WO2019227611A1 - Primer pair for detecting acidithiobacillus, design method and detection method - Google Patents

Primer pair for detecting acidithiobacillus, design method and detection method Download PDF

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WO2019227611A1
WO2019227611A1 PCT/CN2018/095406 CN2018095406W WO2019227611A1 WO 2019227611 A1 WO2019227611 A1 WO 2019227611A1 CN 2018095406 W CN2018095406 W CN 2018095406W WO 2019227611 A1 WO2019227611 A1 WO 2019227611A1
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sequence
thiobacillus
cdss
acidophilus
thiobacillus acidophilus
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毛艳萍
蔡勋超
刘长坤
杨波
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深圳大学
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • the invention relates to the technical field of biological detection, in particular to a primer pair, a design method and a detection method for detecting Thiobacillus acidophilus.
  • Biohydrometallurgical technology has become one of the most promising technologies for leaching and pretreatment of lean ore.
  • the most widely used of leaching microorganisms is Acidithiobacillus sp.
  • the species commonly associated with leaching applications are Thiobacillus acidophilus, Acidithiobacillus thiooxidans, and Acidithiobacillus caldus.
  • the detection and species composition analysis of Thiobacillus acidophilus (species) in environmental samples mainly uses high-throughput sequencing methods of 16S rRNA amplicon sequencing and metagenomic sequencing.
  • these two methods are costly, time-consuming, and require extraction of high-quality total DNA from the sample.
  • the cell concentration of Thiobacillus acidophilus (species) is very low, if the sequencing depth is not enough, its sequence can easily be masked by the abundant species and cause false negative results. Being able to detect it also requires a lot of human and financial resources.
  • an object of the present invention is to provide a primer pair, a design method, and a detection method for detecting Thiobacillus acidophilus, which aims to solve the high cost, time-consuming, and acidophilic sulfur of existing detection methods.
  • the cell concentration of Bacillus (species) is very low, false negative results are prone to occur.
  • a method for designing a primer pair for detecting Thiobacillus acidophilus including the following:
  • Step A1 Extract the CDSs sequence based on the complete genome sequence of the strain of Thiobacillus acidophilus in NCBI;
  • Step B1 Using the extracted CDSs sequence as the query sequence, Blastn compares the NR database that removes the Thiobacillus acidophilus sequence, using the unaligned CDSs sequence as the query sequence, Blastn compares the Thiobacillus acidophilus sequence, and extract The CDSs sequences of all Thiobacillus acidophilus strains on the NR database can be compared to obtain the conservative CDSs sequences of Thiobacillus acidophilus;
  • Step C1 Design a primer pair for detecting Thiobacillus acidophilus according to the conservative CDSs sequence and primer design principles of Thiobacillus acidophilus;
  • step A2 extracting the CDSs sequence according to the complete genome sequence of the target strain of Thiobacillus acidophilus in NCBI;
  • Step B2 Using the extracted CDSs sequence as the query sequence, Blastn compares the NR database that removes the target species sequence of Thiobacillus acidophilus, using the unaligned CDSs sequence as the query sequence, and Blastn compares the target species sequence of Thiobacillus acidophilus.
  • the CDSs sequences of all target strains of Thiobacillus acidophilus on the NR database can be compared to obtain the conserved CDSs sequences of target species of Thiobacillus acidophilus;
  • Step C2 According to the conserved CDSs sequence of the target species of Thiobacillus acidophilus and the principle of primer design, design the primer pair for detecting the target species of Thiobacillus acidophilus.
  • a primer pair for detecting Thiobacillus acidophilus is designed using the design method described above.
  • the primer pair for detecting Thiobacillus acidophilus includes:
  • the upstream primer sequence is 5'-CGAGAGCACAGCCAACTGGAACTC-3 ',
  • the downstream primer sequence is 5'-GCGAGATAG ATGCCATGCGCGA-3 ';
  • the upstream primer sequence is 5'-GGCACATGTGGTAATTTTGGGT-3 ';
  • the downstream primer sequence is 5'-CCCGTAGGCAAAGACGTCC-3 '.
  • a method for detecting Thiobacillus acidophilus including the following:
  • Step A3 extracting the DNA of the sample to be tested, and thermally cleaving the DNA of the sample to obtain a single-stranded DNA;
  • Step B3 Use the primer pair as described above to perform PCR amplification on the DNA single strand to obtain a PCR amplification product
  • Step C3 Perform the agarose gel electrophoresis detection on the PCR amplification product.
  • the step A3 includes the steps of adding a sample to be tested to a TE buffer solution, a water bath at 92 to 98 ° C for 4 to 6 minutes, and then at -18 to -22 ° C. Freeze for 4 to 6 minutes, repeat the steps of water bath and freezing once, and then perform separation treatment to obtain the supernatant containing the DNA single strand.
  • the method for detecting Thiobacillus acidophilus wherein in the step B3, the PCR program is set to 30 cycles.
  • the method for detecting Thiobacillus acidophilus wherein after step C3, the method further comprises:
  • Step D3 Sequencing the amplified product and constructing a molecular evolution tree for determining the composition of the Thiobacillus acidophilus flora in the sample or the evolutionary clustering relationship between the same strains.
  • the present invention provides a method for designing a primer pair for detecting Thiobacillus acidophilus as described above.
  • the primer pair designed by this method detects Thiobacillus acidophilus, it can quickly and accurately detect sulfur acidophilus.
  • Bacillus (species), with strong specificity and high sensitivity, has not yet adopted a similar method to obtain the conserved CDSs sequence of Thiobacillus acidophilus or species and then design corresponding specific primers.
  • FIG. 1 is a flowchart of obtaining a CDSs sequence and designing specific primers of Thiobacillus acidophilus (species) in Examples 1 and 2 of the present invention.
  • FIG. 2 is an electrophoresis diagram of a PCR product for rapid identification of Thiobacillus acidophilus (species) specific primers in Examples 1 and 2 of the present invention.
  • FIG. 3 is an analysis result of the PCR product of the Thiobacillus acidophilus-specific primer in Example 1 of the present invention.
  • FIG. 4 is an analysis result of the PCR product of Thiobacillus ferrooxidans species-specific primers in Example 2 of the present invention.
  • the present invention provides a primer pair, a design method, and a detection method for detecting Thiobacillus acidophilus.
  • the present invention provides a preferred embodiment of a method for designing a primer pair for detecting Thiobacillus acidophilus, including the following steps:
  • a conserved sequence of a specific Thiobacillus acidophilus species can be obtained. Specifically, after determining a Thiobacillus acidophilus species to be detected and identified (ie, a target species of Thiobacillus acidophilus, referred to as a target species), the target CDSs sequence of several model strains. Using this as the query sequence, the Blastn default parameter alignment was used to remove the NR database of the target species model strain sequence, and the unaligned CDSs sequences were extracted. Using this as the query sequence, Blastn compared all the target species in the NR database. Sequence, and extract the CDSs sequences of all target strains that can be compared on the NR library, which is the conserved CDSs sequences of the target species of Thiobacillus acidophilus.
  • the present invention takes the CDS sequence of the permease gene sequence (genus) as an example, and the specific amplified upstream and downstream primer sequences designed to obtain the ion permease gene sequence (genus) are:
  • the upstream primer sequence is 5'-CGAGAGCACAGCCAACTGGAACTC-3 ',
  • the downstream primer sequence is 5'-GCGAGATAG ATGCCATGCGCGA-3 '.
