CN113234839A - Drying-resistant genotyping method for cronobacter sakazakii - Google Patents
Drying-resistant genotyping method for cronobacter sakazakii Download PDFInfo
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Abstract
The invention discloses a method for typing a desiccation-resistant gene of cronobacter sakazakii, which comprises the following steps: (1) acquiring full-length deoxyribonucleic acid sequences of 18 drying-resistant characteristic related genes of cronobacter sakazakii to be typed; (2) combining the 18 drying-resistant gene sequences in different cronobacters sakazakii, and then carrying out typing and phylogenetic tree construction so as to research the genetic relationship and evolutionary relationship of different cronobacters sakazakii. According to the method, the food-borne pathogenic microorganisms and the classification markers associated with the hazard are obtained by setting a reasonable threshold, so that the problems of insufficient MLST resolution and low cgMLST analysis speed are solved, and a pathogenic strain classification technical method giving consideration to both resolution and speed is established. The method can be applied to basic research and clinical research, and can perform accurate and effective typing traceability tracking on the outbreak of Cronobacter sakazakii.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a desiccation-resistant genotyping method for cronobacter sakazakii.
Background
The main genetic material of an organism is DNA, and the genome is a general term for the complete genetic material in a cell or an organism and represents the complete characteristic information and genetic information of the organism. Currently, the research on organisms is increasingly turning to the research on the characteristics of the entire organism genome, the structural characteristics and genetic similarity of each functional gene, and the like. Through the rapid development of the last thirty years, the whole genome sequencing technology is more and more mature. The whole genome sequence of an organism can be sequenced to obtain all information of the organism, and the research of the whole formula can further promote the secret knowledge of human life.
For food-borne pathogenic bacteria at present: on one hand, most databases have the problems of low data volume of food-borne pathogenic bacteria, insufficient annotation, low harmfulness correlation degree between the current microbial polymorphism analysis technology and strains, non-uniform polymorphism analysis technology standard based on genome, overhigh redundant information and the like; on the other hand, the related typing traceability technology of food-borne pathogenic microorganisms based on functional genes is less researched. Typing technical research and development based on the functional genes are additionally carried out, and finally a food-borne pathogenic microorganism pollution traceability network platform is formed, so that real-time seamless butt joint with a food safety risk monitoring network covering China and constructed by a national food safety supervision organization can be realized, and the food safety risk monitoring network can also be butt joint with an international food safety detection traceability network, thereby providing informatization services for prevention, diagnosis and traceability of food-borne pathogenic microorganisms from China to China.
In recent years, cronobacter sakazakii has received increasing attention from researchers, and research on conditions and mechanisms for forming drying-resistant characteristics of cronobacter sakazakii will play an important role in preventing cronobacter sakazakii contamination. In recent years, common phenotyping and molecular typing methods including biochemical typing, matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS), serotype typing, pulsed field gel electrophoresis typing, multi-site sequence typing, random polymorphism amplification DNA genotyping, ribosome typing, special genotyping, and the like have been used in many cases for the typing of cronobacter sakazakii. The method has the advantages that the pathogenicity and stress resistance related genes of pathogenic bacteria, core genomes and whole genomes are not deeply researched, and the finding of more polymorphic markers has important significance for more accurate typing traceability of the cronobacter sakazakii.
The Multiple Locus Sequence Typing (MLST) technique is a bacterial cloning relationship identification method based on determination of transplantable nucleic acid sequences proposed in 1998. Peng Fei performed evolution and retrospective analysis of cronobacter sakazakii and cronobacter proprionate isolates from one infant formula manufacturing plant in china by MLST, Pulsed Field Gel Electrophoresis (PFGE), PCR-based O-antigen serotyping, and ompA and rpoB sequence analysis, and cronobacter sakazakii ST268 in the study differed from the database ST4 only at one site and thus was located within cronobacter sakazakii CC 4. The cronobacter propionate ST201 is located in CC7 because it differs from the database ST7 type by only one site. Thus, not only can MLSTs be identified and genotyped, but they also reflect the potential clinical significance of neonatal infection, and enable accurate source tracking and attribution,
meanwhile, the MLST typing traceability technology is also shown to have advantages in the aspect of evolution analysis of pathogenic bacteria, and the initial infection source of the strain can be judged.
