CN109913526B - Use of microorganisms for identifying and/or differentiating different ethnic groups of individuals - Google Patents
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Abstract
The invention provides application of microorganisms in identifying and/or distinguishing different ethnic groups of individuals and application of products for detecting the abundance of the microorganisms in identifying and/or distinguishing different ethnic groups of individuals, and also provides a method for identifying and/or distinguishing different ethnic groups of individuals, wherein the method comprises the steps of detecting the relative abundance of Acinetobacter in intestinal tracts and/or feces of the individuals and comparing the relative abundance with a threshold value.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to application of microorganisms in identification and/or differentiation of different ethnic groups of individuals, and more particularly relates to a method for detecting relative abundance of Acinetobacter, comparing the relative abundance with a threshold value and identifying ethnic groups of individuals.
Background
Altitude sickness is a natural physiological reaction generated by the body of a person after the person reaches a certain altitude and in order to adapt to changes of air pressure difference, low oxygen content, dry air and the like caused by the altitude. Symptoms of altitude sickness are generally manifested as headache, palpitation, tiredness, chest tightness, shortness of breath, vomiting, anorexia, convulsions, absentmindedness, and sudden decline in cognitive ability. The physical signs include accelerated heart rate, deepened respiration, slight abnormal blood pressure, edema of face or limbs, cyanosis of lips, etc. The plateau heart disease is one of the plateau diseases, and is characterized by the structural function damage of the heart caused by the low-pressure oxygen-poor environment of the plateau, namely the damage of the pulmonary hypertension and the function of the right heart. Tibetan nationality residents of the Qinghai-Tibet plateau are main population at high altitude in the world, carry out long-term physiological and genetic adaptation to the hypoxic and low-air-pressure environment of the plateau, reduce the pulmonary vasoconstriction reactivity in the hypoxic environment, and have better exercise capacity.
There is a large number of commensal flora in the human intestinal tract, which number exceeds 1000 trillion and is about 10 times of the total number of human cells. Meanwhile, the number of microbial genes in the intestinal tract is about 300 ten thousand, which is about 100 times of the number of human genome genes, so that the mass of genes can help the microbes to adapt to changeable environments, and an inseparable symbiotic relationship with a human body is formed. Residents with different genetic backgrounds and living habits have large differences in the composition of intestinal flora, for example, bacteroides is the bacterial genus with the highest relative content in intestinal tracts of western residents and the largest difference among individuals, and korean residents are copromorphus. Differences in the genera also suggest the ability of the individual to tolerate the local environment.
The research is wide at present on the tolerance capability of individuals in the plateau environment or diagnostic markers of the plateau disease. For example, patent CN106248947A discloses the use of lipid and polysaccharide complex lipopolysaccharide as a marker of high altitude cerebral edema of mammals, and experimental tests show that the content of lipopolysaccharide in blood plasma of mammals after entering into high altitude hypoxic environment is increased, and when the content of lipopolysaccharide in blood plasma exceeds 0.5EU/mL, the water content of brain tissue of mammals is increased significantly. Patent CN104614462B discloses that C8-ceramide, sphingosine and glutamine are used as diagnosis markers of high altitude pulmonary edema, and a diagnosis kit for detecting the diagnosis markers has high accuracy of diagnosis results, and provides quantifiable, objective and sensitive clinical diagnosis indexes for diagnosis of high altitude pulmonary edema. Patent CN102605089B discloses a method for detecting susceptibility to excessive high erythrocyte using mononucleotide polymorphism as marker, wherein, primer and kit for detecting mononucleotide polymorphism are provided. Patents CN101135666B, CN101470095B, CN101470096A, CN101135665B, CN101469345B, CN101135667B and CN1898394B all disclose that single nucleotide polymorphism is used as a marker for detecting susceptibility of high altitude pulmonary edema, wherein primers and kits for detecting single nucleotide polymorphism are provided. However, up to now, no technical scheme for identifying and/or distinguishing different ethnic groups of individuals by using Acinetobacter as a marker has been found.
Disclosure of Invention
In a first aspect of the invention, there is provided the use of a microorganism for identifying and/or differentiating between different ethnic groups of individuals.
