CN114965764B - Diagnosis and treatment of constipation - Google Patents

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

Diagnosis and treatment of constipation

Technical Field

The invention belongs to the technical field of biology, and particularly relates to diagnosis and treatment of constipation.

Background

Chronic constipation is a high-incidence chronic disease, the prevalence rate of China is about 4-10%, and the global average prevalence rate is as high as 16%. Patients often improve symptoms through systemic treatment, but a large number of patients remain unhealed for years and repeatedly attack, and almost no treatment is possible except surgery. The cause of the intractable chronic constipation is unknown, the physical and mental health of patients is greatly damaged, and the research of the cause and the pathogenic mechanism is the key for preventing and treating the constipation.

Current methods of clinically diagnosing chronic constipation are still according to roman IV standards. The standard divides the chronic constipation into four types of functional constipation, opioid constipation, constipation-type irritable bowel syndrome and functional bowel movement disorder, which are commonly expressed as difficult defecation, hard defecation, incomplete defecation, obstruction of anus and rectum during defecation, and the need of manual assistance, and the conventional treatment method of the chronic constipation can effectively improve symptoms, such as 1 general treatment, including defecation physiological education, dietary habit change and the like, 2 drug treatment, including bowel movement drugs, bulking agents, permeable bowel movement agents and the like, and 3 mental therapy, including cognitive treatment, stress elimination and the like. Constipation symptoms can be effectively ameliorated by conventional treatment (e.g., high fiber diet, exercise, medication, etc.), pelvic floor biofeedback therapy (biofeedback-AIDED PELVIC floor retraining), and surgical treatment (e.g., rectal anastomosis) for more than 6 months. However, nearly 1/3 of patients still have poor treatment effect and repeated attacks, and are characterized by intractable chronic constipation. The intractable chronic constipation is the most serious functional chronic transmission constipation, the formation cause of which is not clear at present, and a brand new research idea is urgently required to be sought. Earlier studies indicate that the intractable constipation is caused by PIB bacteria (a new species of Shigella) infection, and detection of PIB in fecal bacteria can effectively discover and early warn patients with intractable constipation. However, the direct detection of viable bacteria from feces has the disadvantages of complex sample treatment, high environmental requirements, low positive rate and the like, and a new and simple detection method is very necessary to develop to detect PIB infection.

Disclosure of Invention

In a first aspect the invention provides the use of a reagent comprising (1) a reagent for detecting DPA, optionally further comprising (2) a reagent for detecting PIB, in the manufacture of a test kit for diagnosing constipation.

In one or more embodiments, the constipation is a intractable constipation, preferably a PIB-induced intractable constipation.

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.

In one or more embodiments, the reagent for detecting DPA includes a solvent for DPA, such as methylene chloride and/or chloroform.

In one or more embodiments, the reagent for detecting DPA comprises a reagent for extracting DPA from fecal matter. In one or more embodiments, the reagent for detecting DPA includes one or more of methanol, methylene chloride, water. Preferably, the reagent for detecting DPA comprises methanol, methylene chloride and water mixed in a certain ratio.

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

In one or more embodiments, the DPA derivative is an in vivo metabolite of DPA, including one or more ：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、13,14S- 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, 16,17S-trihydroxy-9, 13, 9Z, 15S-epoxy-DPA, 6272, 16,17S-dihydroxy-7Z, 17S-dihydroxy-7Z, 11E,13E,15Z,19Zn-3, 9 Zn-9, 9Z (p) -DPA, 16,17S-dihydroxy-7A, 37S, 15S-dihydroxy-9, 37Z, 15, 9Z, and the abbreviations (3, 37H, 37P) -DPA).

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 substrate, an enzyme cofactor, an enzyme inhibitor, a fuel, a metal ion, or a ligand (e.g., biotin or hapten).

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

In one or more embodiments, the sample is stool, preferably mammalian stool.

In one or more embodiments, an increase in DPA content in a subject sample as compared to a control level indicates that the subject is suffering from or suspected of suffering from constipation, or is at risk of suffering from constipation. Further, an increase in DPA content in the subject sample, and an increase in PIB content in the subject sample, as compared to a control level, indicates that the subject is suffering from or suspected of suffering from constipation, or is at risk of suffering from constipation.

In some embodiments, the control level is DPA content in the feces of a subject not suffering from constipation.

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

In one or more embodiments, the test kit further comprises one or more substances selected from the group consisting of containers, buffers, adjuvants, solvents, negative controls, positive controls, instructions for use.

In one or more embodiments, the reagent for detecting DPA comprises a DPA standard.

In one or more embodiments, the reagents for detecting PIB include primers or probes that specifically amplify a marker nucleic acid sequence of PIB, antibodies or ligands that specifically bind to a surface protein or secretion of PIB. In one or more embodiments, the marker nucleic acid sequence is a SNP.

The invention also provides a method of extracting DPA from faeces comprising treating a faecal sample obtained from a subject with an extraction solution comprising an alcohol, dichloromethane or chloroform and water.

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

In one or more embodiments, the extract comprises methanol, methylene chloride, and water.

In one or more embodiments, the volume ratio of alcohol, dichloromethane or chloroform to water is 1-10:1-5:1-10, e.g., 2-6:1-4:2-6, preferably 3:2:3.

In another aspect of the application, there is provided a product, such as a test kit or device, for diagnosing constipation, predicting a risk of constipation occurrence or progression in a subject, or for prognosis evaluation of constipation, or for providing a therapeutic regimen, comprising reagents for detecting DPA in a sample of a subject, optionally further comprising reagents for detecting PIB in a sample of a subject.

In one or more embodiments, the sample comprises stool.

In one or more embodiments, the constipation is a intractable constipation, preferably a PIB-induced intractable constipation.

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.

In one or more embodiments, the reagent for detecting DPA comprises a reagent for extracting DPA from fecal matter. In one or more embodiments, the reagent for detecting DPA includes one or more of methanol, methylene chloride, water. Preferably, the reagent for detecting DPA comprises methanol, methylene chloride and water mixed in a certain ratio.

In one or more embodiments, the reagent for detecting DPA includes a substance specific for DPA or a derivative thereof. In one or more embodiments, the DPA derivative is an in vivo metabolite of DPA.

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 substrate, an enzyme cofactor, an enzyme inhibitor, a fuel, a metal ion, or a ligand (e.g., biotin or hapten).

In one or more embodiments, the reagents for detecting PIB include primers or probes that specifically amplify a marker nucleic acid sequence of PIB, antibodies or ligands that specifically bind to a surface protein or secretion of PIB. In one or more embodiments, the marker nucleic acid sequence is a SNP.

In one or more embodiments, the article of manufacture is an apparatus comprising a memory, a processor, and a computer program stored on the memory and operable on the processor, wherein the processor when executing the program performs the steps of:

a) Detecting DPA, optionally also PIB, in a subject sample, and

B) Diagnosing constipation, predicting a risk of constipation occurrence or progression in a subject, or prognostic evaluation of constipation, or providing a therapeutic regimen, by the results of step a).

In one or more embodiments, wherein step a) comprises a 1) detecting DPA in the subject sample, and a 2) determining whether the abundance of DPA detected in step a 1) is above a control level.

In one or more embodiments, wherein step a) comprises a 1) detecting DPA and PIB in the subject sample, and a 2) determining whether the abundance of DPA detected in step a 1) is above a control level, and whether PIB is present or in an amount above the control.

In one or more embodiments, an increase in DPA content in a subject sample as compared to a control level is indicative of the subject suffering from or suspected to suffer from constipation, or is at risk of suffering from constipation, or is poor prognosis of constipation, or is indicative of a need to replace a treatment regimen.

In one or more embodiments, an increase in the DPA content in the subject sample as compared to the control level, and an increase in the PIB content contained in the subject sample, indicates that the subject is suffering from or suspected of suffering from constipation, or is at risk of suffering from constipation, or has a poor prognosis of constipation, or indicates that a treatment regimen needs to be changed.

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

In some embodiments, the reagents used in the products of the application, as well as the characteristics of the products involved, such as the properties of the products, are as defined or set forth herein.

