CN113957165B - Pseudomonas horizontal rapid detection method based on high-throughput sequencing and application thereof - Google Patents
Pseudomonas horizontal rapid detection method based on high-throughput sequencing and application thereof Download PDFInfo
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Abstract
The invention discloses a pseudomonas horizontal rapid detection method based on high-throughput sequencing and application thereof, belonging to the technical field of molecular biology. The invention screens out the segment with specificity on mreB gene of pseudomonas by means of gene marking, evolutionary tree analysis and the like, and can distinguish pseudomonas from other microorganisms, and the resolution of the segment is higher than that of the traditional 16S rRNA gene segment. Based on the specific fragment, the target band obtained by designing primer amplification is 410-462 bp, all the reported Pseudomonas strains can be identified, and the identification accuracy rate of the Pseudomonas strains reaches 100%.
Description
Technical Field
The invention relates to a pseudomonas horizontal rapid detection method based on high-throughput sequencing and application thereof, belonging to the technical field of molecular biology.
Background
Raw milk is contaminated with a range of microorganisms during the production process. Therefore, the cold chain is widely used to inhibit the growth and reproduction of microorganisms in raw milk, ensuring the quality of milk. Psychrophilic bacteria (bacteria with optimal growth temperature of 15-20 ℃ and ubiquitous temperature below 7 ℃) are used as main microorganisms in cold storage raw milk, and grow under a cold chain member and generate heat-resistant hydrolase, so that the psychrophilic bacteria grow to become a 'short plate' for restricting the quality of modern dairy products, and research, detection, prevention and control of the psychrophilic bacteria are important points of attention of modern dairy enterprises and even food enterprises. Pseudomonas is the most dominant genus of psychrophilic milk bacteria, and grows at low temperature, and highly expresses proteases and lipases that are resistant to heat treatment. These enzymes are resistant to heat treatment and become a major factor in milk quality degradation (protein gelation delamination, fat lifting, rancid odor, bitterness, etc.) after pasteurization and ultra high temperature flash sterilization (UHT). In addition, due to the diversity of pseudomonas niches, the different species are different in terms of growth conditions, metabolic characteristics, spoilage potential and the like. Therefore, it is becoming increasingly important to develop a method for rapid detection of Pseudomonas species, even at the level of species, to evaluate their diversity and composition in raw milk to understand the extent of their effect on milk quality (doi: 10.1017/s 0022029919000645).
The methods based on traditional culture methods such as CFC selective media for taxonomic identification can determine species, but the experimental period is long and the relative abundance of pseudomonas in the raw milk cannot be analyzed. Based on this, some psychrophilic bacteria detection means mainly for pseudomonas, such as flow cytometry, direct fluorescence filtration, ATP bioluminescence, aminopeptidase activity assay, ELISA, electronic impedance method and biosensor method, are presented. In recent years, with the development of molecular biology methods, real-time quantitative PCR and fluorescence in situ hybridization are widely used for rapid detection of strains. Particularly, the appearance of a new generation sequencing technology based on the 16S rDNA gene enables a large number of samples to be deeply detected, but the typing method based on the 16S rDNA is insufficient for distinguishing the species level of pseudomonas, and molecular markers with higher resolution are required to meet the identification of different species and subspecies.
There have been previous studies on the division of 227 Pseudomonas into two lineages of Pseudomonas fluorescens and Pseudomonas aeruginosa by means of multiple sequence locus typing (MLST) in system genomics by concatenating 4 housekeeping genes (16 srdna, gyrB, rpoB, rpoD) and further into 14 species populations, (doi: 10.1111/j.1462-2920.2010.02181.X,10.3390/genes 11020139). Furthermore, adnA, filC, aprX isogenes have also been used for predictive analysis of the spoilage potential against Pseudomonas. A recent study (doi: 10.1111/1750-3841.13845, doi: 10.1111/1750-3841.12645) compares the ability of a high throughput sequencing method based on 16S rDNA and a MALDI-TOF MS method to identify Pseudomonas species in milk, both of which have a emphasis on the specific species detected, but cannot compromise the accuracy and speed of detection, revealed that both methods have shortcomings in detection against Pseudomonas species and thus still cannot be used as a complete method for analyzing Pseudomonas species in samples (doi: 10.1016/j.idailyj.2019.06.001). There are studies on the detection of pseudomonas dominance in pork by establishing fluorescent quantitative PCR (qPCR) and real-time fluorescent loop-mediated isothermal amplification (LAMP) detection technique, which can produce results within 2.5h (patent publication No. CN 109593868A). Another study has proposed a quantitative detection method of Pseudomonas bacteria in fresh meat for distinguishing Pseudomonas from other genus (patent publication No. CN 109593868A).
