CN108728557B - Primer pair for detecting thiobacillus acidocaldarius, design method and detection method - Google Patents
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Abstract
The invention discloses a primer pair for detecting thiobacillus acidocaldarius, a design method and a detection method, wherein the design method comprises the following steps: extracting CDSs (sequence variants) according to the complete genome sequence of the Acidithiobacillus model species strain in the NCBI; and obtaining the conservative CDSs (sequence-derived sequences) of the Acidithiobacillus by adopting Blastn comparison, and designing a primer pair for detecting the Acidithiobacillus according to a primer design principle. When the primer pair designed by the method is used for detecting the Acidithiobacillus, the Acidithiobacillus (species) can be quickly and accurately detected, the specificity is strong, the sensitivity is high, and a similar method is not adopted at present to obtain the conservative CDSs (sequence-specific primers) of the Acidithiobacillus or the species so as to design a corresponding specific primer.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a primer pair for detecting Acidithiobacillus thiooxidans, a design method and a detection method.
Background
The biological hydrometallurgical technology has become one of the most potential technologies for leaching and pretreatment of lean ores, and the most widely applied technology in leaching microorganisms is Acidithiobacillus (Sulfobacillus: (Sulfobacillus)Acidithiobacillussp.). Of the Acidithiobacillus genus, the common species related to mineral leaching applications are Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans (S.thiooxidans)Acidithiobacillus thiooxidans) And Acidithiobacillus caldus: (Acidithiobacillus caldus) And the like.
At present, the detection and species composition analysis of Acidithiobacillus (species) in environmental samples mainly adopt a high-throughput sequencing method of 16S rRNA amplicon sequencing and metagenome sequencing. However, both methods are costly, time consuming and require the extraction of high quality sample total DNA. In addition, for environmental samples with abundant species diversity, due to the very low cell concentration of Acidithiobacillus (species), if the sequencing depth is not enough, the sequence is easily covered by the species with high abundance to cause false negative result, and even if the sequence can be detected, a great deal of manpower and financial resources are required to be invested.
Accordingly, the prior art is yet to be improved and developed.
Disclosure of Invention
In view of the above-mentioned shortcomings of the prior art, the present invention aims to provide a primer pair, a design method and a detection method for detecting Acidithiobacillus, which aims to solve the problems of high cost, long time consumption and false negative result when the cell concentration of Acidithiobacillus (species) is very low in the existing detection method.
The technical scheme of the invention is as follows:
a design method of a primer pair for detecting Acidithiobacillus comprises the following steps:
a1, extracting CDSs (CDSs) sequences according to the complete genome sequence of the acidophilic thiobacillus model species strain in NCBI;
step B1, taking the extracted CDSs as a query sequence, aligning with Blastn to remove the NR database of the acidophilus sequences, taking the CDSs which are not aligned as the query sequence, aligning with Blastn to remove the acidophilus sequences, and extracting the CDSs sequences of all the acidophilus strains which can be aligned with the NR database to obtain the acidophilus conservative CDSs sequences;
step C1, designing and obtaining a primer pair for detecting Acidithiobacillus according to the Acidithiobacillus conservative CDSs sequence and a primer design principle;
or step A2, extracting CDSs sequence according to the complete genome sequence of the acidophilic thiobacillus target species model strain in NCBI;
step B2, taking the extracted CDSs as query sequences, conducting Blastn comparison to remove the NR database of the acidophilic thiobacillus target species sequence, taking the CDSs not compared as query sequences, conducting Blastn comparison to the acidophilic thiobacillus target species sequence, and extracting the CDSs sequences of all the acidophilic thiobacillus target species strains which can be compared with the NR database to obtain the acidophilic thiobacillus target species conserved CDSs sequence;
and step C2, designing the primer pair for detecting the acidophilic thiobacillus target species according to the acidophilic thiobacillus target species conservative CDSs sequence and the primer design principle.
