CN112899382A - Detection method for identifying amycolatopsis - Google Patents
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Abstract
The invention discloses a detection method for identifying amycolatopsis, belonging to the field of molecular biology. The invention provides a primer pair capable of specifically identifying amycolatopsis, which is designed based on a specific conserved gene AMETH _3452 in the amycolatopsis and homologous gene fragments in the amycolatopsis, and has strong specificity; the PCR method for rapidly detecting the amycolatopsis is established through optimization of PCR parameters, and repeated multiple times prove that the method is rapid and sensitive, can realize detection of a plurality of strains at the same time, and has the advantages of low detection cost, short time consumption and good effect. Provides a basis for the next step of actinomycete research in detection environment, animals or clinic.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to a primer pair and a detection method for detecting amycolatopsis.
Background
The secondary metabolites produced by microorganisms have various biological activities and are an important repository for drug discovery at present. The microbial medicine has the characteristics of rich sources, unique chemical structure of most secondary metabolites generated by the microbes and difficult synthesis, so that novel compound molecules with medicinal value are mined from the secondary metabolites rich and diverse in the microbes to develop original medicines, and the microbial medicine has important clinical application value.
Amycolatopsis (Amycolatopsis) is an important class of antibiotic-producing actinomycetes, such as the vancomycin-producing bacteria Amycolatopsis orientalis (Amycolatopsis orientalis) and the rifamycin-producing bacteria Amycolatopsis mediterranei (Amycolatopsis mediterranei). Most of the amycolatopsis strains are isolated from soil, and some are isolated from oceans, deserts, sediments, clinical specimens, etc., and belong to the actinomycetes, pseudonocardiales, pseudonocardiaceae. The species of this genus are divided into 2 main branches, the Amycolatopsis orientalis branch (AOS) and the Amycolatopsis methyl branch (AMS). Among them, strains in AOS are often found to produce antibiotics, such as Amycolatopsis orientalis, Amycolatopsis mediterranei. Representative strain of AMS a. methanolica is a facultative methylotrophic actinomycete, no antibiotic production is found and secondary metabolites are minor. The strain in AMS can grow at 45 deg.C or above, and belongs to thermophilic actinomycetes.
At present, the method for identifying the amycolatopsis mainly comprises the following steps of gene sequencing and 16S rDNA comparison: firstly, extracting genome DNA, then carrying out PCR amplification on a 16S rDNA fragment, sequencing the 16S rDNA fragment, and then carrying out sequence comparison, wherein the common bacteria are considered to be the same bacteria if the sequence similarity is more than 99%. The 16S rDNA/RNA gene sequence is widely applied to bacterial identification or construction of phylogenetic relationship of bacteria, and in addition, gyrB and recN genes can be used as phylogenetic identification markers to effectively distinguish strains. However, the above method can identify amycolatopsis, but is costly and time-consuming.
At present, no primer for specific amplification of amycolatopsis exists, a rapid detection method is lacked, and strain identification with large sample amount is not facilitated. Therefore, it is highly desirable to develop a method capable of rapidly identifying amycolatopsis species.
Disclosure of Invention
The purpose of the present invention is to provide a primer set capable of specifically amplifying a gene sequence specific to Amycolatopsis (Amycolatopsis), and to directly and rapidly identify Amycolatopsis by PCR amplification reaction.
In order to achieve the above purpose, the present invention compares the complete genome in Amycolatopsis, and finds that there is a conserved gene sequence in Amycolatopsis corresponding to AMETH _3452 in the whole genome of known Amycolatopsis methanolinica 239, the gene is located in the middle of the circular genome in Amycolatopsis, and the sequence homology between different strains in Amycolatopsis is high. Based on the discovery, the invention designs a primer pair capable of specifically amplifying AMETH _3452 gene and homologous gene fragments in amycolatopsis, and whether the strain to be detected belongs to amycolatopsis can be judged by whether a target product can be amplified.
