WO2023222083A1 - 便秘的诊断和治疗 - Google Patents

便秘的诊断和治疗 Download PDF

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WO2023222083A1
WO2023222083A1 PCT/CN2023/095058 CN2023095058W WO2023222083A1 WO 2023222083 A1 WO2023222083 A1 WO 2023222083A1 CN 2023095058 W CN2023095058 W CN 2023095058W WO 2023222083 A1 WO2023222083 A1 WO 2023222083A1
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dpa
constipation
pib
detecting
subject
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French (fr)
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朱敏生
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陕西安宁云生生物技术有限公司
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    • G—PHYSICS
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    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
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    • A61K35/76—Viruses; Subviral particles; Bacteriophages
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the field of biotechnology, and specifically relates to the diagnosis and treatment of constipation.
  • Chronic constipation is a chronic disease with high incidence, with a prevalence rate of about 4-10% in my country, while the global average prevalence rate is as high as 16%. Symptoms of patients can often be improved through systemic treatment, but a large number of patients still remain unrecovered for many years and have recurring attacks, which are almost impossible to treat except surgery. The cause of this type of refractory chronic constipation is unknown and is extremely harmful to patients' physical and mental health. Exploring its etiology and pathogenic mechanisms is the key to preventing and treating this type of constipation.
  • the current clinical method for diagnosing chronic constipation is still based on the Rome IV criteria.
  • This standard divides chronic constipation into four types: functional constipation, opioid-induced constipation, constipation-predominant irritable bowel syndrome and functional defecation disorder. Their common manifestations are: difficulty in defecation, hard stool, feeling of incomplete defecation, The feeling of anorectal blockage during defecation requires manual assistance.
  • Conventional treatment methods for chronic constipation can often effectively improve symptoms, such as: 1. General treatment: defecation physiology education, changing eating habits, etc.; 2. Drug treatment: laxative drugs, bulking agents, osmotic laxatives, etc.; 3. Psychotherapy: cognitive therapy, eliminating tension, etc.
  • PIB bacteria a new species of Shigella
  • detecting PIB in fecal bacteria can effectively detect and warn patients with refractory constipation.
  • direct detection of live bacteria from feces has shortcomings such as complicated sample processing, high environmental requirements, and low positive rate. It is very necessary to develop new and simple detection methods to detect PIB infection.
  • a first aspect of the present invention provides the use of reagents in preparing a detection kit for diagnosing constipation.
  • the reagents include (1) a reagent for detecting DPA, and the reagent optionally further includes (2) a reagent for detecting PIB. .
  • the constipation is refractory constipation, preferably PIB-induced refractory constipation.
  • 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 immunoassay, ultra-high performance liquid chromatography, capillary gas chromatography and other detection.
  • chromatography e.g., gas chromatography, HPLC
  • mass spectrometry e.g., gas chromatography, HPLC
  • mass spectrometry e.g., gas chromatography, HPLC
  • gas chromatography-mass spectrometry gas chromatography-mass spectrometry
  • liquid chromatography mass spectrometry e.g., colorimetry
  • enzyme-linked immunoassay e.g., enzyme-linked immunoassay
  • ultra-high performance liquid chromatography e.g., capillary
  • the reagent for detecting DPA includes a solvent for DPA, such as methylene chloride and/or chloroform.
  • reagents for detecting DPA include reagents for extracting DPA from feces.
  • the reagent for detecting DPA includes one or more of methanol, methylene chloride, and water.
  • the reagent for detecting DPA includes methanol, methylene chloride and water mixed in a certain proportion.
  • reagents for detecting DPA include substances specific for DPA or derivatives thereof.
  • the DPA derivative is an in vivo metabolite of DPA, including one or more selected from the following: 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, 10E, 12Z
  • the reagent for detecting DPA carries a detectable label, such as a detectable label selected from the group consisting of: radioisotopes, fluorophores, chemiluminescent moieties, enzymes, enzyme substrates, enzyme cofactors , enzyme inhibitors, fuels, metal ions, or ligands (e.g., biotin or haptens).
  • a detectable label selected from the group consisting of: radioisotopes, fluorophores, chemiluminescent moieties, enzymes, enzyme substrates, enzyme cofactors , enzyme inhibitors, fuels, metal ions, or ligands (e.g., biotin or haptens).
  • reagents for detecting DPA are used to qualitatively or quantitatively detect DPA in a subject or a sample thereof.
  • the subject is a mammal suffering from, suspected of suffering from, or at risk of suffering from constipation.
  • the sample is feces, preferably mammalian feces.
  • the DPA content in the subject sample is increased compared to control levels, as shown in It is stated that the subject suffers from or is suspected of suffering from constipation, or is at risk of suffering from constipation. Further, compared with 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.
  • control level is the amount of DPA in the feces of a subject who does not suffer from constipation.
  • 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.
  • the detection kit further includes one or more substances selected from the group consisting of containers, buffers, auxiliaries, solvents, negative controls, positive controls, instructions for use.
  • the reagent for detecting DPA includes a DPA standard.
  • reagents for detecting PIB include primers or probes that specifically amplify the marker nucleic acid sequence of PIB, antibodies or ligands that specifically bind to surface proteins or secretions of PIB.
  • the marker nucleic acid sequence is a SNP.
  • the present invention also provides a method for extracting DPA from stool, the method comprising: treating a stool sample obtained from a subject with an extraction liquid, the extraction liquid containing alcohol, dichloromethane or chloroform and water.
  • the alcohol includes methanol, ethanol, or propanol.
  • the extraction liquid includes methanol, methylene chloride, and water.
  • 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 It is 3:2:3.
  • a product for diagnosing constipation, predicting the risk of occurrence or progression of constipation in a subject, or assessing the prognosis of constipation, or providing a treatment plan is provided, such as a detection kit or device, which includes a method for detecting Reagents for DPA in the subject sample optionally also include reagents for detecting PIB in the subject sample.
  • the sample comprises feces.
  • the constipation is refractory constipation, preferably PIB-induced refractory constipation.
  • 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 Use instrument, liquid chromatography mass spectrometer, colorimetric method, enzyme-linked immunoassay, ultra-high performance liquid chromatography, capillary gas chromatography and other detection.
  • chromatography e.g., gas chromatography, HPLC
  • mass spectrometry e.g., gas chromatography, HPLC
  • gas chromatography mass spectrometry Use instrument e.g., liquid chromatography mass spectrometer
  • colorimetric method e.g., enzyme-linked immunoassay
  • ultra-high performance liquid chromatography e.g., capillary gas chromatography and other detection.
  • reagents for detecting DPA include reagents for extracting DPA from feces.
  • the reagent for detecting DPA includes one or more of methanol, dichloromethane, and water. kind.
  • the reagent for detecting DPA includes methanol, methylene chloride and water mixed in a certain proportion.
  • reagents for detecting DPA include substances specific for DPA or derivatives thereof.
  • the DPA derivative is an in vivo metabolite of DPA.
  • the reagent for detecting DPA carries a detectable label, such as a detectable label selected from the group consisting of: radioisotopes, fluorophores, chemiluminescent moieties, enzymes, enzyme substrates, enzyme cofactors , enzyme inhibitors, fuels, metal ions, or ligands (e.g., biotin or haptens).
  • a detectable label selected from the group consisting of: radioisotopes, fluorophores, chemiluminescent moieties, enzymes, enzyme substrates, enzyme cofactors , enzyme inhibitors, fuels, metal ions, or ligands (e.g., biotin or haptens).
  • reagents for detecting PIB include primers or probes that specifically amplify the marker nucleic acid sequence of PIB, antibodies or ligands that specifically bind to surface proteins or secretions of PIB.
  • the marker nucleic acid sequence is a SNP.
  • the product is a device that includes a memory, a processor, and a computer program stored on the memory and executable on the processor, characterized in that the processor executes the Perform the following steps during the program:
  • step b) Diagnose constipation, predict the subject's risk of occurrence or progression of constipation, or evaluate the prognosis of constipation, or provide a treatment plan based on the results of step a).
  • step a) includes: a1) detecting DPA in the subject sample, and a2) determining whether the abundance of DPA detected in step a1) is higher than a control level.
  • 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 there is Whether PIB or its content is higher than the control.
  • an increase in DPA content in a subject's sample compared to control levels 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 the need for a change in treatment plan.
  • the amount of DPA in the subject's sample is increased compared to control levels, and the subject's sample contains PIB or has an increased amount of PIB, indicating that the subject suffers from, is suspected of suffering from, or is at risk of suffering from constipation. , or the prognosis of constipation is poor, or it may indicate that the treatment plan needs to be changed.
  • control level is the DPA content in a sample from a subject 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.
  • the characteristics involved in the reagents used in the product of the present application and the performance of the product are as defined or described herein.
  • antibiotics include but are not limited to quinolone antibiotics, carbapenem antibiotics, mitomycin, and bleomycin sulfate. Factors, sulfa synergists.
  • the derivative is a hydrate.
  • the constipation is refractory constipation, preferably PIB-induced refractory constipation.
  • the quinolones include, but are not limited to, levofloxacin, balofloxacin, besifloxacin hydrochloride, enrofloxacin, moxifloxacin, pazufloxacin mesylate, gatixacin Star, sparfloxacin, sarafloxacin hydrochloride, norfloxacin, ciprofloxacin, levofloxacin, enoxacin, lomefloxacin, sitafloxacin, difloxacin hydrochloride.
  • Norfloxacin sitafloxacin hydrate, sarafloxacin hydrochloride, besifloxacin hydrochloride, levofloxacin hemihydrate, lomefloxacin hydrochloride, sparfloxacin, lomefloxacin, levofloxacin, and gatifloxacin are preferred.
  • the carbapenems include, but are not limited to, meropenem, doripenem, biapenem, ertapenem sodium; preferably doripenem.
  • Another aspect of the present invention also provides the use of bacteriophage in the preparation of medicines for treating constipation, where the bacteriophage is T4 phage.
  • the constipation is refractory constipation, preferably PIB-induced refractory constipation.
  • the phage is a Myoviridae T4 phage.
  • the phage can be applied in enema, gavage, or injection.
  • the preferred administration route is oral administration, enema, or injection.
  • the preferred administration amount is 1x10 9 phage/time/person.
  • the present invention also provides a pharmaceutical composition for treating constipation, comprising pharmaceutically acceptable excipients and: (1) antibiotics or derivatives thereof, and/or (2) bacteriophages, and the antibiotics include but are not limited to quinolones.
  • antibiotics include but are not limited to quinolones.
  • the derivative is a hydrate.
  • the constipation is refractory constipation, preferably PIB-induced refractory constipation.
  • the quinolones include, but are not limited to, levofloxacin, balofloxacin, besifloxacin hydrochloride, enrofloxacin, moxifloxacin, pazufloxacin mesylate, gatixacin Star, sparfloxacin, salt Salafloxacin acid, norfloxacin, ciprofloxacin, levofloxacin, enoxacin, lomefloxacin, sitafloxacin, difloxacin hydrochloride.
  • Norfloxacin sitafloxacin hydrate, sarafloxacin hydrochloride, besifloxacin hydrochloride, levofloxacin hemihydrate, lomefloxacin hydrochloride, sparfloxacin, lomefloxacin, levofloxacin, and gatifloxacin are preferred.
  • the carbapenems include, but are not limited to, meropenem, doripenem, biapenem, ertapenem sodium; preferably doripenem.
  • the phage is a Myoviridae T4 phage.
  • Figure 1 The chemical substance secreted by PIB is Docosapentaenoic acid (DPA).
  • DPA Docosapentaenoic acid
  • Figure 1 C The elution peak pattern of non-polar substances analyzed by HPLC (C18 column) chromatography. The arrow points to the additional peak appearing in the PIB supernatant.
  • Figure 1 D Additional peaks of PIB were collected and their activity in inhibiting intestinal contractions was determined.
  • Figure 1 E Determination of active peak molecular weight using 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 on inhibiting intestinal contraction. **p ⁇ 0.01.
  • FIG. 1 Colonic administration determines the activity of DPA on colonic evacuation function.
  • Figure 3 DPA content in feces of constipated mice.
  • Figure 4 Patients with refractory chronic constipation have higher levels of DPA in their feces.
  • Figure 4, A and B HPLC analysis of feces from people with refractory chronic constipation (A) and normal healthy people (B). The arrows point to the characteristic peaks 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 refractory chronic constipation and normal healthy people. **p ⁇ 0.01.
  • Figure 5 Determination of PIB bacterial content in feces of mice with PIB constipation treated with norfloxacin.
  • Figure 8 In vitro bactericidal effect of bacteriophages.
  • Figure 9 In vivo therapeutic effect of phage cocktail therapy on PIB constipation model.
  • PIB bacteria CGMCC 20603 are the causative bacteria of refractory chronic constipation, but its pathogenic mechanism is unclear.
  • the inventor speculates that PIB may secrete a certain substance that can directly inhibit the rhythmic contraction of the intestine. This substance may be protein or organic matter.
  • the present invention isolates and identifies the substance, and systematically studies the PIB pathogenic substance and its correlation with the disease.
  • the present invention provides the application of a DPA detection reagent in preparing a pharmaceutical kit for preventing or treating refractory chronic constipation.
  • This application directly measures the specific metabolite of PIB, docosapentaenoic acid (DPA), in feces to determine whether there is PIB infection and refractory constipation.
  • DPA docosapentaenoic acid
  • DPA is a long-chain unsaturated fatty acid that often exists in deep-sea fish, seals, seaweed and other organisms. It is an important component of deep-sea fish oil. 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. However, the effect of these fatty acids on the intestines has not been reported. .
  • the DPA abundance can be determined by chromatography (such as HPLC), mass spectrometry, gas chromatography, gas chromatography mass spectrometry, liquid chromatography mass spectrometry, colorimetry, enzyme-linked immunoassay, ultra-high performance liquid chromatography, Capillary gas chromatography and other detection.
  • chromatography such as HPLC
  • mass spectrometry gas chromatography
  • gas chromatography mass spectrometry gas chromatography mass spectrometry
  • liquid chromatography mass spectrometry colorimetry
  • enzyme-linked immunoassay enzyme-linked immunoassay
  • ultra-high performance liquid chromatography Capillary gas chromatography and other detection.
  • detection substance 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.
  • a 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 Chromatography mass spectrometry, colorimetry, enzyme-linked immunoassay, ultra-high performance liquid chromatography, capillary gas chromatography and other detection.
  • chromatography e.g., gas chromatography, HPLC
  • mass spectrometry e.g., gas chromatography, HPLC
  • mass spectrometry e.g., gas chromatography, HPLC
  • gas chromatography-mass spectrometry gas chromatography-mass spectrometry
  • liquid chromatography Chromatography mass spectrometry e.g., colorimetry
  • enzyme-linked immunoassay e.g., enzyme-linked immunoassay
  • the reagent for detecting DPA may also include a reagent for extracting DPA from feces.
  • the reagent for detecting DPA includes one or more of methanol, methylene chloride, and water.
  • the reagent for detecting DPA includes methanol, methylene chloride and water mixed in a certain proportion.
  • the reagents for detecting DPA include substances specific to DPA or its derivatives. The types and methods of use of these substances are within the scope of common knowledge in the art.
  • the detection reagents of the present invention can also be equipped with detectable labels.
  • the detectable labels include but are not limited to: radioactive isotopes, fluorophores, chemiluminescent moieties, enzymes, enzyme substrates, enzyme cofactors, and enzyme inhibitors. , dyes, metal ions, ligands (such as biotin or hapten), etc.
  • DPA derivatives represent 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, 13,14S-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,
  • reagents for detecting DPA may also include reagents for extracting the above-mentioned derivatives from feces.
  • samples can be pretreated to remove substances that may affect the detection.
  • the sample is a feces sample, which can be derived from human fresh feces, frozen feces, or liquid-diluted feces. Therefore, the present invention also provides a method for extracting DPA from feces, the method comprising: treating a feces sample obtained from a subject with a pretreatment liquid extract, the extract containing alcohol, methylene chloride or chloroform and water.
  • the volume ratio of alcohol, methylene chloride or chloroform to water is 1 to 10:1 to 5:1 to 10, such as 2 to 6:1 to 4:2 to 6, preferably 3:2:3.