However, the bacterial strain MLST typing method based on housekeeping genes usually selects about 7 genes, and although the MLST allele online database is continuously updated, the method is not suitable for a large amount of strain typing analysis due to low resolution and high cost.
The whole Genome multi-locus Sequence Typing is an expanding method of MLST, and is different from MLST which only selects a plurality of conserved genes, and the cgMLST takes a core Genome based on a large number of strains as a Sequence Typing marker. Sarah performed a subtype analysis of cronobacter sakazakii isolates in 77 human subjects in 11 countries and regions of europe by Whole Genome Sequencing (WGS), determined antibiotic resistance and performed a traceability analysis. The cgMLST-based typing method (in which the defined cgMLST gene set consists of 2,831 core genes in total and 1,017 helper target genes) showed high strain diversity, indicating that a cross-country outbreak of the strain did not occur in 2017; and 4 previously unreported historical disease outbreaks were determined based on a traceability analysis of cgMLST. Accurate identification and classification of the outbreak strains using cgMLST is therefore proposed, which is also of great advantage, especially in the context of the analysis of the source of outbreak strains. However, the cgMLST involves a large number of core genes to be analyzed, is highly dependent on whole genome sequencing, and has certain limitation in practical application.
Through searching, no patent publication related to the present patent application has been found.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a desiccation-resistant genotyping method for cronobacter sakazakii.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a desiccation-resistant genotyping method of Cronobacter sakazakii comprises the following steps:
(1) obtaining 18 drying-resistant gene sequences of cronobacter sakazakii
Acquiring full-length deoxyribonucleic acid sequences of 18 drying-resistant characteristic related genes of cronobacter sakazakii to be typed;
(2) desiccation-tolerant genotyping of cronobacter sakazakii
Combining the 18 drying-resistant gene sequences in different cronobacters sakazakii, and then carrying out typing and phylogenetic tree construction so as to research the genetic relationship and evolutionary relationship of different cronobacters sakazakii.
Further, the cronobacter sakazakii in the step (1) includes milk powder, environment, and a clinically isolated cronobacter sakazakii strain.
Further, the 18 cronobacter sakazakii desiccation resistance characteristic genes and corresponding proteins in the step (1) and the NCBI GenBank sequence are as follows: dnaK (AKE93222.1), kefC (AKE93189.1), rpoN (AKE93535.1), rpoS (AKE94754.1), dnaJ (AKE93221.1), grpE (AKE94818.1), proP (AKE93224.1), proV (AKE94787.1), osmY (AKE93274.1), ompW (AKE95806.1), yciT (AKE95832.1), otsA (AKE95599.1), otsB (AKE95598.1), kefB (AKE94234.1), kefG (AKE94233.1), treF (AKE96061.1), opuCA (AKE96003.1), and opuCC (AKE 96005.1).
Further, in the method for obtaining gene sequences based on whole genome sequence alignment in step (1), the BLAST program in NCBI is used; evalue is set to be 1e-3, the result sequence of the comparison pair with the highest Identites value is selected, and the Identites value is not less than 95%.
Further, in the step (1), the full-length deoxyribonucleic acid sequences of the 18 genes related to the drying-resistant property of the cronobacter sakazakii to be typed are obtained by a single-gene PCR amplification method or a whole-genome sequence alignment-based method.
Furthermore, the sequence of the 18 genes with drying resistance characteristics in the step (2) is not required, but when the target strain is typed, the sequence of the genes with drying resistance among strains is strictly unified.
Further, the alignment of the corresponding sequences is performed in the step (2) by a multiple sequence alignment program; and (3) constructing a phylogenetic tree through phylogenetic tree software: performing detection by using a Bootstrap method, wherein the detection times are 1000 times; the Substitution Model uses the kimura 2-parameter Model; Gaps/Missing Data Treatment selects Partial delay with a Sit e Coverage Cutoff value of 95%.
Further, the multiple sequence alignment program is ClustalW and Muscle; the phylogenetic tree software is MEGAX software.
The invention has the advantages and positive effects that:
1. compared with the MLST and cgMLST typing methods, the method obtains the food-borne pathogenic microorganism and the typing marker associated with the hazard by setting a reasonable threshold, solves the problems of insufficient MLST resolution and slow cgMLST analysis speed, and establishes the pathogenic strain typing technical method giving consideration to both resolution and speed. The method establishes a new typing method by utilizing the characteristic functional genes of the cronobacter sakazakii, can be applied to basic research and clinical research, and can carry out accurate and effective typing tracing on the outbreak of the cronobacter sakazakii.