Preferably, the microorganism is Acinetobacter.
Further preferably, the microorganism is Acinetobacter in the intestinal tract or feces.
In one embodiment of the invention, the microorganism is Acinetobacter histaminiferans in the intestine or feces.
Preferably, the different nationalities are a ethnicity in a plateau environment and a ethnicity in a plain environment.
In one embodiment of the present invention, the different ethnic groups are Tibetan and Han.
In one embodiment of the invention, the different ethnic groups are Tibetan and Han nationalities living in a plateau environment.
In a second aspect of the invention, there is provided a method of identifying and/or differentiating different ethnic individuals, said method comprising detecting the abundance of microorganisms in the gut and/or faeces of said different ethnic individuals, comparing said abundance of microorganisms with a threshold value, and determining that said different ethnic individuals are ethnic in a plateau or a plain environment.
The plateau environment is above the altitude of 2000m and has the conditions of low pressure and oxygen deficiency.
Preferably, the plateau environment is above the altitude of 2700m and has low pressure and oxygen deficiency.
In one embodiment of the present invention, the plateau environment is above 3500m in altitude, and has low pressure and oxygen-deficient conditions.
In one embodiment of the present invention, the plateau environment is above 5500m in altitude, and has low pressure and oxygen-deficient conditions.
Preferably, the method comprises the following steps:
1) extracting microbial nucleic acid in feces or intestinal tracts of individuals to be detected;
2) amplifying the microbial nucleic acid extracted in step 1);
3) sequencing the amplified nucleic acid of step 2);
4) calculating the relative abundance of Acinetobacter;
5) the relative abundance value of Acinetobacter genus in the step 4) is compared with 0.91 x 10-4Comparing;
wherein the Acinetobacter genus has a relative abundance value of less than 0.91 × 10-4Determined as a Tibetan person, the Acinetobacter genus has a relative abundance value of greater than 0.91X 10-4Is determined as Han nationality.
In a third aspect of the invention, there is provided the use of a product for detecting the abundance of a microorganism for the identification and/or differentiation of individuals of different ethnic groups.
Preferably, the microorganism is Acinetobacter.
Preferably, the different nationalities are a ethnicity in a plateau environment and a ethnicity in a plain environment.
In a fourth aspect of the invention, there is provided a product for detecting the abundance of a microorganism, said product comprising a reagent and/or a device for detecting the relative abundance of a microorganism, preferably a microorganism of the genus Acinetobacter.
In a fifth aspect of the invention, the application of the reagent for detecting Acinetobacter in preparing the product for evaluating the tolerance capability of individuals in the plateau environment is provided.
Preferably, the Acinetobacter genus is an intestinal Acinetobacter genus.
The reagent for detecting Acinetobacter is a reagent for detecting abundance of Acinetobacter.
Preferably, the reagent for detecting Acinetobacter is a reagent for detecting the abundance of Acinetobacter in the intestinal tract of an individual.
In one embodiment of the invention, the reagent for detecting Acinetobacter is a reagent for detecting the abundance of Acinetobacter in excrement of an individual.
The tolerance capability in the plateau environment is the susceptibility degree of the plateau disease.
The plateau environment is above the altitude of 2000m and has the conditions of low pressure and oxygen deficiency.
Preferably, the plateau environment is above the altitude of 2700m and has low pressure and oxygen deficiency.
In one embodiment of the present invention, the plateau environment is above 3500m in altitude, and has low pressure and oxygen-deficient conditions.
In one embodiment of the present invention, the plateau environment is above 5500m in altitude, and has low pressure and oxygen-deficient conditions.
According to a sixth aspect of the present invention, there is provided a microorganism which identifies and/or distinguishes between individuals of different ethnic groups, said microorganism being of the Acinetobacter genus.
Preferably, the microorganism is Acinetobacter in the intestine or feces.
In one embodiment of the invention, the microorganism is Acinetobacter in feces.
In a seventh aspect of the invention, a marker for evaluating the plateau environment tolerance capacity is provided, and the marker is Acinetobacter.
Preferably, the marker is Acinetobacter in the intestinal tract.