  • the alcohol includes methanol, ethanol or propanol.
  • the extraction liquid includes methanol, methylene chloride and water in a volume ratio of 3:2:3.
  • DPA detection methods include HPLC chromatography, liquid chromatography mass spectrometry, gas chromatography mass spectrometry, enzyme-linked immunoassay, colorimetric method, etc.
  • HPLC chromatography After dissolving the pretreated sample, inject and elute.
  • the chromatographic column usually uses a C18 column, and the mobile phase generally uses acetonitrile/methanol/ammonium formate/isopropanol.
  • the abundance of DPA characteristic peaks is analyzed and compared with the abundance of known concentration standards for quantification.
  • Gas chromatography mass spectrometry Collect 50 mg of feces, methyl esterify it with potassium hydroxide, extract unsaturated fatty acid methyl esters with chloroform or other organic solvents, and drain to prepare samples. Load the sample into a gas chromatograph or gas chromatograph-mass spectrometer and analyze the peak characteristics of DPA methyl ester.
  • Enzyme-linked immunoassay Use anti-DPA or long-chain unsaturated fatty acid antibody as the primary antibody to coat an enzyme-labeled plate, add the sample to be tested (after pretreatment of feces), incubate, wash the plate, add enzyme-labeled secondary antibody, wash the plate, Color development.
  • Colorimetric method Mix potassium iodide solution with stool sample. After the addition reaction is completed, add 1% starch solution Color development.
  • the reagents involved in the above DPA detection method are included in the detection kit of the present invention.
  • This article also provides a product for detecting refractory chronic constipation in patients, such as a kit, which includes reagents for detecting DPA and optionally also includes reagents for detecting PIB.
  • DPA detection reagents can be selected and made into products starting from the detection method used, such as test 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.
  • the kit may also contain one or more substances selected from the group consisting of containers, instructions for use, positive controls, negative controls, buffers, solvents or auxiliaries, such as solutions for suspending or fixing cells. , detectable tags or markers, reagents that remove impurities that affect detection results, such as proteins, nucleic acids, etc., from the sample.
  • a detection kit suitable for detecting DPA and/or PIB in biological samples by chromatography.
  • the detection kit may include: reagents (methanol, acetonitrile, etc.) required for detecting DPA by chromatographic analysis and one or more reagents for detecting PIB; as well as optional containers containing the above reagents and instructions for use.
  • Reagents for detecting PIB include primers or probes that specifically amplify the marker nucleic acid sequence of PIB, and antibodies or ligands that specifically bind to the surface proteins or secretions of PIB.
  • the marker nucleic acid sequence is a SNP.
  • primers or probes see CN202011023676.4, the entire text of which is incorporated herein by reference.
  • the product of the oligonucleotide primer amplifying the genome of Shigella bacteria includes: (1) the genome of the bacteria located in the genome of the bacterial strain with the registration number CGMCC 20603 selected from The nucleotide at the position corresponding to one or more of the following positions: PIB2013.g.331, PIB2324.g.562, PIB2324.g.601, PIB2629.g.1126, PIB3156.g.2396, (2) selected from The nucleotide at one or more of the following positions: the 21st nucleotide of the genomic amplification fragment primed by SEQ ID NO: 9 and 10; the genomic amplification fragment primed by SEQ ID NO: 11 and 12 The 21st nucleotide of the genome amplified fragment primed by SEQ ID NO: 13 and 14; the 20th nucleotide of the genome amplified fragment primed by SEQ ID NO: 15 and 16 Nucleotide
  • the amplification product of the oligonucleotide primer comprises the sequence described 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 of this article can also be accompanied by an instruction manual for the kit, which records how to use the reagents. How to diagnose refractory chronic constipation and choose treatment options.
  • the present invention provides the use of (1) a reagent for detecting DPA, optionally and (2) a reagent for detecting PIB in the preparation of a kit for diagnosing refractory constipation.
  • Reagents for detecting DPA and reagents for detecting PIB are as described in any embodiment herein.
  • the present invention also provides a method for diagnosing refractory constipation, which includes detecting DPA in the feces of a subject, and optionally also includes detecting PIB in the feces of the subject.
  • the DPA content in the subject's feces is greater than or equal to 0.07 ⁇ 0.01ng/mg (for example, 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 ⁇ 0.43ng/mg)
  • the target is a potential patient with refractory constipation.
  • the present invention also provides the use of antibiotics or bacteriophages in preparing medicines for treating refractory constipation.
  • the inventors found that DPA in feces significantly decreased when refractory constipation was treated with PIB-sensitive antibiotics or bacteriophages.
  • the present invention obtains PIB-sensitive antibiotics by screening existing antibiotic drug libraries, and provides pharmaceutical formulas and treatment methods for eliminating PIB and treating refractory constipation.
  • the antibiotics include, but are not limited to, quinolone antibiotics, carbapenem antibiotics, bleomycin sulfate, mitokinase, sulfonamide synergists.
  • the quinolones may include levofloxacin, balofloxacin, besifloxacin hydrochloride, enrofloxacin, moxifloxacin, pazufloxacin mesylate, gatifloxacin, sparfloxacin, and sarafloxacin hydrochloride.
  • the carbapenems may include meropenem, doripenem, biapenem, and ertapenem sodium.
  • norfloxacin is the most sensitive, and the order is: norfloxacin > sitafloxacin hydrate > sarafloxacin hydrochloride > besifloxacin hydrochloride > levofloxacin hemihydrate > hydrochloric acid Lomefloxacin > Sparfloxacin > Lomefloxacin > Levofloxacin > Gatifloxacin > Ciprofloxacin hydrochloride monohydrate > Moxifloxacin > Dilofloxacin hydrochloride > Danofloxacin mesylate > Yes Tosufloxacin tosylate hydrate > Enoxacin sesquihydrate > Balofloxacin.
  • PIB is the most sensitive to doripenem.
  • PIB is the pathogenic bacteria that causes refractory chronic constipation. Removing PIB from the body is an ideal method to treat this disease.
  • Bacteriophages are bacterial viruses that can specifically recognize specific bacteria and invade the bacteria to produce bacterial lytic enzymes and lyse bacteria. body. Bacteriophage can eliminate specific bacteria with high efficiency and specificity. It is a very safe and efficient antibacterial virus. It has been widely used in many countries. Clinical trials have been carried out in the United States and will be put on the market soon. This article screens PIB-specific phages and conducts biological identification to screen out phages that are sensitive to PIB.
  • the phage is a T4 phage, such as a Myoviridae T4 phage.
  • the phage is a PIB-specific phage, which can be obtained by screening through the following steps: using a double-layer plate method to identify the phage stock solution obtained above.
  • the double-layer plate method includes two layers of LB solid culture with upper and lower concentrations. The lower layer is 1.5% agar LB medium to support nutrition; the upper layer is 0.5% agar LB medium to facilitate the growth and diffusion of host bacteria and phages. This method also makes it easier to observe plaques.
  • the specific operation is as follows: first prepare the 1.5% lower agar culture medium, mix 100 ⁇ l logarithmic phase bacterial solution and 100 ⁇ l phage stock solution evenly, let it stand at room temperature for about 10 minutes, and quickly add it to the 0.5% upper culture medium that has been preheated to 48°C. base, mix quickly and pour into the lower culture medium, spread evenly on the lower plate, let stand at room temperature for 20 minutes, and incubate at 37°C at a constant temperature to observe whether there is the formation of plaques. Pick the plaques on the plate, add them to the LB medium, then add the PIB bacterial liquid at a ratio of 5%, and incubate for 6-8 hours at 37°C and 200rpm shaker.
  • the phage treatment plan and efficacy analysis can be as follows: Cocktails of three phages, phage1, 2, and 3, are prepared by co-culture of phage and PIB on a shaker. After thorough mixing, each mouse in the Phage treatment group is gavaged each time. 100 ⁇ l Phage Cocktail.
  • antibiotics and/or bacteriophages described herein can be used in scientific research or in the preparation of therapeutic drugs for corresponding cancers.
  • pharmaceutical composition refers to a composition for administration to an individual and encompasses compositions of antibiotics and/or bacteriophages for treatment.
  • the pharmaceutical composition of the present invention may also contain pharmaceutically acceptable excipients.
  • a "pharmaceutically acceptable” ingredient is one that is suitable for use by humans and/or mammals without undue adverse side effects (e.g., toxicity, irritation, and reactions), that is, a substance that has a reasonable benefit/risk ratio.
  • the pharmaceutical composition of the present invention contains a safe and effective amount of the active ingredient of the present invention and pharmaceutically acceptable excipients.
  • pharmaceutically acceptable excipients refers to excipients used in the administration of therapeutic agents, including various excipients and diluents. Such excipients include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Generally, pharmaceutical preparations match the mode of administration.
  • the dosage forms of the pharmaceutical composition of the present invention are injections, oral preparations (tablets, capsules, oral liquids), transdermal agents, and sustained-release agents.
  • composition can be prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants.
  • Pharmaceutically acceptable excipients of the present invention include antibiotics and/or or excipients for phage delivery.
  • the pharmaceutical composition is preferably manufactured under sterile conditions. Compositions containing these excipients may be formulated by well-known conventional methods.
  • compositions of the present invention can be administered topically or systemically.
  • the compositions provided herein may be administered orally or parenterally, such as intravenously, intraarterially, intrathecally, subdermally, or intramuscularly.
  • Formulations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions.
  • 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, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution, or fixed oils.
  • Intravenous vehicles include fluid and nutritional supplements, electrolyte supplements (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present, such as antimicrobials, antioxidants, chelating agents, inert gases, and the like.
  • the antibiotics described herein can be administered orally, intramuscularly, or intravenously.
  • the phage described herein can be administered orally, enema, gavage, intramuscularly, or intravenously.
  • the pharmaceutical compositions herein may be administered to a subject in appropriate dosages.
  • the active ingredients of the present invention may vary depending on the mode of administration and the severity of the disease to be treated.
  • the dosage regimen can be determined by the attending physician and clinical factors.
  • the dosage for any given patient depends on a variety of factors, including the patient's size, body surface area, age, the specific compound to be administered, sex, time and route of administration, general health and concomitant Other medicines given.
  • several divided doses may be administered daily, or the dosage may be proportionally reduced as dictated by the exigencies of the treatment situation.
  • the dose of phage described herein is 1x10 phage/time/person.
  • the term "subject” or “patient” is a mammal, such as a mouse, rabbit, human, etc.
  • control level refers to a DPA or PIB level used as a reference, which includes, but is not limited to: DPA or PIB levels in a non-constipated subject, DPA or PIB measured in a non-constipated normal biological sample from the same subject, or PIB levels, statistically determined population norm levels, or standardized levels.
  • the control level is determined by (1) detecting DPA or PIB levels in a biological sample from the patient before treatment, (2) DPA or PIB levels in the patient's biological sample after treatment. or PIB level, (3) correlating the patient's response after treatment to 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 can be obtained from commercial sources, or can be synthesized according to disclosed methods or with reference to disclosed methods. Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein.
  • the present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are merely illustrative of the invention and are not intended to limit the scope of the invention, and that the specific examples, materials, quantities, and procedures are to be construed more broadly in accordance with the scope and spirit of the invention set forth herein. No specific conditions are specified in the following examples.
  • the experimental method usually follows conventional conditions such as those described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or conditions recommended by the manufacturer.
  • the reagents include (1) reagents for detecting DPA.
  • the reagents optionally also include (2) reagents for detecting PIB,
  • the constipation includes refractory constipation, more preferably includes refractory constipation caused by PIB,
  • the subject includes a mammal
  • the sample includes feces.
  • 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, gas chromatography mass spectrometry Hydraulic instrument, liquid chromatography mass spectrometer, colorimetric method, enzyme-linked immunoassay, ultra-high performance liquid chromatography, capillary gas chromatography and other detection,
  • Reagents for detecting DPA include solvents for DPA, such as methylene chloride and/or chloroform,
  • Reagents for detecting DPA include reagents for extracting DPA from feces.
  • reagents for detecting DPA include one or more of methanol, methylene chloride, and water; preferably, reagents for detecting DPA include methanol, methylene chloride, and water. mixture,
  • Reagents for detecting DPA include substances specific to DPA or its derivatives,
  • Reagents for detecting PIB include primers or probes that specifically amplify the marker nucleic acid sequence of PIB, and antibodies or ligands that specifically bind to the surface proteins or secretions of PIB.
  • the detection kit also contains one or more substances selected from the following group: containers, buffers, auxiliaries, solvents, negative controls, positive controls, and instructions for use.
  • Item 3 Uses as described in Item 1 or 2, characterized by:
  • the amount of DPA in the subject's sample is increased compared to control levels, indicating that the subject suffers from, is suspected of suffering from, or is at risk of suffering from constipation, or
  • the DPA content in the subject's sample is increased compared to the control level, and the subject's sample contains PIB or has an increased content of PIB, indicating that the subject suffers from or is suspected of suffering from constipation, or is at risk of suffering from constipation,
  • control level is the DPA content in a sample from a subject not suffering from constipation.
  • Item 4 A method of extracting DPA from feces, the method comprising: treating a feces sample obtained from a subject with an extraction liquid, the extraction liquid containing alcohol, methylene chloride or chloroform and water,
  • the alcohol includes methanol, ethanol or propanol,
  • the volume ratio of alcohol, methylene chloride or chloroform to water is 1 ⁇ 10:1 ⁇ 5:1 ⁇ 10,
  • the extraction liquid contains methanol, methylene chloride and water.
  • Item 5 A product for diagnosing constipation, predicting the risk of occurrence or progression of constipation in a subject, or assessing the prognosis of constipation, or providing a treatment plan.
  • the product includes a reagent for detecting DPA in a subject sample, and optionally includes: Reagents for detecting PIB in target samples,
  • the constipation includes refractory constipation, more preferably includes refractory constipation caused by PIB,
  • the subject includes a mammal
  • the sample includes feces.
  • Item 6 The use as described in Item 5, characterized in that the product is a detection kit, and 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, gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, colorimetry, enzyme-linked immunoassay, ultra-high performance liquid chromatography, capillary gas chromatography and other detection,
  • Reagents for detecting DPA include solvents for DPA, such as methylene chloride and/or chloroform,
  • Reagents for detecting DPA include reagents for extracting DPA from feces.
  • reagents for detecting DPA include one or more of methanol, methylene chloride, and water; preferably, reagents for detecting DPA include methanol, methylene chloride, and water. mixture,
  • Reagents for detecting DPA include substances specific to DPA or its derivatives,
  • Reagents for detecting PIB include primers or probes that specifically amplify the marker nucleic acid sequence of PIB, and antibodies or ligands that specifically bind to the surface proteins or secretions of PIB.
  • the detection kit also contains one or more substances selected from the following group: containers, buffers, auxiliaries, solvents, negative controls, positive controls, and instructions for use.
  • Item 7 Use as described in item 5 or 6, characterized in that the product is a device, and the device includes a memory, a processor and a computer program stored in the memory and operable to run on the processor, characterized in that , the processor implements the following steps when executing the program:
  • step b) Diagnose constipation, predict the subject's risk of occurrence or progression of constipation, or evaluate the prognosis of constipation, or provide a treatment plan based on the results of step a),
  • Step a) includes: a1) detecting DPA in the subject sample, and a2) determining whether the abundance of DPA detected in step a1) is higher than the control level, or
  • 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 whether its content is higher than the control level,
  • the DPA content in the subject's sample is increased, indicating 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 for constipation, or indicates that the treatment regimen needs to be changed, or
  • 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 is at risk of suffering from constipation, or has a poor prognosis of constipation, or indicates that the subject needs Change treatment plan, or
  • the control level is the DPA content in samples from subjects who do not suffer from constipation.
  • antibiotics or their derivatives in the preparation of medicines for the treatment of constipation.
  • the antibiotics include but are not limited to quinolone antibiotics, carbapenem antibiotics, mitokines, bleomycin sulfate, and sulfa synergists. agent,
  • the derivative is a hydrate, and/or
  • the constipation includes refractory constipation, preferably including refractory constipation caused by PIB, and/or
  • the quinolones include: levofloxacin, balofloxacin, besifloxacin hydrochloride, enrofloxacin, moxifloxacin, pazufloxacin mesylate, gatifloxacin, sparfloxacin, and sarafloxacin hydrochloride. , norfloxacin, ciprofloxacin, levofloxacin, enoxacin, lomefloxacin, sitafloxacin, difloxacin hydrochloride, and/or
  • the carbapenems include: meropenem, doripenem, biapenem, and ertapenem sodium.
