CN114107531A - Detection and application method of stomach micro-ecological urease positive flora - Google Patents
Detection and application method of stomach micro-ecological urease positive flora Download PDFInfo
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- CN114107531A CN114107531A CN202111552783.0A CN202111552783A CN114107531A CN 114107531 A CN114107531 A CN 114107531A CN 202111552783 A CN202111552783 A CN 202111552783A CN 114107531 A CN114107531 A CN 114107531A
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- urease
- positive bacteria
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- helicobacter pylori
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Abstract
The invention discloses a detection and application method of microecological urease positive flora in stomach, and also discloses a detection related product, wherein the detection related product comprises a reagent for detecting urease positive bacteria, and the product can be used for accurately diagnosing the composition of the flora in stomach of a subject, can dynamically observe the composition change of the flora in stomach of the subject, and guides a clinician to accurately treat the subject, thereby avoiding abuse of antibiotics, and has the advantages of high accuracy, strong specificity, high safety and the like, and is easy for large-scale popularization and application.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to a detection and application method of a stomach micro-ecological urease positive flora.
Background
The human digestive tract contains a large number of bacteria, which is 1.3 times as many as the total number of human cells, and according to the fifth oral microbial standardization workshop, it is reported that 178 bacteria of 38 genera have been detected by the microorganisms in the oral cavity so far, the human intestinal microorganisms contain more than 1000 bacterial species, and the stomach is an important area in the digestive tract micro-ecosystem, which constitutes a unique ecological environment and a characteristic microbial community due to the secretion of gastric acid, and the strong acid environment in the stomach is generally considered not to have the presence of microorganisms in the stomach by the early academic community (Savage D. microbiological technology of the gastrointestinal tract [ J ]. Annual review of microbiology,1977,31(1): 107-. Helicobacter pylori (H.pylori) was first isolated and cultured from human gastric mucosa biopsy tissue by Australian university Warren and Marshall (Warren J R, Marshall B. unidentified cured bacterial on gastric mucosa [ J ] The plant, 1983,321(8336): 1273-.
Since the discovery of helicobacter pylori, intensive research over 20 years has led to the identification of helicobacter pylori as an important pathogen in chronic active gastritis and peptic ulcer, which is also closely related to gastric cancer, and the digestive community gradually has agreed on the presence and pathogenicity of helicobacter pylori in the stomach. However, the current cognition, detection and treatment of the bacteria in the stomach are only limited to helicobacter pylori, the C13 and C14 breath test is adopted as a diagnosis method of helicobacter pylori infection, urea is decomposed by helicobacter pylori urease to generate ammonia and carbon dioxide, and isotope-labeled carbon dioxide can be detected. In recent years, scholars at home and abroad report bacterial and fungal infections except for helicobacter pylori in stomach, if the infection of the helicobacter pylori is judged to be clinically positive according to an expiration test, the helicobacter pylori eradication treatment is blindly carried out on a subject by adopting the existing first-line therapy, namely the quadruple therapy, so that the poor antibiotic treatment effect and the drug-resistant bacterial type of induced infectious bacteria are caused.
In view of the above, in order to solve the above technical problems in the prior art, the invention provides a microecology theory of the stomach for the first time, provides a method for culturing and identifying various pathogens in the stomach, combines the culture result of a gastric tissue sample cleaning solution and the detection result of a metagenome, judges the microecology composition of the stomach of a human body, dynamically observes the change of flora composition, provides a sample for a drug sensitive experiment performed after determining the type of an infected microorganism, guides clinical accurate diagnosis and treatment of a subject through rapid identification of the culture omics, the drug sensitive and the molecular biology, triggers medical and scientific workers to develop a diagnosis and treatment tool which can accurately detect the flora in the stomach compared with an exhalation test, and has great significance for preventing disease occurrence and enhancing physical health of the public.
Disclosure of Invention
The invention aims to provide a method for detecting and applying micro-ecological urease positive flora in stomach.
The above object of the present invention is achieved by the following technical solutions:
in a first aspect of the present invention, a kit for aiding in the diagnosis of helicobacter pylori infection is provided.
Further, the kit comprises a reagent for detecting urease positive bacteria.
Further, the urease-positive bacteria include any one or more of paraporta acidovorans, candida albicans, comamonas testosteroni, stenotrophomonas maltophilia, sphingomonas inevorans, sphingomonas paucimobilis, raoulella terrestris, ralstonia pickettii, saccharomyces assiazei, pseudomonas morganii, rhizomus morganii, pseudomonas putida, klebsiella pneumoniae, tsukamurella slightly-changed, and/or helicobacter pylori.
Further, the reagent comprises a primer, a probe, an antisense oligonucleotide, an aptamer, and/or an antibody specific to the urease-positive bacteria.
Further, the method for detecting urease positive bacteria comprises any one or more of 16S sequencing, whole genome sequencing, quantitative polymerase chain reaction, PCR-pyrosequencing, fluorescence in situ hybridization, microarray and/or PCR-ELISA detection.