In another aspect, the present invention also provides the use of an antibiotic or derivative thereof in the manufacture of a medicament for the treatment of constipation, the antibiotic including, but not limited to, quinolone antibiotics, carbapenem antibiotics, mitomycin, bleomycin sulfate, and a sulfonamide synergist.

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

In one or more embodiments, the constipation is a intractable constipation, preferably a PIB-induced intractable constipation.

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. Preferably norfloxacin, sitafloxacin hydrate, sarafloxacin hydrochloride, besifloxacin hydrochloride, levofloxacin hemihydrate, lomefloxacin hydrochloride, sparfloxacin, lomefloxacin, levofloxacin and gatifloxacin.

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

In another aspect, the invention also provides the use of phage in the manufacture of a medicament for treating constipation, said phage being T4 phage.

In one or more embodiments, the constipation is a intractable constipation, preferably a PIB-induced intractable constipation.

In one or more embodiments, the phage is a myoviridae T4 phage.

In one or more embodiments, the phage may be used for enema, lavage, injection, preferably oral, enema, injection, preferably at 1x10 ⁹ phage/time/person.

In another aspect, the invention also provides a pharmaceutical composition for treating constipation, comprising pharmaceutically acceptable excipients and (1) an antibiotic or derivative thereof, and/or (2) a bacteriophage, wherein the antibiotic comprises, but is not limited to, a quinolone antibiotic, a carbapenem antibiotic, a mitomycin, bleomycin sulfate, and a sulfa synergist, and the bacteriophage is a T4 bacteriophage.

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

In one or more embodiments, the constipation is a intractable constipation, preferably a PIB-induced intractable constipation.

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. Preferably norfloxacin, sitafloxacin hydrate, sarafloxacin hydrochloride, besifloxacin hydrochloride, levofloxacin hemihydrate, lomefloxacin hydrochloride, sparfloxacin, lomefloxacin, levofloxacin and gatifloxacin.

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

In one or more embodiments, the phage is a myoviridae T4 phage.

Drawings

FIG. 1 PIB secreted chemical is docosapentaenoic acid (Docosapentaenoic acid, DPA). FIGS. 1A and B shows the inhibitory activity of polar phase material and nonpolar phase material on intestinal contractility after PIB culture supernatant was extracted with an organic solution (n=3). FIG. 1, C elution peak type of nonpolar material by HPLC (C18 column), arrow indicates additional peak appearing in PIB supernatant. FIG. 1, D, PIB extra peaks were collected and assayed for their activity in inhibiting intestinal contractions. FIG. 1, E, determination of the molecular weight of the active peaks by LC-MASS. FIG. 1, F molecular weight of fragments of the active peak. FIG. 1, G: inhibition effect of DPA pure on intestinal contractions. FIG. 1, H dose effect of DPA neat to inhibit intestinal contractions. * P <0.01.

FIG. 2 colonic administration the activity of DPA on the colonic discharge function was determined.

FIG. 3 DPA content in constipation mice feces.

FIG. 4 shows that the feces of patients with intractable chronic constipation have higher DPA content. FIGS. 4A and B, fecal HPLC analysis of the refractory chronic constipation group (A) and normal healthy group (B), with the arrow indicating the DPA characteristic peak. Figure 4,C:0.03125ng DPA standard HPLC experiment characteristic peak abundance. FIG. 4, D, comparison of fecal DPA content in refractory chronic constipation population and normal healthy population. * P <0.01.

FIG. 5 determination of PIB bacteria content in feces of mice treated with norfloxacin for PIB constipation.

FIG. 6 shows a phage restriction map.

FIG. 7 PCR detection results of T4 g 23.

FIG. 8 in vitro bactericidal effect of phage.

FIG. 9 in vivo therapeutic effect of phage cocktail therapy on PIB constipation model.

Detailed Description

Earlier studies indicate that PIB bacteria (CGMCC 20603) are pathogenic bacteria of intractable chronic constipation, but the pathogenic mechanism is not clear. The inventors hypothesize that PIB may secrete a substance that directly inhibits rhythmic contractions of the gut, which may be either a protein or an organic substance. The invention separates and identifies the substance, and systematically researches the pathogenic substance of PIB bacteria and the correlation of the pathogenic substance with diseases.

In particular to application of a DPA detection reagent in preparing a medicine box for preventing or treating intractable chronic constipation. The application directly measures the PIB specific metabolite, namely docosapentaenoic acid (DPA), in the feces, thereby judging whether PIB infection and intractable constipation occur.

DPA and detection thereof

Earlier studies indicate that PIB bacteria are pathogenic bacteria of intractable chronic constipation, and the secreted DPA can directly inhibit intestinal rhythmic contraction. The inventors have also found that when DPA abundance and PIB are analyzed in combination, both are very relevant to the disease. DPA is a long-chain unsaturated fatty acid, is often present in organisms such as deep sea fish, seal, seaweed and the like, and is an important component of deep sea fish oil. The research shows that DPA and similar long-chain unsaturated fatty acids DHA and EPA thereof are taken as nutritional health food, have obvious beneficial effects on improving cardiovascular diseases, nerve diseases and the like, but the effect of the fatty acids on intestinal tracts is not reported yet.

The invention discovers the relationship between DPA in the feces and constipation for the first time. The DPA abundance can be detected by chromatography (e.g., 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.

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 a derivative thereof.

For example, the reagent for detecting DPA is a reagent for detecting DPA or a derivative thereof 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, ELISA, ultra high performance liquid chromatography, capillary gas chromatography, and the like. Such a reagent may be a solvent for DPA, such as dichloromethane and/or chloroform.

Alternatively, the reagent for detecting DPA may further comprise a reagent for extracting DPA from feces. In one or more embodiments, the reagent for detecting DPA includes one or more of methanol, methylene chloride, water. Preferably, the reagent for detecting DPA comprises methanol, methylene chloride and water mixed in a certain ratio.

For another example, reagents for detecting DPA include substances specific for DPA or derivatives thereof. The types and methods of use of these materials are within the routine knowledge in the art. To facilitate detection, the detection reagents of the present invention may also carry detectable labels including, but not limited to, radioisotopes, fluorophores, chemiluminescent moieties, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, dyes, metal ions, ligands (e.g., biotin or haptens), and the like.

Herein, a DPA derivative means an in vivo metabolite 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、13,14S-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-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 an abbreviation of hydroxy-DPA and H (p) -DPA is an abbreviation of hydroxy (peroxy) -DPA. Typically, the reagent for detecting DPA may also comprise a reagent for extracting the above derivatives from faeces.

Typically, prior to DPA detection, the sample (e.g., stool) may be pretreated to remove substances that may affect the detection. Preferably, the sample is a fecal sample, which may be derived from human fresh fecal matter, frozen fecal matter, fecal matter after dilution with liquid. Accordingly, the present invention also provides a method of extracting DPA from faeces, the method comprising treating a faecal sample obtained from a subject with a pre-treatment solution extract comprising alcohol, dichloromethane or chloroform and water. The volume ratio of the alcohol, dichloromethane or chloroform to water is 1-10:1-5:1-10, for example 2-6:1-4:2-6, preferably 3:2:3. The alcohol includes methanol, ethanol or propanol. In an exemplary embodiment, the extract comprises methanol, methylene chloride, and water in a volume ratio of 3:2:3.

Methods for detecting DPA are conventional in the art. Exemplary DPA detection methods include HPLC chromatography, liquid phase coupled mass spectrometry, gas phase coupled mass spectrometry, enzyme-linked immunosorbent assay, colorimetry, and the like.

HPLC chromatography, after dissolving the pretreated sample, sampling and eluting. The chromatographic column is usually a C18 column, and the mobile phase is usually acetonitrile/methanol/ammonium formate/isopropanol. After fractional elution, DPA characteristic peak abundance is analyzed, compared with the abundance of a standard substance with known concentration, and the DPA characteristic peak abundance is quantified.

Liquid phase combined mass spectrometry, namely collecting 50mg of feces, adding an organic extraction reagent (methanol/dichloromethane/water=420 uL:280uL:420 uL), collecting an organic phase, pumping out, dissolving, preparing a loading sample, and determining the molecular weight and structure by mass spectrometry, and comparing and quantifying the molecular weight and structure with a DPA standard with a known concentration.