2. Aiming at the problems of low harmfulness correlation degree, non-uniform polymorphism analysis technical standards, overhigh redundant information and the like of the current microbial polymorphism analysis technology and bacterial strains, the invention can open a genome database (such as an Assembly database, an EnsemblBacteria database, a GigaDB database and the like of NCBI) as reference, and establish a rapid pathogenic bacterial strain typing technical method with high resolution by selecting a proper genome data set and setting reasonable parameters to obtain a typing marker of the food-borne pathogenic microorganisms.
3. The pollution of cronobacter sakazakii in the milk powder is a great important reason for endangering the life safety of newborns and infants. The applicant carries out separation and identification on the cronobacter sakazakii in domestic milk powder and imported milk powder, so that the pollution condition of the cronobacter sakazakii in the current domestic and foreign markets can be known, and the source and the propagation of the strain can be analyzed, so that the purposes of controlling the pollution source and blocking the propagation path are achieved.
4. Cronobacter sakazakii is a gram-negative bacillus-free bacterium, an opportunistic pathogen, and has been extensively studied because of its unique tolerance in dry environments. The bacteria are more present in the environment and in some foods, especially milk powder, and the life safety of infants can be endangered after the milk powder is polluted by the bacteria. The drying-resistant property of cronobacter sakazakii is related to the drying-resistant gene regulation, so that the strain typing technology can be developed according to the drying-resistant gene regulation. The genetic relationship and the evolutionary relationship among the strains can be analyzed through the difference of the sequences of the drying-resistant genes, and then the strains are accurately tracked and analyzed.
5. The conventional MLST typing method usually selects about 7 genes, has low resolution and high cost, and is not suitable for large-scale strain typing analysis. The deficiency of the MLST typing method can be compensated to a certain extent by the method of desiccation-resistant genotyping of Cronobacter sakazakii. Taking 22 strains of cronobacter sakazakii sequenced in the laboratory as an example, 18 desiccation-tolerant genotyping phylogenetic trees (fig. 1) were better classified into the same branch for SAKA80220 and ENS6106 with closer relationships than MLST genotyping phylogenetic trees (fig. 2), and into the same branch for 11ES102, 11ES89, IQCC10419, 11ES107, and ENS70307-4 with closer relationships among the five strains. Therefore, in general, the method better classifies more strains with similar relatives and has relatively higher resolution.
In order to enrich the food-borne pathogenic microorganism typing traceability network in China, a method for dry-resistant genotyping of cronobacter sakazakii is established. The method is based on the drying-resistant characteristic of the cronobacter sakazakii, and a typing method established according to the sequence difference of the drying-resistant genes can be applied to the traceability research of the cronobacter sakazakii.
Drawings
FIG. 1 shows the 22 Cronobacter sakazakii 18 desiccation-tolerant genotyping phylogenetic trees of the present invention;
FIG. 2 shows a 22-strain MLST-typed phylogenetic tree of Cronobacter sakazakii in the present invention.
Detailed Description
The following detailed description of the embodiments of the present invention is provided for the purpose of illustration and not limitation, and should not be construed as limiting the scope of the invention.
The raw materials used in the invention are conventional commercial products unless otherwise specified; the methods used in the present invention are conventional in the art unless otherwise specified.
A desiccation-resistant genotyping method of Cronobacter sakazakii comprises the following steps:
(1) obtaining 18 drying-resistant gene sequences of cronobacter sakazakii
Acquiring full-length deoxyribonucleic acid sequences of 18 drying-resistant characteristic related genes of cronobacter sakazakii to be typed;
(2) desiccation-tolerant genotyping of cronobacter sakazakii
Combining the 18 drying-resistant gene sequences in different cronobacters sakazakii, and then carrying out typing and phylogenetic tree construction so as to research the genetic relationship and evolutionary relationship of different cronobacters sakazakii.
Preferably, the cronobacter sakazakii in the step (1) includes powdered milk, environment and a clinically isolated cronobacter sakazakii strain.