  • Item 9 Use of bacteriophage in preparing drugs for treating constipation, where the bacteriophage is T4 phage,
  • the constipation includes refractory constipation, preferably including refractory constipation caused by PIB, and/or
  • the phage is Myoviridae T4 phage.
  • Item 10 A pharmaceutical composition for treating constipation, comprising pharmaceutically acceptable excipients and: (1) antibiotics or derivatives thereof, and/or (2) bacteriophages.
  • the antibiotics include but are not limited to quinolone antibiotics, carbon Penem antibiotics, mitokines, bleomycin sulfate, sulfa synergists, the phage is T4 phage,
  • the derivative is a hydrate, and/or
  • the constipation includes refractory constipation, preferably including refractory constipation caused by PIB, and/or
  • the quinolones include: levofloxacin, balofloxacin, besifloxacin hydrochloride, enrofloxacin, moxifloxacin, pazufloxacin mesylate, gatifloxacin, sparfloxacin, and sarafloxacin hydrochloride. , norfloxacin, ciprofloxacin, levofloxacin, enoxacin, lomefloxacin, sitafloxacin, difloxacin hydrochloride, and/or
  • the carbapenems include: meropenem, doripenem, biapenem, ertapenem sodium, and/or,
  • the phage is Myoviridae T4 phage.
  • 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 exist in a variety of tissues.
  • Feces is the main source of DPA produced by PIB, and the sample source is preferably feces.
  • Fecal samples can be derived from human fresh feces, frozen feces, or liquid-diluted feces.
  • Sample processing Prioritize feces samples for pretreatment, which is characterized by mixing a certain amount of feces with organic extraction reagents in proportion to extract organic matter in the organic phase.
  • HPLC purification and separation of DPA The processed samples are separated and purified by HPLC, which is characterized by using a carbon column (preferably using a C18 column), acetonitrile/methanol/ammonium formate/isopropyl alcohol mobile phase elution, and collecting active peaks to prepare samples.
  • LC-MASS liquid chromatography mass spectrometry
  • the kit contains at least the following components: stool collection device, organic solvent, positive standard, negative control, and DPA standard.
  • stool collection device contains at least the following components: stool collection device, organic solvent, positive standard, negative control, and DPA standard.
  • organic solvent positive standard, negative control, and DPA standard.
  • positive standard positive standard
  • negative control negative control
  • DPA standard DPA standard
  • Stool collection device equipment and containers for patients to collect their own stool
  • Negative control negative fecal extract
  • PIB-DPA kit This invention first discovered the relationship between fecal DPA and constipation.
  • gas chromatography or HPLC chromatography analysis methods are commonly used to determine the content of DPA in food and fish oil to evaluate the nutritional content 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 refractory constipation or PIB bacterial infectious constipation.
  • Example 1 It was found that the material basis for PIB bacteria to inhibit intestinal contraction is docosapentaenoic acid (DPA).
  • DPA docosapentaenoic acid
  • mice C57BL/6 mice (half male and half female, 6 weeks to 8 weeks old, weight 20-22 grams)
  • PIB bacteria CGMCC 20603, stored in Zhu Minsheng's laboratory (50% glycerol, -80°C storage).
  • Main reagents Docosapentaenoic acid (DPA) was purchased from Aladdin Company, and proteinase K was purchased from Sigma Company.
  • DPA Docosapentaenoic acid
  • HPLC purification of organic matter The instrument is Agilent 1200 HPLC.
  • the chromatographic column is a C18 column (150mm ⁇ 4.6mm) produced by Yiliteng.
  • the elution mobile phase is A: methanol/acetonitrile/10mM ammonium formate (1:1:1, v/v/v); B: acetonitrile/isopropyl alcohol (1:1, v/v). Collect 20 tubes of eluate and dry.
  • LCMS/MS On-machine analysis: Use hybrid quadrupole time-of-flight mass spectrometry (brand model: AB Sciex Triple TOF 4600 instruments), combined with C18 column (Phenomenex Accucore C18, 150mm ⁇ 2.1mm, 2.6 ⁇ m) for separation.
  • the mobile phases are: : Phase A: acetonitrile/methanol/10mM ammonium formate (1:1:1, v/v/v); Phase B: acetonitrile/isopropanol (1:1, v/v), gradient elution sequence is: 0 -1min, 20%B; 1-4min, 60%B; 4-10min, 70%B; 10-15min, 95%B; 15-20min, 95%B.
  • Mass spectrometry analysis uses DuoSpray Ion Source combined with negative ion mode.
  • the parameter settings are as follows: auxiliary gas pressure, 55psi; atomization gas pressure, 55psi; air curtain gas, 35psi; ion source temperature, 600°C; voltage 4500V; potential energy, 80eV, collision energy 10eV . Peakview software was used for data processing.
  • PIB culture supernatant 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 protein.
  • the organic phase material is taken for activity measurement. The results showed that the organic phase material of the PIB culture supernatant had obvious activity in inhibiting intestinal contraction, while the organic phase material of the control bacteria (DH5a) culture supernatant was inactive, as shown in Figure 1A and B.
  • the active substance of PIB is docosapentaenoic acid
  • the active peak was further analyzed using mass spectrometry, and the molecular weight of the active substance in this peak was 329.25 Daltons; analysis of its fragments revealed that the substance contained two fragments, with molecular weights of 285.26 Daltons and 59.01 Daltons respectively. Enter the above data into the Preview database, and it is found that docosapentaenoic acid is the only candidate molecule.
  • Example 2 Docosapentaenoic acid can inhibit intestinal contraction and intestinal transit speed
  • DPA is a long-chain unsaturated fatty acid that often exists in deep-sea fish, seals, seaweed and other organisms. It is an important component of deep-sea fish oil. 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. However, the effect of these fatty acids on the intestines has not been reported. . The above results suggest that the material basis for PIB's inhibition of intestinal contraction may be DPA, but whether DPA has the effect of directly inhibiting intestinal contraction is unclear. This embodiment intends to determine whether DPA has the function of inhibiting intestinal contraction through in vivo studies.
  • DPA administration and colon motility measurement Inject DPA into the colon through a catheter, add glass microspheres at the same time, and observe the discharge time of the microspheres.
  • Example 3 PIB constipation animal phenotype is related to DPA content in feces
  • Examples 1 and 2 indicate that DPA is the causative molecule responsible for PIB pathogenesis, but it is unclear whether there is a correlation between the DPA content in feces and the constipation phenotype.
  • the present invention analyzed the DPA content in the feces of PIB constipated mice and its correlation with PIB infection through HPLC method.
  • PIB bacteria preserved by Zhu Minsheng Laboratory of Nanjing University
  • mice half male and half female, 6-8 weeks old, weight 20-22 grams
  • LC-MASS analysis of fecal DPA Collect 50 mg of mouse feces, add organic extraction reagent (methanol/dichloromethane/water), collect the organic phase, drain, dissolve, prepare HPLC loading sample, and then use mass spectrometry to determine the molecular weight and structure.
  • organic extraction reagent methanol/dichloromethane/water
  • the feces of 5 cases of PIB constipated mice, 5 cases of C57BL/6 control mice, and 5 cases of constipated mice after phage treatment were collected.
  • the content of DPA was analyzed by LC-MASS, and the results showed that the signal intensity of DPA in the feces of PIB constipated mice was significantly higher than that of the control group and phage treatment group (p ⁇ 0.01) ( Figure 3). Converting the DPA concentration into absolute concentration, the results show that the DPA content in the feces of PIB constipated mice is about 7.51ug/mg, while the DPA content in the feces of control mice is about 0.009ug/mg. There is a very significant difference between the two groups. difference.
  • Example 4 The DPA content in the feces of patients with refractory constipation is significantly increased.
  • Fecal DPA measurement Collect 50 mg of fresh feces, dry it and take 6 mg of dried feces, extract it with an alkane/alcohol organic solvent, collect the organic phase, drain it, redissolve it with an organic solvent, and load it on HPLC.
  • the HPLC analysis steps are the same as those described in Implementation Example 1.
  • This example analyzed the DPA content in the feces of 68 patients with refractory constipation.
  • the sample contains DPA molecules
  • an obvious characteristic peak of DPA will appear at around 10.1-10.4 minutes (verified by LC-MASS), and the peak height is positively correlated with the DPA concentration.
  • the sample does not contain DPA, no characteristic peaks appear ( Figure 4A, 4B).
  • 30 cases were DPA positive, accounting for 44.12%
  • 38 cases were DPA negative, accounting for 55.88%.
  • the PCR method was used to detect PIB bacteria in the feces of 68 cases, and it was found that 38 cases were PIB positive (55.88%) and 30 cases were PIB negative (44.12%).
  • DPA-positive patients were not consistent with PIB-positive patients. 21 of the 38 PIB+ stools were DPA-negative, while 13 of the 30 PIB- stools were DPA-positive. There were 17 samples that were both PIB+ and DPA+, accounting for the total samples. 25%. The same method was used to measure the feces of 30 normal people, and it was found that all samples were PIB negative or DPA negative. The results show that detecting PIB or DPA can detect 44-55% of patients with refractory constipation. If both indicators are detected at the same time, the positive rate can be increased to 75%.
  • 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 stool sample to measure DPA, and indeed observed that some samples had high DPA content, while others had low or undetectable DPA content. In addition, PIB is not detectable in some DPA-positive stools. The PCR method of detecting PIB requires that the bacteria in the feces are alive. If the sampling is not done well or transportation and storage are not good, causing the death of PIB bacteria, the PCR method will not be able to detect it. Therefore, the detection accuracy can be improved by detecting both PIB and DPA indicators simultaneously.
  • the results show that the DPA content in the stool of DPA-positive patients is approximately 2.26 ⁇ 0.43ng/mg; while the DPA content in the stool of normal people or DPA-negative patients is 0.07 ⁇ 0.01ng/mg.
  • the purpose of this embodiment is to obtain PIB-sensitive antibiotics by screening the existing antibiotic drug library, and to provide pharmaceutical formulas and treatment methods for eliminating PIB and treating refractory constipation.
  • Antibiotic drug library purchased from Selleck Company, containing a total of 308 antibiotics.
  • Screening method Dilute each compound in the drug library 200 times with LB culture medium, and the final concentration is 3 to 80 ⁇ g/mL. Add 10 ⁇ l of antibiotics to the PIB bacterial solution wells (96-well plate, 100ul of bacterial solution per well), and after incubation at 37°C for 16 hours, measure the OD600nm.
  • the blank control was LB culture medium.
  • antibiotics are not sensitive to PIB, and the more sensitive ones are: quinolones (levofloxacin, balofloxacin, besifloxacin hydrochloride, enrofloxacin, moxifloxacin, pazufloxacin methanesulfonate) salt, gatifloxacin, sparfloxacin, sarafloxacin hydrochloride, norfloxacin, ciprofloxacin, levofloxacin, enoxacin, lomefloxacin, sitafloxacin, difloxacin hydrochloride) , carbapenems (meropenem, doripenem, biapenem, ertapenem sodium), mitogens, bleomycin sulfate, sulfa synergists.
  • quinolones levofloxacin, balofloxacin, besifloxacin hydrochloride, enrofloxacin, moxifloxaci
  • the present invention conducts concentration gradient dilution of these drugs and determines their dose effects.
  • DH5a Escherichia coli was used as a control bacterium to compare the sensitivity differences of target antibiotics to different intestinal bacteria.
  • norfloxacin is the most sensitive, and the order is: norfloxacin > sitafloxacin hydrate > sarafloxacin hydrochloride > besifloxacin hydrochloride > levofloxacin hemihydrate > rhofloxacin hydrochloride Mefloxacin>Sparfloxacin>Lomefloxacin>Levofloxacin>Gatifloxacin>Ciprofloxacin hydrochloride monohydrate>Moxifloxacin>Difloxacin hydrochloride>Danofloxacin mesylate>p-Toluene Tosufloxacin sulfonate hydrate>enoxacin sesquihydrate>balofloxacin.
  • balofloxacin can sterilize DH5a Escherichia coli
  • the effect is weak and has certain specificity against PIB bacteria.
  • carbapenems particularly, imipenem is the most sensitive to PIB.
  • Sulfate Lymphycin has almost no bactericidal effect on DH5a E. coli. See Table 1 for specific data.
  • Quinolones, carbapenems, and bleomycin sulfate are sensitive to PIB.
  • Example 5 In Example 5, several PIB-sensitive antibiotics were screened. It is unclear whether these antibiotics can clear PIB in the body.
  • the purpose of this example is to evaluate the ability of sensitive antibiotics to eliminate PIB in animals.
  • C57BL/6 animals were purchased from Nanjing Jicui Yaokang Biotechnology Co., Ltd., and the PIB infection model was established in the laboratory.
  • Drug norfloxacin, purchased from CSPC Ouyi Pharmaceutical Co., Ltd.
  • Dosing method Norfloxacin (50 mg/kg) was administered by gavage once a day for 7 consecutive days.
  • Norfloxacin can effectively remove PIB bacteria from the body and can be used to treat constipation.
  • PIB is the pathogenic bacteria that causes refractory chronic constipation. Removing PIB from the body is an ideal method to treat this disease.
  • Bacteriophages are bacterial viruses that can specifically recognize specific bacteria, invade the bacteria, produce bacterial lytic enzymes, and lyse the bacteria. Bacteriophage can eliminate specific bacteria with high efficiency and specificity. It is a very safe and efficient antibacterial virus. It has been widely used in many countries. Clinical trials have been carried out in the United States and will be put on the market soon. In this example, PIB-specific phages will be screened and biologically identified to lay the foundation for drug development.
  • Reagents restriction endonuclease EcoRV, NcoI, SspI, DNase I, RNaseA, sodium dodecyl sulfate (SDS), chloroform, isopropyl alcohol, agarose, agar powder, Trypton, Yeast extract
  • Preparation of host bacteria Take the PIB strain stored in a -80°C refrigerator, culture it on a shaker at 37°C, 200rpm, and detect the OD600 value of the bacterial solution with a spectrophotometer to be between 0.6-0.9 and reach the logarithmic phase.
  • Untreated domestic sewage water samples were collected in Pukou District, Nanjing City. Centrifuge the sewage water sample at 12,000 rpm and 4°C for 10 minutes to remove precipitated impurities and take the supernatant. The supernatant is filtered through a 0.22 ⁇ m filter membrane to remove bacterial impurity contamination and stored in a 4°C refrigerator for later use.
  • Isolation and screening of phage Add the processed sewage sample to an equal volume of 2xLB liquid culture medium, add host bacteria according to an inoculum volume of 5%, inoculate it into a sterile LB liquid culture medium, and cultivate overnight at 37°C and 200rpm. Centrifuge at 12,000 rpm, 4°C for 10 minutes, take the supernatant, and filter through a 0.22 ⁇ m filter membrane to obtain the phage stock solution.
  • the double-layer plate method includes two layers of LB solid culture medium with different concentrations, the lower layer is LB culture medium with 1.5% agar. Mainly to support nutrition; the upper layer is 0.5% agar LB medium to facilitate the growth and diffusion of host bacteria and phages. This method also makes it easier to observe plaques.
  • the specific operation is as follows: first prepare the 1.5% lower agar culture medium, mix 100 ⁇ l logarithmic phase bacterial solution and 100 ⁇ l phage stock solution evenly, let it stand at room temperature for about 10 minutes, and quickly add it to the 0.5% upper culture medium that has been preheated to 48°C. base, mix quickly and pour into the lower culture medium, spread evenly on the lower plate, let stand at room temperature for 20 minutes, and incubate at 37°C at a constant temperature to observe whether there is the formation of plaques.
  • Purification, amplification and preservation of PIB-specific phages Pick the plaques on the plate, add them to LB culture medium, then add PIB bacterial liquid at a ratio of 5%, and incubate for 6-8 hours at 37°C on a 200rpm shaker. After filtration and centrifugation, use the double-layer plate method to coat the plate, and repeat the above operation two to three times until round transparent plaques of uniform size and complete edges are observed. Use a sterilized pipette tip to pick out plaques, and add 5% bacterial solution to the LB culture medium at the same time.