  • the design process of the specific primers of the target species of Thiobacillus acidophilus is to extract all the conserved sequences of a CDS of the target species of Thiobacillus acidophilus, and use Bioedit software to perform an alignment analysis to determine that the CDS is in the CDS sequences of all strains of the species Based on this conserved sequence segment, specific PCR primers are designed to obtain rapid detection primers for the target species of Thiobacillus acidophilus.
  • the present invention takes the CDS sequence of a thioquinone reductase gene (species) as an example, and the specific amplified upstream and downstream primer sequences designed to obtain the thioquinone reductase gene (species) are:
  • the upstream primer sequence is 5'-GGCACATGTGGTAATTTTGGGT-3 ';
  • the downstream primer sequence is 5'-CCCGTAGGCAAAGACGTCC-3 '.
  • the specific primers designed by the above-mentioned design method of the present invention can quickly and accurately detect Thiobacillus acidophilus (species). At present, a similar method has not been adopted to obtain the conserved CDSs sequence of Thiobacillus acidophilus or species for further design. Corresponding specific primers.
  • the present invention also provides a preferred embodiment of a method for detecting Thiobacillus acidophilus, including the following:
  • the test tube is then subjected to a water bath at 92 to 98 ° C (preferably 95 ° C) for 4 to 6 minutes (preferably 5 minutes), and then frozen at -18 to -22 ° C (preferably -20 ° C) for 4 to 6 minutes (preferably 5 minutes).
  • the freezing step is performed once, and then a separation process is performed to obtain a supernatant containing the DNA single strand.
  • the above-mentioned DNA single-stranded supernatant was used as a PCR reaction template, and the synthesized upstream and downstream primers were added, and the PCR reaction system was prepared according to the manufacturer's instructions of the PCR enzyme solution (DNA polymerase, PCR buffer, dNTP).
  • the PCR program was set to 30 cycles, the primer annealing temperature was set according to the software calculated by the software Premier 5 and the extension time was set by the length of the PCR product (1 kb / min).
  • the PCR product is detected by agarose gel electrophoresis. If a single band of the expected size appears, it can be determined that the sample contains a bacterium of the genus Thiobacillus acidophilus or a specific species.
  • the PCR product was cloned by TA, and dozens of clones were selected for Sanger sequencing. The determined sequence was used as the query sequence.
  • the NR database was compared with Blastn, and the sequence on the alignment was extracted to construct a molecular evolution tree. The acidophilicity of the sample can be further determined. Composition of Thiobacillus flora and evolutionary clustering relationships among the same strains.
  • the method for detecting Thiobacillus acidophilus (species) of the present invention does not need to build a library and does not need to extract the total DNA of the sample, and has the advantages of strong specificity, high sensitivity and fast speed.
  • Blastn aligns the acidophilic thiobacillus sequence (Acidithiobacillus (taxid: 119977)), and extract CDSs that can align all thiobacillus acidophilus strains on the NR library. Sequence, this is the Thiobacillus acidophilus conserved CDSs sequence.
  • the design flowchart is shown in Figure 1.
  • the obtained Thiobacillus acidophilus conserved CDSs sequences were imported into the software Bioedit for local rapid alignment (Alignment).
  • the primers for the design of the permease gene sequence (genus) of the iontophoresis were designed.
  • the upstream and downstream primer sequences are:
  • the CDSs sequence of the model strain Thiobacillus acidophilus ATCC23270 was obtained.
  • the NR database of acidothioferillus ferrooxidans sequences (taxid: 920) was removed using Blastn default parameter alignment.
  • the results of the Blastn alignment are the conserved sequences that exist between the species of Acidophilus ferrooxidans and other species. Extract the unaligned CDSs sequence and use it as the query sequence.
  • Blastn aligns the acidophilic ferrooxidans sequences (Acidithiobacillus ferrooxidans (taxid: 920)), and extract all eosinophilic suboxidants that can be compared on the NR library.
  • the CDSs sequence of the Thiobacillus species this is the conserved CDSs sequence of the Thiobacillus acidophilus species.
  • the design flowchart is shown in Figure 1.
  • the PCR system was a 50 ⁇ L system, consisting of 2 ⁇ L total DNA solution, 2 ⁇ L each of the upstream and downstream primers in Example 1, 4 ⁇ L dNTP, 5 ⁇ L of a 10 ⁇ PCR buffer, 0.5 U of DNA polymerase, and 50 ⁇ L of deionized water to supplement the system.
  • the PCR running program is: pre-denaturation (95 ° C, 5min); 30 cycles (denaturation: 95 ° C, 30s; annealing: 55 ° C, 30s; extension: 72 ° C, 1kb / min); full extension (72 ° C, 10min) ; Save (4 ° C) to obtain amplified product A.
  • Example 1 the upstream and downstream primers in Example 1 were replaced with the upstream and downstream primers in Example 2, and other conditions remained unchanged to obtain an amplified product B.
  • the gene-specific primer refers to a Thiobacillus acidophilus-specific primer, that is, an ion permease gene. Sequence amplification primers; species-specific primers refer to the species-specific primers of Thiobacillus acidophilus, that is, the thioquinone reductase gene sequence amplification primers; M1 and M2 represent DL2000 DNA maker respectively; CK1 and CK2 represent sterile In deionized water control, it was determined that both samples contained Thiobacillus acidophilus and Thiobacillus ferrooxidans.
  • the amplified product was TA cloned, and 10 clones were selected from each sample for Sanger sequencing analysis to determine its sequence.
  • the sequence obtained by sequencing was used as the query sequence, and the NR library was used as the database for Blastn alignment. Extract the alignment sequence with high similarity, import it into Bioedit software for local multiple sequence alignment, get rid of heads and tails, obtain multiple sequence files with the same length, and import it into the molecular evolution tree construction software Mega6 to build molecular evolution adjacency trees ( Neighbor joining tree.) Based on the tree diagram, the species composition and abundance of each sample were initially evaluated.
  • the product sequence is mainly similar to the gene sequence of Thiobacillus ferrooxidans thioquinone reductase, but the Thiobacillus ferrooxidans bacteria in the FT and NS samples are clearly divided into two clusters and separated from the known sequence in the database. There may be large differences in the composition of the flora of these individuals, and a new type (species) of Thiobacillus may be present, as shown in FIG. 4.
  • the present invention provides a primer pair for detecting Thiobacillus acidophilus and a design method and detection method.
  • the specific primer designed by the design method of the present invention can quickly and accurately detect Thiobacillus acidophilus.
  • Genus (species), currently has not adopted a similar method to obtain the conserved CDSs sequence of Thiobacillus acidophilus or species and then design the corresponding specific primers; meanwhile, the present invention also provides a method for detecting In the method of Thiobacillus acidi, the detection method does not need to establish a library and does not need to extract the total DNA of the sample, and has the advantages of strong specificity, high sensitivity and fast speed.