In addition, according to investigation, patents reported in China on Pseudomonas detection or methods are only focused on common ones. Pseudomonas aeruginosa (patent No. CN 110470839A), pseudomonas putida (patent No. CN 104374914A), pseudomonas in cow's milk (patent No. CN 101798591B), pseudomonas deformans (patent No. CN 107400650A), pseudomonas coco (patent No. CN 112695075A), pseudomonas fish (patent No. CN 103276093B)), have very few reports on the detection of Pseudomonas species levels internationally. No method can realize rapid, sensitive and specific detection of pseudomonas in other complex samples such as milk and the like.
Disclosure of Invention
The invention obtains the core gene sequence of the pseudomonas by a molecular biology method, compares the core genes, selects a section of core gene segment mreB gene with high resolution as a screening mark, combines with the use of an Illumina Miseq high-throughput sequencing detection technology, and develops a quick detection technology of the pseudomonas for the first time. In order to solve the defects in the prior art, a segment of fragment on the mreB gene (cytoskeletal protein synthesis gene, a housekeeping gene 10.1111/j.1365-2958.2010.07132.X for encoding cell structure formation) is selected, primers are designed based on the segment, and detection and identification of pseudomonas at the species level are performed by a high-throughput sequencing technology. The method can be used for rapidly, sensitively and specifically detecting the composition of different pseudomonas in complex samples.
The invention provides a method for identifying pseudomonas, which takes mreB gene fragments as markers, takes sequences shown as SEQ ID NO.1 and SEQ ID NO.2 as primers, and obtains the pseudomonas strain of the marker gene fragments by amplification.
In one embodiment, the marker gene fragment is 410 to 462bp in size.
In one embodiment, the amplification is performed on a subject containing a genome of a microorganism.
In one embodiment, the subject comprises a genomic DNA extract, a bacterial suspension, or a single colony.
In one embodiment, the genomic DNA includes, but is not limited to, those obtained by extraction from food, air, water, soil, and human.
In one embodiment, the amplification is performed using genomic DNA from a sample to be tested as a template.
In one embodiment, the amplification is performed using a single colony as a template.
The present invention provides a method for identifying the horizontal composition of pseudomonas species in a sample, the method comprising: (1) constructing a pseudomonas mreB gene standard library; (2) Amplifying mreB gene sequences in a complex sample genome by using sequences shown in SEQ ID NO.1 and SEQ ID NO.2 as primers; (3) Recovering the amplification result in the step (2), constructing a library and sequencing; (4) Comparing the sequencing result with the pseudomonas mreB gene standard library constructed in the step (1), and identifying and analyzing the composition characteristics of the pseudomonas strain level in the complex sample.
In one embodiment, the standard library of pseudomonas mreB genes contains mreB gene sequences retrievable in the Genbank database.
In one embodiment, the method comprises the steps of:
(1) Extracting genome DNA of a sample to be detected;
(2) Designing a primer, wherein the forward primer is shown as SEQ ID NO.1, and the reverse primer is shown as SEQ ID NO. 2:
forward primer (SEQ ID No. 1): 5'-ACCCTTATTTACGTGCGCGA-3'
Reverse primer (SEQ ID NO. 2): 5'-ATRTCSACSACCATCGARC-3'
(3) Performing PCR amplification by using the forward primer and the reverse primer extracted in the step (2) and using the genomic DNA extracted in the step (1) as a template, and purifying a PCR product;
(4) Quantifying the PCR product purified in the step (3), mixing the same amount of the PCR products, constructing a Miseq sequencing standard library, and then carrying out high-throughput sequencing;
(5) And (3) obtaining mreB gene sequence information of the complex sample or the mixed bacteria sample to be detected according to the sequencing result in the step (4), and detecting different kinds of pseudomonas in the sample to be detected based on the mreB gene sequence information.