  • Preparation of phage concentrate and phage DNA Mix the preserved phage and PIB strain into LB medium, incubate overnight on a shaker at 37°C, centrifuge at 12000rpm, 5min, 4°C, and filter the supernatant through a 0.22 ⁇ m filter. Obtain the phage stock solution. The phage stock solution is refrigerated and ultracentrifuged at 60,000 rpm, 4°C for 1 hour, remove the supernatant, and dissolve the precipitate with SM buffer to obtain a phage concentrate.
  • Extract phage DNA 1) Add 5 ⁇ l 1mg/ml DNaseI and 2.5 ⁇ l 12.5mg/ml RNaseA to 500 ⁇ l phage concentrate, 37°C, water bath for 30min; 2) Add 25 ⁇ l 10% SDS, add 5 ⁇ l 10mg/ml protein K, 37°C , water bath for 30 min; 3) Add 250 ⁇ l phenol, add 250 ⁇ l chloroform (chloroform), 12000 rpm, 10 min, centrifuge at RT, take 500 ⁇ l of the supernatant of the original lysis solution (aspirate as little as possible, do not aspirate to the middle protein layer); 4) Add an equal volume of 500 ⁇ l chloroform, 12000 rpm, 10 min, and centrifuge at RT; 5) Add 45 ⁇ l 3M NaAc, pH 5.2, 500 ⁇ l isopropyl alcohol, -20°C, 30 min, 12000 rpm, 10 min; 6) Wash twice with 70% ethanol ;7) Dissolve in
  • phage DNA restriction map Take 10 ⁇ l of phage DNA, add restriction endonuclease, for example, EcoRV, NcoI, SSPI, ddH2O, 10x buffer, 37°C, digest in water bath for 6 hours, wait until the reaction is completed, add 2 ⁇ l Terminate the enzyme digestion reaction with 10x loading buffer, prepare a 1% agarose gel, 100v, 1h, and UV-photograph the gel to obtain the phage DNA restriction map, which can be used to distinguish different types of phages.
  • Genomic analysis of phage DNA Genomic DNA samples of phage phage3 and phage4 were sent to Shanghai Tanpu Bio-Sequencing Service Co., Ltd. for whole-genome sequencing. The two phages were sequenced whole-genome using Illumina second-generation sequencing method, and the CARD database (arpcard.mcmaster.ca) was used for resistance analysis. The core of the database is ARO (Antibiotic Resistance Ontology), which includes antibiotic resistance genes, resistance mechanisms, etc. At the same time, virulence genes are also compared, and the database is VFDB (www.mgc.ac.cn/VFs/) to determine whether the phage contains virulence genes and antibiotic resistance genes.
  • ARO Antibiotic Resistance Ontology
  • bacteriophages Since the structure of bacteriophages is relatively simple, usually consisting of a single nucleic acid (DNA/RNA) wrapped by a protein coat, and there is a lack of universal marker genes for virus research, it is currently also selected to target certain types of viruses in order to Some highly conserved structural or functional genes are used as marker genes to study the phylogenetic status of viruses. Common genes include the g23 gene of T4 phage, the g20, psbA and psbD genes of cyanobacterial phage, etc. This example selects the conserved capsid protein gene g23 of T4 phage. Through PCR experiments, the experimental results in Figure 3 also show the identification of the T4g23 gene. The results also show that the screened phages all belong to the T4 phage of the Myoviridae family. It is a relatively common bacteriophage.
  • Phage3/4 are both double-stranded DNA phages. According to the genome, they are both T4 phages of the Myoviridae family. According to the lysis characteristics, both strains are virulent phages.
  • the full genome length of phage3 is 64058bp, GC content is 43.26%.
  • the full genome length of Phage4 is 238877bp, and the GC content is 44.42%.
  • PIB-specific phages were successfully screened, all of which were T4 phages, and no harmful genes or drug-resistant genes were detected.
  • Example 7 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 animals with PIB constipation.
  • PIB bacteria were provided by Zhu Minsheng Laboratory.
  • PIB-specific phages PIB-specific phages phage1, 2, 3, and 4 were stored and provided by Zhu Minsheng's laboratory as described in Example 7.
  • mice C57BL/6 mice, purchased from Jicui Yaokang. Eight-week-old male mice were raised in an SPF environment, the ambient temperature was controlled at 18-24°C, and cultured in a 12-hour light/12-hour dark room cycle.
  • Experimental reagents LB medium; Evans Blue indicator preparation: ready for use, prepare the indicator according to the ratio of 1.5% Evans Blue + 5% methylcellulose. After vortexing to mix thoroughly, ultrasonic at 37°C for 10 minutes; Maixin Bio Ultra Sensitive TMS-P (Mouse/Rabbit) Hypersensitive SP (Mouse/Rabbit) Kit
  • mice 30 C57BL/6 male mice were divided into two groups, one group of 10 mice, as the control group, and one group of 20 mice, as the experimental group, treated by intragastric administration once a week. The amount of each intragastric administration was 100 ⁇ l. The control group was intragastrically administered with LB, and the experimental group was intragastrically administered with PIB bacterial liquid. After 8 intragastric administrations, the constipation model of PIB-infected mice was established.
  • Phage treatment plan and efficacy analysis Prepare three stored phages in advance, culture them on a shaking table with PIB, and prepare a cocktail of three phages: phage1, 2, and 3. After thorough mixing, the mice in each Phage treatment group The mice were gavaged with 100 ⁇ l of phage cocktail each time. After the PIB-infected mouse constipation model was established, the control group was still gavaged with LB once a week. The experimental group was randomly divided into two groups, one group with 10 animals each, and one group was used as PIB. The first group was fed with LB instead; the other group was designated as the Phage group and was instead fed with phage cocktail. Each mouse was gavaged once a week, 100 ⁇ l each time. After continuous gavage for 4 weeks, the defecation frequency and frequency of mice per unit time were also measured. Whole-intestinal transit time was used to determine the therapeutic effect of the phages.
  • the phage and PIB were mixed and added to LB.
  • the five test tubes in A are LB, LB+PIB, and LB+PIB from left to right. +Phage-1, LB+PIB+Phage-2, PIB+Phage3. Judging from the clarity of the test tubes, the clarity of the test tubes with phage and PIB added at the same time was similar to that of the test tubes without PIB, and was significantly clearer than the test tubes with PIB added.
  • Figure 8B shows the initial measurement of the OD600 value of the liquid in the test tube when phage and PIB are added to the shaker.
  • the OD600 value is measured again at regular intervals. As can be seen from the figure, after 100 minutes of co-culture, the OD600 value is the same as the OD600 value. Compared with the test tube that only added PIB, there was a significant decrease, suggesting that during the co-culture process of PIB and phages, the four screened phages have a good in vitro killing effect on PIB.
  • mice in the PIB constipation group had significant differences in intestinal transit time and defecation frequency, indicating that mice in the PIB constipation group still had constipation symptoms, while the phage cocktail treatment group was comparable to the PIB constipation group. than, its Constipation symptoms improved significantly.

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Abstract

一种从粪便中提取并检测PDA的方法，用于检测PDA的试剂在制备诊断便秘的检测试剂盒中的用途，以及用于治疗便秘的药物组合物。通过检测粪便中PDA的量，诊断供试者是否罹患或疑似罹患便秘，或具有罹患便秘的风险，并且采用抗生素和/或T4噬菌体治疗便秘。

Description

便秘的诊断和治疗 技术领域
本发明属于生物技术领域，具体涉及便秘的诊断和治疗。
背景技术
慢性便秘是一种高发慢性病症，我国患病率约4-10％，而全球平均患病率高达16％。患者通过系统治疗常可改善症状，但大量患者仍长年不愈、反复发作，除手术外几乎无法治疗。这类顽固性慢性便秘病因不明，对患者的身心健康危害极大，探讨其病因和致病机制是防治该类便秘的关键。