Preferably, the 18 cronobacter sakazakii desiccation resistance characteristic genes and corresponding proteins in the step (1) and the NCBI GenBank sequence are as follows: dnaK (AKE93222.1), kefC (AKE93189.1), rpoN (AKE93535.1), rpoS (AKE94754.1), dnaJ (AKE93221.1), grpE (AKE94818.1), proP (AKE93224.1), proV (AKE94787.1), osmY (AKE93274.1), ompW (AKE95806.1), yciT (AKE95832.1), otsA (AKE95599.1), otsB (AKE95598.1), kefB (AKE94234.1), kefG (AKE94233.1), treF (AKE96061.1), opuCA (AKE96003.1), and opuCC (AKE 96005.1).
Preferably, in the method for obtaining gene sequences based on whole genome sequence alignment in step (1), the BLAST program in NCBI is used; evalue is set to be 1e-3, the result sequence of the comparison pair with the highest Identites value is selected, and the Identites value is not less than 95%.
Preferably, in the step (1), the full-length deoxyribonucleic acid sequences of the 18 genes related to the drying-resistant property of the cronobacter sakazakii to be typed are obtained by a single-gene PCR amplification method or a whole-genome sequence alignment-based method.
Preferably, the sequence of the 18 genes with drying resistance characteristics in the step (2) is not required, but when the target strain is typed, the sequence of the genes with drying resistance among strains is strictly unified.
Preferably, the alignment of the corresponding sequences in step (2) is performed by a multiple sequence alignment program; and (3) constructing a phylogenetic tree through phylogenetic tree software: performing detection by using a Bootstrap method, wherein the detection times are 1000 times; substistation Mo del uses the kimura 2-parameter model; Gaps/Missing Data Treatment selects Partial delay, and the value of Site C overlap Cutoff is 95%.
Further, the multiple sequence alignment program is ClustalW and Muscle; the phylogenetic tree software is MEGAX software.
Specifically, the preparation and detection examples are as follows:
example 1
A desiccation-resistant genotyping method of Cronobacter sakazakii comprises the following steps:
(1) obtaining 18 drying-resistant gene sequences of cronobacter sakazakii
Acquiring full-length deoxyribonucleic acid sequences of 18 drying-resistant characteristic related genes of cronobacter sakazakii to be typed by a single gene PCR amplification method or a whole genome sequence alignment and other methods;
(2) desiccation-tolerant genotyping of cronobacter sakazakii
Combining the 18 drying-resistant gene sequences in different cronobacters sakazakii, and then carrying out typing and phylogenetic tree construction so as to research the genetic relationship and evolutionary relationship of different cronobacters sakazakii.
Furthermore, the cronobacter sakazakii in the step (1) includes a cronobacter sakazakii strain isolated from milk powder, environment, clinic and the like.
Furthermore, the 18 cronobacter sakazakii desiccation resistance characteristic genes and corresponding proteins in the step (1) and the NCBI GenBank sequence are as follows: dnaK (AKE93222.1), kefC (AKE93189.1), rpoN (AKE93535.1), rpoS (AKE94754.1), dnaJ (AKE93221.1), grpE (AKE94818.1), proP (AKE93224.1), proV (AKE94787.1), osmY (AKE93274.1), ompW (AKE95806.1), yciT (AKE95832.1), otsA (AKE95599.1), otsB (AKE95598.1), kefB (AKE94234.1), kefG (AKE94233.1), treF (AKE96061.1), opuCA (AKE96003.1), and opuCC (AKE 96005.1).
Moreover, in the method for obtaining a gene sequence based on whole genome sequence alignment in the step (1), the BLAST program in NCBI is used; evalue is set to be 1e-3, the result sequence of the comparison pair with the highest Identites value is selected, and the Identites value is not less than 95%.
In the step (2), the order of the 18 genes having the drying-resistant property is not required, but the order of the genes having the drying-resistant property is strictly uniform among strains when the target strain is typed.
Moreover, the alignment of the corresponding sequences in step (2) is performed by a multiple sequence alignment program, including but not limited to ClustalW, Muscle; the phylogenetic tree was constructed by MEGAX software: performing detection by using a Bootstrap method, wherein the detection times are 1000 times; the Substitution Model uses the kimura 2-parameter Model; Gaps/Missing Data Treatment selects Partial delay, and the value of Site Coverage Cutoff is 95%.