The invention provides a kit for identifying pseudomonas, which comprises a primer pair shown as SEQ ID NO.1 and SEQ ID NO. 2.
In one embodiment, the kit contains a forward primer as shown in SEQ ID NO.1 and a reverse primer as shown in SEQ ID NO. 2.
The invention provides a method for screening pseudomonas, which comprises the steps of preparing a sample to be screened into bacterial suspension, diluting, coating on a solid culture medium, culturing until a single colony is formed, and amplifying the sequence of mreB gene by using sequences shown as SEQ ID NO.1 and SEQ ID NO.2 as primers to obtain a bacterial strain with a gene fragment of 410-462 bp.
In one embodiment, the solid medium includes, but is not limited to, CFC selective medium or NB nutrient broth medium.
The invention has the beneficial effects that: compared with the existing method, the method can detect all pseudomonas strains (more than 100 strains) except a few rare strains in a complex sample, and the detection accuracy reaches 100%. The method can realize high-throughput identification, process strain information in batches, comprehensively identify the pseudomonas composition in complex samples without separation and purification, and can identify the pseudomonas composition to a seed level, wherein the resolution is higher than that of a 16SrDNA method. Therefore, the method is based on the detection of pseudomonas, and the result is more accurate than that of the conventional identification method.
The traditional 16S rRNA strip is 1500bp, bidirectional sequencing is needed and splicing is needed, and the target strip is 410-462 bp through the specific primer extension based on mreB gene, so that the sequencing read length requirement of Illumina Miseq is met, and the identification of pseudomonas can be completed through unidirectional identification; furthermore, 16S rRNA is a non-monoclonal copy of a gene in the bacterial genome (i.e., more than one in the genome), so that the same genome may produce multiple copy sequences during amplification; the mreB gene is a monoclonal copy gene (i.e., only one in the genome) in gram-negative bacteria, and only one copy sequence is generated in the same genome in the amplification process, so that the correlation with colony numbers exists. Therefore, when sequencing is carried out in second generation sequencing, the mreB gene is used as a primer for identifying complex samples, so that the pseudomonas identification time and the pseudomonas identification cost can be saved by more than 50%, the detection speed and the detection efficiency are higher, and the detection result is more accurate.
Drawings
FIG. 1 is a phylogenetic tree of Pseudomonas based on the mreB gene.
FIG. 2 is an electrophoretogram of PCR amplification products of selected 13 strains of Pseudomonas and 10 strains of non-Pseudomonas; wherein M represents Marker (100 bp), 1-13 are respectively Pseudomonas aeruginosa (Pseudomonas aeruginosa), pseudomonas fluorescens (Pseudomonas fluorescens), pseudomonas putida (Pseudomonas putida), pseudomonas solanacearum (Pseudomonas solanacearum), pseudomonas alcaligenes (Pseudomonas alcaligenes), pseudomonas oleovorans (Pseudomonas oleovorans), pseudomonas fragi (Pseudomonas fragi), pseudomonas deformans (Pseudomonas plecoglossicida), pseudomonas cold-resistant (Pseudomonas psychrotolerans), pseudomonas monkey (Pseudomonas simiae), pseudomonas longde (Pseudomonas ludensis), pseudomonas grass (Pseudomonas poae), pseudomonas syringae (Pseudomonas syringae); 14-23 are Bacillus licheniformis (Bacillus licheniformis), bacillus subtilis (Bacillus subtilis), bacillus amyloliquefaciens (Bacillus amyloliquefaciens), flavobacterium (Microbacterium maritypicum), escherichia coli (Escherichia coli), lactobacillus fermentum (Lactobacillus fermentum), enterococcus faecalis (Enterococcus faecalis), acinetobacter baumannii (Acinetobacter baumanii), staphylococcus aureus (Staphylococcus aureus), and Bacillus psychrophilus digested (Psychrobacter alimentarius), respectively.