The gas-linked mass spectrometry method comprises collecting 50mg of feces, methyl esterifying with potassium hydroxide and methanol, extracting unsaturated fatty acid methyl ester with chloroform or other organic solvent, and pumping to obtain sample. And (5) analyzing the peak-out characteristics of the DPA methyl ester by using a loading gas chromatograph or a gas chromatograph mass spectrometer.

The ELISA method comprises coating ELISA plate with DPA or long chain unsaturated fatty acid antibody as primary antibody, adding sample to be tested (after pretreatment of feces), incubating, washing plate, adding ELISA secondary antibody, washing plate, and developing.

And (3) colorimetric method, mixing the mixture with the fecal sample by using a potassium iodide solution, and adding a 1% starch solution for color development after the addition reaction is completed.

The reagent involved in the DPA detection method described above is contained in the detection kit of the present invention.

Detection product

Also provided herein is a product, e.g., a kit, for detecting chronic constipation in a patient, comprising reagents for detecting DPA, optionally further comprising reagents for detecting PIB.

Depending on the requirements of the assay method used, the appropriate DPA assay reagent may be selected and formulated into a product, such as a kit, starting from the assay method used. The detection means and reagents contained in the product can be adjusted and modified by a person skilled in the art according to the actual conditions and needs.

The kit may further comprise one or more substances selected from the group consisting of containers, instructions for use, positive controls, negative controls, buffers, solvents or adjuvants, such as solutions for suspending or immobilizing cells, detectable labels or tags, reagents for removing impurities affecting the detection result, such as proteins, nucleic acids, etc., from the sample.

In one example, provided herein is a detection kit suitable for detecting DPA and/or PIB in a biological sample by chromatography. The detection kit can comprise reagents (methanol, acetonitrile and the like) required for detecting DPA by chromatography and one or more reagents for detecting PIB, and an optional container containing the reagents and a use instruction.

Reagents for detecting PIB include primers or probes that specifically amplify a marker nucleic acid sequence of PIB, antibodies or ligands that specifically bind to a surface protein or secretion of PIB. In one or more embodiments, the marker nucleic acid sequence is a SNP. Examples of such primers or probes are described in CN202011023676.4, which is incorporated herein by reference in its entirety. For example, the oligonucleotide primer amplifies a product of the genome of a Shigella bacterium (CGMCC 20603) comprising (1) a nucleotide at a position in the genome of the bacterium corresponding to one or more positions selected from the group consisting of nucleotide at positions PIB2013.g.331, nucleotide at positions PIB2324.g.562, nucleotide at positions PIB2324.g.601, nucleotide at positions PIB2629.g.1126, nucleotide at positions PIB3156.g.2396, (2) a nucleotide at position 21 of a genomic amplified fragment of SEQ ID NO:9 and 10 as primers, nucleotide at position 21 of a genomic amplified fragment of SEQ ID NO:11 and 12 as primers, nucleotide at position 21 of a genomic amplified fragment of SEQ ID NO:13 and 14 as primers, nucleotide at position 20 of a genomic amplified fragment of SEQ ID NO:15 and 16 as primers, nucleotide at least one nucleotide sequence identity of nucleotide sequence 17 and 18 of SEQ ID NO: 8 as primers, or a sequence of nucleotide sequence identity of at least one of nucleotide sequence identity (nucleotide sequence identity of sequence identity (sequence) of sequence (S) of at least one of nucleotide sequence (sequence) shown as SEQ ID NO: 98) or any sequence (sequence) shown as sequence (SEQ ID) of at least one of which is shown in SEQ ID NO: 98). In one or more embodiments, the amplification product of the oligonucleotide primer comprises the sequence set forth in any one of SEQ ID NOs 1-6. In one or more embodiments, the oligonucleotide primer is selected from any one or more of SEQ ID NOS.7-18.

The detection kit can also be provided with a kit using instruction, wherein, the instruction describes how to detect the chronic constipation and how to judge the chronic constipation and select the treatment scheme.

Diagnosis of

The inventor finds that DPA with high concentration exists in the feces of patients with intractable constipation, and the DPA content of the feces of positive patients is 2.26+/-0.43 ng/mg (0.07+/-0.01 ng/mg of normal people). Therefore, the detection of DPA in feces can diagnose intractable constipation, and the positive rate is 44.12%. In addition, the detection of DPA and PIB simultaneously can improve the positive detection rate to 75%.

The invention provides (1) a reagent for detecting DPA, optionally together with (2) the use of a reagent for detecting PIB in the preparation of a kit for diagnosing intractable constipation. Reagents for detecting DPA and reagents for detecting PIB are as described in any of the embodiments herein.

The invention also provides a method of diagnosing obstinate constipation comprising detecting DPA in the subject's stool, optionally further comprising detecting PIB in the subject's stool. Preferably, if the DPA content in the subject's feces is greater than or equal to 0.07.+ -. 0.01ng/mg (e.g., greater than or equal to 0.1ng/mg, greater than or equal to 0.5ng/mg, greater than or equal to 1.0ng/mg, greater than or equal to 1.5ng/mg, greater than or equal to 2.0ng/mg, greater than or equal to 2.26.+ -. 0.43 ng/mg), then the subject is a potential patient for intractable constipation.

Antibiotics or phages

The invention also provides application of the antibiotic or phage in preparing medicines for treating intractable constipation. The inventors found that DPA in feces was significantly reduced in the treatment of intractable constipation with PIB-sensitive antibiotics or phages.

The PIB sensitive antibiotics are obtained by screening the existing anti-biological drug library, and the invention provides a drug formula and a treatment method for eliminating PIB and treating intractable constipation.

As used herein, the antibiotics include, but are not limited to, quinolone antibiotics, carbapenem antibiotics, bleomycin sulfate, mitomycin, sulfonamide potentiators. The quinolone can be levofloxacin, balofloxacin, besifloxacin hydrochloride, enrofloxacin, moxifloxacin, pazufloxacin mesylate, gatifloxacin, sparfloxacin, sarafloxacin hydrochloride, norfloxacin, ciprofloxacin, levofloxacin, enoxacin, lomefloxacin, sitafloxacin, difluorofloxacin hydrochloride and the like. The carbapenem is available is prepared from meropenem doripenem, a biapenem (Biapipenem) ertapenem sodium.

As used herein, norfloxacin is most sensitive among the quinolones and is arranged in the order of norfloxacin > sitafloxacin hydrate > sarafloxacin hydrochloride > besifloxacin hydrochloride > levofloxacin hemihydrate > lomefloxacin hydrochloride > sparfloxacin > lomefloxacin hydrochloride > levofloxacin > gatifloxacin hydrochloride > ciprofloxacin hydrochloride monohydrate > moxifloxacin > difloxacin hydrochloride > danofloxacin mesylate > tosufloxacin hydrate > enoxacin sesquihydrate. As used herein, PIB is most sensitive to doripenem among the carbapenems.

PIB is a pathogenic bacterium causing intractable chronic constipation, and elimination of PIB in vivo is an ideal method for treating the disease. The phage is a bacterial virus, and can specifically recognize a specific bacterium and invade the bacterium to produce bacterial lytic enzymes and lysed cells. The phage can kill specific bacteria with high efficiency and high specificity, is a very safe and efficient antibacterial virus, has been widely applied in a plurality of countries, has been subjected to clinical trial research in the United states, and is about to be put into the market soon. PIB-specific phages were screened for phages sensitive to PIB by biological identification.

As used herein, the phage is a T4 phage, e.g., a myoviridae T4 phage.