Further, 16S sequencing (also known as 16S rDNA sequencing), which is a prokaryotic gene encoding 16S rRNA, has a length of about 1500bp and consists of 10 conserved regions and 9 variable regions (V1-V9) that reflect the inter-species variability. The 16S rDNA sequencing is to extract DNA of microbial flora, select a specific section (V3-V4) of a variable region for PCR amplification, analyze the genetic composition and function of microbial flora and the diversity and abundance of microbial flora in a specific environment sample by a high-throughput sequencing method, further analyze the relationship between the microbes and the environment and between the microbes and a host, and find out genes with specific functions. In the medical field, the method is mainly applied to the correlation analysis of microorganisms and diseases, and the microbial difference between the diseases and healthy individuals is revealed.
Further, Whole Genome Sequencing (WGS) refers to individual genome sequencing of species with unknown genomic sequence, and the sequencing process is as follows: extracting genome DNA, randomly breaking, electrophoretically recovering DNA fragments (0.2-5Kb) with required length, adding a linker, preparing a DNA Cluster (Cluster), sequencing an insert by using a Paired-End (Solexa) or Mate-Pair (SOLID) method, assembling the measured sequences into Contig, and further assembling into Scaffold through the distance of Paired-End so as to assemble into chromosomes and the like. The assembly effect is related to sequencing depth, coverage, sequencing quality and the like; a common assembly is: SOAPdenovo, Trimitty, Abys, etc. The Whole genome sequencing can be mainly divided into Whole genome de novo sequencing (Whole genome de novo sequencing) and Whole genome re-sequencing (Whole genome re-sequencing), the Whole genome de novo sequencing can sequence the genome of a certain species without any reference genome information, and the genome sequence map of the species is obtained by splicing and assembling by using a bioinformatics analysis method, so that the follow-up research of the species is promoted; whole genome re-sequencing is genome sequencing of different individuals with reference genomic species and differential analysis of individuals or populations based on this. The method is mainly used for assisting researchers to discover the variation types such as Single Nucleotide Polymorphism Sites (SNPs), Copy Number Variation (CNV), insertion/deletion (Indel) and the like.
Further, in the embodiment of the present invention, the present invention finds out through experimental studies that more than one helicobacter pylori is in the stomach, but one flora, and there are numerous urease positive bacteria (16 urease positive bacteria), the result indicates that the clinically current positive and negative of the carbon 13 or carbon 14 breath test is inaccurate as the criterion for determining the infection and eradication of helicobacter pylori, the 16 urease positive bacteria exist in the stomach besides helicobacter pylori, the carbon 13 or carbon 14 breath test results of carriers of the urease positive bacteria are positive, which results in false positive result of the infection of helicobacter pylori, and after the helicobacter pylori is eradicated by the antibiotic, the 16 bacteria can also make false determination of the eradication treatment effect.
Further, said 16 urease-positive bacteria are Tricholoma acidovorans, Candida albicans, Comamonas testosteroni, stenotrophomonas maltophilia, Sphingobacterium alcafoensis, Sphingomonas paucimobilis, Sphingomonas oliganpinus, Ralstonia terrestris, Ralstonia pickeri, Sporotrichum assamica, Pseudomonas morganii, Chryseobacterium denitrificans, Morganella morganii, Pseudomonas putida, Klebsiella pneumoniae, Tukamurella slightly variabilis;
further, carbon 13 or carbon 14 breath tests are the most preferred non-invasive methods for the detection of H.pylori at present, and also the most preferred method for the post-treatment review of H.pylori. If helicobacter pylori exists in the stomach of the subject, the bacterium secretes urease into the stomach, and the urea capsule marked by C13 or C14 is orally taken, and after the urea capsule enters the stomach, the urease in the stomach can decompose urea into ammonia and CO marked by C13 or C142Carbon dioxide formed by hydrolysis of urea is absorbed into the blood, carried with the blood stream into the lungs and expelled as gas. The presence of labeled C13 or C14 in the exhaled air of the subject is detected, so that the presence of helicobacter pylori infection in the subject can be judged, and the number of helicobacter pylori can be preliminarily judged according to the detected number of C13 or C14.
The second aspect of the invention provides application of a reagent for detecting urease positive bacteria in preparation of an auxiliary diagnosis of helicobacter pylori infection.
Further, the urease-positive bacteria include any one or more of paraporta acidovorans, candida albicans, comamonas testosteroni, stenotrophomonas maltophilia, sphingomonas inevorans, sphingomonas paucimobilis, raoulella terrestris, ralstonia pickettii, saccharomyces assiazei, pseudomonas morganii, rhizomus morganii, pseudomonas putida, klebsiella pneumoniae, tsukamurella slightly-changed, and/or helicobacter pylori.
Further, the reagent comprises a primer, a probe, an antisense oligonucleotide, an aptamer, and/or an antibody specific to the urease-positive bacteria.
Further, the method for detecting urease positive bacteria comprises any one or more of 16S sequencing, whole genome sequencing, quantitative polymerase chain reaction, PCR-pyrosequencing, fluorescence in situ hybridization, microarray and/or PCR-ELISA detection.
Further, the present invention discovers for the first time that other urease positive flora besides helicobacter pylori exists in the stomach, and the urease positive flora existing in the stomach discovered for the first time comprises Tyrford acidovorans, Candida albicans, Comamonas testosteroni, stenotrophomonas maltophilia, Sphingomonas alcafolivorans, Sphingomonas paucimobilis, Laurushila terrestris, Rosstonia pilei, Spanisia aspalata, Pseudomonas mossambica, Morganella morganii, Pseudomonas putida, Klebsiella pneumoniae, Tsukamurella tersii, and the discovery of these urease positive bacteria lays a foundation for clinically accurately detecting the gastric flora, and provides a brand new idea for assisting accurate diagnosis of helicobacter pylori infection.