FIG. 3 is the accuracy of primer design based on the mreB gene; the accuracy of the method is obtained by mixing different kinds of pseudomonas with representatives in different proportions according to the design, amplifying the primers according to the design, performing high-throughput sequencing, and finally comparing the sequencing result with the proportions of different pre-mixed pseudomonas.
FIG. 4 is a graph showing the level constitution of Pseudomonas species in milk samples in examples.
FIG. 5 is an electrophoresis detection chart of PCR amplified products in milk samples to be tested in examples, and 1 to 8 are different samples.
FIG. 6 is a dissolution profile of qPCR amplified sample genomes.
FIG. 7 is a general P.sp.phylogenetic tree based on the V3-V4 region of the 16S rRNA gene.
Detailed Description
Example 1: construction of Pseudomonas specific primers based on Pseudomonas high-resolution core genes
1. 142 pseudomonas species, which have been reported in the U.S. Biotechnology information center (National Center for Biotechnology Information, NCBI) and European molecular biology laboratory (European Molecular Biology Laboratory, EMBL) databases to have completed whole genome sequencing, were collected and summarized, and a total of 429 whole genome sequences (only one species of one strain was downloaded) were selected for each of their sequencing integrity, re-annotated with Prokka software, and flood genome analysis by Roary software. And respectively carrying out phylogenetic tree construction on the annotated genes by MEGA7 software, and screening to obtain a fragment with the capability of distinguishing different species on the mreB gene. As shown in FIG. 1, it is understood from the figure that in a circular evolutionary tree constructed from this gene, individual species can be separated into different clades.
2. And (3) constructing an mreB gene database, downloading the mreB gene sequences of known pseudomonas from NCBI, EMBL and other databases, and constructing an mreB gene comparison database by using the downloaded sequences. The mreB gene sequence database constructed was applicable to all pseudomonas bacteria known to date (table 1).
TABLE 1 knowledge of the sequence information of all Pseudomonas mreB genes
TABLE 2 mreB Gene (uploaded NCBI) based on the Prokka remarked result
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3. After fully analyzing the conserved sequence and the specific sequence of the mreB gene, selecting a specific sequence (taking the whole-segment mreB gene as a template (full length 1038 bp), selecting a segment with high resolution) to design primers, selecting a primer pair suitable for reading length of an Illumina Miseq sequencing platform from the primer pairs, and finally determining a forward primer shown in SEQ ID NO.1 and a reverse primer shown in SEQ ID NO.2 as final sequencing primers.
Pseudomonas specific primer sequences were designed and synthesized based on the mreB gene:
forward primer (SEQ ID No. 1): 5'-ACCCTTATTTACGTGCGCGA-3' the number of the individual pieces of the plastic,
reverse primer (SEQ ID NO. 2): 5'-ATRTCSACSACCATCGARC-3';
note that: r in the above primer represents bases A and G, and S represents bases G and C.
The Primer-blast (https:// www.ncbi.nlm.nih.gov /) is used for carrying out simulated PCR, and the result shows that the pair of primers only generate amplicons of the pseudomonas genome, which shows that the primers have better pseudomonas specificity, so that the primers designed by the invention ensure the efficiency of subsequent amplification and the accuracy of identification.
4. For the entire included pseudomonas genome database, the authenticity of the specific sequences was verified using BLAST method. After completion of BLAST verification, BLAST search of the target gene sequence based on the whole library of microbial gene information of NR/NT library in the american biological technology information center (National Center for Biotechnology Information, NCBI) database was further verified that the identified strain-specific sequence was present only in the target bacteria and was deleted in other published microbial gene information.