Application of AMETH _3452 gene with a nucleotide sequence shown as SEQ ID No.1 and homologous gene fragment thereof as target in identification of Amycolatopsis (Amycolatopsis).
The conservation of the gene is higher between 300bp and 600bp through sequence analysis, so that degenerate primers are designed in the region, and the genus can be effectively identified through a PCR method.
Further, the invention provides a primer pair for identifying Amycolatopsis (Amycolatopsis), which comprises an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is 5 '-TSGARGGCAANCACGACGC-3', and the nucleotide sequence of the downstream primer is 5 '-AYGTASGGRTGNCCGGTGA-3'.
The invention also provides a PCR detection kit containing the primer pair and used for identifying Amycolatopsis (Amycolatopsis), and the kit also comprises PCR buffer solution, Taq polymerase, dNTP, positive control DNA and negative control DNA.
The invention provides a detection method for identifying Amycolatopsis (Amycolatopsis), which comprises the following steps:
(1) extracting DNA of a strain to be detected;
(2) constructing a PCR reaction system by taking the extracted DNA as a template, wherein the nucleotide sequence of the upstream primer is 5 '-TSGARGGCAANCACGACGC-3', and the nucleotide sequence of the downstream primer is 5 '-AYGTASGGRTGNCCGGTGA-3', and carrying out PCR reaction;
(3) performing gel electrophoresis separation and dyeing on the PCR product, and judging the result: if a 244bp band appears, the strain to be detected is the amycolatopsis, otherwise, the strain to be detected is the non-amycolatopsis.
The PCR reaction system is as follows: in terms of 20. mu.L, 10. mu.L of 2 × Specific Taq Master Mix (Shanghai Bison technology development Co., Ltd.), 0.8. mu.L of 10mmol/L forward primer, 0.8. mu.L of 10mmol/L reverse primer, 1. mu.L of 200. mu.g/. mu.L template DNA, ddH2O 7.4μL。
The PCR reaction conditions are as follows: 1) pre-denaturation at 90-95 ℃; 2) denaturation at 90-95 ℃ for 30s, and annealing at 60-65 ℃ for 30 s; extending for 20s at 72 ℃, and performing 30-40 cycles; 3) extension at 72 ℃.
Preferably, the PCR reaction conditions are: 1) pre-denaturation at 95 ℃ for 2 min; 2) denaturation at 95 ℃ for 30s, annealing at 61 ℃ for 30s, and extension at 72 ℃ for 30s for 35 cycles; 3) extension at 72 ℃ for 12 min. The primer pair provided by the invention has the best specificity for amplifying the target fragment at the annealing temperature of 61 ℃ proved by research.
Further, detecting by a fluorescent quantitative PCR method, specifically, in the step (2), constructing a fluorescent quantitative PCR reaction system, performing qPCR reaction, and judging the result, wherein the Ct value is analyzed, if the Ct value is less than 30, the strain to be detected is amycolatopsis, otherwise, the strain to be detected is not amycolatopsis.
The qPCR reaction system is as follows: in 20. mu.L, 2 XTB Green Fast qPCR Mix 10. mu.L, 10mmol/L upstream primer 0.8. mu.L, 10mmol/L downstream primer 0.8. mu.L, 200. mu.g/. mu.L template DNA 1. mu.L, ddH2O 7.4μL。
The qPCR reaction conditions were: 1) pre-denaturation at 95 ℃ for 2 min; 2) denaturation at 95 ℃ for 30s, annealing at 61 ℃ for 30s, and extension at 72 ℃ for 30s for 40 cycles; 3) extension at 72 ℃ for 12 min.
The invention has the following beneficial effects:
the primer pair is designed based on a specific conserved gene AMETH _3452 in the amycolatopsis and homologous gene segments in the amycolatopsis, and has strong specificity; the PCR method for rapidly detecting the amycolatopsis is established through optimization of PCR parameters, and repeated multiple times prove that the method is rapid and sensitive, can realize detection of a plurality of strains at the same time, and has low detection cost, short time consumption and good effect. Provides a basis for the next step of actinomycete research in detection environment, animals or clinic.