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Abstract

A primer pair for detecting acidithiobacillus, and a design method and a detection method, wherein the design method comprises: extracting CDSs sequences according to complete genome sequences of model species strains of acidithiobacillus sp. in NCBI; and obtaining conserved CDSs sequences of the acidithiobacillus sp. by means of Blastn alignment, and obtaining a primer pair for detecting the acidithiobacillus sp. by design according to a primer design principle. The primer pair designed using the method can quickly and accurately detect the acidithiobacillus sp. (genera) during acidithiobacillus detection, and has high specificity and sensitivity. At present, no similar method has been utilized to obtain the conserved CDSs sequences of the acidithiobacillus sp. or genera so as to design corresponding specific primers.

Description

用于检测嗜酸硫杆菌的引物对及设计方法与检测方法Primer pair for detecting Thiobacillus acidophilus, design method and detection method 技术领域Technical field
本发明涉及生物检测技术领域，尤其涉及一种用于检测嗜酸硫杆菌的引物对及设计方法与检测方法。The invention relates to the technical field of biological detection, in particular to a primer pair, a design method and a detection method for detecting Thiobacillus acidophilus.
背景技术Background technique
生物湿法冶金技术已成为贫矿浸取与预处理最具潜力的技术之一，在浸矿微生物中应用最为广泛的是嗜酸硫杆菌属(Acidithiobacillus sp.)。嗜酸硫杆菌属中，常见的与浸矿应用相关的物种有嗜酸性氧化亚铁硫杆菌、嗜酸性氧化硫硫杆菌(Acidithiobacillus thiooxidans)和嗜酸性喜温硫杆菌(Acidithiobacillus caldus)等。Biohydrometallurgical technology has become one of the most promising technologies for leaching and pretreatment of lean ore. The most widely used of leaching microorganisms is Acidithiobacillus sp. Among the genus Acidophilus, the species commonly associated with leaching applications are Thiobacillus acidophilus, Acidithiobacillus thiooxidans, and Acidithiobacillus caldus.
目前，对环境样品中嗜酸硫杆菌属(种)的检测及物种组成分析，主要采用16S rRNA扩增子测序和宏基因组测序的高通量测序方法。但是这两种方法成本高、耗时长，且需要提取高质量样品总DNA。此外，对于物种多样性丰富的环境样品，由于嗜酸硫杆菌属(种)的细胞浓度非常低，测序深度如果不够，其序列很容易被丰度高的物种掩盖而造成假阴性结果，而且即便能检测出来，也需要投入大量的人力与财力。At present, the detection and species composition analysis of Thiobacillus acidophilus (species) in environmental samples mainly uses high-throughput sequencing methods of 16S rRNA amplicon sequencing and metagenomic sequencing. However, these two methods are costly, time-consuming, and require extraction of high-quality total DNA from the sample. In addition, for environmental samples with rich species diversity, because the cell concentration of Thiobacillus acidophilus (species) is very low, if the sequencing depth is not enough, its sequence can easily be masked by the abundant species and cause false negative results. Being able to detect it also requires a lot of human and financial resources.
因此，现有技术还有待于改进和发展。Therefore, the existing technology needs to be improved and developed.
发明内容Summary of the invention
鉴于上述现有技术的不足，本发明的目的在于提供一种用于检测嗜酸硫杆菌的引物对及设计方法与检测方法，旨在解决现有的检测方法成本高、耗时长以及嗜酸硫杆菌属(种)的细胞浓度非常低时易出现假阴性结果。In view of the above-mentioned shortcomings of the prior art, an object of the present invention is to provide a primer pair, a design method, and a detection method for detecting Thiobacillus acidophilus, which aims to solve the high cost, time-consuming, and acidophilic sulfur of existing detection methods. When the cell concentration of Bacillus (species) is very low, false negative results are prone to occur.
本发明的技术方案如下：The technical scheme of the present invention is as follows:
一种用于检测嗜酸硫杆菌的引物对的设计方法，包括如下：A method for designing a primer pair for detecting Thiobacillus acidophilus, including the following:
步骤A1、根据NCBI中的嗜酸性硫杆菌属模式物种菌株的基因组全序列，提取CDSs序列；Step A1: Extract the CDSs sequence based on the complete genome sequence of the strain of Thiobacillus acidophilus in NCBI;
步骤B1、以提取的CDSs序列为查询序列，Blastn比对除去嗜酸性硫杆菌属序列的NR数据库，以未比对上的CDSs序列为查询序列，Blastn比对嗜酸性硫杆菌属序列，并提取能比对上NR数据库所有嗜酸性硫杆菌属菌株的CDSs序列，得到嗜酸硫杆菌属保守CDSs序列；Step B1: Using the extracted CDSs sequence as the query sequence, Blastn compares the NR database that removes the Thiobacillus acidophilus sequence, using the unaligned CDSs sequence as the query sequence, Blastn compares the Thiobacillus acidophilus sequence, and extract The CDSs sequences of all Thiobacillus acidophilus strains on the NR database can be compared to obtain the conservative CDSs sequences of Thiobacillus acidophilus;
步骤C1、根据所述嗜酸硫杆菌属保守CDSs序列及引物设计原则，设计得到用于检测嗜酸硫杆菌属的引物对；Step C1: Design a primer pair for detecting Thiobacillus acidophilus according to the conservative CDSs sequence and primer design principles of Thiobacillus acidophilus;
或者步骤A2、根据NCBI中的嗜酸性硫杆菌目标种模式菌株的基因组全序列，提取CDSs序列；Or step A2, extracting the CDSs sequence according to the complete genome sequence of the target strain of Thiobacillus acidophilus in NCBI;
步骤B2、以提取的CDSs序列为查询序列，Blastn比对除去嗜酸性硫杆菌目标种序列的NR数据库，以未比对上的CDSs序列为查询序列，Blastn比对嗜酸性硫杆菌目标种序列，并提取能比对上NR数据库所有嗜酸性硫杆菌目标种菌株的CDSs序列，得到嗜酸性硫杆菌目标种保守CDSs序列；Step B2: Using the extracted CDSs sequence as the query sequence, Blastn compares the NR database that removes the target species sequence of Thiobacillus acidophilus, using the unaligned CDSs sequence as the query sequence, and Blastn compares the target species sequence of Thiobacillus acidophilus. The CDSs sequences of all target strains of Thiobacillus acidophilus on the NR database can be compared to obtain the conserved CDSs sequences of target species of Thiobacillus acidophilus;
步骤C2、根据所述嗜酸性硫杆菌目标种保守CDSs序列及引物设计原则，设计得到所述用于检测嗜酸性硫杆菌目标种的引物对。Step C2: According to the conserved CDSs sequence of the target species of Thiobacillus acidophilus and the principle of primer design, design the primer pair for detecting the target species of Thiobacillus acidophilus.
一种用于检测嗜酸硫杆菌的引物对，采用如上所述的设计方法设计而成。A primer pair for detecting Thiobacillus acidophilus is designed using the design method described above.