目前临床上诊断慢性便秘的方法仍是根据罗马IV标准。该标准将慢性便秘分为功能性便秘、阿片药物性便秘、便秘型肠易激综合征和功能性排便障碍四种类型，其共同表现为：排便困难、排干硬便、排便不尽感、排便时肛门直肠堵塞感、需要手法辅助，慢性便秘的常规治疗方法常可有效改善症状，如：1、一般治疗：排便生理教育、改变饮食习惯等；2、药物治疗：通便药物、膨松剂、渗透性通便剂等；3、心理疗法：认知治疗、消除紧张等。病程常6个月以上通过常规治疗方法(如，高纤维饮食、锻炼、药物等)、盆底生物反馈疗法(biofeedback-aided pelvic floor retraining)及手术治疗(如，直肠吻合术)可以有效地改善便秘症状。但仍有近1/3的患者治疗效果不佳、反复发作，表现为顽固性慢性便秘。顽固性慢性便秘是最严重的功能性慢传输性便秘，其形成原因目前还不清楚，迫切需要寻求全新研究思路。前期研究表明，顽固性便秘是由于PIB菌(一种志贺氏的新种)感染引起的，检测粪菌中PIB可以有效的发现和预警顽固性便秘患者。但从粪便中直接检测活菌存在样品处理复杂、环境要求高、阳性率偏低等缺点，非常有必要发展新的、简便的检测方法来检测PIB感染情况。
发明内容
本发明第一个方面提供试剂在制备诊断便秘的检测试剂盒中的用途，所述试剂包括(1)用于检测DPA的试剂，所述试剂任选还包括(2)用于检测PIB的试剂。
在一个或多个实施方案中，所述便秘是顽固性便秘，优选PIB引起的顽固性便秘。
在一个或多个实施方案中，检测DPA的试剂是使用选自下组的一种或多种检测方法检测DPA的试剂：色谱法(例如气相色谱、HPLC)、质谱、气相色谱质谱联用仪、液相色谱质谱联用仪、比色法、酶联免疫法、超高效液相色谱法、毛细管气相色谱等检测。
在一个或多个实施方案中，检测DPA的试剂包括DPA的溶剂，例如二氯甲烷和/或三氯甲烷。
在一个或多个实施方案中，检测DPA的试剂包括从粪便中提取DPA的试剂。在一个或多个实施方案中，检测DPA的试剂包括甲醇、二氯甲烷、水中的一种或多种。优选地，检测DPA的试剂包括以一定比例混合的甲醇、二氯甲烷和水。
在一个或多个实施方案中，检测DPA的试剂包括对DPA或其衍生物具有特异性的物质。
在一个或多个实施方案中，DPA衍生物是DPA的的体内代谢产物，包括选自以下的一种或多种：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-环氧-DPA、7S，14S-二羟基-8E，10E，12Z，16Z，19Z-n-3DPA、13，14-二羟基-7Z，9，11，16Z，19Z-n-3DPA、14，21-二羟基-7Z，10Z，12E，16Z，19Z-n-3DPA、17S-H(p)-DPA、16S，17S-环氧-DPA、10，17S-diHDPA、16，17S-diH-DPA、10R，17S-二羟基-7Z，11E，13E，15Z，19Zn-3DPA、16，17R-二羟基-7Z，10，13，14，19Zn-3DPA、7，17S-diH(p)-DPA、7，8，17S-三羟基-9，11，13，15E，19Z-n-3DPA、7，16，17-三羟基-8，10，12，14E，19Z-n-3DPA、7S，17S-三羟基-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为羟基-DPA缩写，H(p)-DPA为羟基(过氧基)-DPA缩写)。
在一个或多个实施方案中，检测DPA的试剂带有可检测标记物，例如选自下组的可检测标记物：放射性同位素、荧光团、化学发光部分、酶、酶底物、酶辅因子、酶抑制剂、燃料、金属离子、或配体(如，生物素或半抗原)。
在一个或多个实施方案中，检测DPA的试剂用于定性或定量检测对象或其样品中DPA。在一个或多个实施方案中，所述对象是罹患或疑似罹患便秘或具有患便秘风险的哺乳动物。
在一个或多个实施方案中，所述样品是粪便，优选哺乳动物的粪便。
在一个或多个实施方式中，与对照水平相比，对象样品中的DPA含量增加，表 明所述对象罹患或疑似罹患便秘，或具有罹患便秘的风险。进一步地，与对照水平相比，对象样品中的DPA含量增加，并且对象样品中包含PIB或PIB含量增加，表明所述对象罹患或疑似罹患便秘，或具有罹患便秘的风险。
在一些实施方式中，所述对照水平是未罹患便秘的对象粪便中的DPA含量。
在一些实施方式中，所述对照水平为小于或等于0.07±0.01ng/mg，优选为小于或等于0.1±0.01ng/mg。
在一个或多个实施方案中，所述检测试剂盒还包含选自下组的一种或多种物质：容器、缓冲剂、助剂、溶剂、阴性对照物、阳性对照物、使用说明书。
在一个或多个实施方案中，检测DPA的试剂包含DPA标准品。
在一个或多个实施方案中，检测PIB的试剂包括特异性扩增PIB的标志核酸序列的引物或探针、特异性结合PIB的表面蛋白或分泌物的抗体或配体。在一个或多个实施方案中，所述标志核酸序列是SNP。
本发明还提供一种从粪便中提取DPA的方法，所述方法包括：用提取液处理获自对象的粪便样品，所述提取液包含醇、二氯甲烷或三氯甲烷和水。
在一个或多个实施方案中，所述醇包括甲醇、乙醇或丙醇。
在一个或多个实施方案中，所述提取液包含甲醇、二氯甲烷和水。
在一个或多个实施方案中，醇、二氯甲烷或三氯甲烷与水的体积比为1～10∶1～5∶1～10，例如2～6∶1～4∶2～6，优选为3∶2∶3。
在本申请的另一个方面，提供了一种用于诊断便秘、预测对象的便秘发生或进展风险、或便秘预后评估、或提供治疗方案的产品，例如检测试剂盒或装置，其包含用于检测对象样品中DPA的试剂，任选还包括用于检测对象样品中PIB的试剂。
在一个或多个实施方案中，所述样品包含粪便。
在一个或多个实施方案中，所述便秘是顽固性便秘，优选PIB引起的顽固性便秘。
在一个或多个实施方案中，所述检测DPA的试剂是使用选自下组的一种或多种检测方法检测DPA的试剂：色谱法(例如气相色谱、HPLC)、质谱、气相色谱质谱联用仪、液相色谱质谱联用仪、比色法、酶联免疫法、超高效液相色谱法、毛细管气相色谱等检测。
在一个或多个实施方案中，检测DPA的试剂包括从粪便中提取DPA的试剂。在一个或多个实施方案中，检测DPA的试剂包括甲醇、二氯甲烷、水中的一种或多 种。优选地，检测DPA的试剂包括以一定比例混合的甲醇、二氯甲烷和水。
在一个或多个实施方案中，检测DPA的试剂包括对DPA或其衍生物具有特异性的物质。在一个或多个实施方案中，DPA衍生物是DPA的的体内代谢产物。
在一个或多个实施方案中，检测DPA的试剂带有可检测标记物，例如选自下组的可检测标记物：放射性同位素、荧光团、化学发光部分、酶、酶底物、酶辅因子、酶抑制剂、燃料、金属离子、或配体(如，生物素或半抗原)。
在一个或多个实施方案中，检测PIB的试剂包括特异性扩增PIB的标志核酸序列的引物或探针、特异性结合PIB的表面蛋白或分泌物的抗体或配体。在一个或多个实施方案中，所述标志核酸序列是SNP。
在一个或多个实施方案中，所述产品是装置，所述装置包括存储器、处理器以及存储在存储器上并可用在处理器上运行的计算机程序，其特征在于，所述处理器执行所述程序时实现以下步骤：
a)检测对象样品中的DPA，任选还检测PIB，和
b)通过步骤a)的结果诊断便秘、预测对象的便秘发生或进展风险、或便秘预后评估、或提供治疗方案。
在一个或多个实施方案中，其中步骤a)包括：a1)检测对象样品中的DPA，和a2)确定在步骤a1)中检测的DPA的丰度是否高于对照水平。
在一个或多个实施方案中，其中步骤a)包括：a1)检测对象样品中的DPA和PIB，和a2)确定在步骤a1)中检测的DPA的丰度是否高于对照水平，以及是否存在PIB或其含量是否高于对照。
在一个或多个实施方式中，与对照水平相比，对象样品中的DPA含量增加，表明所述对象罹患或疑似罹患便秘、或具有罹患便秘的风险、或便秘预后不良，或表明需更换治疗方案。
在一个或多个实施方案中，与对照水平相比，对象样品中的DPA含量增加，并且对象样品中包含PIB或PIB含量增加，表明所述对象罹患或疑似罹患便秘、或具有罹患便秘的风险、或便秘预后不良，或表明需更换治疗方案。
在一些实施方式中，所述对照水平是未罹患便秘的对象样品中的DPA含量。在一些实施方式中，所述对照水平为小于或等于2.26±0.43ng/mg，例如小于或等于0.07±0.01ng/mg，优选为小于或等于0.05±0.01ng/mg，更优选为小于或等于0.02±0.01ng/mg。
在一些实施方式中，本申请产品中所用试剂以及产品的性能等所涉及的特征如本文中所限定或阐述。
本发明另一方面还提供了抗生素或其衍生物在制备治疗便秘的药物中的用途，所述抗生素包括但不限于喹诺酮类抗生素、碳青霉烯类抗生素、丝裂酶素、硫酸博来霉素、磺胺增效剂。
在一个或多个实施方案中，所述衍生物是水合物。
在一个或多个实施方案中，所述便秘是顽固性便秘，优选PIB引起的顽固性便秘。
在一个或多个实施方案中，所述喹诺酮类包括但不限于左氧氟沙星、巴洛沙星、盐酸贝西沙星、恩诺沙星、莫西沙星、帕珠沙星甲磺酸盐、加替沙星、司帕沙星、盐酸沙拉沙星、诺氟沙星、环丙沙星、左氧氟沙星、依诺沙星、洛美沙星、西他沙星、盐酸二氟沙星。优选诺氟沙星、西他沙星水合物、盐酸沙拉沙星、盐酸贝西沙星、左氧氟沙星半水合物、盐酸洛美沙星、司帕沙星、洛美沙星、左氧氟沙星、加替沙星。
在一个或多个实施方案中，所述碳青霉烯类包括但不限于美罗培南、多尼培南、比阿培南、厄他培南钠；优选多尼培南。
本发明另一方面还提供了噬菌体在制备治疗便秘的药物中的用途，所述噬菌体为T4噬菌体。
在一个或多个实施方案中，所述便秘是顽固性便秘，优选PIB引起的顽固性便秘。
在一个或多个实施方案中，所述噬菌体为肌尾病毒科T4噬菌体。
在一个或多个实施方案中，所述噬菌体可以应用于灌肠、灌胃、注射，优选的施用途径为口服、灌肠、注射，优选的施用量为1x109个噬菌体/次/人。
本发明另一方面还提供了治疗便秘的药物组合物，包含药学上可接受的辅料和：(1)抗生素或其衍生物，和/或(2)噬菌体，所述抗生素包括但不限于喹诺酮类抗生素、碳青霉烯类抗生素、丝裂酶素、硫酸博来霉素、磺胺增效剂，所述噬菌体为T4噬菌体。
在一个或多个实施方案中，所述衍生物是水合物。
在一个或多个实施方案中，所述便秘是顽固性便秘，优选PIB引起的顽固性便秘。
在一个或多个实施方案中，所述喹诺酮类包括但不限于左氧氟沙星、巴洛沙星、盐酸贝西沙星、恩诺沙星、莫西沙星、帕珠沙星甲磺酸盐、加替沙星、司帕沙星、盐 酸沙拉沙星、诺氟沙星、环丙沙星、左氧氟沙星、依诺沙星、洛美沙星、西他沙星、盐酸二氟沙星。优选诺氟沙星、西他沙星水合物、盐酸沙拉沙星、盐酸贝西沙星、左氧氟沙星半水合物、盐酸洛美沙星、司帕沙星、洛美沙星、左氧氟沙星、加替沙星。
在一个或多个实施方案中，所述碳青霉烯类包括但不限于美罗培南、多尼培南、比阿培南、厄他培南钠；优选多尼培南。
在一个或多个实施方案中，所述噬菌体为肌尾病毒科T4噬菌体。
附图说明
图1：PIB分泌的化学物质是二十二碳五烯酸(Docosapentaenoic acid，DPA)。图1，A和B：PIB培养上清用有机溶液萃取后极性相物质和非极性相物质对肠收缩的抑制活性(n＝3)。图1，C：非极性物质经HPLC(C18柱)色谱分析的洗脱峰型，箭头所指为PIB上清中出现的额外峰。图1，D：收集PIB额外峰，并测定其抑制肠收缩的活性。图1，E：用LC-MASS测定活性峰分子量。图1，F：活性峰各碎片的分子量。图1，G：DPA纯品对肠收缩的抑制效应。图1，H：DPA纯品抑制肠收缩的剂量效应。**p＜0.01。
图2：结肠给药测定DPA对结肠排出功能的活性。
图3：便秘小鼠粪便中DPA含量。
图4：顽固性慢性便秘患者粪便中DPA含量较高。图4，A和B：顽固性慢性便秘人群(A)和正常健康人群(B)粪便HPLC分析，箭头所指为DPA特征峰。图4，C：0.03125ng DPA标准品HPLC实验特征峰丰度。图4，D：顽固性慢性便秘人群和正常健康人群粪便DPA含量比较。**p＜0.01。
图5：诺氟沙星治疗PIB便秘小鼠后粪便中PIB菌含量测定。
图6：噬菌体限制性酶切图谱。
图7：T4 g23的PCR检测结果。
图8：噬菌体的体外杀菌效果。
图9：噬菌体cocktail疗法对PIB便秘模型的体内治疗效果。
具体实施方式
前期研究表明，PIB菌(CGMCC 20603)是顽固性慢性便秘的致病菌，但其致病机制不清楚。发明人猜测，PIB可能是分泌了某种物质，能直接抑制肠道节律性收缩，这种物质有可能是蛋白质也有可能是有机物。本发明分离并鉴定了该物质，系统研究了PIB菌致病物质及其与疾病的相关性。
具体地说，本发明提供了一种DPA检测试剂在制备预防或治疗顽固性慢性便秘药盒中的应用。该应用直接测定粪便中的PIB特定代谢产物一一二十二碳五烯酸(DPA)，从而判断是否有PIB感染和顽固性便秘的发生。
DPA及其检测
前期研究表明，PIB菌是顽固性慢性便秘的致病菌，其分泌的DPA能直接抑制肠道节律性收缩。发明人还发现，将DPA丰度和PIB进行联合分析时，两者与疾病的相关性均非常高。DPA是一种长链不饱和脂肪酸，常存在于深海鱼、海豹、海藻等生物内，是深海鱼油的重要成分。已有研究表明，DPA及其类似长链不饱和脂肪酸DHA和EPA作为营养保健食品，对改善心血管疾病、神经疾病等有明显的有益效果，但这类脂肪酸对肠道有何作用还没有报道。
本发明首次发现粪便中DPA与便秘的关系。所述DPA丰度可以由色谱法(例如HPLC)、质谱、气相色谱、气相色谱质谱联用仪、液相色谱质谱联用仪、比色法、酶联免疫法、超高效液相色谱法、毛细管气相色谱等检测。
如本文所用，术语“检测物质”、“检测试剂”或“检测DPA的试剂”可互换使用，包括检测DPA或其衍生物所用的任何物质。
例如，检测DPA的试剂是使用选自下组的一种或多种检测方法检测DPA或其衍生物的试剂：色谱法(例如气相色谱、HPLC)、质谱、气相色谱质谱联用仪、液相色谱质谱联用仪、比色法、酶联免疫法、超高效液相色谱法、毛细管气相色谱等检测。这样的试剂可以是DPA的溶剂，例如二氯甲烷和/或三氯甲烷。
或者，检测DPA的试剂还可包括从粪便中提取DPA的试剂。在一个或多个实施方案中，检测DPA的试剂包括甲醇、二氯甲烷、水中的一种或多种。优选地，检测DPA的试剂包括以一定比例混合的甲醇、二氯甲烷和水。
又如，检测DPA的试剂包括对DPA或其衍生物具有特异性的物质。这些物质的种类和使用方法在本领域常规知识范围内。为了便于检测，本发明的检测试剂还可带有可检测标记，所述可检测标记包括但不限于：放射性同位素、荧光团、化学发光部分、酶、酶底物、酶辅因子、酶抑制剂、染料、金属离子、配体(如，生物素或半抗原)等。
本文中，DPA衍生物表示DPA的的体内代谢产物，包括：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-3DPA、16，17R-dihydroxy-7Z，10，13，14，19Zn-3DPA、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。其中，HDPA为羟基-DPA缩写，H(p)-DPA为羟基(过氧基)-DPA的缩写。通常，检测DPA的试剂还可包括从粪便中提取上述衍生物的试剂。
通常，DPA检测前，样品(例如粪便)可经过预处理以去除可能影响检测的物质。优选地，所述样品为粪便样品，所述粪便样品可以来源于人新鲜粪便、冷冻粪便、液体稀释后粪便。因此，本发明还提供一种从粪便中提取DPA的方法，所述方法包括：用预处理液提取液处理获自对象的粪便样品，所述提取液包含醇、二氯甲烷或三氯甲烷和水。醇、二氯甲烷或三氯甲烷与水的体积比为1～10∶1～5∶1～10，例如2～6∶1～4∶2～6，优选为3∶2∶3。所述醇包括甲醇、乙醇或丙醇。在示例性实施方案中，所述提取液包含体积比为3∶2∶3的甲醇、二氯甲烷和水。
检测DPA的方法是本领域常规知识。示例性DPA检测方法包括HPLC色谱法、液相联用质谱法、气相联用质谱法、酶联免疫法、比色法等。
HPLC色谱法：将预处理样品溶解后，进样、洗脱。色谱柱常采用C18柱，流动相一般用乙腈/甲醇/甲酸铵/异丙醇。经分步洗脱后，分析DPA特征峰丰度，与已知浓度标准品丰度比较，定量。
液相联用质谱法：收集50mg粪便，加入有机物提取试剂(甲醇/二氯甲烷/水＝420uL∶280uL∶420uL)，收集有机相、抽干、溶解、制备上样样品，之后用质谱测定分子量和结构，与已知浓度DPA标准品比较定量。
气相联用质谱法：收集50mg粪便，用氢氧化钾甲醇甲酯化，用三氯甲烷或其他有机溶剂提取不饱和脂肪酸甲酯、抽干制备样品。上样气相色谱或气相色谱质谱联用仪，分析DPA甲酯出峰特征。
酶联免疫法：用抗DPA或长链不饱和脂肪酸抗体为一抗包被酶标板，加入待测样品(粪便经预处理后)、孵育、洗板，加入酶标二抗、洗板、显色。
比色法：用碘化钾溶液与粪便样品混合，待加成反应完成后，加入1％淀粉溶液 显色。
上述DPA检测方法中所涉及的试剂包含在本发明的检测试剂盒中。
检测产品
本文还提供一种用于检测患者顽固性慢性便秘的产品，例如，试剂盒，其包含用于检测DPA的试剂，任选还包括用于检测PIB的试剂。
根据所用检测方法的需要，可选择适当的DPA检测试剂，并将其制成始于所用检测方法的产品，如试剂盒。本领域技术人员可根据实际条件和需要对检测方式和产品中所含试剂进行调整和改变。
所述试剂盒还可包含选自下组的一种或多种物质：容器、使用说明书、阳性对照物、阴性对照物、缓冲剂、溶剂或助剂，例如用于混悬或固定细胞的溶液，可检测的标签或标记，从样品中去除影响检测结果的杂质，例如蛋白质、核酸等的试剂。
在一个示例中，本文提供了一种适于通过色谱法检测生物样品中DPA和/或PIB的检测试剂盒。该检测试剂盒可包含：色谱分析法检测DPA所需试剂(甲醇、乙腈等)和检测PIB的一种或多种试剂；以及可选的装有上述试剂的容器及使用说明书。