In a particular embodiment of the invention, the marker is Acinetobacter in feces.
In an eighth aspect of the present invention, there is provided a product for evaluating the tolerance of an individual in a plateau environment, said product comprising a reagent for detecting Acinetobacter.
Preferably, the reagent for detecting Acinetobacter is a reagent for detecting the abundance of Acinetobacter in the intestinal tract of an individual.
In one embodiment of the invention, the reagent for detecting Acinetobacter is a reagent for detecting the abundance of Acinetobacter in excrement of an individual.
The product of the invention comprises a reagent for detecting Acinetobacter and a reagent for detecting other bacteria.
According to a ninth aspect of the invention, a method for detecting the abundance of Acinetobacter is provided, and the method comprises 16S rRNA sequencing of a sample, OTU clustering of reads obtained by sequencing and species annotation.
Preferably, low-quality partial cutting is carried out on reads obtained by sequencing, data of each sample is split, and/or chimera sequences are removed after 16S rRNA sequencing and among OTU clustering.
Preferably, the clustering method is selected from sortmerna, mothur, trie, uclust _ ref, usearch _ ref, blast, usearch61, usearch61_ ref, sumaclust, swarm, prefix _ suffix, cdhit or uclust.
Preferably, the clustering algorithm is selected from futhest, nerest or average.
Preferably, the annotation algorithm is selected from rdp, blast, rtax, mothur, uclust or sorterna.
In one embodiment of the invention, the OTU clustering clusters sequences with 97% identity.
In a tenth aspect of the present invention, there is provided a method of improving tolerance to a high altitude environment in an individual, the method comprising transplanting Acinetobacter into the individual.
Preferably, the method comprises transplanting Acinetobacter sp.
In one embodiment of the invention, the method comprises transplanting Acinetobacter prior to the removal of the subject from the plateau.
Preferably, the capability of improving the individual plateau environment tolerance is the capability of improving the adaptation of people in plain areas to plateau environments.
The altitude sickness of the invention is selected from acute altitude sickness and/or chronic altitude sickness generated in the altitude environment. The acute altitude disease is selected from altitude coma, altitude cerebral edema, altitude pulmonary edema or mixed diseases with abnormal symptoms of brain and lung; and/or the chronic altitude sickness is selected from the mixed type diseases existing simultaneously in the plateau heart disease, the plateau erythrocytosis, the plateau hypertension, the plateau hypotension, the plateau heart disease and the erythrocytosis. Preferably, the altitude disease is resisting the injury of the heart, the lung and the blood vessels of the altitude disease; further preferably, the altitude sickness is a high altitude heart disease.
In one embodiment of the invention, the altitude disorder is pulmonary hypertension and/or right ventricular hypertrophy.
The clinical manifestations of the altitude sickness are selected from one or more than two of headache, dizziness, palpitation, heart rate acceleration, fatigue, chest distress, short breath, respiration deepening, nausea, vomiting, insomnia, hypodynamia, dim eyesight, somnolence, anorexia, convulsion, consciousness absentmindedness, numbness of hands and feet, cyanosis of lips and fingers, facial edema, edema of limbs or sudden decline of cognitive ability.
The abundance or relative abundance of the Acinetobacter genus of the present invention is the relative abundance of the Acinetobacter genus in a particular flora, and in the examples of the present invention is the degree of abundance of the Acinetobacter genus in the gut flora.
The Acinetobacter of the invention is obtained by separating, purifying or purchasing from an individual.
The Acinetobacter genus of the present invention is provided in a viable form.
The Acinetobacter genus of the present invention is provided in a lyophilized, freeze-dried or spray-dried form.
The Acinetobacter genus of the present invention is provided in the form of spores.
The Acinetobacter genus of the present invention is Acinetobacter histaminiferans.
The term "and/or" as used herein includes a list of items in the alternative as well as any number of combinations of items.
The terms "comprises" and "comprising" as used herein are intended to be open-ended terms that specify the presence of the stated elements or steps, and not substantially affect the presence of other stated elements or steps.