The third aspect of the invention provides a kit for detecting urease-positive bacteria.
Further, the kit comprises a reagent for detecting urease positive bacteria.
Further, the urease-positive bacteria include any one or more of paraporta acidovorans, candida albicans, comamonas testosteroni, stenotrophomonas maltophilia, sphingomonas inevorans, sphingomonas paucimobilis, raoulella terrestris, ralstonia pickettii, saccharomyces assiazei, pseudomonas morganii, rhizomus morganii, pseudomonas putida, klebsiella pneumoniae, tsukamurella slightly-changed, and/or helicobacter pylori.
Further, the reagent comprises a primer, a probe, an antisense oligonucleotide, an aptamer, and/or an antibody specific to the urease-positive bacteria.
Further, the primer refers to a nucleic acid fragment comprising 5 to 100 nucleotides, and preferably, the primer or amplification primer comprises 15 to 30 nucleotides capable of initiating an enzymatic reaction (e.g., an enzymatic amplification reaction).
Further, the probe refers to a nucleic acid sequence comprising at least 5 nucleotides, for example, 5 to 100 nucleotides, which can hybridize with an expression product of a target gene of a target urease-positive bacterium or an amplification product of the expression product under a specified condition to form a complex. The hybridization probes may also include labels for detection. The label includes, but is not limited to, labels for fluorescent quantitative PCR or fluorescent in situ hybridization, including, but not limited to, radioactive labels, non-radioactive labels, the radioactive labels include32P、3H、35S, and the like, and the non-radioactive label includes horseradish peroxidase (HRP), alkaline phosphatase (AKP), Digoxin (DIG), biotin, fluorescein, and the like.
Further, the antibody refers to a specific immunoglobulin directed to an antigenic site. The antibody of the present invention is an antibody that specifically binds to the urease-positive bacteria of the present invention, and can be produced by a conventional method in the art. Forms of antibodies include polyclonal or monoclonal antibodies, antibody fragments (such as Fab, Fab ', F (ab') 2, and Fv fragments), single chain Fv (scfv) antibodies, multispecific antibodies (such as bispecific antibodies), monospecific antibodies, monovalent antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen binding site of an antibody, and any other modified immunoglobulin molecule comprising an antigen binding site, so long as the antibody exhibits the desired biological binding activity.
Further, the kit also comprises an instruction for detecting urease positive bacteria by using the kit.
Further, in certain embodiments, the kit is marketed, distributed, or sold as a unit for performing the methods of the present invention. Such kits may comprise carrier means compartmentalized to receive, in close confinement, one or more container means (e.g., vials, tubes, etc.), each container means comprising one of the separate components to be used in the method. For example, one of the container means may comprise a probe that carries or can carry a detectable label. Such a probe may be a polynucleotide specific for a polynucleotide comprising one or more genes characteristic of the expression of a gene of the urease-positive bacterium. Where the kit utilizes nucleic acid hybridization to detect a target nucleic acid, the kit can also have a container containing one or more nucleic acids for amplifying the target nucleic acid sequence and/or a container containing a reporter means.
Further, the kit will typically comprise the above-described container and one or more other containers containing commercially and user-desired materials, including buffers, diluents, filters, needles, syringes, and the like. A label may be present on the container to indicate the particular application of the composition, and may also indicate the direction of in vivo or in vitro use, such as those described above. Other optional components of the kit include one or more buffers (e.g., blocking buffer, wash buffer, substrate buffer, etc.), other reagents (e.g., substrate chemically altered by enzymatic labeling), epitope retrieval solutions, control samples (positive and/or negative controls), control sections, and the like.
In a fourth aspect of the invention, a system or device is provided for aiding in the diagnosis of H.pylori infection.
Further, the system or apparatus comprises:
(1) nucleic acid sample separation unit: a nucleic acid sample for isolating the intragastric flora from gastric mucosal tissue of a subject;
(2) a sequencing unit: the nucleic acid sample sequencing unit is used for sequencing the nucleic acid sample of the gastric flora obtained by the separation of the nucleic acid sample separation unit to obtain a sequencing result;
(3) a data processing unit: detecting urease positive bacteria in the gastric flora according to the sequencing result obtained by the sequencing unit, wherein the urease positive bacteria comprise any one or more of paraporta acidovorans, candida albicans, comamonas testosteroni, stenotrophomonas maltophilia, sphingosine eating bacillus, sphingosine paucimobilis, lawsonia terrestris, rhostonia pickettii, pseudomonas asarosii, pseudomonas moxidensis, pseudomonas moxidens, tsukamurella slightly changed and helicobacter pylori;
(4) a result determination unit: based on the results of the data processing unit, a diagnosis of the subject is derived and the results are stored in a data carrier.
Further, the data carrier is a computer readable medium.
Further, the nucleic acid sample separation unit, the sequencing unit, the data processing unit and the result determination unit in the system or the device are operatively connected to each other. How the units are operatively linked will depend on the type of unit contained in the device. For example, in case a tool for automatically quantitatively sequencing the gastric flora is applied in the sequencing unit, the data obtained by the automatic operation unit may be processed by the data processing unit.