A primer pair for detecting the thiobacillus acidocaldarius is designed by adopting the design method.
The primer pair for detecting the Acidithiobacillus comprises:
primer pairs for the detection of Acidithiobacillus:
the sequence of the upstream primer is 5'-CGAGAGCACAGCCAACTGGAACTC-3',
the sequence of the downstream primer is 5'-GCGAGATAG ATGCCATGCGCGA-3';
or a primer pair for detecting an acidophilic thiobacillus ferrooxidans species:
the sequence of the upstream primer is 5'-GGCACATGTGGTAATTTTGGGT-3';
the sequence of the downstream primer is 5'-CCCGTAGGCAAAGACGTCC-3'.
A method of detecting thiobacillus acidocaldarius, comprising:
a3, extracting DNA of a sample to be detected, and thermally cracking the DNA of the sample to obtain a DNA single strand;
step B3, carrying out PCR amplification on the DNA single strand by adopting the primer pair to obtain a PCR amplification product;
and step C3, carrying out agarose gel electrophoresis detection on the PCR amplification product.
The method for detecting Acidithiobacillus, wherein the step A3 comprises the following steps: adding a sample to be detected into TE buffer solution, carrying out water bath for 4-6 min at 92-98 ℃, then freezing for 4-6 min at-18 to-22 ℃, repeating the water bath and freezing steps once, and then carrying out separation treatment to obtain supernatant containing the DNA single strand.
The method for detecting Acidithiobacillus, wherein in the step B3, the PCR program is set to 30 cycles.
The method for detecting Acidithiobacillus, wherein, after the step C3, the method further comprises:
and D3, sequencing the amplification product and constructing a molecular evolution tree for judging the composition of the Acidithiobacillus flora in the sample or the evolutionary clustering relationship among the same strains.
Has the advantages that: the invention provides a design method of the primer pair for detecting the Acidithiobacillus, when the primer pair designed by the method is used for detecting the Acidithiobacillus, the Acidithiobacillus (species) can be quickly and accurately detected, the specificity is strong, the sensitivity is high, and a similar method is not adopted at present for obtaining the conservative CDSs (sequence-specific primers) of the Acidithiobacillus or the species so as to design the corresponding specific primer.
Drawings
FIG. 1 is a flowchart of the sequence acquisition and specific primer design of Acidithiobacillus (species) conserved CDSs in examples 1 and 2 of the present invention.
FIG. 2 is the electrophoresis chart of the PCR product for rapid identification of Acidithiobacillus (species) -specific primers in example 1 and example 2 of the present invention.
FIG. 3 shows the result of PCR product analysis of Acidithiobacillus specific primers in example 1 of the present invention.
FIG. 4 shows the result of PCR product analysis of a thiobacillus acidophilus species-specific primer in example 2 of the present invention.
Detailed Description
The invention provides a primer pair for detecting thiobacillus acidocaldarius, a design method and a detection method, and the invention is further described in detail below in order to make the purpose, technical scheme and effect of the invention clearer and clearer. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The invention provides a preferred embodiment of a design method of a primer pair for detecting Acidithiobacillus, which comprises the following steps:
(1) acquisition of conserved sequence
The complete sequence of The genome of a strain of The Acidithiobacillus sp was downloaded from The National Center for Biotechnology Information database (NCBI) and The CDSs (coding sequences, in The form of CDS) sequences were extracted. The extracted CDSs were then used as query sequences (query sequences) to align the NR database with the eosinophilia-depleted sequences using the Blastn default parameters. And (3) drawing a perl language script to extract the CDSs sequences which are not aligned, taking the CDSs sequences as query sequences, aligning all the acidophilic thiobacillus sequences in an NR database by Blastn, and extracting the sequences appearing in each species, namely obtaining the acidophilic thiobacillus conserved CDSs sequences.