The term "identify" as used herein refers to the determination or determination of one or more individuals as one or more attributes, for example, the term "identify" in the embodiments of the present invention refers to the determination or determination of one or more individuals as having a source of Han nationality or Tibetan nationality.
The term "distinguish" as used herein means to separate two or more individuals into different attributes, for example, the term "distinguish" as used in the embodiments of the present invention refers to distinguish two or more individuals into those whose sources are Han nationalities or those whose sources are Tibetan nationalities.
Drawings
Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1: the difference in the abundance of Acinetobacter in feces between Tibetan soldier group (Zang) and Han soldier group (Han).
FIG. 2: the relative abundance of Acinetobacter species identifies and/or distinguishes ROC curves for sensitivity and specificity where the individual source is Han or Tibetan.
FIG. 3: is formed by the colony in the excrement of the Tibetan soldier group (Zang).
FIG. 4: colony composition in feces of Han group of soldiers (Han).
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
1.1 study population
According to STROBE statement design case contrast research, taking 128 Tibetan male soldiers with habitual height above 3500m serving in a certain army in China from a certain month to a certain month in a certain year as an experimental group, and matching 128 plain Han soldiers according to the age of the Tibetan soldiers for contrast. Strictly controlling two groups of people to perform the same mixed diet of Chinese troops, keeping the same training environment and training intensity at the altitude of 3500m, and stopping smoking and drinking for 3 months. Soldiers with chronic inflammatory disease, oral antibiotics, acute infection and gastrointestinal disease were excluded.
1.2 fecal sample Collection
After each study object is brought in, each study object receives a closestool excrement collector, excrement is put into an excrement collecting pipe after the excrement is discharged, the excrement is immediately stored in a refrigerator at the temperature of minus 20 ℃, excrement specimens are transferred to a refrigerator at the temperature of minus 80 ℃ for storage the next day, and the excrement specimens are stored for later use in a unified mode.
1.3 DNA extraction
Pipette 1000. mu.L of cetyltrimethylammonium Bromide (CTAB) lysate into a 2.0mL EP tube, add lysozyme, add about 500. mu.L of sample to the lysate, and mix several times in a 65 ℃ water bath, reversing the process to thoroughly lyse the sample. Centrifuging to obtain supernatant, adding phenol (pH8.0): chloroform: isoamyl alcohol (25: 24: 1), reverse mixing, and centrifuging at 12000rpm for 10 min. Taking the supernatant, adding chloroform: isoamyl alcohol (24: 1), reverse mixing, and centrifuging at 12000rpm for 10 min. The supernatant was aspirated into a 1.5mL centrifuge tube, isopropanol was added, shaken up and down, and precipitated at-20 ℃. Centrifuge at 12000rpm for 10 minutes and pour out the liquid, taking care not to pour out the pellet. The column was washed 2 times with 1mL of 75% ethanol, and the remaining small amount of liquid was collected by centrifugation again and then aspirated out with a pipette tip. And drying the clean bench or airing the clean bench at room temperature. Addition of ddH2O dissolving the DNA sample, adding RNaseA 1. mu.L of digested RNA, and standing at 37 ℃ for 15 min. Then, the purity and concentration of the DNA are detected by agarose gel electrophoresis, an appropriate amount of sample DNA is taken out to a centrifuge tube, and the sample DNA is diluted to 1 ng/. mu.L by sterile water.
1.4 PCR amplification, sample mixing and purification
1.3 diluted genome DNA is taken as a template, specific primers 515F and 806R with Barcode, Phusion High-Fidelity PCR Master Mix with GC Buffer from New England Biolabs and High-efficiency High-Fidelity enzyme (TransFast Taq DNA Polymerase) are selected for carrying out PCR according to a sequencing region 16S V3-V4, and the amplification efficiency and accuracy are ensured;
wherein,
the specific primer 515F is: 5 '-GTGCCAGCMGCCGCGGTAA-3' (SEQ ID NO: 1)
The specific primer 806R is: 5 '-GGACTACHVGGGTWTCTAAT-3' (SEQ ID NO: 2).