Drawings
FIG. 1 shows the conserved region sequence alignment of AMETH _3452 gene in Amycolatopsis.
FIG. 2 shows the optimal temperature for screening PCR amplification.
FIG. 3 is a qPCR amplification curve diagram for detecting a strain to be detected by using primers.
FIG. 4 is a gel electrophoresis image after qPCR amplification of a strain to be detected by using primers.
Detailed Description
The present invention will be further described with reference to the following examples and accompanying drawings, which are not intended to limit the scope of the invention.
The methods in the following examples are conventional in the art unless otherwise specified. The strains used were purchased from the German Collection of microorganisms and cell cultures (DSMZ) and deposited in the laboratory. The environmental wastewater is diluted by taking sewage from the vicinity of a farm, the matrix is complex, and the structure of bacterial flora is various.
AMETH — 3452 gene analysis:
through the comparison of the complete genome of the Amycolatopsis, a conserved gene is found to exist at the replication termination position of the genome of the genus, which corresponds to AMETH _3452 in the complete genome of the known Amycolatopsis methanolica239, and the nucleotide sequence is shown in SEQ ID No. 1. The protein encoded by the gene was found to be present in various genera of actinomycetes by alignment with the bacterial genome. However, the gene in amycolatopsis has low homology (< 30%) with other genus nucleic acids, and there is no matching length of more than 100 bp. The GenBank database found 18 strains of amycolatopsis, found by BLAST alignment with each other, to have a nucleic acid homology of between 80.75% and 85.20%. Further, alignment analysis of the sequence is carried out by Clustal W, and the conservation between 300bp and 600bp of the gene is higher, as shown in figure 1, so that a degenerate primer is designed in the region, and the genus can be effectively identified by a PCR (polymerase chain reaction) and qPCR (quantitative polymerase chain reaction) method.
Example 1
1. Primer synthesis
The AMETH _3452 gene and the homologous sequence thereof are analyzed, and PCR primers are designed, and the details are shown in Table 1. The primers were synthesized according to the sequence of the sequence listing, Veneziton Biotechnology engineering (Shanghai) Ltd.
TABLE 1
In the table, S ═ C or G; r is Aor G; n ═ a, C, G or T; y ═ C or T.
2. Optimization of PCR reaction conditions
2.1 environmental wastewater DNA was used as a blank control, and the DNA of Amycolatopsis (A. methanolica)239 was known as a positive control. The strain was isolated and cultured, and mycelia were scraped directly from the culture dish, and mycelia genomic DNA was extracted using a bacterial DNA extraction kit (Shanghai, Czejust bioengineering, Ltd.).
2.2 the extracted DNA is used as a template, and the amplification is carried out by the PCR method using the above primers. The annealing temperature of PCR was optimized by gradient PCR, and the optimal annealing temperature was 61 deg.C, and the specific gel diagram is shown in FIG. 2.
3. Authentication
3.1 Amycolatopsis strains A.methanolica 239, A.plurensis, A.pretriensis, A.sulphouruea, A.decaplanin, A.kentuckyensis 9, A.kentuckyensis 303, A.tolypomycena, A.mediterranei U32 are used as positive controls, and Campylobacter C.jejuni, Escherichia coli E.coli, Streptomyces S.coelicolor, S.griseus, S.are used as positive controls.
natalensis was negative control.
Colonies from each culture dish were scraped and genomic DNA was extracted using a bacterial DNA extraction kit (Shanghai Czeri bioengineering Co., Ltd.).