所述的用于检测嗜酸硫杆菌的引物对，包括：The primer pair for detecting Thiobacillus acidophilus includes:
用于检测嗜酸硫杆菌属的引物对：Primer pairs for detecting Thiobacillus acidophilus:
上游引物序列为5’-CGAGAGCACAGCCAACTGGAACTC-3’，The upstream primer sequence is 5'-CGAGAGCACAGCCAACTGGAACTC-3 ',
下游引物序列为5’-GCGAGATAG ATGCCATGCGCGA-3’；The downstream primer sequence is 5'-GCGAGATAG ATGCCATGCGCGA-3 ';
或者用于检测嗜酸性氧化亚铁硫杆菌种的引物对：Or primer pairs for the detection of acidophilus Thiobacillus ferrooxidans species:
上游引物序列为5’-GGCACATGTGGTAATTTTGGGT-3’；The upstream primer sequence is 5'-GGCACATGTGGTAATTTTGGGT-3 ';
下游引物序列为5’-CCCGTAGGCAAAGACGTCC-3’。The downstream primer sequence is 5'-CCCGTAGGCAAAGACGTCC-3 '.
一种检测嗜酸硫杆菌的方法，包括如下：A method for detecting Thiobacillus acidophilus, including the following:
步骤A3、提取待测样品的DNA，并将样品的DNA热裂解，得到DNA单链；Step A3, extracting the DNA of the sample to be tested, and thermally cleaving the DNA of the sample to obtain a single-stranded DNA;
步骤B3、采用如上所述的引物对，对所述DNA单链进行PCR扩增，得到PCR扩增产物；Step B3: Use the primer pair as described above to perform PCR amplification on the DNA single strand to obtain a PCR amplification product;
步骤C3、将所述PCR扩增产物进行琼脂糖凝胶电泳检测。Step C3: Perform the agarose gel electrophoresis detection on the PCR amplification product.
所述的检测嗜酸硫杆菌的方法，其中，所述步骤A3包括步骤：将待测样品加入到TE缓冲液中，于92～98℃下水浴4～6min，然后于-18～-22℃下冷冻4～6min，重复水浴和冷冻的步骤一次，再进行分离处理得到含有所述DNA单链的上清液。In the method for detecting Thiobacillus acidophilus, the step A3 includes the steps of adding a sample to be tested to a TE buffer solution, a water bath at 92 to 98 ° C for 4 to 6 minutes, and then at -18 to -22 ° C. Freeze for 4 to 6 minutes, repeat the steps of water bath and freezing once, and then perform separation treatment to obtain the supernatant containing the DNA single strand.
所述的检测嗜酸硫杆菌的方法，其中，所述步骤B3中，PCR程序设置为30个循环。The method for detecting Thiobacillus acidophilus, wherein in the step B3, the PCR program is set to 30 cycles.
所述的检测嗜酸硫杆菌的方法，其中，所述步骤C3之后，还包括：The method for detecting Thiobacillus acidophilus, wherein after step C3, the method further comprises:
步骤D3、将所述扩增产物进行测序并构建分子进化树，用于判断样品中的嗜酸硫杆菌属菌群的组成或同种菌株之间的进化聚类关系。Step D3: Sequencing the amplified product and constructing a molecular evolution tree for determining the composition of the Thiobacillus acidophilus flora in the sample or the evolutionary clustering relationship between the same strains.
有益效果：本发明提供了一种如上所述的用于检测嗜酸硫杆菌的引物对的设计方法，本方法设计的引物对检测嗜酸硫杆菌时，能够快速、准确地检测出嗜酸硫杆菌属(种)，特异性强、灵敏度高，目前还没有采用类似的方法来获取嗜酸硫杆菌属或种的保守CDSs序列进而设计相应的特异性引物。Beneficial effect: The present invention provides a method for designing a primer pair for detecting Thiobacillus acidophilus as described above. When the primer pair designed by this method detects Thiobacillus acidophilus, it can quickly and accurately detect sulfur acidophilus. Bacillus (species), with strong specificity and high sensitivity, has not yet adopted a similar method to obtain the conserved CDSs sequence of Thiobacillus acidophilus or species and then design corresponding specific primers.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1为本发明实施例1和实施例2中嗜酸硫杆菌属(种)保守CDSs序列获取及特异性引物设计流程图。FIG. 1 is a flowchart of obtaining a CDSs sequence and designing specific primers of Thiobacillus acidophilus (species) in Examples 1 and 2 of the present invention.
图2为本发明实施例1和实施例2中嗜酸硫杆菌属(种)特异性引物快速鉴定PCR产物电泳图。FIG. 2 is an electrophoresis diagram of a PCR product for rapid identification of Thiobacillus acidophilus (species) specific primers in Examples 1 and 2 of the present invention.
图3为本发明实施例1中的嗜酸硫杆菌属特异性引物PCR产物分析结果。FIG. 3 is an analysis result of the PCR product of the Thiobacillus acidophilus-specific primer in Example 1 of the present invention.
图4为本发明实施例2中的嗜酸性氧化亚铁硫硫杆菌种特异性引物PCR产物分析结果。FIG. 4 is an analysis result of the PCR product of Thiobacillus ferrooxidans species-specific primers in Example 2 of the present invention.
具体实施方式Detailed ways
本发明提供了一种用于检测嗜酸硫杆菌的引物对及设计方法与检测方法，为使本发明的目的、技术方案及效果更加清楚、明确，以下对本发明进一步详细说明。应当理解，此处所描述的具体实施例仅用以解释本发明，并不用于限定本发明。The present invention provides a primer pair, a design method, and a detection method for detecting Thiobacillus acidophilus. In order to make the purpose, technical solution, and effect of the present invention clearer and more specific, the present invention is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.
本发明提供了一种用于检测嗜酸硫杆菌的引物对的设计方法的较佳实施例，包括如下步骤：The present invention provides a preferred embodiment of a method for designing a primer pair for detecting Thiobacillus acidophilus, including the following steps:
(1)保守序列的获取(1) Acquisition of conservative sequences
从美国国家生物技术信息中心(NCBI，The National Center for Biotechnology Information)数据库下载嗜酸性硫杆菌属模式物种菌株的基因组全序列，并提取其CDSs(编码序列，为CDS的复数形式)序列。然后以提取的CDSs为查询序列(query sequence)，使用Blastn默认参数比对除去嗜酸性硫杆菌属序列的NR数据库。撰写perl语言脚本提取未比对上的CDSs序列，并以其为查询序列，Blastn 比对NR数据库中所有嗜酸性硫杆菌属序列，提取在每个物种中出现的序列，即获得嗜酸硫杆菌属保守CDSs序列。Download the complete genome sequence of the Thiobacillus acidophilus model species strain from the National Center for Biotechnology Information (NCBI) database and extract its CDSs (coding sequence, the plural form of CDS) sequence. Then, the extracted CDSs are used as a query sequence, and the Blastn default parameters are used to compare and remove the NR database of Thiobacillus acidophilus sequences. Write a Perl script to extract unaligned CDSs sequences and use them as query sequences. Blastn compares all Thiobacillus acidophilus sequences in the NR database and extracts the sequences that appear in each species to obtain Thiobacillus acidophilus. Is a conserved CDSs sequence.