检测PIB的试剂包括特异性扩增PIB的标志核酸序列的引物或探针、特异性结合PIB的表面蛋白或分泌物的抗体或配体。在一个或多个实施方案中，所述标志核酸序列是SNP。所述引物或探针的示例参见CN202011023676.4，其全文通过引用纳入本文。例如，所述寡核苷酸引物扩增志贺氏菌属细菌(CGMCC 20603)基因组的产物包含：(1)所述细菌的基因组中位于与登记号为CGMCC 20603的细菌菌株的基因组的选自以下的一个或多个位置对应的位置的核苷酸：PIB2013.g.331、PIB2324.g.562、PIB2324.g.601、PIB2629.g.1126、PIB3156.g.2396，(2)选自以下的一个或多个位置的核苷酸：由SEQ ID NO：9和10作为引物的基因组扩增片段的第21个核苷酸；由SEQ ID NO：11和12作为引物的基因组扩增片段的第21个核苷酸；由SEQ ID NO：13和14作为引物的基因组扩增片段的第21个核苷酸；由SEQ ID NO：15和16作为引物的基因组扩增片段的第20个核苷酸；由SEQ ID NO：17和18作为引物的基因组扩增片段的第21个核苷酸；(3)选自SEQ ID NO：2-6的任意一个或多个序列或其具有(2)中所述核苷酸的片段或与它们具有至少98％序列相同性的变体，或(4)SEQ ID NO：1所示16S rDNA序列或与其具有至少98％序列相同性的序列。在一个或多个实施方案中，所述寡核苷酸引物的扩增产物包含SEQ ID NO：1-6中任一所述序列。在一个或多个实施方案中，所述寡核苷酸引物选自SEQ ID NO：7-18中任一种或多种。
本文的检测试剂盒中还可附有试剂盒的使用说明书，其中记载了如何采用试剂 盒进行检测，以及如何对顽固性慢性便秘进行判断、对治疗方案进行选择。
诊断
发明人发现，顽固性便秘患者粪便中存在有高浓度的DPA，阳性患者粪便的DPA含量为2.26±0.43ng/mg(正常人群为0.07±0.01ng/mg)。因此，检测粪便中的DPA，可以诊断顽固性便秘，阳性率为44.12％。此外，同时测定DPA和PIB两个指标能提高阳性检出率至75％。
本发明提供(1)检测DPA的试剂，任选和(2)检测PIB的试剂在制备诊断顽固性便秘的试剂盒中的用途。检测DPA的试剂和检测PIB的试剂如本文任一实施方案所述。
本发明还提供诊断顽固性便秘的方法，包括检测对象粪便中的DPA，任选还包括检测对象粪便中的PIB。优选地，若对象粪便中DPA含量大于或等于0.07±0.01ng/mg(例如大于或等于0.1ng/mg、大于或等于0.5ng/mg、大于或等于1.0ng/mg、大于或等于1.5ng/mg、大于或等于2.0ng/mg、大于或等于2.26±0.43ng/mg)，则对象是顽固性便秘的潜在患者。
抗生素或噬菌体
本发明还提供了抗生素或噬菌体在制备治疗顽固性便秘药物中的应用。发明人发现，PIB敏感性抗生素或噬菌体在治疗顽固性便秘时，粪便中的DPA显著下降。
本发明通过对现有抗生物药库进行筛选获得PIB敏感性抗生素，为清除PIB和治疗顽固性便秘提供药物配方和治疗方法。
如本文所用，所述抗生素包括但不限于喹诺酮类抗生素、碳青霉烯类抗生素、硫酸博来霉素、丝裂酶素、磺胺增效剂。所述喹诺酮类可以有左氧氟沙星、巴洛沙星、盐酸贝西沙星、恩诺沙星、莫西沙星、帕珠沙星甲磺酸盐、加替沙星、司帕沙星、盐酸沙拉沙星、诺氟沙星、环丙沙星、左氧氟沙星、依诺沙星、洛美沙星、西他沙星、盐酸二氟沙星等。所述碳青霉烯类可以有美罗培南、多尼培南、比阿培南、厄他培南钠。
如本文所用，所述喹诺酮类中，诺氟沙星最敏感，其排列顺序为：诺氟沙星＞西他沙星水合物＞盐酸沙拉沙星＞盐酸贝西沙星＞左氧氟沙星半水合物＞盐酸洛美沙星＞司帕沙星＞洛美沙星＞左氧氟沙星＞加替沙星＞环丙沙星盐酸盐一水合物＞莫西沙星＞盐酸二氟沙星＞甲磺酸达氟沙星＞对甲苯磺酸妥舒沙星水合物＞依诺沙星倍半水合物＞巴洛沙星。如本文所用，所述碳青霉烯类中，PIB对多尼培南最为敏感。
PIB是引起顽固性慢性便秘的致病菌，清除体内PIB是治疗该疾病的理想方法。噬菌体是细菌病毒，能特异性地识别特定菌，并侵入该细菌制造细菌裂解酶、裂解菌 体。噬菌体能高效高特异性地消灭特定细菌，是非常安全高效的抗菌病毒，已在许多国家广泛应用，在美国已开展临床试验研究，不久即将投入市场。本文筛选PIB特异性噬菌体，并进行生物学鉴定，筛选到对PIB敏感的噬菌体。
如本文所用，所述噬菌体为T4噬菌体，例如肌尾病毒科T4噬菌体。
如本文所用，所述噬菌体为PIB特异性噬菌体，可以通过以下步骤筛选获得：采用双层平板法对上述获得的噬菌体原液进行鉴定，双层平板法包含为上下不同浓度的两层的LB固体培养基，其下层为1.5％琼脂的LB培养基，以支撑营养为主；上层为0.5％琼脂LB培养基，以利于宿主菌和噬菌体生长扩散。这种方法也更便于噬菌斑的观察。具体操作为：首先准备好1.5％的下层琼脂培养基，将100μl对数期菌液和100μl噬菌体原液混合均匀后，室温静置约10min，快速加入到预先加热至48℃的0.5％的上层培养基中，快速混合后倒入下层培养基中，均匀铺平在下层平板中，室温静置20min，正置37℃恒温培养，观察是否有噬菌斑的形成。挑取平板上的噬菌斑，加入至LB培养基中，再按5％的比例加入PIB菌液，37℃，200rpm摇床培养6-8h。过滤离心后，用双层平板法涂布平板，重复上述操作两至三次，直至观察到大小均一，边缘完整的圆形透明状斑块。用灭好菌的枪头挑取斑块，同时加入5％菌液至LB培养基中，37℃，200rpm，摇床过夜培养扩增，12000rpm，5min，4℃离心，再用0.22μm滤膜过滤，加入到10％的SM buffer中，于-80℃冰箱保存。
如本文所用，噬菌体治疗方案和疗效分析可以为通过噬菌体与PIB共摇床培养，制备phage1，2，3这三种噬菌体的cocktail，充分混合后，每只Phage治疗组的小鼠每次灌胃100μl phage cocktail。
药物组合物
本文所述抗生素和/或噬菌体可以用于科学研究或用于制备相应癌症的治疗药物。本文中，“药物组合物”指用于给予个体的组合物且涵盖用于治疗的抗生素和/或噬菌体的组合物。本发明的药物组合物还可包含药学上可接受的辅料。
如本文中所用，“药学上可接受的”的成分是适用于人和/或哺乳动物而无过度不良副反应(如毒性、刺激和反应)的，即具有合理的效益/风险比的物质。
本发明的药物组合物含有安全有效量的本发明的活性成分以及药学上可接受的辅料。术语“药学上可接受的辅料”指用于治疗剂给药的辅料，包括各种赋形剂和稀释剂。这类辅料包括(但不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。通常药物制剂与给药方式匹配，本发明的药物组合物的剂型为注射剂、口服制剂(片剂、胶囊、口服液)、透皮剂、缓释剂。例如用生理盐水或含葡萄糖和其他辅剂的水溶液通过常规方法进行制备。本发明的药学上可接受的辅料包括适用于抗生素和/ 或噬菌体递送的辅料。所述的药物组合物宜在无菌条件下制造。可通过熟知的常规方法配制包含这些辅料的组合物。
本发明的组合物可局部或全身性给予。在某些实施方式中，本发明提供的组合物可通过口服或胃肠道外给予，例如静脉内、动脉内、鞘内、真皮下或肌内给予。胃肠道外给药制剂包括无菌的水性或非水性溶液、悬液和乳液。非水性溶剂的示例是丙二醇、聚乙二醇、植物油如橄榄油，和可注射有机酯如油酸乙酯。水性运载体包括水、醇溶液/水溶液、乳液或悬液，包括盐水和缓冲介质。胃肠道外载剂包括氯化钠溶液、林格右旋糖、右旋糖和氯化钠、乳酸林格溶液或固定油。静脉内载剂包括液体和营养补充剂、电解质补充剂(例如基于林格右旋糖的那些物质)等。也可存在防腐剂和其它添加剂，例如，抗微生物剂、抗氧化剂、螯合剂和惰性气体等。本文所述抗生素可通过口服、肌内或静脉内给予。本文所述噬菌体可以通过口服、灌肠、灌胃、肌内或静脉内给予。
本文的药物组合物可以合适剂量给予对象。本发明所述的活性成分可随给药的模式和待治疗的疾病的严重程度等而变化。可由主治医师和临床因素确定剂量方案。如医学领域所熟知，用于任何一个患者的剂量取决于多种因素，包括患者的体型、体表面积、年龄、待给予的特定化合物、性别、给药时间和给药途径、总体健康状况和同时给予的其他药物。例如，由治疗状况的迫切要求，可每天给予若干次分开的剂量，或将剂量按比例地减少。示例性地，本文所述噬菌体的剂量为1x109个噬菌体/次/人。
如本文所用，术语“对象”或“患者”是哺乳动物，例如鼠、兔、人等。
如本文所用，术语“对照水平”是指用作参照的DPA或PIB水平，其包括但不限于：非便秘对象的DPA或PIB水平、由同一对象的非便秘正常生物样品中测得的DPA或PIB水平、通过统计学确定的群体标准水平、或经标准化的水平。在一个或多个实施方案中，所述对照水平通过下述步骤确定：(1)治疗前，在来自患者的生物样品中检测DPA或PIB水平，(2)治疗后所述患者生物样品中DPA或PIB水平，(3)将治疗后所述患者的应答与在步骤(2)种确定的DPA或PIB水平相关联，然后对比(1)的DPA或PIB生物水平来确定对照水平。
本发明的化合物可从市售途经获得，或者可按照已披露的方法或参照已披露的方法合成得到。本发明的其它方面由于本文的公开内容，对本领域的技术人员而言是显而易见的。下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围，特定实施例、材料、数量和操作步骤将根据本文所阐述的本发明的范围和精神进行更宽泛的解释。下列实施例中未注明具体条件 的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York：Cold Spring Harbor Laboratory Press，1989)中所述的条件，或按照制造厂商所建议的条件。
本发明的一些具体实施方案
项目1、试剂在制备诊断便秘的检测试剂盒中的用途，所述试剂包括(1)用于检测DPA的试剂，所述试剂任选还包括(2)用于检测PIB的试剂，
优选地，所述便秘包括顽固性便秘，更优选包括由PIB引起的顽固性便秘，
优选地，所述对象包括哺乳动物，
优选地，所述样品包括粪便。
项目2、如项目1所述的用途，其特征在于，所述检测DPA的试剂是使用选自下组的一种或多种检测方法检测样品中DPA的试剂：色谱法、质谱、气相色谱质谱联用仪、液相色谱质谱联用仪、比色法、酶联免疫法、超高效液相色谱法、毛细管气相色谱等检测，
优选地，
检测DPA的试剂包括DPA的溶剂，例如二氯甲烷和/或三氯甲烷，
检测DPA的试剂包括从粪便中提取DPA的试剂，例如，检测DPA的试剂包括甲醇、二氯甲烷、水中的一种或多种；优选地，检测DPA的试剂包括甲醇、二氯甲烷和水的混合物，
检测DPA的试剂包括对DPA或其衍生物具有特异性的物质，
检测PIB的试剂包括特异性扩增PIB的标志核酸序列的引物或探针、特异性结合PIB的表面蛋白或分泌物的抗体或配体，
所述检测试剂盒还包含选自下组的一种或多种物质：容器、缓冲剂、助剂、溶剂、阴性对照物、阳性对照物、使用说明书。
项目3、如项目1或2所述的用途，其特征在于，
与对照水平相比，对象样品中的DPA含量增加，表明所述对象罹患或疑似罹患便秘，或具有罹患便秘的风险，或
与对照水平相比，对象样品中的DPA含量增加，并且对象样品中包含包含PIB或PIB含量增加，表明所述对象罹患或疑似罹患便秘，或具有罹患便秘的风险，
优选地，所述对照水平是未罹患便秘的对象样品中的DPA含量。
项目4、一种从粪便中提取DPA的方法，所述方法包括：用提取液处理获自对象的粪便样品，所述提取液包含醇、二氯甲烷或三氯甲烷和水，
优选地，所述醇包括甲醇、乙醇或丙醇，
优选地，醇、二氯甲烷或三氯甲烷与水的体积比为1～10∶1～5∶1～10，
更优选地，所述提取液包含甲醇、二氯甲烷和水。
项目5、一种用于诊断便秘、预测对象的便秘发生或进展风险、或便秘预后评估、或提供治疗方案的产品，所述产品包含用于检测对象样品中DPA的试剂，任选还包括用于检测对象样品中PIB的试剂，
优选地，所述便秘包括顽固性便秘，更优选包括由PIB引起的顽固性便秘，
优选地，所述对象包括哺乳动物，
优选地，所述样品包括粪便。
项目6、如项目5所述的用途，其特征在于，所述产品是检测试剂盒，所述检测DPA的试剂是使用选自下组的一种或多种检测方法检测样品中DPA的试剂：色谱法、质谱、气相色谱质谱联用仪、液相色谱质谱联用仪、比色法、酶联免疫法、超高效液相色谱法、毛细管气相色谱等检测，
优选地，
检测DPA的试剂包括DPA的溶剂，例如二氯甲烷和/或三氯甲烷，
检测DPA的试剂包括从粪便中提取DPA的试剂，例如，检测DPA的试剂包括甲醇、二氯甲烷、水中的一种或多种；优选地，检测DPA的试剂包括甲醇、二氯甲烷和水的混合物，
检测DPA的试剂包括对DPA或其衍生物具有特异性的物质，
检测PIB的试剂包括特异性扩增PIB的标志核酸序列的引物或探针、特异性结合PIB的表面蛋白或分泌物的抗体或配体，
所述检测试剂盒还包含选自下组的一种或多种物质：容器、缓冲剂、助剂、溶剂、阴性对照物、阳性对照物、使用说明书。
项目7、如项目5或6所述的用途，其特征在于，所述产品是装置，所述装置包括存储器、处理器以及存储在存储器上并可用在处理器上运行的计算机程序，其特征在于，所述处理器执行所述程序时实现以下步骤：
a)检测对象样品中的DPA，任选还检测PIB，和
b)通过步骤a)的结果诊断便秘、预测对象的便秘发生或进展风险、或便秘预后评估、或提供治疗方案，
优选地，
步骤a)包括：a1)检测对象样品中的DPA，和a2)确定在步骤a1)中检测的DPA的丰度是否高于对照水平，或
其中步骤a)包括：a1)检测对象样品中的DPA和PIB，和a2)确定在步骤a1)中检测的DPA的丰度是否高于对照水平，以及是否存在PIB或其含量是否高于对照，
更优选地，
与对照水平相比，对象样品中的DPA含量增加，表明所述对象罹患或疑似罹患便秘、或具有罹患便秘的风险、或便秘预后不良，或表明需更换治疗方案，或
与对照水平相比，对象样品中的DPA含量增加，并且对象样品中包含PIB或PIB含量增加，表明所述对象罹患或疑似罹患便秘、或具有罹患便秘的风险、或便秘预后不良，或表明需更换治疗方案，或
所述对照水平是未罹患便秘的对象样品中的DPA含量。
项目8、抗生素或其衍生物在制备治疗便秘的药物中的用途，所述抗生素包括但不限于喹诺酮类抗生素、碳青霉烯类抗生素、丝裂酶素、硫酸博来霉素、磺胺增效剂，
优选地，
所述衍生物是水合物，和/或
所述便秘包含顽固性便秘，优选包含PIB引起的顽固性便秘，和/或
所述喹诺酮类包括：左氧氟沙星、巴洛沙星、盐酸贝西沙星、恩诺沙星、莫西沙星、帕珠沙星甲磺酸盐、加替沙星、司帕沙星、盐酸沙拉沙星、诺氟沙星、环丙沙星、左氧氟沙星、依诺沙星、洛美沙星、西他沙星、盐酸二氟沙星，和/或
所述碳青霉烯类包括：美罗培南、多尼培南、比阿培南、厄他培南钠。
项目9、噬菌体在制备治疗便秘的药物中的用途，所述噬菌体为T4噬菌体，
优选地，
所述便秘包含顽固性便秘，优选包含PIB引起的顽固性便秘，和/或
所述噬菌体为肌尾病毒科T4噬菌体。
项目10、一种治疗便秘的药物组合物，包含药学上可接受的辅料和：(1)抗生素或其衍生物，和/或(2)噬菌体，所述抗生素包括但不限于喹诺酮类抗生素、碳青霉烯类抗生素、丝裂酶素、硫酸博来霉素、磺胺增效剂，所述噬菌体为T4噬菌体，
优选地，
所述衍生物是水合物，和/或
所述便秘包含顽固性便秘，优选包含PIB引起的顽固性便秘，和/或
所述喹诺酮类包括：左氧氟沙星、巴洛沙星、盐酸贝西沙星、恩诺沙星、莫西沙星、帕珠沙星甲磺酸盐、加替沙星、司帕沙星、盐酸沙拉沙星、诺氟沙星、环丙沙星、左氧氟沙星、依诺沙星、洛美沙星、西他沙星、盐酸二氟沙星，和/或
所述碳青霉烯类包括：美罗培南、多尼培南、比阿培南、厄他培南钠，和/或，
所述噬菌体为肌尾病毒科T4噬菌体。
示例性实施方案
样本来源：PIB是一种肠道菌，显而易见具有感染多组织的可能性。DPA是PIB特征性代谢产物，因此，有可能存在于多种组织。粪便是PIB产生DPA的主要产地，样本来源优先为粪便。粪便样本可来源于人新鲜粪便、冷冻粪便、液体稀释后粪便。
样品处理：优先选择粪便样本进行预处理，其特征是将一定量的粪便与有机提取试剂按比例混合，提取有机相中的有机物。
HPLC纯化分离DPA：将处理好的样品用HPLC分离纯化，其特征是用碳柱(优先使用C18柱)、乙腈/甲醇/甲酸铵/异丙醇流动相洗脱、收集活性峰制备样品。
LC-MASS(液质联用质谱)确证分子结构：用活性峰上样LC-MASS分析，确定分子量为330道尔顿的物质峰，再次确定其碎片分子量，然后提取物质的结构。
制作粪便DPA检测试剂盒：试剂盒至少包含以下组成部分：粪便收集装置、有机溶剂、阳性标准品、阴性对照品、DPA标准品。具体用途如下：
粪便收集装置：患者自行采集粪便设备和容器
有机溶剂：用于样品萃取
阳性标准品：阳性粪便提取物
标准品：DPA纯品
阴性对照品：阴性粪便提取物
PIB-DPA试剂盒的用途：本发明首次发现粪便DPA与便秘的关系。目前常利用气相色谱或HPLC色谱分析的方法测定食物、鱼油中DPA的含量，用以评价食物的营养成分。食物中DPA主要在小肠吸收，不会到达结肠，因此不会影响粪便DPA含量而干扰诊断。本发明所描述的粪便DPA检测方法可用于诊断和筛查顽固性便秘或PIB菌感染性便秘。
以下将以具体实施例的方式对本发明作进一步说明。应理解，这些实施例仅仅是阐述性的，并非用于限制本发明的范围。实施例中所用到的方法和试剂，除非另有说明，否则为本领域的常规方法和试剂。
实施例1、发现PIB菌抑制肠道收缩的物质基础是二十二碳五烯酸(DPA)
材料与方法
动物：C57BL/6小鼠(雌雄各半、6周-8周龄、体重20-22克)
PIB菌：CGMCC 20603，由朱敏生实验室保存(50％甘油、-80℃保存)主要试剂：二十二碳五烯酸(DPA)购自Aladdin公司，蛋白酶K购自Sigma公司.