Furthermore, all units in the system or the device are connected in a wired mode and/or a wireless mode; the analysis module comprises a computer host, a central processing unit and a network server; the output module is a display, a printer and an audio output device.
Further, the wireless connection mode can be wireless local area network, Bluetooth, infrared ray and the like; the wired connection mode can be a fixed telephone network and the like. The use of the system/device by the user is greatly facilitated by the aforementioned connection means, while accurate diagnosis of the presence of H.pylori infection in a subject can be made by means of increasingly developed information technology and increasingly popular network resources.
In a fifth aspect, the invention provides a method of detecting the gastric flora.
Further, the method comprises the steps of:
(1) collecting a gastric mucosa tissue sample of a subject, and cleaning the sample by using sterile normal saline to obtain a gastric mucosa tissue sample cleaning solution for culture;
(2) grinding the cleaned gastric mucosa tissue sample to obtain a bacterial suspension, and culturing in a sterile culture medium;
(3) carrying out strain purification and strain identification on the strains obtained by culturing in the steps (1) and (2) to obtain gastric mucosa tissue sample cleaning solution and gastric mucosa tissue sample bacterial suspension culture results;
(4) and (3) carrying out metagenome sequencing analysis on the sample obtained in the step (2) to obtain a metagenome sequencing result, and judging the microecological composition of the gastric flora of the subject by combining the culture result of the gastric mucosa tissue sample cleaning solution obtained in the step (1).
Further, the microecological structure of the finally obtained gastric flora is the result of the culture with the sample polishing solution — the result of the culture with the sample washing solution.
Furthermore, the detection method combines the culture result of the gastric tissue sample cleaning solution and the detection result of the metagenome, can accurately judge the microecological composition of the flora in the stomach of the subject, and dynamically observe the composition change of the flora.
Preferably, the subject is a human.
Further, the metagenomic sequencing analysis in step (4) can be replaced by mass spectrometry to determine the species of infection in the stomach, and further by drug susceptibility testing to determine the appropriate antibiotic to guide the clinical doctor in diagnosis and treatment of the subject.
In a sixth aspect the invention provides a method for producing a urease.
Further, the method comprises the steps of performing purification culture on urease positive bacteria, and extracting urease from the 16 urease positive bacteria respectively after the purification culture;
preferably, said 16 urease-positive bacteria comprise any one or more of paraporta acidovorans, candida albicans, comamonas testosteroni, stenotrophomonas maltophilia, sphingomonas vorax, sphingomonas paucimobilis, raoultella terrestris, ralstonia pickettii, myceliophthora assiazakii, pseudomonas morganii, rhizomus morganii, pseudomonas putida, klebsiella pneumoniae, tsukamurella slightly-changed, and/or helicobacter pylori.
After 16 urease positive bacteria except helicobacter pylori exist in the stomach and are detected by the method, a drug sensitivity experiment is carried out according to a strain identification result, clinical treatment is carried out according to a drug sensitivity experiment result, the helicobacter pylori can be prevented from being detected according to an expiration test, the helicobacter pylori tetranectic therapy is blindly applied to antibiotic treatment, the urease positive bacteria except the helicobacter pylori are prevented from generating drug-resistant strains, and the side effects caused by taking various antibiotics by a patient are avoided. China is a big antibiotic-resistant country, according to the current helicobacter pylori diagnosis standard, if only positive carbon 13 or carbon 14 breath experiments are judged to be helicobacter pylori infection, a first-line clinical treatment method is an antibiotic quadruple therapy, and a group of urease positive bacteria provided by the invention can cause the positive carbon 13 or carbon 14 breath experiments, so that the false positive judgment is caused on the clinical helicobacter pylori diagnosis, the blind application of antibiotics is caused, the harm and the burden are caused to the health and the economy of people, the infection rate of the helicobacter pylori in China is reported to be 50%, the infection rate is obtained according to the carbon 13 or carbon 14 breath diagnosis, the positive results of the breath experiments caused by all the urease positive bacteria in the stomach are judged to be helicobacter pylori infection, therefore, the invention provides a group of urease positive flora as the first interference bacteria for distinguishing and judging the helicobacter pylori, with the progress of research work, urease positive bacteria of different regions and crowds can be detected, and the detection, diagnosis and treatment methods of the intragastric infectious bacteria in clinic are further enriched.
Compared with the prior art, the invention has the advantages and beneficial effects that:
according to the invention, other urease positive bacteria except helicobacter pylori exist in the stomach for the first time, wherein the other urease positive bacteria comprise Tyrfurin acidovorans, Candida albicans, Comamonas testosteroni, stenotrophomonas maltophilia, Sphingomonas alchagoides, Sphingomonas paucimobilis, Ralstonia terrestris, Ralstonia pickettii, Sphaeria assiacicola, Pseudomonas morbid, Morganella denitrificans, Morganella morganii, Pseudomonas putida, Klebsiella pneumoniae and Tsukamurella tip change.