The PCR product is detected by electrophoresis by using agarose gel with 2 percent concentration; the PCR products were mixed in equal amounts according to the concentration of the PCR products, and after mixing well, the PCR products were purified by agarose gel electrophoresis using 1 XTAE 2%, and the target band was recovered by shearing. Product purification kit used was the recovery of PCR products using the Thermo Scientific GeneJET gel recovery kit.
1.5 library construction and on-machine sequencing
Constructing a Library by using an Ion Plus Fragment Library Kit 48 rxns Library construction Kit of Thermofisoher company, and using Ion S5 of Thermofisoher after the constructed Library is qualified by Qubit quantification and Library detectionTMXL was sequenced on machine.
1.6 data analysis
1.6.1 sequencing data processing
Cutadapt (V1.9.1, http:// cutapt. readthetadocs. io/en/stable /) is used for carrying out low-quality partial shearing on Reads, then each sample data is split from the obtained Reads according to Barcode, the Barcode and primer sequence are cut off for preliminary quality control to obtain original data, the Reads obtained after the treatment needs to be treated for removing a chimera sequence, the Reads sequence is compared with a species annotation database to detect the chimera sequence, and finally the chimera sequence is removed, so that the final effective data are obtained.
1.6.2 clustering of operational taxonomic unit (OUT) and species annotation
All effective data of all samples are clustered by using Upearse software (Upearse v7.0.1001, http:// www.drive5.com/Uparse /), sequences are clustered into OTUs (operational Taxonomic units) by default with 97% consistency, representative sequences of the OTUs are selected at the same time, and the sequences with the highest frequency of occurrence in the OTUs are selected as the representative sequences of the OTUs according to the algorithm principle. Species annotation is carried out on OTUS sequences, species annotation analysis is carried out by a Mothur method and an SSUrRNA database of SILVA (http:// www.arb-SILVA. de /) (the threshold value is set to be 0.8-1), taxonomic information is obtained, and the community composition of Han soldier groups and Tibetan soldier groups is counted at the genus level respectively (see figures 3 and 4).
1.6.3 calculation of relative abundance of genera and comparison of differences
Relative abundance of the genus was expressed as Acinetobacter OTU values/all the genus OTU values detected in the test samples, and the difference was compared by Mann-Whitney test because the samples were not normally distributed. And drawing an ROC curve to identify/distinguish individual ethnic sources or evaluate the sensitivity and specificity of relative abundance of the genus of the bacteria to the diagnosis of the tolerance capability in the plateau environment, calculating a threshold value, wherein P is less than 0.05, the difference has statistical significance, and performing statistical processing by adopting an SPSS22.0 software package.
Second, experimental results
2.1 the difference in the relative abundance of Acinetobacter in fecal specimens from Han and Tibetan populations
Relative abundance of Acinetobacter in the Han population [ median (quartile)]Is [ 2.32X 10 ]-4（0.93×10-4，4.70×10-4）]Relative abundance of Acinetobacter genus in the Tibetan population (median (quartile)]Is 0.01X 10-4（0，0.09×10-4) It is shown that the relative abundance of Acinetobacter in Tibetan is significantly lower than that of Han, and the difference is significant, as shown in FIG. 1.
2.2 Acinetobacter genus relative abundance identification/differentiation of sensitivity and specificity of Hanzang origin
As shown in FIG. 2, the ROC curve shows the relative abundance of Acinetobacter to identify/distinguish Hanzan origin or evaluate plateau environmentSensitivity and specificity of lower tolerance, Area Under ROC Curve (AUC) value of 0.965, relative abundance of genus 0.91X 10-4The maximum john index of 0.879 was obtained with an Accuracy (Accuracy) of 0.941, a specificity of 0.968, a sensitivity of 0.911, and a F1-Score value of 0.944.
The results show that the relative abundance of Acinetobacter in the fecal specimen is detected by using 16SrRNA sequencing, whether the source of the crowd is Tibetan or Han can be identified, or the tolerance of the individual to the plateau environment can be evaluated, and when the relative abundance is more than 0.91 multiplied by 10-4The individual can be judged to be a Han group of people and has strong tolerance to the plateau environment.