结肠收缩张力测定：
PIB分泌有机物的提取：将冻存的PIB菌接种于LB中，37℃培养过夜(12-16小时)，收集培养上清；同时，将DH5a大肠杆菌也接种于LB中，37℃培养过夜(12-16小时)，收集培养上清得到对照上清。将15ml上清用提取液分装10管，用SpeedVac仪(Thermofisher)抽干。用预冷的甲醇溶液(410ul甲醇+210ul水)充分溶解，然后加入280ul二氯甲烷和210ul水。震荡混匀后，离心收集非极性相，并抽干备用。
HPLC纯化有机物：仪器为Agilent 1200 HPLC。色谱柱为C18柱(150mm×4.6mm)为Yiliteng产品。洗脱流动相为A：甲醇/乙腈/10mM甲酸铵(1∶1∶1， v/v/v)；B：乙腈/异丙醇(1∶1，v/v)。收集20管洗脱液并干燥。
LCMS/MS：上机分析：选用混合四极杆时间飞行质谱(品牌型号为AB Sciex Triple TOF 4600 instruments)，结合C18柱子(Phenomenex Accucore C18，150mm×2.1mm，2.6μm)分离，流动相分别是：A相：乙腈/甲醇/10mM甲酸铵(1∶1∶1，v/v/v)；B相：乙腈/异丙醇(1∶1，v/v)，梯度洗脱顺序为：0-1min，20％B；1-4min，60％B；4-10min，70％B；10-15min，95％B；15-20min，95％B。质谱分析使用DuoSpray Ion Source结合负离子模式，参数设置如下：辅助气压力，55psi；雾化气压力，55psi；空气幕气体，35psi；离子源温度，600℃；电压4500V；势能，80eV，碰撞能量10eV。数据处理用Peakview软件。
结果
一、PIB分泌物存在于有机相
首先排除PIB所分泌的活性物是否是蛋白质。用蛋白酶K处理PIB培养上清(37℃、60分钟)，处理后上清仍然有抑制肠道收缩的活性，由此活性产物不可能是蛋白质。当用二氯甲烷/甲醇/水提取液处理PIB培养上清，取有机相物质进行活性测定。结果表明，PIB培养上清的有机相物质具有明显的抑制肠收缩的活性，而对照细菌(DH5a)培养上清的有机相物质无活性，如图1A、B所示。
二、PIB活性物质为二十二碳五烯酸
为确定PIB上清活性物质的化学结构，用液质联用质谱分析上述有机相混合物质。当样品经液相色谱时，PIB培养上清样品在洗脱后期出现一个特异性峰，而对照样品不出现(图1C)。收集该峰样品，并测定其生物学活性。结果发现：该峰具有显著的生物学活性，而其他洗脱峰没有明显的肠收缩抑制活性(图1D)，提示该活性峰是PIB培养上清的特征性活性峰。用质谱进一步分析该活性峰，该峰的活性物质的分子量为329.25道尔顿；对其碎片进行分析，发现该物质含有两个片段，分子量分别为285.26道尔顿和59.01道尔顿。将以上数据输入Preview数据库，结果发现二十二碳五烯酸是唯一候选分子。
结论：本研究首次发现，PIB分泌的肠抑制活性物质是DPA。
实施例2、二十二碳五烯酸可抑制肠收缩和肠传输速度
DPA是一种长链不饱和脂肪酸，常存在于深海鱼、海豹、海藻等生物内，是深海鱼油的重要成分。已有研究表明，DPA及其类似长链不饱和脂肪酸DHA和EPA作为营养保健食品，对改善心血管疾病、神经疾病等有明显的有益效果，但这类脂肪酸对肠道有何作用还没有报道。以上结果提示，PIB抑制肠收缩的物质基础可能是 DPA，但DPA是否具有直接抑制肠收缩的作用还不清楚。本实施例拟通过体内研究确定DPA是否具有肠收缩抑制功能。
材料与方法
DPA：购自Aladdin公司
DPA给药和结肠动力测定：将DPA通过导管输入结肠，同时加入玻璃微球，观察微球的排出时间。
结果：
用商业化的DPA纯品具有直接抑制结肠收缩的体内活性：为证实DPA在体内是否具有肠道功能，将DPA输注到结肠，然后测定结肠传输时间。结果表明：当肠道输注DPA后，肠传输时间延长了150％左右(图2)。以上结果证实，DPA具有抑制肠收缩、肠传输的功能，是PIB致病分子。
结论：该研究首先发现DPA能显著抑制肠道收缩、降低肠动力，由此导致便秘的发生。
实施例3、PIB便秘动物表型与粪便中DPA的含量相关
实施例1和2的结果表明DPA是PIB致病的致病分子，但粪便中DPA含量与便秘表型是否存在相关性还不清楚。为此，本发明通过HPLC的方法分析了PIB便秘小鼠粪便中DPA的含量及其与PIB感染的相关性。
材料与方法
PIB菌：由南京大学朱敏生实验室保存
动物：C57BL/6小鼠(雌雄各半、6-8周龄、体重20-22克)
LC-MASS分析粪便DPA：收集50mg小鼠粪便，加入有机物提取试剂(甲醇/二氯甲烷/水)，收集有机相、抽干、溶解、制备HPLC上样样品，然后用质谱测定分子量和结构。
实验结果：
本实施例收集了5例PIB便秘小鼠的粪便、5例C57BL/6对照小鼠的粪便、5例经噬菌体治疗后的便秘小鼠。经LC-MASS分析了DPA的含量，结果发现，PIB便秘小鼠的粪便DPA的信号强度明显高于对照组和噬菌体治疗组(p＜0.01)(图3)。将DPA浓度换算成绝对浓度，结果表明：PIB便秘小鼠粪便中的DPA含量约为7.51ug/mg，而对照小鼠粪便中的DPA含量约为0.009ug/mg，两组之间存在极显著差异。
结论
PIB便秘小鼠粪便中DPA含量升高，DPA与便秘有明显相关性。
实施例4、顽固性便秘患者粪便中DPA含量显著升高
根据以前研究，约56％的顽固性便秘患者粪便中是PIB菌阳性，但DPA的含量如何、与疾病的相关性如何还不清。阐明这一问题是评价DPA诊断价值的重要依据，本实施例的目的是通过HPLC测定人粪便中DPA含量，比较便秘组和正常组间的差别。
材料与方法
人群资料：顽固性便秘病例来自于南京金陵医院，所有病人均经罗马III标准诊断为便秘、所有病人均为慢传输便秘、排除肠易激综合症性便秘、所有病人愿意进行手术治疗并已实施手术治疗；正常人群来自于健康志愿者，身体健康，无主诉便秘。
粪便DPA测定：收集50mg新鲜粪便，烘干后取6mg干燥粪便，用烷/醇有机溶解剂提取后，收集有机相，抽干后用有机溶剂重新溶解、HPLC上样。HPLC分析步骤同实施实例一所述。
粪便PIB测定：参见CN202011023676。
结果
本实施例分析了68例顽固性便秘患者粪便中DPA的含量。当样本中含有DPA分子时，在出峰时间为10.1-10.4min左右可出现明显的DPA特征峰(经LC-MASS验证)，峰高与DPA浓度呈正相关。当样品中不含有DPA时，则不出现特征峰(图4A、4B)。68例中30例为DPA阳性，占比44.12％，38例为DPA阴性，占比55.88％。同时，用PCR方法测定68例粪便中PIB菌的情况，发现38例为PIB阳性(55.88％)，30例为PIB阴性(44.12％)。DPA阳性患者与PIB阳性患者并不一致，38例PIB+粪便中有21例为DPA阴性，而30例PIB-粪便中有13例为DPA阳性，同时为PIB+和DPA+的样本有17例，占总样本的25％。采用同样的方法测定了30例正常人的粪便，结果发现所有样本均为PIB阴性或DPA阴性。该结果表明：检测PIB或DPA可检测出44-55％的顽固性便秘患者，如果同时检测两个指标，检出阳性率可提高到75％。
部分PIB阳性患者粪便检测不到DPA，可能原因是DPA在粪便中不是均分布的，当取样量小时，可能丢失了DPA阳性区。发明人在同一粪便样品中，在不同部分取小样测定DPA，的确观察到有的样品DPA含量高，有的样品含量低或检测不到。另外，有些DPA阳性粪便中检测不到PIB。PCR检测PIB的方法要求粪便中细菌是活的，如果取样不好或运输存放不好，导致PIB细菌死亡，PCR方法将检测不到。因此，可以通过同时检测PIB和DPA两个指标来提高检测精度。
如果将以上测定DPA的信号强度换算成绝对浓度，结果显示：DPA阳性患者粪便的DPA含量约为2.26±0.43ng/mg；而正常人群或DPA阴性粪便中DPA含量为0.07±0.01ng/mg。
结论
顽固性便秘患者粪便中存在有高浓度的DPA，阳性率为44.12％，测定粪便中DPA浓度可以诊断顽固性便秘。同时测定DPA和PIB两个指标能提高阳性检出率至75％。
实施例5、PIB敏感性抗生素的筛选
往期研究已发现，PIB感染是导致顽固性便秘的重要原因，但如何清除体内的PIB还不清楚。本实施例目的在于通过对现有抗生素药库进行筛选获得PIB敏感性抗生素，为清除PIB和治疗顽固性便秘提供药物配方和治疗方法。
材料与方法
抗生素药库：购自Selleck公司，共含308种抗生素。
筛选方法：用LB培养液将药库每一化合物稀释200倍，最终浓度为3～80μg/mL。取10μl抗生素分别加入PIB菌液孔(96孔板，每孔100ul菌液)，37℃培养16小时后，测定OD600nm。空白对照为LB培养液。
结果
通过筛选结果发现，绝大部分抗生素对PIB均不敏感，较敏感的有：喹诺酮类(左氧氟沙星、巴洛沙星、盐酸贝西沙星、恩诺沙星、莫西沙星、帕珠沙星甲磺酸盐、加替沙星、司帕沙星、盐酸沙拉沙星、诺氟沙星、环丙沙星、左氧氟沙星、依诺沙星、洛美沙星、西他沙星、盐酸二氟沙星)、碳青霉烯类(美罗培南、多尼培南、比阿培南、厄他培南钠)、丝裂酶素、硫酸博来霉素、磺胺增效剂。
为进一步评价敏感药物的抗菌效价，本发明将这些药物作浓度梯度稀释，测定其剂量效应。同时，用DH5a大肠杆菌为对照菌，比较目标抗生素对肠道不同细菌的敏感性差异。结果表明，在喹诺酮类药物中，诺氟沙星最敏感，其排列顺序为：诺氟沙星＞西他沙星水合物＞盐酸沙拉沙星＞盐酸贝西沙星＞左氧氟沙星半水合物＞盐酸洛美沙星＞司帕沙星＞洛美沙星＞左氧氟沙星＞加替沙星＞环丙沙星盐酸盐一水合物＞莫西沙星＞盐酸二氟沙星＞甲磺酸达氟沙星＞对甲苯磺酸妥舒沙星水合物＞依诺沙星倍半水合物＞巴洛沙星。其中巴洛沙星、盐酸贝西沙星、诺氟沙星、盐酸洛美沙星、依诺沙星倍半水合物、对甲苯磺酸妥舒沙星水合物、洛美沙星对DH5a大肠杆菌的杀菌效果较弱，对PIB菌有一定的特异性。碳青霉烯类中，多尼培南对PIB最为敏感。硫酸博 莱霉素对DH5a大肠杆菌几乎无杀菌效果。具体数据见表1。
表1抗菌素对PIB的MIC及MBC测定(mg/L)PIB和DH5α


注：“无”表示在本实施例中药物梯度稀释浓度下，检测菌对药物不敏感。
结论
喹诺酮类、碳青霉烯类、硫酸博来霉素对PIB敏感。
实施例6、诺氟沙星抗生素清除体内PIB的效果评价
实例五筛选得到若干PIB敏感的抗生素，这些抗生素能不能清除体内PIB尚且不清楚。本实施例目的在于评价敏感抗生素清除动物体内PIB的能力。
材料与方法
动物：C57BL/6动物购于南京集萃药康生物技术有限公司，实验室建立PIB感染模型。
药物：诺氟沙星，购于石药集团欧意药业有限公司。
给药方式：灌胃诺氟沙星(50mg/kg)，每天一次，连续7天。
效果观察：取动物粪便培养、收获菌液，用PCR检测PIB菌。
结果
用诺氟沙星治疗PIB便秘动物1周后，动物粪便中的PIB菌开始转阴，停止用药1月后，粪便中仍检测不出PIB(图5)。
结论
诺氟沙星能有效的清除体内PIB菌，可用于便秘治疗。
实施例7、PIB特异性噬菌体的筛选与鉴定
PIB是引起顽固性慢性便秘的致病菌，清除体内PIB是治疗该疾病的理想方法。噬菌体是细菌病毒，能特异性地识别特定菌，并侵入该细菌制造细菌裂解酶、裂解菌体。噬菌体能高效高特异性地消灭特定细菌，是非常安全高效的抗菌病毒，已在许多国家广泛应用，在美国已开展临床试验研究，不久即将投入市场。本实施例将筛选PIB特异性噬菌体，并进行生物学鉴定，为药物开发打下基础。
材料与方法
菌株：PIB菌、K12大肠杆菌由朱敏生实验室提供
培养基：LB液体培养基，1.5％固体LB培养基
试剂：限制性内切酶EcoRV，NcoI，SspI，DNase I，RNaseA，十二烷基硫酸钠(SDS)，三氯甲烷，异丙醇，琼脂糖，琼脂粉，Trypton，Yeast extract
仪器设备：超低温冰箱，4℃冰箱，电泳仪，恒温摇床，生物安全柜，低温离心机，灭菌锅，分光光度计，Beckman Conlter超速离心机
宿主菌的准备：取保藏于-80℃冰箱的PIB菌种，37℃，200rpm，摇床培养，分光光度计检测菌液OD600值为0.6-0.9之间，到达对数期。
污水水样采集与处理：于南京市浦口区采集未经处理的生活污水水样。将污水水样12000rpm，4℃离心，10min，去除沉淀杂质，取上清，上清通过0.22μm滤膜过滤，去除细菌杂质污染，存于4℃冰箱中保存备用。
噬菌体的分离筛选：处理好的污水样品，加入到等体积的2xLB液体培养基中，按照5％的接种量加入宿主菌，接种于无菌LB液体培养基中，37℃，200rpm过夜培养，次日12000rpm，4℃，离心10min，取上清，经0.22μm滤膜过滤，即获得噬菌体原液。
PIB特异性噬菌体的筛选：采用双层平板法对上述获得的噬菌体原液进行鉴定，双层平板法包含为上下不同浓度的两层的LB固体培养基，其下层为1.5％琼脂的LB培养基，以支撑营养为主；上层为0.5％琼脂LB培养基，以利于宿主菌和噬菌体生长扩散。这种方法也更便于噬菌斑的观察。具体操作为：首先准备好1.5％的下层琼脂培养基，将100μl对数期菌液和100μl噬菌体原液混合均匀后，室温静置约10min，快速加入到预先加热至48℃的0.5％的上层培养基中，快速混合后倒入下层培养基中，均匀铺平在下层平板中，室温静置20min，正置37℃恒温培养，观察是否有噬菌斑的形成。
PIB特异性噬菌体的纯化、扩增与保存：挑取平板上的噬菌斑，加入至LB培养基中，再按5％的比例加入PIB菌液，37℃，200rpm摇床培养6-8h。过滤离心后，用双层平板法涂布平板，重复上述操作两至三次，直至观察到大小均一，边缘完整的圆形透明状斑块。用灭好菌的枪头挑取斑块，同时加入5％菌液至LB培养基中，37℃，200rpm，摇床过夜培养扩增，12000rpm，5min，4℃离心，再用0.22μm滤膜过滤，加入到10％的SM buffer中，于-80℃冰箱保存。