Example 2: verification of Pseudomonas specific primers at the genus level
More 13 species of Pseudomonas are reported in raw milk, namely Pseudomonas aeruginosa (Pseudomonas aeruginosa), pseudomonas fluorescens (Pseudomonas fluorescens), pseudomonas putida (Pseudomonas putida), pseudomonas solanacearum (Pseudomonas solanacearum), pseudomonas alcaligenes (Pseudomonas alcaligenes), pseudomonas putida (Pseudomonas oleovorans), pseudomonas fragilis (Pseudomonas fragi), pseudomonas proteus (Pseudomonas plecoglossicida), pseudomonas cold-resistant (Pseudomonas psychrotolerans), pseudomonas monkey (Pseudomonas simiae), pseudomonas longde (Pseudomonas ludensis), pseudomonas poae (Pseudomonas poae), pseudomonas syringae (Pseudomonas syringae), and bacillus licheniformis (Bacillus licheniformis) which are common pollutants of 10 species of non-Pseudomonas raw milk, bacillus subtilis (Bacillus subtilis), bacillus amyloliquefaciens (Bacillus amyloliquefaciens), xanthobacter (Microbacterium maritypicum), escherichia coli (Escherichia coli), lactobacillus fermentum (Lactobacillus fermentum), enterococcus faecalis (Enterococcus faecalis), acinetobacter baumannii (Acinetobacter baumanii), staphylococcus aureus (Staphylococcus aureus) and psychrophilic digestion bacteria (Psychrobacter alimentarius).
Extracting genome DNA of the strain respectively, extracting genome of microorganism, referring to the instruction in a bacterial genome DNA extraction kit (Tiangen Biochemical technology (Beijing) limited company, beijing, china), and carrying out PCR amplification by using the extracted genome as a template by using primers with sequences shown as SEQ ID NO.1 and SEQ ID NO.2, wherein the amplification conditions are as follows:
(1) the PCR amplification reaction system comprises the following components: 1. Mu.L of genomic DNA template, 25. Mu.L of 2×taq mix (TaKaRa), 10. Mu.M forward and reverse primers, and ddH were added 2 O to 50. Mu.L.
(2) The PCR amplification reaction conditions were: pre-denaturation at 95℃for 8min, then denaturation at 95℃for 40s, annealing at 59℃for 40s, extension at 72℃for 40s as one cycle, and 34 cycles were performed, followed by extension at 72℃for 8min.
As shown in FIG. 3, the PCR results showed that Bacillus licheniformis (Bacillus licheniformis), bacillus subtilis (Bacillus subtilis), bacillus amyloliquefaciens (Bacillus amyloliquefaciens), flavobacterium (Microbacterium maritypicum), escherichia coli (Escherichia coli), lactobacillus fermentum (Lactobacillus fermentum), enterococcus faecalis (Enterococcus faecalis), acinetobacter baumannii (Acinetobacter baumanii), staphylococcus aureus (Staphylococcus aureus), and Bacillus stearothermophilus (Psychrobacter alimentarius) could not amplify the target bands, pseudomonas aeruginosa (Pseudomonas aeruginosa), pseudomonas fluorescens (Pseudomonas fluorescens), pseudomonas putida (Pseudomonas putida), pseudomonas solanacearum (Pseudomonas solanacearum), pseudomonas alcaligenes (Pseudomonas alcaligenes), pseudomonas fragi (Pseudomonas oleovorans), pseudomonas fragi (Pseudomonas fragi), pseudomonas fragi (Pseudomonas plecoglossicida), pseudomonas psychrophila (Pseudomonas psychrotolerans), pseudomonas monkey (Pseudomonas simiae), pseudomonas longde (Pseudomonas ludensis), pseudomonas Pseudomonas (Pseudomonas poae), and Pseudomonas syringae (Pseudomonas syringae) were amplified to obtain the target bands having a size of 410 to 462bp.
The PCR product was gel recovered (gel recovery kit was purchased from Tiangen Biotechnology Co., ltd.), sequenced, quantified using a Varioskan LUX multifunctional microplate reader, mixed in equal amounts, constructed with a IlluminaTruSeq DNA LT Sample Preparation Kit kit, sequenced on-line on an Illumina Miseq sequencing platform, and compared with the gene library constructed in example 1, and the genus Pseudomonas in the sample to be tested was detected.
