CN102703592A - Methods for acquiring gap gene sequence of Staphylococcus chromogenes and primers of gap gene sequence - Google Patents

Abstract

The present invention relates to a gene sequence, in particular to a method for obtaining the gap gene sequence of Staphylococcus chromogenes and its primers. It adopts the method of combining plate streaking and dilution coating to treat freshwater fish intestinal bacteria in mannitol high-salt Separation and screening were carried out on agar (MSA) medium, and a strain was obtained. After culturing in a special medium, systematic research was carried out on morphological observation, physiological and biochemical characteristics, and molecular identification of gene sequences, and it was determined that the strain was Staphylococcus chromogenes; After extracting the genomic DNA of the strain, perform BLAST nucleotide sequence comparison based on the conserved sequence, and use PCR technology to amplify Staphylococcus chromogenes with reference to the specificity of the Staphylococcus conserved sequence designed to amplify the gap gene fragment The genome gap sequence, the screened pair of primers obtained the primers used to detect the PCR amplification fragment size of 931bp, the primer sequence is forward gap-F (5'-3'): ATGGTTTTGGTAGAATTGGTCGTTTA, reverse gap-R (5'-3'): GACATTTCGTTATCATACCAAGCTG.

Description

A method for obtaining the gap gene sequence of Staphylococcus chromogenes and its primers

technical field

The invention relates to a gene sequence, in particular to a method for obtaining the gap gene sequence of Staphylococcus chromogenes and a primer thereof, and belongs to the technical field of biological detection.

Background technique

Staphylococcus is widely distributed in nature and on the surface of human and animal skin and mucous membranes. It is the most common pyogenic coccus and often causes various suppurative infections, sepsis and urinary tract infections. It is one of the important pathogens of hospital infections and community-acquired infections. . The genus Staphylococcus contains at least 39 known species, 11 of which can be divided into two or more subspecies, and more than 50 species have been discovered so far. Bacteria of the genus Staphylococcus are a type of spherical bacteria that are Gram-positive. There is no report about Staphylococcus causing fish diseases, and many reports have found that Staphylococcus bacteria are the dominant flora of some fish intestinal bacteria. Zhao Qingxin found that Staphylococcus bacteria existed in the intestinal flora of bream, and some studies found that the dominant flora in the gut of grass carp was Staphylococcus bacteria; Sun Yunzhang et al. The normal flora in the intestinal tract of fish is an important part of the intestinal tract, which is not pathogenic to fish itself, but may be pathogenic to other organisms.

Due to the extremely high similarity (90%-99%) of staphylococci, 16S rDNA cannot be used to accurately identify them at the species level. determination. The staphylococcal gap gene encodes glyceraldehyde-3-phosphate dehydrogenase (glyceraldehyde-3-phosphate dehydrogenase), which is a housekeeping gene. Studies have shown that this gene can be detected in at least 12 strains of Staphylococcus subspecies. Yugueros et al. used the method of hybridization to detect all PCR-gap positive genes with a 279bp DNA fragment, and the results were all positive; the primers were used to amplify other bacterial species, and no target bands appeared in the results. Staphylococcus chromogenes is a kind of Gram-positive bacteria that does not move and does not produce spores. It exists widely in the environment and shows strong adaptability and heterogeneity. It is an opportunistic pathogen for humans and animals. Exudative dermatitis in pigs, septic polyarthritis and mastitis in cows. So far, there are no literature reports and invention patents at home and abroad for the rapid detection method of Staphylococcus chromogenes in freshwater fish and its aquatic products.

Contents of the invention

The purpose of the present invention is to provide a method for obtaining the sequence of the Staphylococcus chromogenes gap gene and its primers in order to quickly detect Staphylococcus chromogens in the intestinal tract of freshwater fish and its aquatic products, from the conserved sequence of the known sequence gene A method for detecting the gap gene by PCR using primers. The main content is as follows : adopt the method of plate streaking and dilution coating to isolate and screen the intestinal bacteria of freshwater fish on the mannitol high-salt agar (MSA) medium, obtain a strain, and carry out the test after culturing in a special medium. Morphological observation, physiological and biochemical characteristics, and gene sequence molecular identification and other systematic studies confirmed that the strain was Staphylococcus chromogenes; after extracting the genomic DNA of the strain through culture, BLAST nucleotide sequence comparison was carried out according to the conserved sequence, referring to To amplify the specificity of the gap gene fragment designed by the conservative sequence of Staphylococcus, use the primer design software primer5.0 to design primers; send the designed primers to the company for synthesis, and use PCR technology to amplify the genome gap of Staphylococcus chromogenes Sequence, the screened pair of primers obtained the primers used to detect the PCR amplification fragment size of 931bp, the primer sequence is forward gap-F (5'-3'): ATGGTTTTGGTAGAATTGGTCGTTTA, reverse gap-R (5'-3'): GACATTTCGTTATCATACCAAGCTG.