3.2qPCR reaction
PCR reaction (20. mu.L): 10 μ L of 2 XTB Green Fast qPCR Mix, 0.8 μ L of 10mmol/L forward primer, 0.8 μ L of 10mmol/L reverse primer, 1 μ L of 200 μ g/μ L template DNA, 7.4 μ L ddH2O。
And (3) PCR reaction conditions: pre-denaturation at 95 ℃ for 2 min; denaturation at 95 ℃ for 30s, annealing at 61 ℃ for 30s, and extension at 72 ℃ for 30s for 40 cycles; finally, extension is carried out for 12min at 72 ℃.
The fluorescence quantitative PCR was performed using Bio-Rad (CFX96) as an apparatus, and the positive control 239 of Amycolatopsis was diluted in a gradient manner to obtain a fluorescence quantitative amplification curve as shown in FIG. 3, with a minimum detection limit of 200X 10-4μg/μL。
The DNA of the cultured colony was subjected to fluorescent quantitative PCR, and the specific Ct values are shown in Table 2.
It is known that the Ct value of the positive control of the genus Amycolatopsis is 30 or less, and the Ct value of the genus Amycolatopsis is 30 or less. These strains were described as amycolatopsis. Ct values of streptomycete S.coelicolor, S.griseus, S.natalensis, campylobacter C.jejuni, escherichia coli E.coli and environmental wastewater are all above 30.
TABLE 2
The PCR product was electrophoresed using 2% agarose gel (120V,30 min). The product bands were observed.
As shown in FIG. 4, the amycolatopsis bacteria A.methanolica 239, A.plurensis, A.pretriensis, A.sulphourea, A.decaplanin, A.kentuckyensis 9, A.kentuckyensis 303, A.tolypomycena and A.mediterranei U32 all amplified a 244bp band, whereas none of the amycolatopsis bacteria amplified a 244bp band.
Sequence listing
<110> Zhejiang province academy of agricultural sciences
<120> detection method for identifying amycolatopsis
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 810
<212> DNA
<213> Amycolatopsis methanolica)
<400> 1
gtgcgctccc acagctatga cgacatcctc gccggcccgc gcagacgcaa gatccccgag 60
gtcgtggcgg agcgcgggct ggtggtcgag gaaccgggaa gcggtttctg cggcgcggtg 120
gtccggttcg agcagggcaa cgtggtgctg gaggaccgcc acggccgcca ccgcgtgttc 180
ccgctggagc ccgccgggtt cctgctcgag ggcaagccgg tgacgctggt acggccgaag 240
gccgcgccgc cgtccccggc ttcggcgcgt tcggcgtccg ggtcggtgaa ggtgcagggg 300
ctgcgggccc gcaccgcgcg tgactcgcgc atctgggtcg agggcaagca cgacgccgaa 360
ctggtggaac gcgtgtgggg gcacgacctg cgcgtcgagg gcgtcgtcgt ggaaccgctc 420
gacggcgtgg acgtgctggc cgaggcgatc gaggagttcg gcaccggccc cggcaggcgg 480
ctgggcgtcc tggtcgacca cctggtgccg ggcagcaagg aatcccgcct ggtggacgcg 540
atccgcgacg agaacgtgct ggtcaccggc cacccctacg tcgacgtgtg gcaggcggtg 600
aaaccgtccg cggtcggcat ccgggcgtgg ccggcggtgc cgcgcggcac gccgtggaag 660
gagggcgtgt gcgcggcgct gggctggggc gagacctacg agggctggca gcgggtgctg 720
gccgcggtga gcagcttccg cgacctggag acgccgctga tcggcgccgt cgagcggctg 780
atcgacttcg tgaccgaccc ggaaggctga 810
<210> 2
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tsgarggcaa ncacgacgc 19
<210> 3
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
aygtasggrt gnccggtga 19
Claims (8)
1. Application of AMETH _3452 gene with a nucleotide sequence shown as SEQ ID No.1 and homologous gene fragment thereof as target in identification of Amycolatopsis (Amycolatopsis).
2. The application of the primer pair in identifying Amycolatopsis (Amycolatopsis) is characterized in that the primer pair comprises an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is 5 '-TSGARGGCAANCACGACGC-3', and the nucleotide sequence of the downstream primer is 5 '-AYGTASGGRTGNCCGGTGA-3'.