As used herein, the phage is PIB-specific phage and can be obtained by screening the phage stock solution obtained by a double-layer plate method, wherein the double-layer plate method comprises two layers of LB solid culture media with different concentrations, namely a lower layer of LB solid culture media with 1.5% agar for supporting nutrition, and an upper layer of LB solid culture media with 0.5% agar for facilitating growth and diffusion of host bacteria and phage. This approach also facilitates plaque observation. The preparation method comprises the steps of firstly preparing a lower agar culture medium with the concentration of 1.5%, uniformly mixing 100 mu l of log phase bacterial liquid and 100 mu l of phage stock solution, standing at room temperature for about 10min, rapidly adding the mixture into an upper culture medium with the concentration of 0.5% which is preheated to 48 ℃, rapidly mixing, pouring the mixture into the lower culture medium, uniformly paving the mixture in a lower flat plate, standing at room temperature for 20min, standing at the constant temperature for 37 ℃ and observing whether plaque is formed. Plaques on the plates were picked up, added to LB medium, and PIB broth was added at a ratio of 5%, shake cultured at 37℃at 200rpm for 6-8h. After centrifugation by filtration, the plates were coated by a double-plate method and the above procedure was repeated two to three times until round transparent plaques of uniform size and complete edges were observed. Picking up plaque with sterilized gun head, adding 5% bacterial solution into LB culture medium, culturing and amplifying at 37 deg.C and 200rpm overnight with shaking table, centrifuging at 12000rpm and 5min at 4 deg.C, filtering with 0.22 μm filter membrane, adding into 10% SM buffer, and storing at-80 deg.C in refrigerator.

As used herein, phage therapy protocol and efficacy analysis can be performed by co-shaking Phage with PIB to prepare cocktail of three Phage, phage1,2,3, and after thorough mixing, 100 μl Phage cocktail per lavage of mice in each Phage treatment group.

Pharmaceutical composition

The antibiotics and/or phages described herein can be used in scientific research or in the preparation of therapeutic drugs for the corresponding cancers. Herein, "pharmaceutical composition" refers to a composition for administration to an individual and encompasses a composition of antibiotics and/or phages for use in therapy. The pharmaceutical composition of the present invention may further comprise pharmaceutically acceptable excipients.

As used herein, a "pharmaceutically acceptable" ingredient is a substance that is suitable for use in humans and/or mammals without undue adverse side effects (such as toxicity, irritation, and response), i.e., a reasonable benefit/risk ratio.

The pharmaceutical composition of the invention contains safe and effective amount of the active ingredients of the invention and pharmaceutically acceptable auxiliary materials. The term "pharmaceutically acceptable excipients" refers to excipients used for administration of therapeutic agents, including various excipients and diluents. Such excipients include, but are not limited to, saline, buffers, dextrose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation is matched with the administration mode, and the dosage forms of the pharmaceutical composition are injection, oral preparation (tablet, capsule and oral liquid), transdermal agent and sustained release agent. For example with physiological saline or an aqueous solution containing glucose and other adjuvants by conventional methods. Pharmaceutically acceptable excipients according to the invention include excipients suitable for antibiotic and/or phage delivery. The pharmaceutical compositions are preferably manufactured under sterile conditions. The compositions comprising these adjuvants may be formulated by well known conventional methods.

The compositions of the present invention may be administered locally or systemically. In certain embodiments, the compositions provided herein may be administered orally or parenterally, such as intravenously, intraarterially, intrathecally, subdermally, or intramuscularly. Parenteral formulations include sterile aqueous or nonaqueous 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/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, lin Geyou dextrose, dextrose and sodium chloride, ringer's lactate solution or fixed oils. Intravenous vehicles include liquid and nutritional supplements, electrolyte supplements (such as those based on Yu Linge dextrose), and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like. The antibiotics described herein may be administered orally, intramuscularly or intravenously. The phage described herein may be administered orally, enema, intragastrically, intramuscularly or intravenously.

The pharmaceutical compositions herein may be administered to a subject in a suitable dosage. The active ingredients of the present invention may vary depending on the mode of administration, the severity of the disease to be treated, etc. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, the dosage for any one patient depends on a variety of factors including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs administered simultaneously. For example, separate doses may be administered several times per day, or the dose may be proportionally reduced, as dictated by the urgent need for the treatment of the condition. Illustratively, the phage dosage described herein is 1x10 ⁹ phage/second/person.

As used herein, the term "subject" or "patient" is a mammal, e.g., a mouse, rabbit, human, etc.

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

The compounds of the present invention may be obtained commercially or may be synthesized according to or with reference to the disclosed methods. Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein. The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the invention, and that specific embodiments, materials, amounts, and steps of operation are to be construed in a broader sense in light of the scope and spirit of the invention as set forth herein. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by conventional conditions such as those described in Sambrook et al, molecular cloning, A laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or by the manufacturer's recommendations.

Exemplary embodiments

PIB is an enterobacteria and has obvious possibility of infecting multiple tissues. DPA is a characteristic metabolite of PIB and, therefore, is likely to be present in a variety of tissues. Faeces are the main source of DPA from PIB, with the sample source being preferentially faeces. The fecal sample may be derived from human fresh feces, frozen feces, and feces after dilution with liquid.

The sample treatment is that the fecal sample is preferentially selected for pretreatment, and is characterized in that a certain amount of feces and an organic extraction reagent are mixed in proportion to extract organic matters in an organic phase.

HPLC purification and separation DPA the treated sample is separated and purified by HPLC, characterized in that the sample is prepared by eluting with a carbon column (preferably a C18 column), acetonitrile/methanol/ammonium formate/isopropanol mobile phase and collecting the active peaks.

LC-MASS (liquid Mass Spectrometry) confirms the molecular structure by loading the active peaks into LC-MASS analysis, determining the peak of the substance with molecular weight of 330 daltons, determining the molecular weight of the fragments again, and then extracting the structure of the substance.

The kit for preparing the fecal DPA detection at least comprises a fecal collecting device, an organic solvent, a positive standard substance, a negative control substance and a DPA standard substance. The specific application is as follows:

excrement collector, and apparatus and container for collecting excrement by patient

Organic solvent for sample extraction

Positive standard substance, positive fecal extract

Standard substance DPA pure product

Negative control, negative fecal extract

The PIB-DPA kit is used for finding the relation between fecal DPA and constipation for the first time. Currently, the DPA content in foods and fish oils is commonly measured by using a gas chromatography or HPLC chromatography method to evaluate the nutritional ingredients of foods. DPA in food is mainly absorbed in small intestine and does not reach colon, so that the DPA content of feces is not affected to interfere with diagnosis. The fecal DPA detection method disclosed by the invention can be used for diagnosing and screening intractable constipation or PIB bacterial infection constipation.

The invention will be further illustrated by means of specific examples. It should be understood that these examples are illustrative only and are not intended to limit the scope of the invention. The methods and reagents used in the examples are, unless otherwise indicated, conventional in the art.

Example 1 the substance base for inhibiting intestinal contractions by PIB was found to be docosapentaenoic acid (DPA)

Materials and methods

Animals C57BL/6 mice (half of male and female, age of 6-8 weeks, weight 20-22 g)

PIB strain CGMCC 20603, which is a main reagent of Zhu Minsheng laboratory preservation (50% glycerol and-80 ℃ preservation), and docosapentaenoic acid (DPA) is purchased from Aladin company and proteinase K is purchased from Sigma company.

Determination of colonic contraction tension:

PIB secretion organic matter extraction, namely inoculating frozen PIB bacteria into LB, culturing at 37 ℃ overnight (12-16 hours), collecting culture supernatant, simultaneously inoculating DH5a escherichia coli into LB, culturing at 37 ℃ overnight (12-16 hours), and collecting culture supernatant to obtain control supernatant. 15ml of the supernatant was dispensed into 10 tubes and dried by a SpeedVac instrument (Thermofisher). The solution was thoroughly dissolved with a pre-chilled methanol solution (410 ul methanol +210ul water) and then 280ul dichloromethane and 210ul water were added. And after shaking and mixing uniformly, centrifugally collecting a nonpolar phase, and pumping for later use.

The organics were purified by HPLC using an Agilent 1200HPLC. The chromatographic column was C18 column (150 mm. Times.4.6 mm) product Yiliteng. The elution mobile phase was A methanol/acetonitrile/10 mM ammonium formate (1:1:1, v/v/v) and B acetonitrile/isopropanol (1:1, v/v). The 20-tube eluate was collected and dried.