The comparison result is verified to be consistent with the information of the initially selected strain species classification. The method can effectively distinguish the bacterial strains of the pseudomonas from the bacterial strains of other genera, and the comparison result after sequencing can identify the pseudomonas by 100 percent.
Meanwhile, the sample genome is amplified by a qPCR method, and a single dissolution curve proves that the specificity of the primer and the phenomenon that the primer does not have mismatch, primer dimer and the like and influence the amplification result occur, as shown in figure 6.
Example 3: construction of Pseudomonas rapid detection method and verification of Pseudomonas rapid detection method on species level
To verify the accuracy of primers SEQ ID No.1 and SEQ ID No.2 in analyzing Pseudomonas, 7 kinds of Pseudomonas aeruginosa (Pseudomonas aeruginosa), pseudomonas fluorescens (Pseudomonas fluorescens), pseudomonas putida (Pseudomonas putida), pseudomonas fragi (Pseudomonas fragi), pseudomonas proteida (Pseudomonas plecoglossicida), pseudomonas syringae (Pseudomonas syringae) and Pseudomonas monkey (Pseudomonas simiae) genomes were mixed according to the concentration of 0.003-25 ng/. Mu.L for simulating the detection of different kinds of Pseudomonas in complex samples. Based on the newly designed Pseudomonas identification primers and in combination with a second generation sequencer, then simulated sample sequencing was performed according to the method steps described in example 2, and finally the mreB gene sequence information obtained by sequencing was compared with the constructed database (constructed in example 1). As the concentration of the DNA of each strain is detected before mixing, specifically shown in Table 3, the DNA content of each strain is determined, and the DNA is detected by a Pseudomonas detection method, the quantity of each strain which can be read by the DNA concentration of each strain theoretically has a certain correlation with the DNA concentration in the corresponding mixed sample. In addition, since the DNA concentration of the strain is measured, the final comparison result is consistent with the strain with the corresponding concentration, and thus the accuracy of the detection method can be detected in the step.
TABLE 3 construction of the Rapid detection methods for Pseudomonas and verification thereof at the seed level
Comparing the content of different species of Pseudomonas obtained according to the high throughput sequencing method with the content of pre-mixed Pseudomonas in the simulated sample to obtain the results as shown in FIG. 2, it can be seen from FIG. 2 that the content of Pseudomonas species sequenced using primers SEQ ID NO.1 and SEQ ID NO.2 has the same content as the pre-mixed Pseudomonas speciesGood consistency (y= 1.0076x-0.1012, r 2 =0.9974), the high throughput sequencing method is therefore highly accurate in detecting pseudomonas species.
Therefore, the method for detecting different species of pseudomonas based on high-throughput sequencing is suitable for comparing mreB gene sequence information of complex samples with mreB gene sequences of known species in a constructed database, so as to obtain different species of pseudomonas in the complex samples.
Example 4: application of pseudomonas rapid detection method in raw milk sample
For the specific embodiment, see example 2, the sample is genomic DNA extracted from raw milk obtained from a milk factory, and the extraction is performed by using a DNA extraction kit from MPbiomedicals company of America, and specific operation steps are performed according to the operation instructions of the kit.
Comparing the sequence information of the mreB gene obtained by sequencing with the constructed database (constructed in example 1), the detection result is shown in figure 4, and figure 4 shows the composition structure of pseudomonas in a sample. Based on the sequence (assigned) of the existing library aligned to more than 99%, the fluorescent pseudomonas has obvious abundance advantage, the abundance of the grass pseudomonas, the longde pseudomonas, the copper pseudomonas and the malodor pseudomonas is sequentially reduced, and the other 16 kinds of the fluorescent pseudomonas are detected to exist in a small amount, which accords with the literature report. The data show that the method can rapidly and accurately detect the pseudomonas composition in the raw milk.