Example 2 verification experiment
According to STROBE statement design case contrast study, 5 volunteers are selected, 5 volunteers are strictly controlled to carry out the same mixed diet of Chinese troops, the same altitude of 3500m environment and exercise intensity are kept, and smoking cessation and drinking are continued for 3 months. All 5 volunteers excluded chronic inflammatory disease, oral antibiotics, acute infection and gastrointestinal disease.
The experimental steps are as follows:
1. collecting a fecal sample;
2. extracting DNA;
3. PCR amplification, sample mixing and purification;
4. constructing a library and sequencing on a computer;
5. processing sequencing data;
6. clustering of the classification units of the operation and annotation of the species. Steps 1-6 were performed as described in example 1.
Results of the experiment
3 of 5 volunteers, namely, Acinetobacter genus relative abundance values are lower than a threshold value, and the relative abundance values are judged to be Tibetan; the relative abundance value of Acinetobacter of 2 volunteers is higher than the threshold value, and the volunteers are judged as Han nationality. After experimental detection, inquiry and investigation prove that 3 volunteers are in the Tibetan true, 2 volunteers are in the Han nationality true, and the experimental result is accurate.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.
Sequence listing
<110> general hospital of liberation military of Chinese people
<120> use of microorganisms for identifying and/or differentiating individuals of different ethnic groups
<130> 1
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 19
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
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<210> 2
<211> 20
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
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ggactachvg ggtwtctaat 20
Claims (3)
1. The application of the microorganism in identifying and/or distinguishing different ethnic groups of individuals is characterized in that the microorganism is Acinetobacter in intestinal tracts or excrement, the different ethnic groups are Tibetan and Han nationalities on plateau, the plateau is over 3500m in altitude, the relative abundance value of the Acinetobacter of the individuals is compared with a threshold value, and the threshold value is 0.91 multiplied by 10-4When the relative abundance value of the individual Acinetobacter genus is less than 0.91X 10-4The individual is a Tibetan patient,when the relative abundance value of individual Acinetobacter is more than 0.91X 10-4The individual is Han nationality, and the relative abundance value is represented by "Acinetobacter OTU (operational Taxomic Unit) value"/"ratio of all the OTU values of the genera detected by the test sample".
2. A method for identifying and/or differentiating different ethnic groups of individuals, wherein the method comprises detecting the relative abundance of microorganisms in the intestines and/or feces of the different ethnic groups of individuals, comparing the relative abundance value of the microorganisms with a threshold value, determining that the different ethnic groups of individuals are ethnic groups in a plateau environment or ethnic groups in a plain environment, wherein the microorganisms are Acinetobacter in the intestines or feces, wherein the different ethnic groups are Tibetan and Han nations in the plateau, wherein the plateau is at an altitude of more than 3500m, comparing the relative abundance value of the individual Acinetobacter with the threshold value, and wherein the threshold value is 0.91 x 10-4When the relative abundance value of the individual Acinetobacter genus is less than 0.91X 10-4The individual is Tibetan, and when the relative abundance value of the individual Acinetobacter genus is more than 0.91 × 10-4The individual is Han nationality, and the relative abundance value is represented by "Acinetobacter OTU (operational Taxomic Unit) value"/"ratio of all the OTU values of the genera detected by the test sample".
3. The method of claim 2, wherein the method comprises the steps of:
1) extracting microbial nucleic acid in feces or intestinal tracts of individuals to be detected;
2) amplifying the microbial nucleic acid extracted in step 1);
3) sequencing the amplified nucleic acid of step 2);
4) calculating the relative abundance of Acinetobacter;
5) the relative abundance value of Acinetobacter genus in the step 4) is compared with 0.91 x 10-4And (4) comparing.
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| CN101248188A (en) * | 2005-06-28 | 2008-08-20 | 株式会社东芝 | Method, array, apparatus and test system to discriminate individuals |
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| EP1083234A1 (en) * | 1998-05-27 | 2001-03-14 | Mitsubishi Chemical Corporation | Process for producing l-ribose |
| CN101248188A (en) * | 2005-06-28 | 2008-08-20 | 株式会社东芝 | Method, array, apparatus and test system to discriminate individuals |
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