LCMS/MS on-machine analysis by mixing quadrupole time-of-flight mass spectrometry (model number AB Sciex Triple TOF 4600.4630 instruments) combined with C18 column (Phenomenex Accucore C18,150 mM. Times.2.1 mM,2.6 μm) separation, mobile phases respectively: A phase: acetonitrile/methanol/10 mM ammonium formate (1:1, v/v/v), B phase: acetonitrile/isopropanol (1:1, v/v), gradient elution order: 0-1min,20% B, 1-4min,60% B, 4-10min,70% B, 10-15min,95% B, 15-20min,95% B. Mass spectrometry used DuoSpray Ion Source in combination with negative ion mode, the parameters were set as assist gas pressure, 55psi, atomizing gas pressure, 55psi, air curtain gas, 35psi, ion source temperature, 600 ℃, voltage 4500V, potential energy, 80eV, collision energy 10eV. Data processing is performed using Peakview software.

Results

1. PIB secretion is present in the organic phase

It is first excluded whether the active secreted by PIB is a protein. The PIB culture supernatant was treated with proteinase K (37 ℃ for 60 minutes) and the treated supernatant still had activity to inhibit intestinal contractions, whereby the active product was unlikely to be a protein. When PIB culture supernatant was treated with methylene chloride/methanol/water extract, the organic phase was taken for activity measurement. The results showed that the organic phase material of the PIB culture supernatant had a significant activity to inhibit intestinal contractions, whereas the organic phase material of the control bacteria (DH 5 a) culture supernatant was inactive, as shown in FIG. 1A, B.

2. The PIB active substance is docosapentaenoic acid

To determine the chemical structure of the PIB supernatant active material, the organic phase mixture was analyzed by liquid chromatography-mass spectrometry. When the sample was subjected to liquid chromatography, the PIB culture supernatant sample showed a specific peak at the late eluting period, whereas the control sample did not (fig. 1C). The peak samples were collected and assayed for biological activity. As a result, it was found that this peak had a remarkable biological activity, while the other elution peaks had no remarkable intestinal contraction inhibition activity (FIG. 1D), suggesting that this activity peak was a characteristic activity peak of PIB culture supernatant. The active peak was further analyzed by mass spectrometry and the molecular weight of the active substance of the peak was 329.25 daltons, and its fragments were analyzed and found to contain two fragments having molecular weights of 285.26 daltons and 59.01 daltons, respectively. The above data were entered into the Preview database and as a result, docosapentaenoic acid was found to be the only candidate molecule.

Conclusion this study for the first time found that the intestinal inhibitory active secreted by PIB was DPA.

Example 2 docosapentaenoic acid inhibits intestinal contractility and intestinal transit Rate

DPA is a long-chain unsaturated fatty acid, is often present in organisms such as deep sea fish, seal, seaweed and the like, and is an important component of deep sea fish oil. The research shows that DPA and similar long-chain unsaturated fatty acids DHA and EPA thereof are taken as nutritional health food, have obvious beneficial effects on improving cardiovascular diseases, nerve diseases and the like, but the effect of the fatty acids on intestinal tracts is not reported yet. The above results suggest that the material basis for PIB to inhibit intestinal contractions may be DPA, but it is not clear whether DPA has a direct effect of inhibiting intestinal contractions. This example is intended to determine whether DPA has an intestinal contractile inhibition function by in vivo studies.

Materials and methods

DPA available from Aladin Co

DPA administration and colon motility measurements DPA was delivered to the colon via a catheter while glass microspheres were added and the time of expulsion of the microspheres was observed.

Results:

DPA was infused into the colon and then colon transit time was measured in order to confirm whether DPA has intestinal function in vivo. The results show that intestinal transit time is prolonged by about 150% after intestinal infusion of DPA (FIG. 2). The above results confirm that DPA has the function of inhibiting intestinal contractions and intestinal transport and is a PIB pathogenic molecule.

Conclusion this study first found that DPA significantly inhibited intestinal contractions, decreasing intestinal motility, thereby leading to constipation.

Example 3 PIB Constipation animal phenotype was related to DPA content in feces

The results of examples 1 and 2 show that DPA is a causative molecule of PIB disease, but it is not clear whether there is a correlation between DPA content in stool and constipation phenotype. For this purpose, the present invention analyzed the content of DPA in the feces of mice with PIB constipation and its correlation with PIB infection by HPLC.

Materials and methods

PIB bacteria, stored in Zhu Minsheng laboratory from Nanjing university

Animals C57BL/6 mice (half of the male and female, 6-8 weeks old, weight 20-22 g)

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

Experimental results:

In this example, feces from 5 PIB constipation mice, feces from 5C 57BL/6 control mice, and constipation mice after phage treatment were collected. DPA levels were analyzed by LC-MASS and found to be significantly higher in fecal DPA signal intensity in PIB constipation mice than in control and phage treatment groups (p < 0.01) (FIG. 3). The DPA concentration is converted into absolute concentration, and the result shows that the DPA content in the PIB constipation mouse excrement is about 7.51ug/mg, and the DPA content in the control mouse excrement is about 0.009ug/mg, and the two groups have extremely obvious difference.

Conclusion(s)

DPA content in the feces of mice with PIB constipation is increased, and DPA has obvious correlation with constipation.

Example 4 significant increase in DPA content in feces of patients with intractable Constipation

According to the previous study, about 56% of patients with intractable constipation were positive for PIB bacteria in their feces, but how the DPA content was related to the disease was not clear. The elucidation of this problem is an important basis for evaluating the diagnostic value of DPA, and the object of this example is to measure the DPA content in human feces by HPLC and compare the difference between constipation group and normal group.

Materials and methods

Crowd data is that intractable constipation cases are from Nanjing Jinling hospitals, all patients are diagnosed as constipation by Roman III standard, all patients are constipation with slow transmission, constipation with irritable bowel syndrome is eliminated, all patients are willing to perform operation treatment and have been subjected to operation treatment, normal crowd is from healthy volunteers, and the patients are healthy and have no complaint of constipation.

The DPA measurement of the excrement comprises the steps of collecting 50mg of fresh excrement, drying, taking 6mg of dry excrement, extracting with an alkyl/alcohol organic solvent, collecting an organic phase, re-dissolving with the organic solvent after pumping, and loading the sample by HPLC. HPLC analysis steps are described in example one.

Fecal PIB assay, see CN202011023676.

Results

The present example analyzed the DPA content in the feces of 68 patients with intractable constipation. When DPA molecules are contained in the sample, obvious DPA characteristic peaks (verified by LC-MASS) can appear at about 10.1-10.4min of peak-out time, and the peak height is positively correlated with the DPA concentration. When no DPA is contained in the sample, no characteristic peak appears (fig. 4A, 4B). Of 68, 30 were positive for DPA, with a 44.12% duty cycle, 38 were negative for DPA, and with a 55.88% duty cycle. Meanwhile, in 68 cases of feces, PIB bacteria were measured by PCR, 38 cases were found to be PIB positive (55.88%), and 30 cases were found to be PIB negative (44.12%). The DPA positive patients were not consistent with the PIB positive patients, 21 out of 38 PIB+ feces were DPA negative, 13 out of 30 PIB-feces were DPA positive, and 17 samples of PIB+ and DPA+ accounted for 25% of the total samples. The same procedure was used to determine the faeces of 30 normal persons, and as a result, it was found that all samples were PIB negative or DPA negative. The result shows that 44-55% of patients with intractable constipation can be detected by detecting PIB or DPA, and if two indexes are detected simultaneously, the detection positive rate can be increased to 75%.

Part of PIB positive patients showed no detectable DPA, probably because DPA was not evenly distributed in the feces, and DPA positive areas could be lost when the sampling amount was small. The inventors did observe that some samples were high in DPA content and some samples were low or undetectable in DPA from the same fecal sample, as measured by taking small samples at different portions. In addition, PIB was not detected in some DPA positive feces. The method for detecting PIB by PCR requires that bacteria in feces are alive, and if sampling is not good or transportation and storage are not good, the PIB bacteria are dead, and the PCR method cannot detect the PIB bacteria. Therefore, the detection accuracy can be improved by detecting both the PIB and DPA indexes simultaneously.

If the signal intensity of the DPA measured above is converted to an absolute concentration, the result shows that the DPA content of the feces of the patient positive for DPA is about 2.26+/-0.43 ng/mg, and the DPA content of the feces of the normal group or the feces negative for DPA is 0.07+/-0.01 ng/mg.

Conclusion(s)

The high concentration of DPA exists in the feces of patients with intractable constipation, the positive rate is 44.12%, and the intractable constipation can be diagnosed by measuring the concentration of DPA in the feces. And meanwhile, the detection rate of the positive detection rate can be improved to 75% by measuring two indexes of DPA and PIB.