噬菌体浓缩液与噬菌体DNA制备：将保存的噬菌体与PIB菌株混合加入到LB培养基中，37℃摇床过夜培养，12000rpm，5min，4℃离心，取上清过0.22μm滤膜， 得噬菌体原液，噬菌体原液60000rpm，4℃，1h冷冻超速离心，去除上清，用SM buffer溶解沉淀得噬菌体浓缩液。提取噬菌体DNA：1)500μl噬菌体浓缩液中加入5μl 1mg/ml DNaseI、2.5μl 12.5mg/ml RNaseA，37℃，水浴30min；2)加入25μl 10％SDS，加入5μl 10mg/ml protein K，37℃，水浴30min；3)加入250μl酚，加入250μl氯仿(三氯甲烷)，12000rpm，10min，RT离心，取原有裂解液的量的上清500μl(尽量少吸，不要吸到中间蛋白层)；4)加入等量体积500μl氯仿，12000rpm，10min，RT离心；5)加入45μl 3M NaAc，PH5.2，500μl异丙醇，-20℃，30min，12000rpm，10min；6)70％乙醇清洗两次；7)10-20μl H2O溶解；8)测定浓度；9)存于-20℃。
噬菌体DNA限制性酶切图谱的制作：取10μl噬菌体DNA，加入限制性内切酶，例如，EcoRV，NcoI，SSPI，ddH2O，10x buffer，，37℃，水浴酶切6h，待反应结束，加入2μl 10x loading buffer终止酶切反应，配制1％的琼脂糖凝胶，100v，1h，紫外照胶，得噬菌体DNA限制性酶切图谱，据此可区分噬菌体的不同类型。
噬菌体DNA的基因组学分析：将噬菌体phage3和phage4的基因组DNA样品送至上海探普生物测序服务有限公司进行全基因组测序。通过Illumina二代测序的方法对这两株噬菌体进行全基因组测序，并使用CARD数据库(arpcard.mcmaster.ca)进行耐药分析，数据库核心是ARO(Antibiotic Resistance Ontology)，ARO包含了与抗生素抗性基因，抗性机制等。同时，也进行毒力基因比对，数据库为VFDB(www.mgc.ac.cn/VFs/)，判断噬菌体是否含有毒力基因和抗生素抗性基因。
实验结果
1、筛选获得四种特异性PIB噬菌体：
通过用噬菌体原液与PIB共培养的方法，再通过双层平板法反复筛选纯化后，可以观察到平板表面存在大量半透明状的近圆形斑块，即噬菌斑的存在。挑取纯化后的噬菌斑再次与PIB共培养，通过清除宿主菌株和使用超高速离心的方法，获得了富集后的噬菌体浓缩液，通过酚氯仿法提取制备噬菌体DNA，再通过限制性片段多态性的方法，使用限制性内切酶EcoRV，NcoI，SSPI对噬菌体的全基因组进行酶切后，得到了多种不同类型的噬菌体(图6)。从图6可以得知，当用SSPI限制性内切酶酶切时，可以区分出phage1和phage2、3是不同种类的噬菌体，于是又再次用EcoRV和NcoI做酶切，可以得知phage2、phage3和phage4也是不同种类的噬菌体，由此可以得知，筛选分离出了四种不同种类的PIB特异性噬菌体。
2、四种噬菌体的遗传学特征
由于噬菌体的结构比较简单，通常是由蛋白质外壳包裹着单一核酸(DNA/RNA)，同时对于病毒的研究缺乏通用的标记基因，因此目前也选择针对某种类型的病毒，以 一些高度保守的结构或功能基因作为标记基因，研究病毒的系统发育地位，常见的基因有T4噬菌体的g23基因，蓝藻噬菌体的g20、psbA和psbD基因等。本实施例选取了T4常用的噬菌体保守的衣壳蛋白基因g23，通过PCR实验，图3的实验结果也表明T4g23基因的鉴别，结果也表明所筛选的噬菌体均属于肌尾病毒科的T4噬菌体，是一种较为常见的噬菌体。
全基因组测序结果显示，Phage3/4均为双链DNA噬菌体，按照基因组来看，均为肌尾病毒科的T4噬菌体，根据裂解特性来分，两株均为烈性噬菌体，phage3的基因组全长为64058bp，GC含量是43.26％。Phage4的基因组全长为238877bp，GC含量为44.42％。
结论
成功筛选获得了4种PIB特异性噬菌体，均为T4噬菌体，且未检测到有害基因和抗药基因。
实施例8、PIB特异性噬菌体对PIB便秘的治疗作用
实施例7筛选得到的噬菌体在体外具有良好的杀菌作用，但在体内是否有杀菌作用还不清楚。本实施例的目的在于在PIB便秘动物中评价本发明筛选的3种PIB特异性噬菌体的药效。
材料与方法
菌株：PIB菌由朱敏生实验室提供。
PIB特异性噬菌体：PIB特异性噬菌体phage1、2、3、4如实施例7所述，由朱敏生实验室保存、提供。
实验动物：C57BL/6小鼠，购于集萃药康。8周雄鼠，在SPF环境下饲养，环境温度控制在18-24℃，12h光照/12h暗室循环培养。
实验试剂：LB培养基；Evans Blue指示剂配制：现配现用，按照1.5％Evans Blue+5％甲基纤维素的比例配制指示剂，涡旋充分混匀后，37℃超声10min；迈新生物Ultra Sensitive TMS-P(Mouse/Rabbit)超敏SP(鼠/兔)试剂盒
PIB体外杀菌效果分析：
1、细菌准备：1)提前准备好PIB菌液，37℃，200rpm，摇床过夜培养；2)按照菌液∶LB＝1∶500，噬菌体原液∶LB＝1∶50的比例，加入到LB中，摇晃均匀，取60μl管内液体，分光光度计测量OD600值；3)以相同倾斜角度放入摇床培养，同样的条件，37℃，200rpm，摇床培养，每间隔1h观察试管内液体的清晰度，并测量管内液体的OD600值。
2、PIB感染便秘模型的制作及鉴定：C57BL/6雄鼠30只，分为两组，一组10只，作为对照组，一组20只，作为实验组，每周一次灌胃处理，每次灌胃量为100μl，对照组用LB灌胃，实验组用PIB菌液灌胃，8次灌胃后，PIB感染小鼠便秘模型建成。
3、PIB便秘模型鉴定：在灌胃过程中，测定小鼠是否成功感染PIB菌，取小鼠的新鲜粪便，加入带有amp抗性的LB培养基中，震荡捣碎摇匀，37℃摇床过夜培养，次日，取菌液做PCR检测。
4、肠传输速度的测定：连续八次灌胃，PIB感染小鼠便秘模型建成，测定小鼠的排便频率和小鼠的全肠通过时间，小鼠灌胃前一天禁食，次日，每只小鼠灌胃100μl Evans Blue指示剂凝胶，记录每只鼠的灌胃时间以及排出带有蓝色指示剂的粪便的时间，计算二者的时间差，即为小鼠的全肠通过时间。
5、噬菌体治疗方案和疗效分析：预先准备好已保藏的三种噬菌体，与PIB共摇床培养，制备phage1，2，3这三种噬菌体的cocktail，充分混合后，每只Phage治疗组的小鼠每次灌胃100μl phage cocktail，在PIB感染小鼠便秘模型建成后，对照组依旧每周一次给其灌胃LB，将实验组随机分成两组，一组各10只，其中一组作为PIB组，改灌LB；另一组作为Phage组，改灌phage cocktail，每只鼠每周灌胃一次，每次100μl，连续灌胃4周后，同样通过测定小鼠单位时间内的排便频率和全肠通过时间来判定噬菌体的治疗效果。
实验结果
1、四种噬菌体体外杀灭细菌的效果
为了进一步验证筛选到的噬菌体是否能够重复对PIB的杀灭作用，噬菌体与PIB混合加入到LB中，图8，A中的五个试管从左到右依次是LB，LB+PIB，LB+PIB+Phage-1，LB+PIB+Phage-2，PIB+Phage3。从试管的清晰程度来看，同时加有噬菌体和PIB的试管，其清晰度与未加PIB的试管近似，与加有PIB的试管相比，明显更清晰。图8B是在phage与PIB共同加入到摇床时，初始测量试管中液体的OD600值，之后每隔一段时间，再次测量OD600值，从图中可以看出，在共培养100min后，OD600值与只加PIB的试管相比，有显著性的降低，提示：在PIB与噬菌体共培养的过程中，所筛选的这四种噬菌体对PIB具有很好的体外杀灭效果。
2、噬菌体治疗PIB感染动物的效果
图9显示的三组从左到右依次为对照组、PIB便秘组和噬菌体治疗组。PIB便秘组的小鼠与对照组小鼠相比，其全肠通过时间、排便频率都具有显著性的差异，表明PIB便秘组小鼠仍然存在便秘症状，而噬菌体cocktail治疗组与PIB便秘组相比，其 便秘症状有明显的恢复。
结论
四种噬菌体均具有高效的杀灭PIB的能力；PIB噬菌体对PIB引起的便秘症状具有明显的治疗效果。

Claims (10)

  1. 试剂在制备诊断便秘的检测试剂盒中的用途，所述试剂包括(1)用于检测DPA的试剂，所述试剂任选还包括(2)用于检测PIB的试剂，
    优选地，所述便秘包括顽固性便秘，更优选包括由PIB引起的顽固性便秘，
    优选地，所述对象包括哺乳动物，
    优选地，所述样品包括粪便。
  2. 如权利要求1所述的用途，其特征在于，所述检测DPA的试剂是使用选自下组的一种或多种检测方法检测样品中DPA的试剂：色谱法、质谱、气相色谱质谱联用仪、液相色谱质谱联用仪、比色法、酶联免疫法、超高效液相色谱法、毛细管气相色谱等检测，
    优选地，
    检测DPA的试剂包括DPA的溶剂，例如二氯甲烷和/或三氯甲烷，
    检测DPA的试剂包括从粪便中提取DPA的试剂，例如，检测DPA的试剂包括甲醇、二氯甲烷、水中的一种或多种；优选地，检测DPA的试剂包括甲醇、二氯甲烷和水的混合物，
    检测DPA的试剂包括对DPA或其衍生物具有特异性的物质，
    检测PIB的试剂包括特异性扩增PIB的标志核酸序列的引物或探针、特异性结合PIB的表面蛋白或分泌物的抗体或配体，
    所述检测试剂盒还包含选自下组的一种或多种物质：容器、缓冲剂、助剂、溶剂、阴性对照物、阳性对照物、使用说明书。
  3. 如权利要求1或2所述的用途，其特征在于，
    与对照水平相比，对象样品中的DPA含量增加，表明所述对象罹患或疑似罹患便秘，或具有罹患便秘的风险，或
    与对照水平相比，对象样品中的DPA含量增加，并且对象样品中包含包含PIB或PIB含量增加，表明所述对象罹患或疑似罹患便秘，或具有罹患便秘的风险，
    优选地，所述对照水平是未罹患便秘的对象样品中的DPA含量。
  4. 一种从粪便中提取DPA的方法，所述方法包括：用提取液处理获自对象的粪便样品，所述提取液包含醇、二氯甲烷或三氯甲烷和水，
    优选地，所述醇包括甲醇、乙醇或丙醇，
    优选地，醇、二氯甲烷或三氯甲烷与水的体积比为1～10∶1～5∶1～10，
    更优选地，所述提取液包含甲醇、二氯甲烷和水。
  5. 一种用于诊断便秘、预测对象的便秘发生或进展风险、或便秘预后评估、或提供治疗方案的产品，所述产品包含用于检测对象样品中DPA的试剂，任选还包括用于检测对象样品中PIB的试剂，
    优选地，所述便秘包括顽固性便秘，更优选包括由PIB引起的顽固性便秘，
    优选地，所述对象包括哺乳动物，
    优选地，所述样品包括粪便。
  6. 如权利要求5所述的用途，其特征在于，所述产品是检测试剂盒，所述检测DPA的试剂是使用选自下组的一种或多种检测方法检测样品中DPA的试剂：色谱法、质谱、气相色谱质谱联用仪、液相色谱质谱联用仪、比色法、酶联免疫法、超高效液相色谱法、毛细管气相色谱等检测，
    优选地，
    检测DPA的试剂包括DPA的溶剂，例如二氯甲烷和/或三氯甲烷，
    检测DPA的试剂包括从粪便中提取DPA的试剂，例如，检测DPA的试剂包括甲醇、二氯甲烷、水中的一种或多种；优选地，检测DPA的试剂包括甲醇、二氯甲烷和水的混合物，
    检测DPA的试剂包括对DPA或其衍生物具有特异性的物质，
    检测PIB的试剂包括特异性扩增PIB的标志核酸序列的引物或探针、特异性结合PIB的表面蛋白或分泌物的抗体或配体，
    所述检测试剂盒还包含选自下组的一种或多种物质：容器、缓冲剂、助剂、溶剂、阴性对照物、阳性对照物、使用说明书。
  7. 如权利要求5或6所述的用途，其特征在于，所述产品是装置，所述装置包括存储器、处理器以及存储在存储器上并可用在处理器上运行的计算机程序，其特征在于，所述处理器执行所述程序时实现以下步骤：
    a)检测对象样品中的DPA，任选还检测PIB，和
    b)通过步骤a)的结果诊断便秘、预测对象的便秘发生或进展风险、或便秘预后评估、或提供治疗方案，
    优选地，
    步骤a)包括：a1)检测对象样品中的DPA，和a2)确定在步骤a1)中检测的DPA的丰度是否高于对照水平，或
    其中步骤a)包括：a1)检测对象样品中的DPA和PIB，和a2)确定在步骤a1)中检测的DPA的丰度是否高于对照水平，以及是否存在PIB或其含量是否高于对照，
    更优选地，
    与对照水平相比，对象样品中的DPA含量增加，表明所述对象罹患或疑似罹患便秘、或具有罹患便秘的风险、或便秘预后不良，或表明需更换治疗方案，或
    与对照水平相比，对象样品中的DPA含量增加，并且对象样品中包含PIB或PIB含量增加，表明所述对象罹患或疑似罹患便秘、或具有罹患便秘的风险、或便秘预后不良，或表明需更换治疗方案，或
    所述对照水平是未罹患便秘的对象样品中的DPA含量。
  8. 抗生素或其衍生物在制备治疗便秘的药物中的用途，所述抗生素包括但不限于喹诺酮类抗生素、碳青霉烯类抗生素、丝裂酶素、硫酸博来霉素、磺胺增效剂，
    优选地，
    所述衍生物是水合物，和/或
    所述便秘包含顽固性便秘，优选包含PIB引起的顽固性便秘，和/或
    所述喹诺酮类包括：左氧氟沙星、巴洛沙星、盐酸贝西沙星、恩诺沙星、 莫西沙星、帕珠沙星甲磺酸盐、加替沙星、司帕沙星、盐酸沙拉沙星、诺氟沙星、环丙沙星、左氧氟沙星、依诺沙星、洛美沙星、西他沙星、盐酸二氟沙星，和/或
    所述碳青霉烯类包括：美罗培南、多尼培南、比阿培南、厄他培南钠。
  9. 噬菌体在制备治疗便秘的药物中的用途，所述噬菌体为T4噬菌体，
    优选地，
    所述便秘包含顽固性便秘，优选包含PIB引起的顽固性便秘，和/或
    所述噬菌体为肌尾病毒科T4噬菌体。
  10. 一种治疗便秘的药物组合物，包含药学上可接受的辅料和：(1)抗生素或其衍生物，和/或(2)噬菌体，所述抗生素包括但不限于喹诺酮类抗生素、碳青霉烯类抗生素、丝裂酶素、硫酸博来霉素、磺胺增效剂，所述噬菌体为T4噬菌体，
    优选地，
    所述衍生物是水合物，和/或
    所述便秘包含顽固性便秘，优选包含PIB引起的顽固性便秘，和/或
    所述喹诺酮类包括：左氧氟沙星、巴洛沙星、盐酸贝西沙星、恩诺沙星、莫西沙星、帕珠沙星甲磺酸盐、加替沙星、司帕沙星、盐酸沙拉沙星、诺氟沙星、环丙沙星、左氧氟沙星、依诺沙星、洛美沙星、西他沙星、盐酸二氟沙星，和/或
    所述碳青霉烯类包括：美罗培南、多尼培南、比阿培南、厄他培南钠，和/或，
    所述噬菌体为肌尾病毒科T4噬菌体。
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