The present invention is realized by the following technical scheme: a method for obtaining the gap gene sequence of Staphylococcus chromogenes, which is characterized in that: the method of combining plate marking and dilution coating is used to treat freshwater fish intestinal bacteria in mannitol high-salt A strain was isolated and screened on agar (MSA) medium, and was confirmed to be Staphylococcus chromogenes through systematic studies such as morphological observation, physiological and biochemical characteristics, and gene sequence molecular identification.

A PCR-specific primer design method for the gap gene sequence of Staphylococcus chromogenes, which is characterized in that: through BLAST nucleotide sequence comparison, find the conserved sequence of the target gene that needs to be detected in this species; according to the conserved sequence, design for amplification To increase the specific primers for the gap gene fragments, use the primer design software primer5.0 to design primers; compare the primers with nucleotide sequences to ensure the high conservation of the 3' end of the primers; the specific steps are as follows:

(1) Screening of primers for Staphylococcus chromogenes: send the designed primers to Sangon Bioengineering (Shanghai) Co., Ltd. for synthesis, use PCR technology to amplify the genomic DNA of Staphylococcus chromogenes for the obtained primers, and detect each pair of primers Whether it is available, that is, whether a clear, single, and regular band can be amplified. Among the primers screened, the primer used to detect the PCR amplification fragment size of the bacterium is 931bp, and the gap gene encodes an amino acid sequence of 310aa. The primer sequence is forward gap-F (5'-3'): ATGGTTTTGGTAGAATTGGTCGTTTA, reverse gap-R (5'-3'): GACATTTCGTTATCATACCAAGCTG;

(2) Optimization of PCR amplification conditions for Staphylococcus chromogenes: PCR conditions were optimized for the gap gene-specific primers obtained from the isolated strains of this genus, and the best PCR amplification system and amplification conditions were obtained as follows: the best PCR amplification The amplification system is 25 μl, including 2.5 μl 10×Reaction Buffer (with Mg2+), 0.5 μl dNTP (including dATP, dTTP, dCTP, dGTP, each 2.5 mM), forward and reverse primers (10 μM) each 0.5 μl, Taq enzyme 0.3 μl , the extracted bacterial genomic DNA template was 2.0 μl, and the total volume of 25 μl was made up with sterilized double distilled water ddH ₂ O 17.2 μl; the optimal PCR reaction conditions were: pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 30s, annealing at 52°C for 50s, Extend at 72°C for 45s, then extend again at 72°C for 10min after 30 cycles, and store the PCR product at 4°C for later use;

(3) Molecular sequencing identification of Staphylococcus chromogenes: 10g/L agarose gel electrophoresis was used to detect PCR products, the target band was excised, and the gel recovery kit was recovered. After the recovered products were cloned, positive clones were selected and sent to Shanghai Sangon for sequencing; After sequencing, the BLAST and ClustalX programs were used for sequence comparison analysis, and the Neighbor-joining method was used to construct a phylogenetic tree, which showed that the strain was 98.82% homologous to the type strain (CCM 3387) of Staphylococcus chromogenes , and thus identified The bacterium is Staphylococcus chromogenes.

The present invention screens out special primers for Staphylococcus genus through PCR amplification technology, and establishes a molecular detection technology suitable for detecting freshwater fish-derived Staphylococcus chromogenes, which has the following advantages:

1. The cost is low, and the experimental operation is safe and reliable. The strain is isolated and screened from the intestinal bacteria of freshwater fish by means of plate streaking and dilution coating, and is obtained after culturing in a special medium, which can be carried out in general microbiological laboratories; when molecular identification of the strain is carried out, The general PCR method is adopted, using conventional reagents and instruments, and does not require probes and gene library construction, so isotope contamination is avoided.

2. The technology is simple and feasible. The present invention makes full use of the existing information provided by bioinformatics, and finds the conserved sequence of the target gene that needs to be detected in this species through BLAST nucleotide sequence comparison; according to the conserved sequence, it is designed to amplify the specificity of the gap gene fragment For primers, primers were designed with the primer design software primer5.0; the primers were compared with the nucleotide sequence to ensure the high conservation of the 3' end of the primers.