Drawings
Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1 shows graphs of the results of culturing the same sample (52#) on medium I (left dish) and medium II (right dish);
FIG. 2 is a graph showing the results of the culture of the same sample (52#) with the addition of the antibiotic mixed liquid culture medium III (right dish in FIG. 2) and the addition of the antibiotic mixed liquid culture medium IV (left dish in FIG. 2);
FIG. 3 is a graph showing the results of culturing the same sample (94#) on medium I (right dish) and medium III (left dish);
FIG. 4 is a graph showing the results of culturing the same sample (54#) on medium III (right dish) and medium IV (left dish);
FIG. 5 is a graph showing the results of culturing the same sample (68#) on medium I (left dish) and medium II (right dish);
FIG. 6 is a graph showing the results of culturing the same sample (68#) on medium III (right dish) and medium IV (left dish);
FIG. 7 is a diagram showing the genus composition of the strain of the present invention detected from 100 specimens of gastric mucosal tissue;
fig. 8 shows a metagenomic map of 35 gastric mucosal tissue samples.
Detailed Description
The present invention is further illustrated below with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention. As will be understood by those of ordinary skill in the art: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents. The following examples are examples of experimental methods not indicating specific conditions, and the detection is usually carried out according to conventional conditions or according to the conditions recommended by the manufacturers.
Example detection and identification of the gastric flora
1. Tissue source
Gastric mucosa tissues of 100 clinical subjects collected by using a common endoscope, a painless endoscope and a magnetic control capsule capable of taking tissues are put into a BRAIN HEART INFUSION (brand OXOID, goods number CM1135) 30% glycerol preservation solution EP tube after the tissues are collected, and the tissues are sent to a laboratory for culture as soon as possible within 2 hours, and are recommended to be preserved by freezing at a temperature of between 20 ℃ below zero and 80 ℃ below zero within 2 hours.
2. Sample cleaning
In order to remove oral bacteria brought into the stomach by the liquid medicine, the stomach tissue sample is placed into an EP tube filled with 1mL of sterile physiological saline for mid-point vibration for 5 seconds, a suction tube is replaced to move the stomach tissue sample to another EP tube filled with 1mL of sterile physiological saline for mid-point vibration for 5 seconds, after three times of cleaning, the stomach tissue sample is taken out and placed into a grinder, and 0.4mL of sterile physiological saline is added for grinding to obtain tissue grinding liquid;
1-3 EP tubes of the gastric tissue lavage fluid were streaked and inoculated into two dishes containing 10% sterile defibrinated sheep BLOOD, CampyLOBACTER AGAR BASE and 10% sterile defibrinated sheep BLOOD, and cultured in a three-gas (10% carbon dioxide, 5% oxygen, 85% nitrogen) incubator for 48 hours, and the culture results were recorded.
3. Test method
3.1 tissue treatment and culture
Taking out gastric mucosa tissue from the preservation solution by using a disposable sterile straw, cleaning according to the sample cleaning method described in the embodiment 2, putting the tissue into a sterilization grinder after cleaning, adding 0.4mL of sterile physiological saline, fully grinding to obtain tissue grinding fluid, sucking the grinding fluid, coating the grinding fluid on the surfaces of four culture media of 3.2.1 and 3.2.2, and putting the tissue into a three-gas incubator for culturing for 48 hours;
the culture media 3.2.1 and 3.2.2 are four culture media with different components, and the aim is to simultaneously apply the culture media with different components to be suitable for the growth of various bacteria with different types and detect the gastric flora as comprehensively as possible.
3.2 preparation of the culture Medium
3.2.1 Medium I: campylobert AGAR BASE (Karmali), cat # CM0935, brand OXOID + sterile defibrinated sheep blood (10%); and (3) a culture medium II: COLUMBIA BLOOD AGAR BASE, cat # CM0331, brand OXOID + sterilized defibrinated sheep BLOOD (10%).
3.2.2 Medium III: campylobert AGAR BASE (Karmali), cat # CM0935, brand OXOID + sterile defibrinated sheep blood (10%) + antibiotic cocktail (2%); culture medium IV: COLUMBIA BLOOD AGAR BASE, cat # CM0331, brand OXOID + sterile defibrinated sheep BLOOD (10%) + antibiotic cocktail (2%).
3.2.3 preparation of antibiotic mixture: an antibiotic mixture solution was prepared from 0.5mL of vancomycin (10mg/L), 0.25mL of polymyxin B (5mg/L), 0.25mL of amphotericin B (5mg/L), 1.5mL of TMP (10mg/L) and 97.5mL of sterile pure water.
3.3 Strain purification
Single colonies from four different culture dishes of culture media I-IV after 48 hours of culture are smeared on the culture medium I or the culture medium II, and the three-gas incubator is used for 48 hours of culture.
3.4 identification of the Strain
(i) V2 operating protocol of full-automatic bacteria identification and drug sensitivity analysis system
i-1) identification card and concentration
GN gram negative bacilli identification card 0.5-0.63 McLee's concentration.
GP gram positive identification card concentration is 0.5-0.63 McLeod.
YST yeast identification card concentration of 1.8-2.2 McLee.
Neisseria NH, Haemophilus and other fastidious bacteria at concentrations of 2.7-3.3 Maifang.
i-2) drug sensitivity card and concentration
AST-GNxx/Nxxx gram-negative bacilli susceptibility card, 3.0mL saline + 145. mu.L of 0.5-0.63M M.unit suspension.
AST-GPxx/Pxxx gram-positive cocci drug sensitive card, 3.0mL saline + 280. mu.L of 0.5-0.63M cell suspension.
i-3) preparation of bacterial suspension and dilution of 0.45% NaCl solution, pH 4.5-7.2
Selecting the 3.3 purified strains according to a gram staining microscopy identification card, operating according to an operating guide, and selecting a drug sensitive card according to an identification result to perform a drug sensitive experiment.