按照类似的方法，可以获得特定的嗜酸性硫杆菌种的保守序列，具体为，确定将要检测、鉴定的嗜酸性硫杆菌种(即嗜酸性硫杆菌目标种，简称目标种)后，获得该目标种模式菌株的CDSs序列。以其为查询序列，使用Blastn默认参数比对除去该目标种模式菌株序列的NR数据库，并提取未比对上的CDSs序列，以其为查询序列，Blastn比对NR数据库中所有该目标种的序列，并提取能比对上NR库所有该目标种菌株的CDSs序列，此即该嗜酸性硫杆菌目标种的保守CDSs序列。According to a similar method, a conserved sequence of a specific Thiobacillus acidophilus species can be obtained. Specifically, after determining a Thiobacillus acidophilus species to be detected and identified (ie, a target species of Thiobacillus acidophilus, referred to as a target species), the target CDSs sequence of several model strains. Using this as the query sequence, the Blastn default parameter alignment was used to remove the NR database of the target species model strain sequence, and the unaligned CDSs sequences were extracted. Using this as the query sequence, Blastn compared all the target species in the NR database. Sequence, and extract the CDSs sequences of all target strains that can be compared on the NR library, which is the conserved CDSs sequences of the target species of Thiobacillus acidophilus.
(2)特异性引物的设计(2) Design of specific primers
提取嗜酸硫杆菌属的某一CDS的所有保守序列，使用Bioedit软件进行比对分析，确定该CDS在嗜酸硫杆菌属所有物种CDS序列中的保守序列区段，根据该保守序列区段设计特异性PCR引物，获得嗜酸硫杆菌属的快速检测引物。All conserved sequences of a certain CDS of Thiobacillus acidophilus were extracted and compared using Bioedit software to determine the conserved sequence segments of the CDS in the CDS sequences of all species of Thiobacillus acidophilus. Designed based on the conserved sequence segments Specific PCR primers for rapid detection of Thiobacillus acidophilus.
本发明以离子外流通透酶基因序列(属)的CDS序列为例，设计得到离子外流通透酶基因序列(属)的特异扩增上、下游引物序列分别为：The present invention takes the CDS sequence of the permease gene sequence (genus) as an example, and the specific amplified upstream and downstream primer sequences designed to obtain the ion permease gene sequence (genus) are:
上游引物序列为5’-CGAGAGCACAGCCAACTGGAACTC-3’，The upstream primer sequence is 5'-CGAGAGCACAGCCAACTGGAACTC-3 ',
下游引物序列为5’-GCGAGATAG ATGCCATGCGCGA-3’。The downstream primer sequence is 5'-GCGAGATAG ATGCCATGCGCGA-3 '.
嗜酸性硫杆菌目标种的特异性引物的设计过程为：提取嗜酸性硫杆菌目标种的某一CDS的所有保守序列，使用Bioedit软件进行比对分析，确定该CDS在该种所有菌株CDS序列中的保守序列区段，根据该保守序列区段设计特异性PCR引物，获得嗜酸性硫杆菌目标种的快速检测引物。The design process of the specific primers of the target species of Thiobacillus acidophilus is to extract all the conserved sequences of a CDS of the target species of Thiobacillus acidophilus, and use Bioedit software to perform an alignment analysis to determine that the CDS is in the CDS sequences of all strains of the species Based on this conserved sequence segment, specific PCR primers are designed to obtain rapid detection primers for the target species of Thiobacillus acidophilus.
本发明以硫醌还原酶基因(种)的CDS序列为例，设计得到硫醌还原酶基因(种)的特异扩增上、下游引物序列分别为：The present invention takes the CDS sequence of a thioquinone reductase gene (species) as an example, and the specific amplified upstream and downstream primer sequences designed to obtain the thioquinone reductase gene (species) are:
上游引物序列为5’-GGCACATGTGGTAATTTTGGGT-3’；The upstream primer sequence is 5'-GGCACATGTGGTAATTTTGGGT-3 ';
下游引物序列为5’-CCCGTAGGCAAAGACGTCC-3’。The downstream primer sequence is 5'-CCCGTAGGCAAAGACGTCC-3 '.
本发明通过上述设计方法设计的特异性引物，能够快速、准确地检测出嗜酸硫杆菌属(种)，目前还没有采用类似的方法来获取嗜酸硫杆菌属或种的保守CDSs序列进而设计相应的特异性引物。The specific primers designed by the above-mentioned design method of the present invention can quickly and accurately detect Thiobacillus acidophilus (species). At present, a similar method has not been adopted to obtain the conserved CDSs sequence of Thiobacillus acidophilus or species for further design. Corresponding specific primers.
本发明还提供了一种检测嗜酸硫杆菌的方法的较佳实施例，包括如下：The present invention also provides a preferred embodiment of a method for detecting Thiobacillus acidophilus, including the following:
(1)样品总DNA热裂解(1) The total DNA of the sample is thermally lysed
取环境样品于无菌离心管中，并向其中加入TE缓冲液。然后将试管于92～98℃(优选95℃)下水浴4～6min(优选5min)，然后于-18～-22℃(优选-20℃)下冷冻4～6min(优选5min)，重复水浴和冷冻的步骤一次，再进行分离处理得到含有所述DNA单链的上清液。Take environmental samples in sterile centrifuge tubes and add TE buffer to them. The test tube is then subjected to a water bath at 92 to 98 ° C (preferably 95 ° C) for 4 to 6 minutes (preferably 5 minutes), and then frozen at -18 to -22 ° C (preferably -20 ° C) for 4 to 6 minutes (preferably 5 minutes). The freezing step is performed once, and then a separation process is performed to obtain a supernatant containing the DNA single strand.
(2)PCR与样品物种组成分析(2) PCR and sample composition analysis
吸取上述DNA单链的上清液作为PCR反应模板，加入合成好的上下游引物，按照PCR酶液(DNA聚合酶、PCR缓冲液、dNTP)厂家说明书配制PCR反应体系。PCR程序设置为30个循环，引物退火温度按软件Premier Primer5计算所得设置，延伸时间由PCR产物长度设置(1kb/min)。The above-mentioned DNA single-stranded supernatant was used as a PCR reaction template, and the synthesized upstream and downstream primers were added, and the PCR reaction system was prepared according to the manufacturer's instructions of the PCR enzyme solution (DNA polymerase, PCR buffer, dNTP). The PCR program was set to 30 cycles, the primer annealing temperature was set according to the software calculated by the software Premier 5 and the extension time was set by the length of the PCR product (1 kb / min).
PCR产物经琼脂糖凝胶电泳检测，如出现预期大小的单一条带，则可判断该样品中含有嗜酸硫杆菌属或特定种的菌群。PCR产物经TA克隆，挑取数十条克隆进行Sanger测序，以测定序列为查询序列，用Blastn比对NR数据库，并提取比对上的序列构建分子进化树，即可进一步判断样品的嗜酸硫杆菌属菌群组成及同种菌株之间进化聚类关系。The PCR product is detected by agarose gel electrophoresis. If a single band of the expected size appears, it can be determined that the sample contains a bacterium of the genus Thiobacillus acidophilus or a specific species. The PCR product was cloned by TA, and dozens of clones were selected for Sanger sequencing. The determined sequence was used as the query sequence. The NR database was compared with Blastn, and the sequence on the alignment was extracted to construct a molecular evolution tree. The acidophilicity of the sample can be further determined. Composition of Thiobacillus flora and evolutionary clustering relationships among the same strains.