Comparative example 1: accuracy verification of 16S rRNA V3-V4 for Pseudomonas identification
The 16S rRNA is used as a marker to construct a phylogenetic tree of common pseudomonas in cow milk (the 16S rRNA gene sequences of different strains of the pseudomonas are downloaded by using NCBI and EMBL databases respectively, and the phylogenetic tree is constructed by using a neighbor-joining distance algorithm through MEGA software). As can be seen from FIG. 7, the classification result of the V3-V4 region identification of the 16Sr RNA gene is not accurate enough to distinguish some species, such as Pseudomonas taiwanensis and Pseudomonas veronii.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of Jiangnan
<120> Pseudomonas horizontal rapid detection method based on high-throughput sequencing and application thereof
<130> BAA211085A
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<170> PatentIn version 3.3
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Claims (8)
1. A method for identifying pseudomonas, which is not used for diagnosing diseases, and is characterized in that mreB gene is used as a marker, sequences shown as SEQ ID NO.1 and SEQ ID NO.2 are used as primers, and a strain for amplifying to obtain marker gene fragments is pseudomonas; the gene fragment size of the marker is 410-462 bp.
2. The method of claim 1, wherein the amplification is performed using a sample containing the genome of the microorganism as a template.
3. The method of claim 2, wherein the sample comprises genomic DNA, a bacterial suspension, or a single colony.
4. The method of claim 2, wherein the amplification is performed using genomic DNA comprising the mixture to be tested as a template.
5. A method for identifying a pseudomonas horizontal composition in a sample, said method not being useful for diagnosis of a disease, said method comprising: (1) constructing a pseudomonas mreB gene database; (2) Amplifying the mreB gene sequence in the genome of the sample by using the sequences shown in SEQ ID NO.1 and SEQ ID NO.2 as primers; (3) recovering and sequencing the amplification result of the step (2); (4) Comparing the sequencing result with the pseudomonas mreB gene database constructed in the step (1), and identifying and analyzing the composition characteristics of the pseudomonas strain level in the sample; the pseudomonas mreB gene standard database contains a pseudomonas mreB sequence which can be searched in a Genbank database.
6. A kit for identifying pseudomonas species, which is characterized by comprising a primer pair shown as SEQ ID NO.1 and SEQ ID NO. 2.
7. A method for screening pseudomonas is characterized in that genomic DNA is extracted from a sample to be screened, sequences shown in SEQ ID NO.1 and SEQ ID NO.2 are used as primers, a bacterial solution is utilized to amplify the sequence of mreB gene, and a bacterial strain with a 410-462 bp gene fragment is obtained by amplification;
or preparing a sample to be screened into bacterial suspension, diluting the bacterial suspension, coating the bacterial suspension on a solid culture medium, culturing the bacterial suspension to form single colonies, shaking the single colonies to obtain bacterial liquid, and amplifying the sequence of the mreB gene by using the bacterial liquid with the sequences shown as SEQ ID NO.1 and SEQ ID NO.2 as primers to obtain the bacterial strain with the gene fragment of 410-462 bp as pseudomonas.
8. The method of claim 7, wherein the medium comprises, but is not limited to, CFC selective medium or NB nutrient broth.
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| CN107653306A (en) * | 2017-11-13 | 2018-02-02 | 江南大学 | A kind of Bifidobacterium quick determination method and application based on high-flux sequence |
| WO2020069397A1 (en) * | 2018-09-27 | 2020-04-02 | Cortexyme, Inc. | Methods for detection of microbial nucleic acids in body fluids |
| CN111534622A (en) * | 2020-05-19 | 2020-08-14 | 江南大学 | Bacteroides rapid detection method based on high-throughput sequencing and application |
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| CN107653306A (en) * | 2017-11-13 | 2018-02-02 | 江南大学 | A kind of Bifidobacterium quick determination method and application based on high-flux sequence |
| WO2020069397A1 (en) * | 2018-09-27 | 2020-04-02 | Cortexyme, Inc. | Methods for detection of microbial nucleic acids in body fluids |
| CN111534622A (en) * | 2020-05-19 | 2020-08-14 | 江南大学 | Bacteroides rapid detection method based on high-throughput sequencing and application |
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