Example 2
A desiccation-resistant genotyping method of Cronobacter sakazakii comprises the following steps:
(1) obtaining 17 drying-resistant gene sequences of cronobacter sakazakii
Acquiring full-length deoxyribonucleic acid sequences of 17 drying-resistant characteristic related genes of cronobacter sakazakii to be typed by a single gene PCR amplification method or a whole genome sequence alignment and other methods;
(2) desiccation-tolerant genotyping of cronobacter sakazakii
Combining the 17 drying-resistant gene sequences in different cronobacters sakazakii, and then carrying out typing and phylogenetic tree construction so as to research the genetic relationship and evolutionary relationship of different cronobacters sakazakii.
Furthermore, the cronobacter sakazakii in the step (1) includes a cronobacter sakazakii strain isolated from milk powder, environment, clinic and the like.
Furthermore, the 17 cronobacter sakazakii desiccation resistance characteristic genes and the corresponding proteins in the step (1) and the NCBI GenBank sequence are as follows: kefC (AKE93189.1), rpoN (AKE93535.1), rpoS (AKE94754.1), dnaJ (AKE93221.1), grpE (AKE94818.1), proP (AKE93224.1), proV (AKE94787.1), osmY (AKE93274.1), ompW (AKE95806.1), yceT (AKE95832.1), otsA (AKE95599.1), otsB (AKE95598.1), kefB (AKE94234.1), kefG (AKE94233.1), treF (AKE96061.1), opuCA (AKE96003.1), and opuCC (AKE 96005.1).
Moreover, in the method for obtaining a gene sequence based on whole genome sequence alignment in the step (1), the BLAST program in NCBI is used; evalue is set to be 1e-3, the result sequence of the comparison pair with the highest Identites value is selected, and the Identites value is not less than 95%.
In the step (2), the order of the 17 genes having the drying-resistant property is not required, but the order of the genes having the drying-resistant property is strictly uniform among strains when the target strain is typed.
Moreover, the alignment of the corresponding sequences in step (2) is performed by a multiple sequence alignment program, including but not limited to ClustalW, Muscle; and (3) constructing a phylogenetic tree through phylogenetic tree software: performing detection by using a Bootstrap method, wherein the detection times are 1000 times; the Substitution Model uses the kimura 2-parameter Model; Gaps/Missing Data Treatment selects Partial delay, and the value of Site Coverage Cutoff is 95%.
Example 3
A desiccation-resistant genotyping method of Cronobacter sakazakii comprises the following steps:
(1) obtaining 18 drying-resistant gene sequences of cronobacter sakazakii
Acquiring full-length deoxyribonucleic acid sequences of 18 drying-resistant characteristic related genes of cronobacter sakazakii to be typed by a single gene PCR amplification method or a whole genome sequence alignment and other methods;
(2) desiccation-tolerant genotyping of cronobacter sakazakii
Combining the 18 drying-resistant gene sequences in different cronobacters sakazakii, and then carrying out typing and phylogenetic tree construction so as to research the genetic relationship and evolutionary relationship of different cronobacters sakazakii.
Furthermore, the cronobacter sakazakii in the step (1) includes a cronobacter sakazakii strain isolated from milk powder, environment, clinic and the like.
Furthermore, the 18 cronobacter sakazakii desiccation resistance characteristic genes and corresponding proteins in the step (1) and the NCBI GenBank sequence are as follows: dnaK (AKE93222.1), kefC (AKE93189.1), rpoN (AKE93535.1), rpoS (AKE94754.1), dnaJ (AKE93221.1), grpE (AKE94818.1), proP (AKE93224.1), proV (AKE94787.1), osmY (AKE93274.1), ompW (AKE95806.1), yciT (AKE95832.1), otsA (AKE95599.1), otsB (AKE95598.1), kefB (AKE94234.1), kefG (AKE94233.1), treF (AKE96061.1), opuCA (AKE96003.1), and opuCC (AKE 96005.1).
Moreover, in the method for obtaining a gene sequence based on whole genome sequence alignment in the step (1), the BLAST program in NCBI is used; evalue is set to be 1e-5, the result sequence of the comparison pair with the highest Identites value is selected, and the Identites value is not less than 95%.
In the step (2), the order of the 18 genes having the drying-resistant property is not required, but the order of the genes having the drying-resistant property is strictly uniform among strains when the target strain is typed.