Example 5 screening for PIB-sensitive antibiotics

Previous studies have found that PIB infection is a significant cause of intractable constipation, but it is not clear how PIB is cleared in the body. The aim of the embodiment is to obtain PIB sensitive antibiotics by screening the existing antibiotic drug library, and provide a drug formula and a treatment method for eliminating PIB and treating intractable constipation.

Materials and methods

The antibiotic medicine warehouse is purchased from Selleck company and contains 308 antibiotics.

The screening method comprises the steps of diluting each compound in the drug library by 200 times by using LB culture solution, wherein the final concentration is 3-80 mug/mL. Mu.l of the antibiotics were added to each of PIB liquid wells (96-well plates, 100ul of liquid per well), and after culturing at 37℃for 16 hours, OD600nm was measured. The blank is LB medium.

Results

Most antibiotics are not sensitive to PIB, and more sensitive ones are 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 and ertapenem sodium), mitomycin, bleomycin sulfate and sulfanilamide synergists.

To further evaluate the antibacterial potency of sensitive drugs, the invention makes concentration gradient dilutions of these drugs and measures the dose effect. Meanwhile, DH5a escherichia coli is used as a control bacterium, and the sensitivity difference of the target antibiotics to different bacteria in the intestinal tract is compared. The results show that norfloxacin is most sensitive in quinolone drugs, and the arrangement sequence of norfloxacin hydrate, sarafloxacin hydrochloride, besifloxacin hydrochloride, levofloxacin hemihydrate, lomefloxacin hydrochloride, sparfloxacin, lomefloxacin, gatifloxacin, ciprofloxacin hydrochloride monohydrate, moxifloxacin hydrochloride, danofloxacin mesylate, tosufloxacin hydrate, enoxacin sesquihydrate and balofloxacin are shown. Wherein, the balofloxacin, the besifloxacin hydrochloride, the norfloxacin, the lomefloxacin hydrochloride, the enoxacin sesquihydrate, the tosufloxacin paratoluenesulfonate hydrate and the lomefloxacin have weaker sterilization effect on DH5a escherichia coli and certain specificity on PIB bacteria. Of carbapenems, doripenem is most sensitive to PIB. Bleomycin sulfate has little bactericidal effect on DH5a E.coli. The specific data are shown in Table 1.

TABLE 1 MIC of antibiotics for PIB and MBC assay (mg/L) for PIB and DH 5. Alpha

Note that "none" means that the test bacteria are not sensitive to the drug at the drug gradient dilution concentration in this example.

Conclusion(s)

Quinolones, carbapenems, bleomycin sulfate are sensitive to PIB.

Example 6 evaluation of the Effect of norfloxacin antibiotics on eliminating PIB in vivo

Example five screening several PIB-sensitive antibiotics that could not clear PIB in vivo was not clear. This example aims at evaluating the ability of sensitive antibiotics to clear PIB in animals.

Materials and methods

Animals C57BL/6 were purchased from Nanjing Jixiaokang biotechnology Co., ltd, and PIB infection model was established in the laboratory.

The medicine norfloxacin is purchased from European Italian pharmaceutical Co., ltd.

The administration mode is norfloxacin (50 mg/kg) administered once a day for 7 days.

And (3) observing the effect, namely culturing animal feces, harvesting bacterial liquid, and detecting PIB bacteria by using PCR.

Results

PIB bacteria in the animal feces began to turn negative after 1 week of treatment of PIB constipation animals with norfloxacin, and PIB was not detected in the feces after 1 month of drug administration was stopped (FIG. 5).

Conclusion(s)

Norfloxacin can effectively remove PIB bacteria in the body, and can be used for treating constipation.

EXAMPLE 7 selection and identification of PIB-specific phages

PIB is a pathogenic bacterium causing intractable chronic constipation, and elimination of PIB in vivo is an ideal method for treating the disease. The phage is a bacterial virus, and can specifically recognize a specific bacterium and invade the bacterium to produce bacterial lytic enzymes and lysed cells. The phage can kill specific bacteria with high efficiency and high specificity, is a very safe and efficient antibacterial virus, has been widely applied in a plurality of countries, has been subjected to clinical trial research in the United states, and is about to be put into the market soon. The embodiment screens PIB specific phage and carries out biological identification, thus laying foundation for drug development.

Materials and methods

The strain PIB and K12 E.coli are provided by Zhu Minsheng laboratory

Culture medium LB liquid culture medium, 1.5% solid LB culture medium

Reagent(s) of restriction enzyme EcoRV, ncoI, sspI, DNase I, RNaseA, sodium Dodecyl Sulfate (SDS), chloroform, isopropanol, agarose, agar powder, trypton, yeast extract

The instrument and equipment comprises an ultralow temperature refrigerator, a 4 ℃ refrigerator, an electrophoresis apparatus, a constant temperature shaking table, a biosafety cabinet, a low temperature centrifuge, a sterilizing pot, a spectrophotometer and a Beckman Conlter ultracentrifuge

Preparing host bacteria by collecting PIB strain stored in-80 deg.C refrigerator, culturing at 37 deg.C and 200rpm in shaking table, and detecting bacterial liquid OD600 value of 0.6-0.9 by spectrophotometer to logarithmic phase.

And (3) collecting and treating the sewage water sample, namely collecting an untreated domestic sewage water sample in a Nanjing city and Pu' er mouth area. Centrifuging the sewage water sample at 12000rpm and 4 ℃ for 10min, removing precipitated impurities, taking supernatant, filtering the supernatant by a 0.22 mu m filter membrane, removing bacterial impurity pollution, and storing in a 4 ℃ refrigerator for standby.

Separating and screening phage, namely adding the treated sewage sample into an equal volume of 2xLB liquid medium, adding host bacteria according to an inoculum size of 5%, inoculating into the sterile LB liquid medium, culturing at 37 ℃ and 200rpm overnight, centrifuging at 12000rpm and 4 ℃ for 10min the next day, taking the supernatant, and filtering through a 0.22 mu m filter membrane to obtain phage stock solution.

Screening PIB specific phage, namely identifying the obtained phage stock solution by adopting a double-layer flat plate method, wherein the double-layer flat plate method comprises two layers of LB solid culture mediums with different concentrations, the lower layer of LB solid culture medium is 1.5% of agar, the support nutrition is taken as the main material, and the upper layer of LB solid culture medium is 0.5% of agar, so that the growth and the diffusion of host bacteria and phage are facilitated. This approach also facilitates plaque observation. The preparation method comprises the steps of firstly preparing a lower agar culture medium with the concentration of 1.5%, uniformly mixing 100 mu l of log phase bacterial liquid and 100 mu l of phage stock solution, standing at room temperature for about 10min, rapidly adding the mixture into an upper culture medium with the concentration of 0.5% which is preheated to 48 ℃, rapidly mixing, pouring the mixture into the lower culture medium, uniformly paving the mixture in a lower flat plate, standing at room temperature for 20min, standing at the constant temperature for 37 ℃ and observing whether plaque is formed.

The purification, amplification and preservation of PIB specific phage, namely picking plaque on a flat plate, adding the plaque into LB culture medium, adding PIB bacterial liquid according to the proportion of 5%, and carrying out shaking culture at 37 ℃ and 200rpm for 6-8h. After centrifugation by filtration, the plates were coated by a double-plate method and the above procedure was repeated two to three times until round transparent plaques of uniform size and complete edges were observed. Picking up plaque with sterilized gun head, adding 5% bacterial solution into LB culture medium, culturing and amplifying at 37 deg.C and 200rpm overnight with shaking table, centrifuging at 12000rpm and 5min at 4 deg.C, filtering with 0.22 μm filter membrane, adding into 10% SM buffer, and storing at-80 deg.C in refrigerator.