传统的16S rRNA扩增子测序和宏基因组测序的高通量方法，不仅成本高、耗时长，且需要提取高质量样品总DNA。本发明的嗜酸硫杆菌属(种)的检测方法，无需建库，不需提取样品总DNA，具有特异性强、灵敏度高及速度快的优点。Traditional 16S rRNA amplicon sequencing and metagenomic sequencing high-throughput methods are not only costly and time-consuming, but also require the extraction of high-quality total DNA from the sample. The method for detecting Thiobacillus acidophilus (species) of the present invention does not need to build a library and does not need to extract the total DNA of the sample, and has the advantages of strong specificity, high sensitivity and fast speed.
下面通过实施例对本发明进行详细说明。The present invention is described in detail below through examples.
实施例1嗜酸硫杆菌属特异性引物设计Example 1 Design of Thiobacillus acidophilus-specific primers
(1)嗜酸硫杆菌属保守CDSs序列的获得(1) Obtaining the conserved CDSs sequence of Thiobacillus
从NCBI中下载模式菌株嗜酸性氧化亚铁硫杆菌ATCC23270的基因组全序列(ftp://ftp.ncbi.nlm.ni-h.gov/genomes/all/GCF/000/021/485/GCF_000021485.1_ASM2148v1)，并提取其CDSs序列。以提取的CDSs为查询序列，使用Blastn默认参数比对除去嗜酸性硫杆菌属序列(exclude Acidithiobacillus(taxid:119977))的NR数据库。Blastn比对上的结果即为嗜酸性硫杆菌属与其他物种之间存在的保守序列。提取未比对上的CDSs序列，以其为查询序列，Blastn比对嗜酸性硫杆菌属序列(Acidithiobacillus(taxid:119977))，并提取能比对上NR库所有嗜酸 性硫杆菌属菌株的CDSs序列，此即嗜酸硫杆菌属保守CDSs序列。设计流程图见图1。Download the full genome sequence of the model strain Thiobacillus acidophilus ATCC23270 from NCBI (ftp://ftp.ncbi.nlm.ni-h.gov/genomes/all/GCF/000/021/485/GCF_000021485.1_ASM2148v1 ), And extract its CDSs sequence. With the extracted CDSs as the query sequence, the NR database that excludes Acidithiobacillus sequences (taxid: 119977) was removed using Blastn default parameter alignment. The result of the Blastn alignment is the conserved sequence between Thiobacillus acidophilus and other species. Extract the unaligned CDSs sequence and use it as the query sequence. Blastn aligns the acidophilic thiobacillus sequence (Acidithiobacillus (taxid: 119977)), and extract CDSs that can align all thiobacillus acidophilus strains on the NR library. Sequence, this is the Thiobacillus acidophilus conserved CDSs sequence. The design flowchart is shown in Figure 1.
(2)嗜酸硫杆菌属特异性引物设计(2) Design of Thiobacillus acidophilus-specific primers
将获得的嗜酸硫杆菌属保守CDSs序列导入软件Bioedit，进行本地快速比对(Alignment)。以离子外流通透酶基因序列(属)的CDS序列为例，根据引物设计的基本原则，设计离子外流通透酶基因序列(属)扩增引物。上、下游引物序列分别为：The obtained Thiobacillus acidophilus conserved CDSs sequences were imported into the software Bioedit for local rapid alignment (Alignment). Taking the CDS sequence of the permease gene sequence (genus) as an example, according to the basic principles of primer design, the primers for the design of the permease gene sequence (genus) of the iontophoresis were designed. The upstream and downstream primer sequences are:
5’-CGAGAGCACAGCCAACTGGAACTC-3’，5’-CGAGAGCACAGCCAACTGGAACTC-3 ’,
5’-GCGAGATAG ATGCCATGCGCGA-3’，PCR扩增产物长度为421bp。5'-GCGAGATAG ATGCCATGCGCGA-3 ', and the PCR amplification product was 421 bp in length.
实施例2嗜酸性氧化亚铁硫杆菌种特异性引物设计Example 2 Design of Specific Primers for Acidophilus Thiobacillus Ferrooxidans
(1)嗜酸性氧化亚铁硫杆菌种保守CDSs序列的获得(1) Obtaining conserved CDSs sequences of Thiobacillus acidophilus
参照实施例1的方法，获得模式菌株嗜酸性氧化亚铁硫杆菌ATCC23270的CDSs序列。以其为查询序列，使用Blastn默认参数比对除去嗜酸性氧化亚铁硫杆菌种序列(exclude Acidithiobacillus ferrooxidans(taxid:920))的NR数据库。Blastn比对上的结果即为嗜酸性氧化亚铁硫杆菌种与其他物种之间存在的保守序列。提取未比对上的CDSs序列，以其为查询序列，Blastn比对嗜酸性氧化亚铁硫杆菌种序列(Acidithiobacillus ferrooxidans(taxid:920))，并提取能比对上NR库所有嗜酸性氧化亚铁硫杆菌种菌株的CDSs序列，此即嗜酸性氧化亚铁硫杆菌种保守CDSs序列。设计流程图见图1。Referring to the method of Example 1, the CDSs sequence of the model strain Thiobacillus acidophilus ATCC23270 was obtained. Using this as the query sequence, the NR database of acidothioferillus ferrooxidans sequences (taxid: 920) was removed using Blastn default parameter alignment. The results of the Blastn alignment are the conserved sequences that exist between the species of Acidophilus ferrooxidans and other species. Extract the unaligned CDSs sequence and use it as the query sequence. Blastn aligns the acidophilic ferrooxidans sequences (Acidithiobacillus ferrooxidans (taxid: 920)), and extract all eosinophilic suboxidants that can be compared on the NR library. The CDSs sequence of the Thiobacillus species, this is the conserved CDSs sequence of the Thiobacillus acidophilus species. The design flowchart is shown in Figure 1.
(2)嗜酸性氧化亚铁硫杆菌种特异性引物设计(2) Design of species-specific primers for Thiobacillus ferrooxidans
将获得的嗜酸性氧化亚铁硫杆菌种保守CDSs序列导入软件Bioedit，进行本地快速比对(Alignment)。以硫醌还原酶基因(种)的CDS序列为例，根据引物设计的基本原则，设计硫醌还原酶基因(种)扩增引物。硫醌还原酶基因(种)的特异扩增上、下游引物序列分别为：The obtained conserved CDSs of Thiobacillus ferrooxidans species were imported into the software Bioedit for rapid local alignment (Alignment). Taking the CDS sequence of the thioquinone reductase gene (species) as an example, according to the basic principles of primer design, a thioquinone reductase gene (species) amplification primer was designed. The specific amplified upstream and downstream primer sequences of the thioquinone reductase gene (species) are:
5’-GGCACATGTGGTAATTTTGGGT-3’，5’-GGCACATGTGGTAATTTTGGGT-3 ’,
5’-CCCGTAGGCAAA GACGTCC-3’，PCR扩增产物长度为1150bp。5'-CCCGTAGGCAAA GACGTCC-3 ', and the PCR amplification product was 1150bp in length.
