product according to claim 20, characterized in that The control level is the amount of DPA in a sample from a subject not suffering from constipation.

22. Use of an antibiotic or a derivative thereof in the preparation of a medicament for treating constipation, wherein the antibiotic is selected from the group consisting of levofloxacin, balofloxacin, besifloxacin hydrochloride, enrofloxacin, moxifloxacin, pazufloxacin mesylate, gatifloxacin, sparfloxacin, sarafloxacin hydrochloride, norfloxacin, ciprofloxacin, levofloxacin, enoxacin, lomefloxacin, sitafloxacin, difloxacin hydrochloride, meropenem, doripenem, biapenem, and ertapenem sodium, and the constipation is intractable constipation caused by PIB.

23. The use according to claim 22, characterized in that The derivative is a hydrate.

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