In a similar manner, the conserved sequences of a particular Acidithiobacillus species can be obtained, specifically, after determining the Acidithiobacillus species to be detected and identified (i.e., Acidithiobacillus target species, simply referred to as target species), the CDSs of the model strain of the target species can be obtained. Taking the obtained sequence as a query sequence, using Blastn default parameter alignment to remove the NR database of the target species model strain sequence, extracting the CDSs sequence which is not aligned, taking the obtained sequence as the query sequence, using Blastn to align the sequences of all the target species in the NR database, and extracting the CDSs sequence which can be aligned with all the strains of the target species in the NR database, namely the conserved CDSs sequence of the target species of the acidophilic thiobacillus.
(2) Design of specific primers
Extracting all conserved sequences of a certain CDS of the Acidithiobacillus, performing comparison analysis by using Bioedit software, determining conserved sequence sections of the CDS in CDS sequences of all species of the Acidithiobacillus, and designing a specific PCR primer according to the conserved sequence sections to obtain a rapid detection primer of the Acidithiobacillus.
The invention takes the CDS sequence of the gene sequence (genus) of the ion efflux permease as an example, and the sequence of the specific amplification upstream primer and the sequence of the specific amplification downstream primer of the gene sequence (genus) of the ion efflux permease are respectively designed as follows:
the sequence of the upstream primer is 5'-CGAGAGCACAGCCAACTGGAACTC-3',
the sequence of the downstream primer is 5'-GCGAGATAG ATGCCATGCGCGA-3'.
The design process of the specific primer of the acidophilic thiobacillus target species is as follows: extracting all conserved sequences of a certain CDS of the acidophilic thiobacillus target species, carrying out alignment analysis by using Bioedit software, determining conserved sequence sections of the CDS in CDS sequences of all strains, designing a specific PCR primer according to the conserved sequence sections, and obtaining a rapid detection primer of the acidophilic thiobacillus target species.
The CDS sequence of the sulfide quinone reductase gene (seed) is taken as an example, the sequence of the specific amplification upstream primer and the sequence of the specific amplification downstream primer of the sulfide quinone reductase gene (seed) are respectively designed as follows:
the sequence of the upstream primer is 5'-GGCACATGTGGTAATTTTGGGT-3';
the downstream primer sequence is 5'-CCCGTAGGCAAAGACGTCC-3'.
The specific primer designed by the design method can quickly and accurately detect the Acidithiobacillus (species), and a similar method is not adopted to obtain the conservative CDSs (sequence-dependent sequences) of the Acidithiobacillus or the species so as to design the corresponding specific primer at present.
The invention also provides a preferred embodiment of the method for detecting the acidithiobacillus acidophilus, which comprises the following steps:
(1) total DNA thermal lysis of samples
Environmental samples were taken in sterile centrifuge tubes and TE buffer was added thereto. And then, carrying out water bath on the test tube for 4-6 min (preferably 5 min) at 92-98 ℃ (preferably 95 ℃), then freezing for 4-6 min (preferably 5 min) at-18 ℃ -22 ℃ (preferably-20 ℃), repeating the steps of water bath and freezing once, and then carrying out separation treatment to obtain the supernatant containing the DNA single strand.
(2) PCR and sample species composition analysis
And (3) taking the supernatant of the DNA single strand as a PCR reaction template, adding synthesized upstream and downstream primers, and preparing a PCR reaction system according to the instructions of PCR enzyme liquid (DNA polymerase, PCR buffer solution and dNTP) manufacturers. The PCR program was set to 30 cycles, the Primer annealing temperature was set as calculated by the software Premier Primer5, and the extension time was set by the PCR product length (1 kb/min).
The PCR product is detected by agarose gel electrophoresis, if a single band with expected size appears, the sample can be judged to contain the bacteria of Acidithiobacillus or specific species. And (3) cloning the PCR product by TA, selecting dozens of clones for Sanger sequencing, comparing the determined sequences with an NR database by using Blastn by using a query sequence, and extracting the compared sequences to construct a molecular evolution tree, so that the composition of the Acidithiobacillus flora of the sample and the evolutionary clustering relation among the same strains can be further judged.