实施例3热裂解法提取环境样品总DNA及嗜酸硫杆菌菌群的快速检测Example 3 Rapid extraction of total DNA from environmental samples and rapid detection of Thiobacillus acidophilus flora
取采集自两个水质净化厂曝气池的活性污泥样品(代号分别为NS和FT)100mg，放入1.5mL的无菌离心管中，并向其中加入1mL pH＝7的TE缓冲液。 在95℃的废水中水浴5min，然后将其转入-20℃冰箱，冷冻5min，重复上述步骤一次。将处理后的样品1,2000×g离心1min，并将离心后获得的上清液转移到新的1.5mL的无菌离心管中，即获得样品总DNA溶液。PCR体系为50μL体系，组成为：2μL总DNA溶液，实施例1中的上、下游引物各2μL，4μL dNTP，10×PCR缓冲液5μL，DNA聚合酶0.5U，去离子水补充体系至50μL。PCR运行程序为：预变性(95℃，5min)；30个循环(变性：95℃，30s；退火：55℃，30s；延伸：72℃，1kb/min)；充分延伸(72℃，10min)；保存(4℃)，得到扩增产物A。Take 100 mg of activated sludge samples (codes NS and FT) collected from the aeration tanks of two water purification plants, put them into 1.5 mL sterile centrifuge tubes, and add 1 mL of TE buffer solution with pH = 7. Water bath in 95 ° C waste water for 5min, then transfer it to -20 ° C refrigerator, freeze for 5min, and repeat the above steps once. The processed sample was centrifuged at 1,2000 × g for 1 min, and the supernatant obtained after centrifugation was transferred to a new 1.5 mL sterile centrifuge tube to obtain a total DNA solution of the sample. The PCR system was a 50 μL system, consisting of 2 μL total DNA solution, 2 μL each of the upstream and downstream primers in Example 1, 4 μL dNTP, 5 μL of a 10 × PCR buffer, 0.5 U of DNA polymerase, and 50 μL of deionized water to supplement the system. The PCR running program is: pre-denaturation (95 ° C, 5min); 30 cycles (denaturation: 95 ° C, 30s; annealing: 55 ° C, 30s; extension: 72 ° C, 1kb / min); full extension (72 ° C, 10min) ; Save (4 ° C) to obtain amplified product A.
依照上述过程，将实施例1中的上、下游引物替换为实施例2中的上、下游引物，其它条件均不变，得到扩增产物B。According to the above process, the upstream and downstream primers in Example 1 were replaced with the upstream and downstream primers in Example 2, and other conditions remained unchanged to obtain an amplified product B.
将扩增产物A和扩增产物B，分别经0.8％琼脂糖凝胶电泳检测，如图2所示，属特异性引物是指嗜酸硫杆菌属特异性引物，即离子外流通透酶基因序列扩增引物；种特异性引物是指嗜酸性氧化亚铁硫杆菌种特异性引物，即硫醌还原酶基因序列扩增引物；M1、M2分别代表DL2000 DNA maker；CK1、CK2分别代表无菌去离子水对照，可以确定两个样品中均含有嗜酸硫杆菌属菌群及嗜酸性氧化亚铁硫杆菌菌群。Amplified product A and amplified product B were respectively detected by 0.8% agarose gel electrophoresis. As shown in FIG. 2, the gene-specific primer refers to a Thiobacillus acidophilus-specific primer, that is, an ion permease gene. Sequence amplification primers; species-specific primers refer to the species-specific primers of Thiobacillus acidophilus, that is, the thioquinone reductase gene sequence amplification primers; M1 and M2 represent DL2000 DNA maker respectively; CK1 and CK2 represent sterile In deionized water control, it was determined that both samples contained Thiobacillus acidophilus and Thiobacillus ferrooxidans.
实施例4环境样品嗜酸硫杆菌菌群的物种组成分析Example 4 Analysis of Species Composition of Thiobacillus acidophilus Bacteria in Environmental Samples
将扩增产物进行TA克隆，每个样品各挑取10个克隆子进行Sanger测序分析，以确定其序列。以测序获得序列为查询序列，NR库为数据库，进行Blastn比对。提取相似度高的比对结果序列，导入Bioedit软件进行本地多重序列比对，掐头去尾，获得长度一致的多重序列文件，并将其导入分子进化树构建软件Mega6，构建分子进化邻接树(Neighbor joining tree)。根据树形图，初步评估各样品的物种组成及丰度。结果显示，使用离子外流通透酶基因(属)特异引物PCR得到的产物序列主要与硫杆菌三个种的离子外流通透酶基因序列相似，包括A.ferrivorans、嗜酸性氧化亚铁硫杆菌和喜温嗜酸硫杆菌，且富集到的硫杆菌菌群里面可能喜温嗜酸硫杆菌的丰度较高，如图3所示；使用硫醌还原酶基因(种)特异引物PCR得到的产物序列主要与嗜酸性氧化亚铁硫杆菌硫醌还原酶基因序列相似，但是FT和NS样品中的嗜酸性氧化亚铁硫杆菌菌群明显分为 两簇且与数据库已知序列分开，表明二者的菌群组成可能存在较大差别，且可能有新型(种)的硫杆菌存在，如图4所示。The amplified product was TA cloned, and 10 clones were selected from each sample for Sanger sequencing analysis to determine its sequence. The sequence obtained by sequencing was used as the query sequence, and the NR library was used as the database for Blastn alignment. Extract the alignment sequence with high similarity, import it into Bioedit software for local multiple sequence alignment, get rid of heads and tails, obtain multiple sequence files with the same length, and import it into the molecular evolution tree construction software Mega6 to build molecular evolution adjacency trees ( Neighbor joining tree.) Based on the tree diagram, the species composition and abundance of each sample were initially evaluated. The results showed that the product sequences obtained by PCR using specific primers of the iontophoretase gene (genus) were mainly similar to the sequences of the iontophoretase genes of the three species of Thiobacillus, including A. ferrivorans, acidophilus Thiobacillus ferrooxidans, and Thiobacillus acidophilus is warm, and the enriched Thiobacillus flora may have a higher abundance of Thiobacillus acidophilus, as shown in FIG. 3; PCR obtained using specific primers of the thioquinone reductase gene (species) The product sequence is mainly similar to the gene sequence of Thiobacillus ferrooxidans thioquinone reductase, but the Thiobacillus ferrooxidans bacteria in the FT and NS samples are clearly divided into two clusters and separated from the known sequence in the database. There may be large differences in the composition of the flora of these individuals, and a new type (species) of Thiobacillus may be present, as shown in FIG. 4.
综上所述，本发明提供了一种用于检测嗜酸硫杆菌的引物对及设计方法与检测方法，本发明的设计方法设计的特异性引物，能够快速、准确地检测出嗜酸硫杆菌属(种)，目前还没有采用类似的方法来获取嗜酸硫杆菌属或种的保守CDSs序列进而设计相应的特异性引物；同时本发明还基于设计的特异性引物，提供了一种检测嗜酸硫杆菌的方法，本检测方法无需建库，不需提取样品总DNA，具有特异性强、灵敏度高及速度快的优点。In summary, the present invention provides a primer pair for detecting Thiobacillus acidophilus and a design method and detection method. The specific primer designed by the design method of the present invention can quickly and accurately detect Thiobacillus acidophilus. Genus (species), currently has not adopted a similar method to obtain the conserved CDSs sequence of Thiobacillus acidophilus or species and then design the corresponding specific primers; meanwhile, the present invention also provides a method for detecting In the method of Thiobacillus acidi, the detection method does not need to establish a library and does not need to extract the total DNA of the sample, and has the advantages of strong specificity, high sensitivity and fast speed.