Moreover, the alignment of the corresponding sequences in step (2) is performed by a multiple sequence alignment program, including but not limited to ClustalW, Muscle; phylogenetic trees were constructed by MEGA7 software: performing detection by using a Bootstrap method, wherein the detection times are 1000 times; the Substitution Model uses the kimura 2-parameter Model; Gaps/Missing Data Treatment selects Partial delay, with a Site Coverage Cutoff value of 90%.
Although the embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the embodiments disclosed.
Claims (8)
1. A desiccation-tolerant genotyping method for Cronobacter sakazakii is characterized by comprising the following steps: the method comprises the following steps:
(1) obtaining 18 drying-resistant gene sequences of cronobacter sakazakii
Acquiring full-length deoxyribonucleic acid sequences of 18 drying-resistant characteristic related genes of cronobacter sakazakii to be typed;
(2) desiccation-tolerant genotyping of cronobacter sakazakii
Combining the 18 drying-resistant gene sequences in different cronobacters sakazakii, and then carrying out typing and phylogenetic tree construction so as to research the genetic relationship and evolutionary relationship of different cronobacters sakazakii.
2. The method of dry-tolerant genotyping of cronobacter sakazakii according to claim 1, wherein: the cronobacter sakazakii in the step (1) comprises milk powder, environment and a clinically isolated cronobacter sakazakii strain.
3. The method of dry-tolerant genotyping of cronobacter sakazakii according to claim 1, wherein: the 18 cronobacter sakazakii desiccation-resistant characteristic genes in the step (1), the corresponding proteins and the NCBI GenBank sequence are as follows: dnaK (AKE93222.1), kefC (AKE93189.1), rpoN (AKE93535.1), rpoS (AKE94754.1), dnaJ (AKE93221.1), grpE (AKE94818.1), proP (AKE93224.1), proV (AKE94787.1), osmY (AKE93274.1), ompW (AKE95806.1), yciT (AKE95832.1), otsA (AKE95599.1), otsB (AKE95598.1), kefB (AKE94234.1), kefG (AKE94233.1), treF (AKE96061.1), opuCA (AKE96003.1), and opuCC (AKE 96005.1).
4. The method of dry-tolerant genotyping of cronobacter sakazakii according to claim 1, wherein: in the method for obtaining gene sequences based on whole genome sequence alignment in the step (1), a BLAST program in NCBI is used; evalue is set to be 1e-3, the result sequence of the comparison pair with the highest Identites value is selected, and the Identites value is not less than 95%.
5. The method of dry-tolerant genotyping of cronobacter sakazakii according to claim 1, wherein: in the step (1), the full-length deoxyribonucleic acid sequences of 18 genes related to the drying-resistant property of the cronobacter sakazakii to be typed are obtained by a single-gene PCR amplification method or a whole-genome sequence alignment method.
6. The method of dry-tolerant genotyping of cronobacter sakazakii according to claim 1, wherein: the sequence of the 18 genes with the drying resistance characteristics in the step (2) is not required, but when the target strains are typed, the sequence of the genes with the drying resistance among the strains is strictly unified.
7. The method for dry-tolerant genotyping of cronobacter sakazakii according to any one of claims 1 to 6, wherein: aligning corresponding sequences in the step (2) through a multi-sequence alignment program; and (3) constructing a phylogenetic tree through phylogenetic tree software: performing detection by using a Bootstrap method, wherein the detection times are 1000 times; the Substistation Model uses the kimur a 2-parameter Model; Gaps/Missing Data Treatment selects Partial delay, and the value of Site Coverage Cutoff is 95%.
8. The method of dry-tolerant genotyping of cronobacter sakazakii of claim 7, wherein: the multiple sequence alignment programs are ClustalW and Muscle; the phylogenetic tree software is MEGAX software.
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| CN113512554A (en) * | 2021-07-09 | 2021-10-19 | 合肥工业大学 | Protein for regulating sakazakii high-pressure stress resistance, encoding gene and application thereof |
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| CN105950744A (en) * | 2016-06-01 | 2016-09-21 | 东北农业大学 | Tracing method of cronobacter spp. in infant formula milk powder |
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| CN113512554A (en) * | 2021-07-09 | 2021-10-19 | 合肥工业大学 | Protein for regulating sakazakii high-pressure stress resistance, encoding gene and application thereof |
| CN113512554B (en) * | 2021-07-09 | 2022-07-12 | 合肥工业大学 | Protein for regulating sakazakii cronobacter sakazakii pressure-resistant strong stress, encoding gene thereof and application thereof |
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