The traditional high-throughput method for sequencing 16S rRNA amplicon and metagenome sequencing has high cost and long time consumption, and needs to extract high-quality total DNA of a sample. The detection method of Acidithiobacillus (species) does not need to build a library, does not need to extract total DNA of a sample, and has the advantages of strong specificity, high sensitivity and high speed.
The present invention will be described in detail below with reference to examples.
Example 1 Acidithiobacillus specific primer design
(1) Obtaining of Acidithiobacillus conserved CDSs sequence
The complete genome sequence of the model strain Thiobacillus acidophilus ATCC23270 was downloaded from NCBI (ftp:// ftp. NCBI. nlm. ni-h. gov/genes/all/GCF/000/021/485/GCF _000021485.1_ \\/u
ASM2148v 1), and extracts its CDSs sequences. With the extracted CDSs as query sequences, the eosinophil thiobacillus sequences (excludes) are removed by using Blastn default parameter alignmentAcidithiobacillus(taxi: 119977)). The result of the Blastn alignment is the conserved sequence between the Acidithiobacillus and other species. Extracting CDSs sequences not aligned, using the CDSs sequences as query sequences, and aligning eosinophilia thiobacillus sequences by Blastn (b)Acidithiobacillus(taxi: 119977)), and extracting the CDSs sequences of all the Acidithiobacillus strains aligned to the NR pool, namely the Acidithiobacillus conserved CDSs sequences. The design flow chart is shown in figure 1.
(2) Acidithiobacillus specific primer design
The obtained Acidithiobacillus conserved CDSs sequences were introduced into the software Bioedit and subjected to local rapid Alignment (Alignment). Taking the CDS sequence of the gene sequence (genus) of the ion efflux permease as an example, the amplification primer of the gene sequence (genus) of the ion efflux permease is designed according to the basic principle of primer design. The sequences of the upstream primer and the downstream primer are respectively as follows:
5’-CGAGAGCACAGCCAACTGGAACTC-3’，
5'-GCGAGATAG ATGCCATGCGCGA-3', PCR the length of the amplified product is 421 bp.
Example 2 design of primers specific for Acidithiobacillus ferrooxidans species
(1) Obtaining of conservative CDSs sequences of Acidithiobacillus ferrooxidans
With reference to the procedure of example 1, CDSs sequences of the model strain Thiobacillus acidophilus ATCC23270 were obtained. Using this as a query sequence, the eosinophilic Thiobacillus species sequence (excclude) was eliminated using a Blastn default parameter alignmentAcidithiobacillus ferrooxidans(taxi: 920)). The result of the Blastn alignment is the conserved sequence between the Acidithiobacillus ferrooxidans species and other species. Extracting the CDSs sequences not aligned, using the CDSs sequences as query sequences, and Blastn aligning acidophilic thiobacillus ferrooxidans sequences (Acidithiobacillus ferrooxidans(taxi: 920)), and extracting the CDSs sequences which can be aligned with all strains of the Acidithiobacillus ferrooxidans species in the NR library, namely the conserved CDSs sequences of the Acidithiobacillus ferrooxidans species. The design flow chart is shown in figure 1.
(2) Design of acidophilic thiobacillus ferrooxidans species-specific primers
The obtained acidophilic thiobacillus ferrooxidans conserved CDSs sequences were introduced into the software Bioedit for local rapid Alignment (Alignment). Taking the CDS sequence of the sulfide quinone reductase gene (species) as an example, the amplification primer of the sulfide quinone reductase gene (species) is designed according to the basic principle of primer design. The specific amplification upstream primer sequence and the specific amplification downstream primer sequence of the sulfide quinone reductase gene (species) are respectively as follows:
5’-GGCACATGTGGTAATTTTGGGT-3’，
5'-CCCGTAGGCAAA GACGTCC-3', PCR the length of the amplified product is 1150 bp.