(ii) Microorganism mass spectrum identification system (IVD MALDI Biotypeer)
ii-1) sample preparation: and uniformly smearing a single colony or 3.4 purified strains of a sample to be detected in sample hole sites of the MALDI target plate, and recording the position and the number of the sample in a recording table.
ii-2) standard: add 1. mu.L of the standard solution to 1 well of the sample and dry it naturally.
ii-3) adding 1 μ L matrix solution to the dried standard or sample (if the sample is a bacterium which is difficult to break the wall, adding 1 μ L70% formic acid, air drying, adding the matrix solution), air drying, and performing strain identification according to IVD MALDI Biotyper standard operating procedure.
3.5 metagenomic analysis of stomach tissue
In order to more fully understand the microbial composition in the stomach tissue, this example performed metagenomic monitoring analysis on 35 samples while performing a culture study.
4. Results of the culture
As shown in FIG. 1, the same sample (52#) was cultured on medium I (left dish) and medium II (right dish) under the same culture conditions, with different results.
As can be seen from comparison of FIGS. 1 and 2, the same sample (52#), the same medium (COLUMBIA BLOOD AGE BASE + sterilized defibrinated sheep BLOOD), and the medium without antibiotic mixture (II) (right dish in FIG. 1) and the medium with antibiotic mixture (IV) (left dish in FIG. 2) were cultured under the same conditions.
As shown in FIG. 3, the same sample (94#) obtained different culture results on medium I (right dish) and medium III (left dish) under the same culture conditions. It is shown that the strains grown in the same sample under the same culture conditions, in the same culture medium (campylobster AGAR BASE + sterile defibrinated sheep blood) with and without the addition of antibiotic mixtures are different.
As shown in FIG. 4, the same sample (54#) obtained different culture results on medium III (right dish) and medium IV (left dish) under the same culture conditions. It was shown that the same sample grew different strains in two different media, both with antibiotic mixtures, under the same culture conditions.
The culture results show that different culture results can be obtained on the same tissue on the culture medium I, the culture medium II, the culture medium III and the culture medium IV, so that when the intragastric flora is detected, the four culture media are simultaneously used for respectively culturing the same tissue sample, and the aim of more closely detecting all the intragastric flora is fulfilled.
5. Identification results
(1) The identification of the bacterial colonies cultured in the medium shown in FIGS. 1 to 4 was carried out by the identification method of 3.4, and the results are shown in Table 1.
TABLE 1 statistics of the identification results
As can be seen from Table 1, the same sample (No. 52) was different in the species grown in culture medium I (left dish in FIG. 1) and culture medium II (right dish in FIG. 1) under the same culture conditions; under the same culture conditions, the same sample (No. 52) has different strains grown in culture medium II (right dish in FIG. 1) and culture medium IV (left dish in FIG. 2); under the same culture conditions, the same sample (94#) was different in the strains grown in the culture medium III (left dish in FIG. 3) and the culture medium I (right dish in FIG. 3); the same sample (54#) grew different strains in medium III (right dish) and medium IV (left dish) under the same culture conditions.
(2) Comparison of culture results for different media
A comparison of the species obtained by culturing the same sample (68#) on media I-II with the species obtained by culturing on media I-IV under the same culture conditions is shown in Table 2.
TABLE 2 statistics of culture results for different media
As can be seen from Table 2, the same sample was cultured under the same culture conditions in the presence of four culture media to obtain more strains than culture media I-II (containing no antibiotics).
(3) Detection result of gastric flora
The inventor detects helicobacter pylori, 84 species of 23 genera and 96 strains of other 9 bacilli, schaalia odorotodytica and mycobacterium abstinella strain in total from 100 gastric mucosa tissue samples, wherein the genera are shown in the following figure 7:
after carrying out acid resistance (fast urease test) tests on the 96 bacteria, a stomach helicobacter pylori detection kit (urease method-Anxin biotechnology, Inc., Sanming City) and a urease gene detection method are adopted, and the adopted UreA primer sequences are as follows:
a forward primer: 5'-GCCAATGGTAAATTAGTTCC-3' (SEQ ID NO: 1);
reverse primer: 5'-CTCCTTAATTGTTTTTACAT-3' (SEQ ID NO: 2).
