CN-119432831-B - Composite preservative and same-tube multi-group combined detection integrated technology for fecal microorganisms and metabolites thereof

Abstract

The invention provides a compound preservative for fecal microorganisms and metabolites thereof and a common-tube multi-group combined detection integrated technology, wherein the compound preservative contains cetyltrimethylammonium bromide (CTAB), ammonium chloride (NH 4 Cl) and Phosphate Buffer (PBS) as main components, can keep the stability of the microorganisms and the metabolites thereof in a fecal sample in the room-temperature storage and transportation process, and can ensure the extraction efficiency of deoxyribonucleic acid (DNA) of the microorganisms in the fecal sample. The invention also eliminates the influence of the preservative component on metabolite detection by adding the treatment reagent, and obviously improves the response and ionization efficiency of the fecal sample metabolites in mass spectrum, thereby improving the sensitivity and accuracy of the detection result. The invention not only solves the problem of fecal sampling timeliness, but also provides a technical foundation and thought for the feasibility research of fecal sample same-tube multiunit science, and has wide scientific research and clinical application prospect.

Inventors

• NI YAN
• YANG CHUANHAO

Assignees

• 浙江大学

Dates

Publication Date

20260508

Application Date

20240827

Claims (2)

1. 1. A method for detecting metabolites in a fecal sample containing a complex preservative for non-disease diagnosis purposes, comprising the steps of: (1) Taking 5-50 mu L of fecal sample containing the composite preservative, and freeze-drying at low temperature; (2) Sequentially adding a mixed organic solvent and magnetic beads into the freeze-dried sample, and homogenizing, wherein the mixed organic solvent is methanol/acetonitrile mixed solution; (3) Centrifuging the homogenized sample at a low temperature and a high speed, taking 40-80 mu L of supernatant, and measuring the accurate content of CTAB in the sample; (4) According to the content of CTAB, accurately preparing a sodium dodecyl sulfate SDS solution with corresponding proportion; (5) Coprecipitation elimination, namely adding 40-80 mu L of the SDS solution prepared in the step (4) into a proper amount of supernatant obtained in the step (3), vibrating and incubating for a certain time, centrifuging at a low temperature and high speed again, and taking the final supernatant for metabolite detection; in the step (5), SDS is added according to the mole ratio of SDS to CTAB of 1:1, the reaction temperature is 10 ℃, and the reaction time is 20 min; The complex preservative consisted of 2.0% CTAB, 20% NH 4 Cl and 10% PBS.
2. 2. The method of claim 1, wherein the metabolites in the fecal sample comprise GCA, TCA, GUDCA, TUDCA, TDCA, GDCA, TCDCA, GCDCA, TLCA, LCA, bCA, CA, UDCA, DCA and CDCA.

Description

Composite preservative and same-tube multi-group combined detection integrated technology for fecal microorganisms and metabolites thereof Technical Field The invention relates to the field of chemical analysis and medical inspection, in particular to a compound preservative for fecal microorganisms and metabolites thereof and a common-tube multi-group chemical combination inspection integrated technology. Background Fecal samples are an important source for directly acquiring intestinal microbial information and are considered to contain the most abundant metabolites, and therefore, fecal samples play a vital role in the research of intestinal microbial and its metabolite combination analysis. The noninvasive collection mode of the feces makes the feces sample pay attention to early cancer risk assessment and screening, and particularly in the last two years, the national drug administration approves a plurality of novel diagnosis products based on the feces, proves the feasibility and the effectiveness of the feces sample in the field of medical innovation, and provides a more convenient and practical disease diagnosis and prevention means for clinic. However, stool samples are characterized by timeliness and heterogeneity, which presents challenges for sample collection, storage, transportation, multiple-study, and clinical testing. The collected fecal samples were stored at room temperature or 4 ℃ for more than 12 hours resulting in significant changes in fecal microorganisms and their metabolites. Thus, collection and storage of stool samples is widely recommended as immediate frozen at-80 ℃ conditions and transported using dry ice. However, this harsh storage and shipping condition makes patient home sampling, large-scale community screening, trans-regional transportation, and even hospitalized patient sampling difficult to implement. In order to solve the above problems, the use of various preservative solutions is an ideal solution. At present, various DNA preservation solutions can effectively maintain the DNA room temperature stability of a fecal sample, maintain the integrity of microorganism information in the sample, and avoid the severe requirements on sample freezing and transportation. Common DNA preservation solutions include ethanol, RNAlater, EDTA salts, and commercial kit products OMNIgene GUT, stool DNA Stabilizer, etc., and DNA preservation agents are used in many clinical studies. Although the existing DNA exclusive preservative has the technical advantages of room temperature storage, convenient transportation and the like, the existing DNA exclusive preservative has limitations. The biggest disadvantage is that the feces sample added with the DNA preservative cannot be qualitatively and quantitatively detected by liquid chromatography mass spectrometry, because the high-salt component, chelating agent or surfactant contained in the DNA preservative can significantly inhibit or destroy chromatographic column separation and mass spectrometry ionization. In summary, the clinical research queue widely adopts the DNA preservative to protect the fecal sample, and although this measure solves the challenges of fecal sample storage and transportation, it limits the detection of metabolomics based on mass spectrometry, and finally makes the clinical sample multiple-study association analysis only use samples with different preservation modes, which results in greatly weakening the information association among multiple studies. Therefore, the invention provides a compound preservative for fecal sample microorganisms and metabolites thereof, which is convenient for collecting, storing and transporting clinical samples. In addition, the invention realizes the detection of microorganisms and metabolites simultaneously in the same solution system (simultaneous joint inspection) by properly disposing mass spectrometry detection interfering components of the preservative. Disclosure of Invention Aiming at the problems existing in the prior art, the invention provides a compound preservative for fecal microorganisms and metabolites thereof and a common-tube multi-group combined detection integrated technology, and a method for preprocessing fecal samples containing the preservative and quantitatively detecting the metabolites, wherein the compound preservative comprises CTAB, NH 4 Cl and PBS components, and can effectively keep the stability of fecal sample DNA in room temperature storage and transportation. The invention provides a preservative treatment and metabolite detection method, which can effectively eliminate the detection influence of the preservative on the metabolites, remarkably improve the response and ionization efficiency of the metabolites of the stool sample in mass spectrum and ensure the sensitivity and accuracy of detection. In one aspect, the invention provides a fecal composite preservative comprising CTAB, NH 4 Cl and PBS. In some embodiments, the components of the fecal co