Example 3 Rapid detection of Total DNA and Acidithiobacillus flora of environmental sample extracted by thermal cracking method
100 mg of activated sludge samples (code numbers NS and FT, respectively) collected from aeration tanks of two water purification plants were taken and put into a 1.5 mL sterile centrifuge tube, and 1 mL of TE buffer solution of pH = 7 was added thereto. Bathing in 95 deg.C wastewater for 5min, transferring into-20 deg.C refrigerator, freezing for 5min, and repeating the above steps once. The treated sample 1,2000 Xg was centrifuged for 1 min, and the supernatant obtained after centrifugation was transferred to a new 1.5 mL sterile centrifuge tube to obtain a sample total DNA solution. The PCR system is a 50 mu L system and comprises the following components: mu.L of total DNA solution, 2. mu.L of each of the upstream and downstream primers in example 1, 4. mu.L of dNTP, 5. mu.L of 10 XPCR buffer, 0.5U of DNA polymerase, and 50. mu.L of deionized water. The PCR run program was: pre-denaturation (95 ℃, 5 min); 30 cycles (denaturation: 95 ℃ C., 30 s; annealing: 55 ℃ C., 30 s; extension: 72 ℃ C., 1 kb/min); fully extending (72 ℃, 10 min); the resulting product was stored (4 ℃ C.) to obtain an amplification product A.
The amplification product B was obtained by replacing the upstream and downstream primers in example 1 with those in example 2 according to the above procedure, and keeping the other conditions unchanged.
Respectively detecting the amplification product A and the amplification product B by 0.8% agarose gel electrophoresis, wherein the genus specific primer is a specific primer of Acidithiobacillus, namely an ion efflux permease gene sequence amplification primer, as shown in figure 2; the species-specific primer refers to an acidophilic thiobacillus ferrooxidans species-specific primer, namely a thioquinone reductase gene sequence amplification primer; m1 and M2 represent DL2000 DNA marker respectively; CK1 and CK2 represent sterile deionized water controls, respectively, and it was confirmed that both samples contained Acidithiobacillus bacteria and Acidithiobacillus ferrooxidans bacteria.
Example 4 species composition analysis of Acidithiobacillus flora of environmental samples
The amplified products were TA cloned and 10 clones were picked for each sample for Sanger sequencing analysis to determine their sequence. And performing Blastn comparison by taking the sequence obtained by sequencing as a query sequence and the NR library as a database. Extracting the alignment result sequence with high similarity, introducing Bioedit software to perform local multiple sequence alignment, removing heads and tails to obtain multiple sequence files with consistent lengths, introducing the multiple sequence files into molecular evolution tree construction software Mega6, and constructing a molecular evolution junction tree (Neighbor junction tree). And preliminarily evaluating the species composition and abundance of each sample according to the dendrogram. The results show that the product sequences obtained by using the specific primers PCR of the ion efflux permease gene (genus) are mainly similar to the gene sequences of the ion efflux permease of three species of thiobacillus, including A. ferrivorans, Acidithiobacillus ferrooxidans and Acidithiobacillus caldus, and the abundance of the Acidithiobacillus caldus which may be enriched in the Acidithiobacillus caldus flora is high, as shown in FIG. 3; the product sequence obtained by PCR using the thioquinone reductase gene (species) -specific primers was mainly similar to the thioquinone reductase gene sequence of Thiobacillus acidophilus, but the Acidithiobacillus acidophilus flora in the FT and NS samples was clearly divided into two clusters and separated from the known sequences in the database, indicating that there may be a large difference in flora composition between the two and that there may be a new type (species) of Thiobacillus, as shown in FIG. 4.