And (3) detecting 16 urease positive bacteria except helicobacter pylori in the stomach tissue sample, wherein the 16 urease positive bacteria are respectively: halofuginia acidovora, candida albicans, comamonas testosteroni, stenotrophomonas maltophilia, sphingomonas inevorans, sphingomonas paucimobilis-like, raoulus terrestris, ralstonia pickettii, trichosporon assiazella ansamilara, pseudomonas morganii, golardia denitrificans, morganella morganii, pseudomonas putida, klebsiella pneumoniae, tsukamurella slightly;
the invention discovers that the 16 urease positive bacteria have gastric detection reports except that pseudomonas putida and gold denitrifying bacteria have gastric detection reports, and other 14 urease positive bacteria have no gastric tissue detection related reports, and the invention discovers for the first time; the 16 urease positive bacteria are detected to be negative in helicobacter pylori detection, the clinical diagnosis result is shown in table 3, the 16 urease positive bacteria are also pathogenic bacteria, not only can cause interference on carbon 13 or carbon 14 breath tests, but also can cause influence on treatment of the subjects, if the subjects are judged to be infected by the helicobacter pylori only according to the positive breath tests, and then the existing first-line therapy, namely the quadruple therapy, is adopted to carry out helicobacter pylori eradication treatment on the subjects blindly, so that the antibiotic treatment effect is poor and the drug-resistant bacteria type of the infectious bacteria is induced, the discovery provides an important theoretical basis for accurate diagnosis and treatment of the subjects, and the method can accurately diagnose which pathogenic bacteria in the stomach, further accurately treat the subjects and avoid abuse of antibiotics;
the finding that the presence of the above 16 urease-positive bacteria in the stomach of the present invention can cause human diseases is also confirmed in the prior art which discloses that acid-fed bacteria can cause lung infection (see document: acid-fed bacteria causes lung infection in 1 case); the prior art discloses that candida albicans is a conditional pathogen, and can cause candidiasis in organisms when the immune function of the organisms is reduced or the mutual restriction action among microorganisms is disordered (see the literature: clinical analysis of candida albicans infection); the prior art discloses that Comamonas testosteroni can cause intracranial infection (see literature: one example of intracranial infection with Comamonas testosteroni); the prior art discloses that stenotrophomonas maltophilia can cause infectious diseases of body parts such as pulmonary infection, burn infection, urinary tract infection, skin wound infection and the like; the prior art discloses that sphingosine bacillus alcalopecuronide can cause the body to produce infectious related diseases (see the literature: Sphingobacterium multivorum infection in a child with an extensive burn); the prior art discloses that sphingomonas paucimobilis can cause nosocomial infection, and further causes the body to generate infection-related diseases (see the literature: 116 cases of nosocomial infection and drug resistance characteristics of sphingomonas paucimobilis); the prior art discloses that sphingomonas paucimobilis is a gram-negative non-zymocyte, which can cause human body to infect, especially the crowd with low immunity; the prior art discloses that Laurushibara terrestris can cause body bloodstream infection (see the literature: 1 case of Laurushibara terrestris causing bloodstream infection); the prior art discloses that the ralstonia pickettii can cause the organism to generate related symptoms such as sepsis (see 5 cases of sepsis of patients in ICU caused by the ralstonia pickettii and review of the literature); the prior art discloses that the Ashbya isattva can cause Urinary tract infection of the body (see the literature: Urinary track infection by Trichosporon asahii in a case); the prior art discloses that the denitrifying bacteria can cause the imbalance of the flora in the stomach and further cause the body to generate relevant disease symptoms (see the literature: the infection of an acid resistant Kingella denitrificans in the stored mass nutrient to gastric dysbiosis by Helicobacter pylori); the prior art discloses that Morganella morganii can cause liver abscess (see a document: one example of liver abscess caused by Morganella morganii and review of the document); the prior art discloses that pseudomonas putida belongs to a conditional pathogen in a human body, and can infect the urethra or skin of the body to cause infection when the body is weak in resistance; the prior art discloses that Klebsiella pneumoniae can cause intraocular inflammation (see the literature: endogenesis Klebsiella Endophylla Associated with Klebsiella pneumoniae Pneumonia); the prior art discloses tsukamurella slightly changed as a pathogenic microorganism (see literature: tsukamurella potentially water-borne pathogenic microorganism), and further shows that the 16 urease-positive bacteria present in the stomach of the present invention can cause the occurrence of human-related diseases;
when the carbon 13 or carbon 14 breath test is carried out, 16 urease positive bacteria detected by the invention interfere the judgment of the helicobacter pylori, and especially when 16 bacteria are detected in the stomach tissue of a tested person, the helicobacter pylori is not detected, so that a positive result is also obtained when the carbon 13 or carbon 14 breath test is carried out, if the helicobacter pylori antibiotic treatment is carried out on the tested person according to the positive carbon breath test result, a drug-resistant strain is induced to be generated by a non-helicobacter pylori bacteria strain, the subsequent treatment difficulty of the carried person is increased, and therefore, the diagnosis and eradication diagnosis of the helicobacter pylori by the current carbon 13 or carbon 14 breath test are necessary to be completed.
Statistics of clinical diagnosis results of subjects with urease-positive bacteria listed in Table 316
(4) Metagenomic analysis of gastric tissue
In order to understand the microbial composition in the stomach tissue more comprehensively, the present embodiment performs metagenomic monitoring analysis on the gastric mucosal tissue samples of 35 clinical subjects while performing the omics, and the results show that in the stomach, besides helicobacter pylori, also a plurality of microorganisms can parasitize, both urease-positive strains and urease-negative strains exist, and reveal the microbial composition of the stomach, and the main strains are found in 35 metagenomic maps (see fig. 8).
The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.
Sequence listing
<110> Shijiazhuang city disease prevention and control center (Shijiazhuang city sanitation detection center)
<120> detection and application method of stomach micro-ecological urease positive flora
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Claims (10)
1. A kit for aiding in the diagnosis of helicobacter pylori infection, said kit comprising reagents for detecting urease-positive bacteria comprising any one or more of paraporta acidovorans, candida albicans, comamonas testosteroni, stenotrophomonas maltophilia, sphingomonas inedibromicola, sphingomonas paucimobilis, raoultella terrestris, rhostonia pickettii, myceliophthora assamii, pseudomonas morgans, pseudomonas putida, klebsiella pneumoniae, tsukamurella slightly catarrhalis, and/or helicobacter pylori.