3. Fast, accurate and reliable. Compared with the traditional detection method, large-scale sequencing with the genome is avoided, which saves a lot of time and financial resources. As long as the conserved sequence of the gene is found, the gene of any species can be detected by cross-species PCR; at the same time, the primers of the present invention are specific Strong performance, which greatly enhances the specificity and efficiency of amplification.

Description of drawings

The present invention will be described in further detail below in conjunction with the accompanying drawings and embodiments.

Fig. 1 is the electrophoresis picture of the PCR product of the gap gene of the present invention.

In Figure 1: M is DL-2000 DNA marker, and the length of the gap fragment is 931bp.

Detailed ways

The present invention can be realized through following several operation steps:

1. Isolation and culture of Staphylococcus chromogenes:

Disinfect the surface of the freshwater fish with 75% alcohol, take the middle part of the intestinal tract aseptically, place it in a centrifuge tube filled with 10mL sterile normal saline, cut it into pieces and mix well, take an appropriate amount and evenly spread it on the mannitol high On the salt agar medium, keep the temperature in the medium at a constant temperature of 37°C, and cultivate for 18-24 hours; select typical colonies and transfer them to a new mannitol high-salt agar medium plate, and pass them repeatedly for 2-3 times to screen out the target Bacteria were inoculated onto the slope for later use; the colony grew on the plate to form a single colony, and the characteristics of the colony were round protrusions, neat edges, smooth surface, moist, opaque colonies, and the microscopic observation of the bacteria showed Staphylococcus aureus; The composition and preparation method of the mannitol high-salt agar medium described are (g/L): peptone 10.0, beef powder 1.0, D-mannitol 10.0, sodium chloride 100.0, phenol red 0.025, agar 13.0, pH 7.4 ± 0.2; When preparing, weigh 109.0g of this product, add it to 1000ml of distilled water, autoclave at 121°C for 15 minutes, and pour it into a sterile plate when it is cooled to about 45-50°C.

2. Physiological and biochemical identification of Staphylococcus chromogenes:

After the four physiological and biochemical characteristics of the cultured strain were identified, it was shown that the strain was negative for oxidase, positive for catalase, unable to move, and positive for nitrate reduction. Consult the "Bergey's Handbook of Systematic Bacteriology". The morphological characteristics and the results of various physiological and biochemical tests basically conformed to the characteristics of the genus Staphylococcus , and it was initially determined to be a genus of Staphylococcus.

A PCR-specific primer design method for the gap gene sequence of Staphylococcus chromogenes, which is characterized in that: through BLAST nucleotide sequence comparison, find the conserved sequence of the target gene that needs to be detected in this species; according to the conserved sequence, design for amplification To increase the specific primers for the gap gene fragments, use the primer design software primer5.0 to design primers; compare the primers with nucleotide sequences to ensure the high conservation of the 3' end of the primers; the specific steps are as follows:

(1) Screening of primers for Staphylococcus chromogenes: send the designed primers to Sangon Bioengineering (Shanghai) Co., Ltd. for synthesis, use PCR technology to amplify the genomic DNA of Staphylococcus chromogenes for the obtained primers, and detect each pair of primers Whether it is available, that is, whether a clear, single, and regular band can be amplified. Among the primers screened, the primer used to detect the PCR amplification fragment size of the bacterium is 931bp, and the gap gene presumably encodes an amino acid sequence of 310aa , the primer sequence is forward gap-F (5'-3'): ATGGTTTTGGTAGAATTGGTCGTTTA, reverse gap-R (5'-3'): GACATTTCGTTATCATACCAAGCTG;

(2) Optimization of PCR amplification conditions for Staphylococcus chromogenes: PCR conditions were optimized for the gap gene-specific primers obtained from the isolated strains of this genus, and the best PCR amplification system and amplification conditions were obtained as follows: the best PCR amplification The amplification system is 25 μl, including 2.5 μl 10×Reaction Buffer (with Mg2+), 0.5 μl dNTP (including dATP, dTTP, dCTP, dGTP, 2.5 mM each), 0.5 μl each of forward and reverse primers (10 μM), 0.3 μl Taq enzyme , the extracted bacterial genomic DNA template was 2.0 μl, and the total volume of 25 μl was made up with sterilized double distilled water ddH ₂ O 17.2 μl; the optimal PCR reaction conditions were: pre-denaturation at 94°C for 5 minutes, denaturation at 94°C for 30s, annealing at 52°C for 50s, Extend at 72°C for 45s, then extend again at 72°C for 10min after 30 cycles, and store the PCR product at 4°C for later use;