In conclusion, the invention provides a primer pair for detecting Acidithiobacillus, a design method and a detection method, the specific primer designed by the design method can quickly and accurately detect Acidithiobacillus (species), and a similar method is not adopted to obtain the conserved CDSs (sequence-specific primers) of the Acidithiobacillus or the species so as to design a corresponding specific primer at present; meanwhile, the invention also provides a method for detecting the thiobacillus acidocaldarius based on the designed specific primer, and the detection method does not need to build a library, does not need to extract the total DNA of a sample, and has the advantages of strong specificity, high sensitivity and high speed.
It is to be understood that the invention is not limited to the examples described above, but that modifications and variations may be effected thereto by those of ordinary skill in the art in light of the foregoing description, and that all such modifications and variations are intended to be within the scope of the invention as defined by the appended claims.
Claims (2)
1. A primer set for detecting Acidithiobacillus is characterized in that,
the method comprises the following steps:
primer pairs for the detection of Acidithiobacillus:
the sequence of the upstream primer is 5'-CGAGAGCACAGCCAACTGGAACTC-3',
the sequence of the downstream primer is 5'-GCGAGATAG ATGCCATGCGCGA-3';
the design method adopted by the primer pair for detecting the Acidithiobacillus comprises the following steps:
step A1: extracting CDSs (sequence variants) according to the complete genome sequence of the Acidithiobacillus model species strain in the NCBI;
step B1: taking the extracted CDSs as query sequences, performing Blastn comparison on an NR database with the eosinophilia sequences removed, taking the CDSs not compared as query sequences, performing Blastn comparison on the eosinophilia sequences, and extracting CDSs sequences capable of being compared with all eosinophilia strains in the NR database to obtain eosinophilia conservative CDSs sequences;
step C1: and designing a primer pair for detecting the Acidithiobacillus according to the conservative CDSs of the Acidithiobacillus and a primer design principle.
2. A method for detecting Acidithiobacillus, comprising:
step A3: extracting DNA of a sample to be detected, and thermally cracking the DNA of the sample to obtain a DNA single chain;
step B3: performing PCR amplification on the DNA single strand by using the primer pair of claim 1 to obtain a PCR amplification product;
step C3: carrying out agarose gel electrophoresis detection on the PCR amplification product;
wherein the step A3 includes the steps of: adding a sample to be detected into a TE buffer solution, performing water bath at 92-98 ℃ for 4-6 min, then freezing at-18-22 ℃ for 4-6 min, repeating the water bath and freezing steps once, and performing separation treatment to obtain a supernatant containing the DNA single strand;
in the step B3, the PCR program is set to 30 cycles;
after the step C3, the method further includes:
step D3: sequencing the amplification product and constructing a molecular evolution tree for judging the composition of the Acidithiobacillus flora in the sample.
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| "Gene Turnover Contributes to the Evolutionary Adaptation of Acidithiobacillus caldus：Insights from Comparative Genomics";Xian Zhang等;《Frontiers in Microbiology》;20161206;第7卷;摘要，第2页右栏第3段-第10页右栏第2段，图1-4 * |
| "嗜酸硫杆菌属物种的比较基因组学分析";李净净 等;《生物技术》;20141231;第26卷(第6期);摘要，第1.1-1.2节、2.1节，图4 * |
| "源自嗜酸氧化亚铁硫杆菌的硫化物：醌氧化还原酶的特征、结构和机理研究";张燕飞;《中国博士学位论文全文数据库 基础科学辑》;20150115(第1期);摘要，正文第2.1节、2.2.6节、2.3.1-2.3.3节、6.1-6.3节 * |
| "食源性致病菌特异性靶点发掘系统及数据库的构建";余水静;《中国博士学位论文全文数据库 医药卫生科技辑》;20120715(第07期);摘要，正文第3.1.6-3.1.7节、3.2.1节、3.3节、4.1.7-4.1.8节、4.2.1节、4.3节、5.1.2节、5.3节 * |
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| Publication number | Publication date |
|---|---|
| WO2019227611A1 (en) | 2019-12-05 |
| CN108728557A (en) | 2018-11-02 |
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