2. The kit of claim 1, wherein the reagents comprise primers, probes, antisense oligonucleotides, aptamers, and/or antibodies specific for the urease-positive bacteria.
3. The kit of claim 1, wherein the method for detecting urease-positive bacteria comprises any one or more of 16S sequencing, whole genome sequencing, quantitative polymerase chain reaction, PCR-pyrosequencing, fluorescence in situ hybridization, microarray, and/or PCR-ELISA detection.
4. Use of a reagent for the detection of urease-positive bacteria in the manufacture of a kit for the assisted diagnosis of helicobacter pylori infection, wherein said urease-positive bacteria comprise any one or more of paraporta acidovorans, candida albicans, comamonas testosteroni, stenotrophomonas maltophilia, sphingomonas inevorans, sphingomonas paucimobilis, raoulus terrestris, rosstonia pickettii, saccharomyces assidus assamica, pseudomonas moschata, pseudomonas putida, klebsiella pneumoniae, tsukamurella slightly altered, and/or helicobacter pylori.
5. The use of claim 4, wherein the reagent comprises a primer, probe, antisense oligonucleotide, aptamer, and/or antibody specific for the urease-positive bacteria.
6. The use of claim 4, wherein the method for detecting urease-positive bacteria comprises any one or more of 16S sequencing, whole genome sequencing, quantitative polymerase chain reaction, PCR-pyrosequencing, fluorescence in situ hybridization, microarray, and/or PCR-ELISA detection.
7. A kit for detecting urease positive bacteria, which is characterized in that the kit comprises a reagent for detecting urease positive bacteria, wherein the urease positive bacteria comprise any one or more of Tricholoma foeniculi, Candida albicans, Comamonas testosteroni, stenotrophomonas maltophilia, Sphingobacterium alcfeeding, Sphingomonas paucimobilis, Sphingomonas oliganomonas paucimobilis, Laurus terrestris, Rosstonia pickettii, Spirospora assiazeri, Pseudomonas mossambica, Pseudomonas putida, Klebsiella pneumoniae, Tukamurella slightly changed, and/or helicobacter pylori;
preferably, the reagent comprises a primer, probe, antisense oligonucleotide, aptamer, and/or antibody specific for the urease-positive bacteria.
8. A system or device for aiding in the diagnosis of helicobacter pylori infection, the system or device comprising:
(1) nucleic acid sample separation unit: a nucleic acid sample for isolating the intragastric flora from gastric mucosal tissue of a subject;
(2) a sequencing unit: the nucleic acid sample sequencing unit is used for sequencing the nucleic acid sample of the gastric flora obtained by the separation of the nucleic acid sample separation unit to obtain a sequencing result;
(3) a data processing unit: detecting urease positive bacteria in the gastric flora according to the sequencing result obtained by the sequencing unit, wherein the urease positive bacteria comprise any one or more of paraporta acidovorans, candida albicans, comamonas testosteroni, stenotrophomonas maltophilia, sphingomonas inevorans, sphingomonas paucimobilis, raoulus terrestris, rhostonia pickettii, candida assamica, pseudomonas moxidens, pseudomonas denitrificans, morganella morgana, pseudomonas putida, klebsiella pneumoniae, tsukamurella slightly changed, and/or helicobacter pylori;
(4) a result determination unit: obtaining a diagnosis result of the subject according to the result of the data processing unit, and storing the result in the data carrier;
preferably, the data carrier is a computer readable medium.
9. A method for detecting the gastric flora, comprising the steps of:
(1) collecting a gastric mucosa tissue sample of a subject, and cleaning the sample by using sterile normal saline to obtain a gastric mucosa tissue sample cleaning solution for culture;
(2) grinding the cleaned gastric mucosa tissue sample to obtain a bacterial suspension, and culturing in a sterile culture medium;
(3) carrying out strain purification and strain identification on the strains obtained by culturing in the steps (1) and (2) to obtain gastric mucosa tissue sample cleaning solution and gastric mucosa tissue sample bacterial suspension culture results;
(4) and (3) carrying out metagenome sequencing analysis on the sample obtained in the step (2) to obtain a metagenome sequencing result, and judging the microecological composition of the gastric flora of the subject by combining the culture result of the gastric mucosa tissue sample cleaning solution obtained in the step (1).
10. A method for producing and preparing urease is characterized by comprising the steps of performing purification culture on urease positive bacteria, and extracting the urease from 16 urease positive bacteria respectively after the purification culture;
preferably, said 16 urease-positive bacteria comprise any one or more of paraporta acidovorans, candida albicans, comamonas testosteroni, stenotrophomonas maltophilia, sphingomonas vorax, sphingomonas paucimobilis, raoultella terrestris, ralstonia pickettii, myceliophthora assiazakii, pseudomonas morganii, rhizomus morganii, pseudomonas putida, klebsiella pneumoniae, tsukamurella slightly-changed, and/or helicobacter pylori.
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| CN101019764A (en) * | 2007-03-05 | 2007-08-22 | 青岛科技大学 | Pyloric helicobacter detecting method |
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