3. A PCR detection kit for identifying Amycolatopsis (Amycolatopsis) is characterized by comprising an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is 5 '-TSGARGGCAANCACGACGC-3', and the nucleotide sequence of the downstream primer is 5 '-AYGTASGGRTGNCCGGTGA-3'.
4. An assay for identifying Amycolatopsis (Amycolatopsis), comprising the steps of:
(1) extracting DNA of a strain to be detected;
(2) constructing a PCR reaction system by taking the extracted DNA as a template, wherein the nucleotide sequence of the upstream primer is 5 '-TSGARGGCAANCACGACGC-3', and the nucleotide sequence of the downstream primer is 5 '-AYGTASGGRTGNCCGGTGA-3', and carrying out PCR reaction;
(3) performing gel electrophoresis separation and dyeing on the PCR product, and judging the result: if a 244bp band appears, the strain to be detected is the amycolatopsis, otherwise, the strain to be detected is the non-amycolatopsis.
5. The detection method for identifying Amycolatopsis (Amycolatopsis) according to claim 4, wherein in the step (2), a fluorescent quantitative PCR reaction system is constructed to perform qPCR reaction, and the result judgment further comprises analyzing the Ct value, wherein if the Ct value is less than 30, the strain to be detected is Amycolatopsis, otherwise, the strain to be detected is non-Amycolatopsis.
6. The assay for identifying Amycolatopsis (Amycolatopsis) according to claim 5, wherein the PCR reaction system is: in 20. mu.L, 2 XTB Green Fast qPCR Mix 10. mu.L, 10mmol/L upstream primer 0.8. mu.L, 10mmol/L downstream primer 0.8. mu.L, 200. mu.g/. mu.L template DNA 1. mu.L, ddH2O 7.4μL。
7. The assay for identifying Amycolatopsis (Amycolatopsis) according to claim 5, wherein the PCR reaction conditions are: 1) pre-denaturation at 90-95 ℃; 2) denaturation at 90-95 ℃ for 30s, and annealing at 60-65 ℃ for 30 s; extending for 30s at 72 ℃, and performing 30-40 cycles; 3) extension at 72 ℃.
8. The assay for identifying Amycolatopsis (Amycolatopsis) according to claim 7, wherein the PCR reaction conditions are: 1) pre-denaturation at 95 ℃ for 2 min; 2) denaturation at 95 ℃ for 30s, annealing at 61 ℃ for 30s, and extension at 72 ℃ for 30s for 40 cycles; 3) extension at 72 ℃ for 12 min.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113628684A (en) * | 2021-08-06 | 2021-11-09 | 苏州鸿晓生物科技有限公司 | Sample bacterial species detection methods and systems |
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| CN105874073A (en) * | 2013-12-03 | 2016-08-17 | 西姆莱斯股份公司 | Shuttle vectors and expression vectors for amycolatopsis |
| CN109609537A (en) * | 2018-12-18 | 2019-04-12 | 上海交通大学 | A kind of application of gene editing method in Amycolatopsis orientalis |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105874073A (en) * | 2013-12-03 | 2016-08-17 | 西姆莱斯股份公司 | Shuttle vectors and expression vectors for amycolatopsis |
| CN109609537A (en) * | 2018-12-18 | 2019-04-12 | 上海交通大学 | A kind of application of gene editing method in Amycolatopsis orientalis |
Non-Patent Citations (3)
| Title |
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| BIAO TANG等: "A systematic study of the whole genome sequence of Amycolatopsis methanolica strain 239T provides an insight into its physiological and taxonomic properties which correlate with its position in the genus", 《SYNTHETIC AND SYSTEMS BIOTECHNOLOGY》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113628684A (en) * | 2021-08-06 | 2021-11-09 | 苏州鸿晓生物科技有限公司 | Sample bacterial species detection methods and systems |
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