应当理解的是，本发明的应用不限于上述的举例，对本领域普通技术人员来说，可以根据上述说明加以改进或变换，所有这些改进和变换都应属于本发明所附权利要求的保护范围。It should be understood that the application of the present invention is not limited to the above examples. For those of ordinary skill in the art, improvements or changes can be made according to the above description, and all these improvements and changes should fall within the protection scope of the appended claims of the present invention.

Claims (7)

  1. 一种用于检测嗜酸硫杆菌的引物对的设计方法，其特征在于，包括如下：步骤A1、根据NCBI中的嗜酸性硫杆菌属模式物种菌株的基因组全序列，提取CDSs序列；A method for designing a primer pair for detecting Thiobacillus acidophilus, which comprises the following steps: Step A1: extracting a CDSs sequence based on the entire genome sequence of a strain of Thiobacillus acidophilus in the NCBI;
    步骤B1、以提取的CDSs序列为查询序列，Blastn比对除去嗜酸性硫杆菌属序列的NR数据库，以未比对上的CDSs序列为查询序列，Blastn比对嗜酸性硫杆菌属序列，并提取能比对上NR数据库所有嗜酸性硫杆菌属菌株的CDSs序列，得到嗜酸硫杆菌属保守CDSs序列；Step B1: Using the extracted CDSs sequence as the query sequence, Blastn compares the NR database that removes the Thiobacillus acidophilus sequence, using the unaligned CDSs sequence as the query sequence, Blastn compares the Thiobacillus acidophilus sequence, and extract The CDSs sequences of all Thiobacillus acidophilus strains on the NR database can be compared to obtain the conservative CDSs sequences of Thiobacillus acidophilus;
    步骤C1、根据所述嗜酸硫杆菌属保守CDSs序列及引物设计原则，设计得到用于检测嗜酸硫杆菌属的引物对；Step C1: Design a primer pair for detecting Thiobacillus acidophilus according to the conservative CDSs sequence and primer design principles of Thiobacillus acidophilus;
    或者步骤A2、根据NCBI中的嗜酸性硫杆菌目标种模式菌株的基因组全序列，提取CDSs序列；Or step A2, extracting the CDSs sequence according to the complete genome sequence of the target strain of Thiobacillus acidophilus in NCBI;
    步骤B2、以提取的CDSs序列为查询序列，Blastn比对除去嗜酸性硫杆菌目标种序列的NR数据库，以未比对上的CDSs序列为查询序列，Blastn比对嗜酸性硫杆菌目标种序列，并提取能比对上NR数据库所有嗜酸性硫杆菌目标种菌株的CDSs序列，得到嗜酸性硫杆菌目标种保守CDSs序列；Step B2: Using the extracted CDSs sequence as the query sequence, Blastn compares the NR database that removes the target species sequence of Thiobacillus acidophilus, using the unaligned CDSs sequence as the query sequence, and Blastn compares the target species sequence of Thiobacillus acidophilus. The CDSs sequences of all target strains of Thiobacillus acidophilus on the NR database can be compared to obtain the conserved CDSs sequences of target species of Thiobacillus acidophilus;
    步骤C2、根据所述嗜酸性硫杆菌目标种保守CDSs序列及引物设计原则，设计得到所述用于检测嗜酸性硫杆菌目标种的引物对。Step C2: According to the conserved CDSs sequence of the target species of Thiobacillus acidophilus and the principle of primer design, design the primer pair for detecting the target species of Thiobacillus acidophilus.
  2. 一种用于检测嗜酸硫杆菌的引物对，其特征在于，采用权利要求1所述的设计方法设计而成。A primer pair for detecting Thiobacillus acidophilus, which is designed by using the design method of claim 1.
  3. 根据权利要求2所述的用于检测嗜酸硫杆菌的引物对，其特征在于，包括：用于检测嗜酸硫杆菌属的引物对：The primer pair for detecting Thiobacillus acidophilus according to claim 2, further comprising: a primer pair for detecting Thiobacillus acidophilus:
    上游引物序列为5’-CGAGAGCACAGCCAACTGGAACTC-3’，The upstream primer sequence is 5'-CGAGAGCACAGCCAACTGGAACTC-3 ',
    下游引物序列为5’-GCGAGATAG ATGCCATGCGCGA-3’；The downstream primer sequence is 5'-GCGAGATAG ATGCCATGCGCGA-3 ';
    或者用于检测嗜酸性氧化亚铁硫杆菌种的引物对：Or primer pairs for the detection of acidophilus Thiobacillus ferrooxidans species:
    上游引物序列为5’-GGCACATGTGGTAATTTTGGGT-3’；The upstream primer sequence is 5'-GGCACATGTGGTAATTTTGGGT-3 ';
    下游引物序列为5’-CCCGTAGGCAAAGACGTCC-3’。The downstream primer sequence is 5'-CCCGTAGGCAAAGACGTCC-3 '.
  4. 一种检测嗜酸硫杆菌的方法，其特征在于，包括如下：A method for detecting Thiobacillus acidophilus, which comprises the following features:
    步骤A3、提取待测样品的DNA，并将样品的DNA热裂解，得到DNA单链； 步骤B3、采用权利要求2或3所述的引物对，对所述DNA单链进行PCR扩增，得到PCR扩增产物；Step A3: Extract the DNA of the sample to be tested, and thermally lyse the DNA of the sample to obtain a single-stranded DNA; Step B3: Use the primer pair according to claim 2 or 3 to perform PCR amplification on the single-stranded DNA to obtain PCR amplification products;
    步骤C3、将所述PCR扩增产物进行琼脂糖凝胶电泳检测。Step C3: Perform the agarose gel electrophoresis detection on the PCR amplification product.
  5. 根据权利要求4所述的检测嗜酸硫杆菌的方法，其特征在于，所述步骤A3包括步骤：将待测样品加入到TE缓冲液中，于92～98℃下水浴4～6min，然后于-18～-22℃下冷冻4～6min，重复水浴和冷冻的步骤一次，再进行分离处理得到含有所述DNA单链的上清液。The method for detecting Thiobacillus acidophilus according to claim 4, wherein the step A3 comprises the steps of: adding a sample to be tested into a TE buffer solution, and bathing at 92-98 ° C for 4-6 minutes, and then Freeze at -18 to -22 ° C for 4 to 6 minutes, repeat the steps of water bath and freezing once, and then perform separation treatment to obtain the supernatant containing the DNA single strand.
  6. 根据权利要求4所述的检测嗜酸硫杆菌的方法，其特征在于，所述步骤B3中，PCR程序设置为30个循环。The method for detecting Thiobacillus acidophilus according to claim 4, wherein in step B3, the PCR program is set to 30 cycles.
  7. 根据权利要求4所述的检测嗜酸硫杆菌的方法，其特征在于，所述步骤C3之后，还包括：The method for detecting Thiobacillus acidophilus according to claim 4, wherein after step C3, further comprising:
    步骤D3、将所述扩增产物进行测序并构建分子进化树，用于判断样品中的嗜酸硫杆菌属菌群的组成或同种菌株之间的进化聚类关系。Step D3: Sequencing the amplified product and constructing a molecular evolution tree for determining the composition of the Thiobacillus acidophilus flora in the sample or the evolutionary clustering relationship between the same strains.
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