(3) Molecular sequencing identification of Staphylococcus chromogenes: 10g/L agarose gel electrophoresis was used to detect PCR products, the target band was excised, recovered by gel recovery kit, and after the recovered products were cloned, positive clones were selected and sent to Shanghai Sangon for sequencing; After sequencing, the BLAST and ClustalX programs were used for sequence comparison analysis, and the Neighbor-joining method was used to construct a phylogenetic tree, which showed that the strain was 98.82% homologous to the type strain (CCM 3387) of Staphylococcus chromogenes ( S.chromogenes ), thus The bacterium was identified as Staphylococcus chromogenes.

Chromogenic Staphylococcus (Staphylococcus chromogenes) gap gene upstream and downstream primer sequences:

the

gap-F: ATGGTTTTGGTAGAATTGGTCGTTTA

gap-R: GACATTTCGTTATCATACCAAGCTG

the

The gap gene sequence is 931 bp long:

the

ATGGTTTTGGTAGAATTGGTCGTTTAGCATTCAGAAGAATTCAAGACGTAGAAAATATTGAGGTTGTAGCTGTAAACGATTTAACAGACGACGATATGCTTGCACATTTATTAAAATATGACACAATGCAAGGTCGTTTTACTGAAGAAGTAGATGTAATTGATGGTGGTTTCCGCGTAAATGGTAAAGAAGTGAAATCATTCTCTGAACCAGAACCATCAAAATTACCATGGAAAGATCTTGACGTAGATGTTGTTTTAGAATGTACAGGTTTCTTTACATCAAAAGAAAAAGCAGAAGCTCACATTGAAGCGGGTGCTAAAAAAGTATTAATTTCTGCACCAGGAACTGGCGATCTTAAAACAATCGTATATAATGTCAACCATGAAGAATTAGACGGTTCTGAAACAGTTGTATCAGGTGCTTCTTGTACAACAAACTCATTAGCACCAGTAGCAAAAACTTTAAACGATGAATTTGGTATCGTTGAAGGTTTAATGACTACAATTCACGCATACACTGGTGACCAAAATACACAAGACTCACCACACAGAAAAGGTGACAAACGTCGTGCACGTGCAGCTGCAGAAAACATTATTCCTAACTCAACTGGTGCTGCGAAAGCAATCGGTTTAGTTATCCCTGAAATCGATGGAAAATTAGACGGTGGCGCACAACGTGTACCAGTAGCAACAGGTTCATTAACTGAATTAACAGTTGTCTTAGATGAAGAAGTATCAGTAGAAGACGTTAACAATGCAATGAAAAATGCAACAAACGAATCATTCGGTTACACTGAAGACGAAATCGTATCTTCAGATGTTGTAGGCATGACTCTCGGTGCATTATTTGATGCAACTCAAACACGTGTCATGACTGTTGGCGACCGTCAATTAGTTAAAGTAGCAGCTTGGTATGATAACGAAATGTC

the

the

the

The single-letter amino acid sequence of the gap gene is as follows:

the

GFGRIGRLAFRRIQDVENIEVVAVNDLTDDDMLAHLLKYDTMQGRFTEEVDVIDGGFRVNGKEVKSFSEPEPSKLPWKDLDVDVVLECTGFFTSKEKAEAHIEAGAKKVLISAPGTGDLKTIVYNVNHEELDGSETVVSGASCTTNSLAPVAKTLNDEFGIVEGLMTTIHAYTGDQNTQDSPHRKGDKRRARAAAENIIPNSTGAAKAIGLVIPEIDGKLDGGAQRVPVATGSLTELTVVLDKEVSVEDVNNAMKNATNESFGYTEDEIVSSDVVGMTFGALFDATQTRVMTVGDRQLVKVAAWYDNEMS

the

The three-letter amino acid sequence of the gap gene is as follows:

the

GlyPheGlyArgIleGlyArgLeuAlaPheArgArgIleGlnAspValGluAsnIleGluValValAlaValAsnAspLeuThrAspAspAspMetLeuAlaHisLeuLeuLysTyrAspThrMetGlnGlyArgPheThrGluGluValAspValIleAspGlyGlyPheArgValAsnGlyLysGluValLysSerPheSerGluProGluProSerLysLeuProTrpLysAspLeuAspValAspValValLeuGluCysThrGlyPhePheThrSerLysGluLysAlaGluAlaHisIleGluAlaGlyAlaLysLysValLeuIleSerAlaProGlyThrGlyAspLeuLysThrIleValTyrAsnValAsnHisGluGluLeuAspGlySerGluThrValValSerGlyAlaSerCysThrThrAsnSerLeuAlaProValAlaLysThrLeuAsnAspGluPheGlyIleValGluGlyLeuMetThrThrIleHisAlaTyrThrGlyAspGlnAsnThrGlnAspSerProHisArgLysGlyAspLysArgArgAlaArgAlaAlaAlaGluAsnIleIleProAsnSerThrGlyAlaAlaLysAlaIleGlyLeuValIleProGluIleAspGlyLysLeuAspGlyGlyAlaGlnArgValProValAlaThrGlySerLeuThrGluLeuThrValValLeuAspLysGluValSerValGluAspValAsnAsnAlaMetLysAsnAlaThrAsnGluSerPheGlyTyrThrGluAspGluIleValSerSerAspValValGlyMetThrPheGlyAlaLeuPheAspAlaThrGlnThrArgValMetThrValGlyAspArgGlnLeuValLysValAlaAlaTrpTyrAspAsnGluMetSer

Claims (2)

1. A method for obtaining the gap gene sequence of Staphylococcus chromogenes, which is characterized in that: take the method of combining plate streaking and dilution coating to carry out freshwater fish intestinal bacteria on mannitol high-salt agar (MSA) medium After isolation and screening, a strain was obtained, and through systematic research such as morphological observation, physiological and biochemical characteristics, and gene sequence molecular identification, it was confirmed that the strain was Staphylococcus chromogenes. 2. A method for designing PCR-specific primers of Staphylococcus chromogenes gap gene sequence, which is characterized in that: by BLAST nucleotide sequence alignment, find the conserved sequence of the target gene that this species needs to detect; according to the conserved sequence, design To amplify the specific primers of the gap gene fragment, use the primer design software primer5.0 to design primers; compare the primers with nucleotide sequences to ensure the high conservation of the 3' end of the primers; the specific steps are as follows: (1) Screening of primers for Staphylococcus chromogenes: send the designed primers to Sangon Bioengineering (Shanghai) Co., Ltd. for synthesis, use PCR technology to amplify the genomic DNA of Staphylococcus chromogenes for the obtained primers, and detect each pair of primers Whether it is available, that is, whether a clear, single, and regular band can be amplified. Among the primers screened, the primer used to detect the PCR amplification fragment size of the bacterium is 931bp, and the gap gene encodes an amino acid sequence of 310aa. The primer sequence is forward gap-F (5'-3'): ATGGTTTTGGTAGAATTGGTCGTTTA, reverse gap-R (5'-3'): GACATTTCGTTATCATACCAAGCTG; (2) Optimization of PCR amplification conditions for Staphylococcus chromogenes: PCR conditions were optimized for the gap gene-specific primers obtained from the isolated strains of this genus, and the best PCR amplification system and amplification conditions were obtained as follows: the best PCR amplification The amplification system is 25 μl, including 2.5 μl 10×Reaction Buffer (with Mg2+), 0.5 μl dNTP (including dATP, dTTP, dCTP, dGTP, each 2.5 mM), forward and reverse primers (10 μM) each 0.5 μl, Taq enzyme 0.3 μl , the extracted bacterial genomic DNA template was 2.0 μl, and the total volume of 25 μl was made up with sterilized double distilled water ddH ₂ O 17.2 μl; the optimal PCR reaction conditions were: pre-denaturation at 94°C for 5 minutes, denaturation at 94°C for 30s, annealing at 52°C for 50s, Extend at 72°C for 45s, then extend again at 72°C for 10min after 30 cycles, and store the PCR product at 4°C for later use; (3) Molecular sequencing identification of Staphylococcus chromogenes: 10g/L agarose gel electrophoresis was used to detect PCR products, the target band was excised, and the gel recovery kit was recovered. After the recovered products were cloned, positive clones were selected and sent to Shanghai Sangon for sequencing; After sequencing, the BLAST and ClustalX programs were used for sequence comparison analysis, and the Neighbor-joining method was used to construct a phylogenetic tree, which showed that the strain was 98.82% homologous to the type strain (CCM 3387) of Staphylococcus chromogenes , and thus identified The bacterium is Staphylococcus chromogenes.

Images