Phage concentrate and phage DNA are prepared by mixing stored phage and PIB strain, adding into LB culture medium, shaking overnight culturing at 37deg.C, centrifuging at 12000rpm for 5min at 4deg.C, collecting supernatant, filtering with 0.22 μm filter membrane to obtain phage stock solution, freezing and ultracentrifugating at 60000rpm for 1 hr, removing supernatant, and dissolving precipitate with SM buffer to obtain phage concentrate. Phage DNA was extracted 1) 5. Mu.l 1mg/ml DNaseI, 2.5. Mu.l 12.5mg/ml RNaseA,37℃in a water bath for 30min, 2) 25. Mu.l 10% SDS, 5. Mu.l 10mg/ml protein K,37℃in a water bath for 30min, 3) 250. Mu.l phenol, 250. Mu.l chloroform (chloroform), 12000rpm,10min, RT centrifugation, 500. Mu.l of supernatant from the original lysate (with minimal aspiration, no aspiration to the intermediate protein layer), 4) equal volumes of 500. Mu.l chloroform, 12000rpm,10min, RT centrifugation, 5) 45. Mu.l 3M NaAc,PH5.2,500. Mu.l isopropanol, -20℃30min,12000rpm,10min, 6) 70% ethanol washing twice, 7) 10-20. Mu. l H2O solubilization, 8) concentration determination, 9) storage at-20 ℃.

Preparation of phage DNA restriction enzyme map, namely, taking 10 μl of phage DNA, adding restriction enzyme (such as EcoRV, ncoI, SSPI, ddH2O,10x buffer, 37 ℃ and water bath enzyme cutting for 6 h), adding 2 μl of 10x loading buffer to terminate enzyme cutting reaction after the reaction is finished, preparing 1% agarose gel, 100v,1h and ultraviolet irradiation gel to obtain phage DNA restriction enzyme map, thereby distinguishing different types of phages.

Genomic analysis of phage DNA genomic DNA samples of phage3 and phage4 were sent to Shanghai Probep Biosequencing services Co., ltd for whole genome sequencing. Both phages were sequenced through the Illumina second generation sequencing method and were subjected to resistance analysis using CARD database (arpcard. Mcmaster. Ca), centered at ARO (Antibiotic Resistance Ontology), containing genes for resistance to antibiotics, resistance mechanisms, etc. Meanwhile, virulence gene comparison is also carried out, a database is VFDB (www.mgc.ac.cn/VFs /), and whether the phage contains virulence genes and antibiotic resistance genes is judged.

Experimental results

1. Four specific PIB phages were obtained by screening:

By the method of co-culturing phage stock solution and PIB and repeatedly screening and purifying by a double-layer plate method, a large amount of semitransparent nearly circular plaques, namely plaques, can be observed on the surface of the plate. The purified plaques were picked up again to co-culture with PIB, the enriched phage concentrate was obtained by removing the host strain and using ultra-high speed centrifugation, phage DNA was prepared by phenol chloroform extraction, and the whole genome of phage was digested with restriction enzyme EcoRV, ncoI, SSPI by restriction fragment polymorphism (FIG. 6). As can be seen from FIG. 6, when the SSPI restriction enzyme was used, it was found that there were different types of phages in phase 1, phase 2 and 3, and then, when EcoRV and NcoI were used again for the enzyme digestion, it was found that there were also different types of phages in phase 2, phase 3 and phase 4, and thus, it was found that four different types of PIB-specific phages were isolated by screening.

2. Genetic characterization of four phages

Because the phage has a relatively simple structure, a single nucleic acid (DNA/RNA) is usually wrapped by a protein shell, and a general marker gene is lacked in the research of viruses, so that the phylogenetic status of viruses is studied by taking some highly conserved structural or functional genes as marker genes for certain types of viruses, and the general genes include the g23 gene of T4 phage, the g20, psbA and psbD genes of cyanobacteria phage and the like. In the embodiment, the capsid protein gene g23 conserved by the phage commonly used in T4 is selected, and the experimental result of FIG. 3 also shows the identification of the T4g23 gene by PCR experiment, and the result also shows that the selected phage belongs to T4 phage of the Myoviridae family, and is a more common phage.

The whole genome sequencing result shows that Phage3/4 are double-stranded DNA phages, T4 phages of the myoviridae are obtained according to genome, the two phages are virulent phages according to the splitting characteristic, the genome total length of Phage3 is 64058bp, and the GC content is 43.26%. Phage4 has a genome of 238877bp in full length and a GC content of 44.42%.

Conclusion(s)

The 4 PIB specific phages obtained by successful screening are all T4 phages, and no harmful genes and drug resistance genes are detected.

EXAMPLE 8 therapeutic Effect of PIB-specific phages on PIB constipation

The phage selected in example 7 had good bactericidal effect in vitro, but it was not clear whether there was bactericidal effect in vivo. The purpose of this example was to evaluate the efficacy of the 3 PIB-specific phages screened according to the invention in PIB constipation animals.

Materials and methods

PIB strain is supplied by Zhu Minsheng laboratories.

PIB-specific phages phage1, 2, 3, 4 were stored and supplied by Zhu Minsheng laboratories as described in example 7.

The experimental animals, C57BL/6 mice, were purchased from Jizhikang. 8 weeks male mice are bred in an SPF environment, the environmental temperature is controlled at 18-24 ℃, and the mice are circularly cultured in a dark room under 12h illumination/12 h.

The experimental reagent comprises LB culture medium, evans Blue indicator preparation, a Michaux Ultra SENSITIVE TMS-P (Mouse/Rabbit) hypersensitive SP (Mouse/Rabbit) kit, wherein the Evans Blue indicator is prepared according to the proportion of 1.5% Evans blue+5% methylcellulose, and the Evans Blue and methylcellulose are mixed uniformly by vortex for 10min at 37 ℃ after the mixture is stirred fully

PIB in vitro sterilization effect analysis:

1. Bacterial preparation 1) PIB bacterial liquid is prepared in advance, at 37 ℃ and 200rpm, shaking is carried out overnight, 2) phage stock solution is added into LB according to the ratio of LB=1:500 and LB=1:50, shaking is carried out uniformly, 60 mu l of liquid in a tube is taken, an OD600 value is measured by a spectrophotometer, 3) shaking culture is carried out under the same inclined angle, and the conditions are the same, at 37 ℃,200rpm, shaking culture, the definition of the liquid in the tube is observed every 1h interval, and the OD600 value of the liquid in the tube is measured.

2. C57BL/6 male mice are divided into two groups, one group is divided into 10, the other group is used as a control group, the other group is used as an experimental group, the gastric lavage is carried out once a week, the gastric lavage amount is 100 mu l each time, the control group is subjected to gastric lavage by LB, the experimental group is subjected to gastric lavage by PIB bacterial liquid, and the constipation model of the PIB infected mice is built after 8 gastric lavages.

3. And (3) identifying a PIB constipation model, namely determining whether the mice are successfully infected with PIB bacteria in the stomach filling process, taking fresh feces of the mice, adding the fresh feces into an LB culture medium with amp resistance, vibrating, mashing and shaking the mice uniformly, culturing the mice by a shaking table at 37 ℃ overnight, and taking bacterial liquid for PCR detection the next day.

4. Measurement of intestinal transport speed, namely, constructing a constipation model of a PIB infected mouse, measuring the defecation frequency of the mouse and the total intestinal transit time of the mouse, fasted the day before the mouse is filled with stomach, filling 100 mu lEvans Blue of indicator gel into each mouse the next day, recording the gastric filling time of each mouse and the time for discharging the excrement with a blue indicator, and calculating the time difference of the two, namely, the total intestinal transit time of the mouse.

5. Phage treatment protocol and efficacy analysis, three preserved phages are prepared in advance, and co-shaking culture is carried out with PIB, so that cocktail of three phages of Phage1,2 and 3 is prepared, after thorough mixing, 100 mu l Phage cocktail is filled into mice of each Phage treatment group each time, after a constipation model of PIB infected mice is built, a control group is still filled with stomach LB once every week, an experimental group is randomly divided into two groups, one group is used as PIB group and is changed into LB, the other group is used as Phage group and is changed into Phage cocktail, each mouse is filled with stomach once every week, and after continuous gastric filling is carried out for 4 weeks, and the therapeutic effect of the phages is judged by measuring the defecation frequency and the total intestinal transit time of the mice in unit time.

Experimental results

1. Effect of killing bacteria of four kinds of bacteriophage in vitro

To further verify whether the screened Phage was able to replicate the killing effect on PIB, phage was added to LB in admixture with PIB, five tubes in FIG. 8, A were LB, LB+PIB, LB+PIB+Phage-1, LB+PIB+Phage-2, PIB+Phage3 in order from left to right. From the perspective of the clarity of the tubes, the tubes with phage and PIB added at the same time were similar in clarity to the tubes without PIB and significantly clearer than the tubes with PIB added. FIG. 8B shows that when phage and PIB are added together into a shaker, the OD600 value of the liquid in the test tube is initially measured, and then the OD600 value is measured again at intervals, and as can be seen from the figure, after 100min of co-culture, the OD600 value is remarkably reduced compared with that of the test tube only added with PIB, and the four selected phages have good in-vitro killing effect on PIB in the process of co-culture of the PIB and the phages.

2. Effect of phage on treating PIB infected animals

The three groups shown in fig. 9 are, from left to right, a control group, a PIB constipation group, and a phage-treated group. Compared with the mice in the control group, the mice in the PIB constipation group have remarkable differences in the total intestinal transit time and defecation frequency, which indicates that the mice in the PIB constipation group still have constipation symptoms, and the phage cocktail treatment group has remarkable recovery of the constipation symptoms compared with the mice in the PIB constipation group.

Conclusion(s)

The four phages have high-efficiency PIB killing capability, and the PIB phages have obvious treatment 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 agaaatcagc ttgctgattt gctgacgagt ggcggacggg tgagtaatgt 1560

ctgggaaact gcctgatgga gggggataac tactggaaac ggtagctaat accgcataac 1620

gtcgcaagac caaagagggg gaccttcggg cctcttgcca tcggatgtgc ccagatggga 1680

ttagctagta ggtggggtaa cggctcacct aggcgacgat ccctagctgg tctgagagga 1740

tgaccagcca cactggaact gagacacggt ccagactcct acgggaggca gcagtgggga 1800

atattgcaca atgggcgcaa gcctgatgca gccatgccgc gtgtatgaag aaggccttcg 1860

ggttgtaaag tactttcagc ggggaggaag ggagtaaagt taataccttt gctcattgac 1920

gttacccgca gaagaagcac cggctaactc cgtgccagca gccgcggtaa tacggagggt 1980

gcaagcgtta atcggaatta ctgggcgtaa agcgcacgca ggcggtttgt taagtcagat 2040

gtgaaatccc cgggctcaac ctgggaactg catctgatac tggcaagctt gagtctcgta 2100

gaggggggta 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 taacagaaat cagcttgctg atttgctgac 3000

gagtggcgga cgggtgagta atgtctggga aactgcctga tggaggggga taactactgg 3060

aaacggtagc taataccgca taacgtcgca agaccaaaga gggggacctt cgggcctctt 3120

gccatcggat gtgcccagat gggattagct agtaggtggg gtaacggctc acctaggcga 3180

cgatccctag ctggtctgag aggatgacca gccacactgg aactgagaca cggtccagac 3240

tcctacggga ggcagcagtg gggaatattg cacaatgggc gcaagcctga tgcagccatg 3300

ccgcgtgtat gaagaaggcc ttcgggttgt aaagtacttt cagcggggag gaagggagta 3360

aagttaatac ctttgctcat tgacgttacc cgcagaagaa gcaccggcta actccgtgcc 3420

agcagccgcg gtaatacgga gggtgcaagc gttaatcgga attactgggc gtaaagcgca 3480

cgcaggcggt ttgttaagtc agatgtgaaa tccccgggct caacctggga actgcatctg 3540

atactggcaa gcttgagtct cgtagagggg ggtagaattc 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 (23)

1. Use of a reagent comprising a reagent for detecting DPA in a sample in the manufacture of a test kit for diagnosing constipation in a subject,

The constipation is intractable constipation caused by PIB,

The subject is a human or a mouse and,

The sample is stool.

2. The use of claim 1, wherein the reagent further comprises a reagent for detecting PIB.

3. The method according to claim 2, wherein the reagent for detecting DPA is a reagent for detecting DPA in a sample by one or more detection methods selected from the group consisting of chromatography, mass spectrometry, colorimetry, and ELISA.

4. The method according to claim 2, wherein the reagent for detecting DPA is a reagent for detecting DPA in a sample by one or more detection methods selected from the group consisting of gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, ultra performance liquid chromatography, and capillary gas chromatography.

5. The use according to claim 3 or 4, further comprising one or more of the following features:

reagents for detecting DPA include solvents for DPA,

Reagents for detecting DPA include reagents for extracting DPA from fecal matter;

reagents for detecting DPA include substances specific for DPA or derivatives thereof,

Reagents for detecting PIB include primers or probes that specifically amplify a marker nucleic acid sequence of PIB, antibodies or ligands that specifically bind to a surface protein or secretion of PIB,

The detection kit further comprises one or more substances selected from the group consisting of containers, buffers, adjuvants, solvents, negative controls, positive controls, and instructions for use.

6. The use according to claim 5, wherein the reagent for detecting DPA comprises dichloromethane and/or chloroform.

7. The use according to claim 5, wherein the reagent for detecting DPA comprises one or more of methanol, dichloromethane, water.

8. The use according to any one of claims 6 to 7, wherein the reagent for detecting DPA comprises a mixture of methanol, dichloromethane and water.

9. The use according to claim 8, wherein,

An increase in DPA content in a sample of a subject compared to a control level, indicating that the subject is suffering from or suspected of suffering from constipation, or is at risk of suffering from constipation, or

An increase in DPA content in the subject sample, and an increase in PIB or PIB content in the subject sample, as compared to a control level, indicates that the subject is suffering from or suspected of suffering from constipation, or is at risk of suffering from constipation.

10. The use of claim 9, wherein the control level is DPA content in a subject sample 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 and,

The sample is stool.

12. The product of claim 11, wherein the product is a detection kit and the reagents for detecting DPA and PIB are reagents for detecting DPA and PIB in a sample using one or more detection methods selected from the group consisting of chromatography, mass spectrometry, colorimetry, and enzyme-linked immunosorbent assay.

13. The product of claim 11, wherein the product is a detection kit and the reagents for detecting DPA and PIB are reagents for detecting DPA in a sample using one or more detection methods selected from the group consisting of gas chromatography mass spectrometry, liquid chromatography mass spectrometry, ultra performance liquid chromatography, capillary gas chromatography.

14. The product of claim 12 or 13, further comprising one or more of the following features:

reagents for detecting DPA include solvents for DPA,

Reagents for detecting DPA include reagents for extracting DPA from fecal matter;

reagents for detecting DPA include substances specific for DPA or derivatives thereof,

Reagents for detecting PIB include primers or probes that specifically amplify a marker nucleic acid sequence of PIB, antibodies or ligands that specifically bind to a surface protein or secretion of PIB,

The detection kit further comprises one or more substances selected from the group consisting of containers, buffers, adjuvants, solvents, negative controls, positive controls, and instructions for use.

15. The product of claim 14, wherein the reagent for detecting DPA comprises methylene chloride and/or chloroform.

16. The product of claim 14, wherein the reagent for detecting DPA comprises one or more of methanol, methylene chloride, and water.

17. The product of any one of claims 15-16, wherein the reagent for detecting DPA comprises a mixture of methanol, methylene chloride, and water.

18. The article of claim 11, wherein the article is an apparatus that comprises a memory, a processor, and a computer program stored on the memory and operable on the processor, wherein the processor, when executing the program, performs the steps of:

a) Detecting DPA and PIB in a subject sample, and

B) And diagnosing constipation by the result of step a).

19. The product of claim 18, wherein step a) comprises a 1) detecting DPA and PIB in the subject sample, and a 2) determining whether the abundance of DPA detected in step a 1) is above a control level, and whether PIB is present or in an amount above the control.

20. The article of claim 19, wherein the article of manufacture comprises,

An increase in DPA content in the subject sample, and an increase in PIB or PIB content in the subject sample, as compared to a control level, indicates that the subject is suffering from or suspected of suffering from constipation, or is at risk of suffering from constipation.

21. The product of claim 20, wherein the control level is DPA content in a sample of the subject not suffering from constipation.

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

23. The use according to claim 22, wherein the derivative is a hydrate.