CN105567810A - Method for detecting flora composition in phylum classification level - Google Patents
Method for detecting flora composition in phylum classification level Download PDFInfo
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Abstract
The present invention relates to a flora detection method in the technical field of biology, and especially relates to a quantitative method for detecting flora composition on the basis of a phylum specific primer. The method includes phylum specific primer design process; qPCR system configuration; qPCR condition setting and operating and other procedures. A macrostructure of flora in a sample and differences of the flora in the different samples can be quickly examined in phylum classification level on the basis quantitative amplification of the phylum specific primer. The quantitative method aims at total number of copies of ribosomal genes of each phylum, but not at detailed number of each species cells of each phylum.
Description
Technical field
The present invention relates to the flora method of operating in biological technical field, especially measure the quick qPCR quantivative approach of flora composition structure based on door Auele Specific Primer.
Background technology
Natural complicated flora generally by tens, the even more not same species of microorganism of hundreds of form, and this composition can occurrence dynamics change under different envrionment conditionss.Most of mankind of the bacterial classification included by nearly all natural flora understand very few; often have bacterial classification over half in flora sample belong to unknown bacterium and still do not know how to cultivate, so it is also more difficult to carry out structure composition quantitative assay fast to natural flora.
Several Bacterial community determination techniques common are at present summarized as follows both at home and abroad: (1) classical culture protocols; In such as soil, most of microorganism belongs to and does not cultivate bacterium, and use conventional medium culture soil microorganism speed slow, qualification workload is very large; (2) tetra-sodium high-flux sequence method; This is at present popular both at home and abroad a kind of structure of community method for quantitatively determining, has generally been come by biotech company's Delegation Server, expensive, the order-checking number that can return up to hundreds of thousands of bar, generally can qualitative, quantitative to belonging to; (3) FISH technology; (4) DGGE/TGGE technology; (5) T-RFLP technology; (6) RAPD technology; (7) SSCP technology; (8) AFLP technology etc.
Above-mentioned all technology are all difficult to accomplish that on-the-spot (2 hours) fast measures the composition structure of flora.
Summary of the invention
The object of the invention is to overcome deficiency of the prior art, a kind of optimization method of the real-time quantitative polymerase chain reaction (PCR) amplification based on door Auele Specific Primer is provided.Amplified target sequence is ribosomal gene sequence.Although primer specificity provided by the invention is very high, but the copy number of ribosomal gene in each bacterial classification genome included by each is not all clear, what calculate is total copy number of the ribosomal gene of whole species that certain comprises, so, the present invention be directed to the quantivative approach of total copy number of each ribosomal gene, but not the detailed number of each each Species Cell comprised.The method reaches to certain specific amplified of microorganism species concrete in arbitrary sample by using door Auele Specific Primer in PCR reaction system, and uses qPCR to increase.Because qPCR technology both domestic and external out can control within 2 hours from sample process to quantitative result, after design of primers is also verified, practical application is got up and will be possessed quantitative judge Be very effective, polyvinyl chloride, effect with low cost.
The present invention is achieved through the following technical solutions:
Should based on the optimization method of the amplification of chain reaction of nucleic acid polymerase of door Auele Specific Primer, the method comprises the following steps:
The design of door Auele Specific Primer: by the amplification of ribosomal gene in sample (such as 16SrDNA amplimer can be 27F (5 '-AGAGTTTGATCCTGGCTCAG-3 ') and 1492R (5 '-GGTTACCTTGTTACGACTT-3 ')) and TA cloning and sequencing, obtain a collection of effective clone, checked order by colony clone, information biology sequential analysis, obtains species information corresponding to each clone (Jie Men detailed outline section genus kind).By to the Multiple Sequence Alignment of the ribosomal gene sequence in whole effectively order-checking clones with repeatedly compare, find out several characteristic SNP (single nucleotide polymorphism) site of respective door.This site uses as 3 ' least significant end position of primer.The base principle of design of 3 ' penultimate of primer is, does not have mispairing or form a mispairing (weak mispairing: A-C, T-G between it and target sequence; Or the mispairing of medium tenacity: A-A, C-C, G-G), also form a mispairing (strong mispairing: T-T, T-C, A-G with non-target sequences; Or the mispairing of medium tenacity: A-A, C-C, G-G).So, primer and target sequence match completely or only have the mispairing of a 3 ' penultimate, generally do not affect expanding effect, and have continuous two mispairing of two 3 ' least significant ends with non-target sequences, generally cause increasing unsuccessfully.So can obtain the door Auele Specific Primer of single base identification.Design amplification length generally in 80-500 base so that for qPCR.These primer pairs carry out pcr amplification with the TA of the actual ribosomal gene sequence of each clone pure plasmid for template and (use NPK02 test kit, the large positive GREDBIO in Shandong) verify and hybrid template checking, and qPCR (uses NPK62 test kit, the large positive GREDBIO in Shandong) (requirement is an amplification out special object band only in checking, the analysis of Tm value only has master tape (not containing primer dimer or little dimer)), determine the specificity of each pair of primer.It is each 1 right that each door selects the primer of best specific amplification, but best primer also may have several right.Above-mentioned method of design is not limited to which door specific, goes for the specific primer design of in any (natural or artificial) flora sample any.First the door Auele Specific Primer of design is applicable to sample used.If the species coverage of certain is enough large in certain sample, such as include more than 95% of this known species both at home and abroad, then the door Auele Specific Primer designed just possesses the versatility of 95%, can also be applicable to other samples a lot.The acquisition of sample ribosomal gene sequence information need not be confined to TA clone sequencing.
Obtain a specific primer sequence by above-mentioned specific optimizing process, carry out synthesis preparation by concerns about bio technology company afterwards;
The configuration of qPCR system: any qPCR test kit and qPCR instrument can be used.QPCR instrument requirements can detect the fluorescent substance that corresponding test kit comprises, as EvaGreen, SYBRgreen etc.;
The setting of qPCR condition and operation; The adjusting and optimizing of amplification condition is carried out according to different flora and different doors; The primer designed during the different door of the same group of quantitative judge possesses close Tm value, to test at identical amplification condition as far as possible;
QPCR product detects: can be detected by agarose gel electrophoresis.After amplified conditions optimization actual when measuring without the need to carrying out the detection of product more at every turn; The Tm value of qPCR product is analyzed also can as the criterion of amplification quality; Amplified production equal one time Tm value analysis can see single product peak;
Advantage of the present invention and beneficial effect are:
1. in natural flora sample, quite a few or most of bacterium are often unknown microorganism, directly in kind of aspect such as classification such as grade, a collection of bacterial classification of quantitative judge is also more difficult, and in the aspect of door, macrostructure and the structural changes at different conditions thereof of sample flora can be investigated fast based on the quantitative amplification of door Auele Specific Primer, not only simple but also practical.
2. because present method can complete mensuration at 2 hours scenes, can be used for accumulating Bacterial community quantitative data and its biological function in target sample show between relation data.If use state-of-the-art miniature portable qPCR instrument, can also be shorter to finally completing the quantitatively required time from sample process.
3. the consumptive material involved by enforcement of the present invention is all common molecular biology reagents, is conveniently easy to get.Described qPCR system configurations can use any common test kit.For the NPK62 test kit of Shandong large just (GREDBIO), the composition of qPCR system is shown in form 1.
4., compared with existing method, the present invention can the content of the quantitative main door of sample flora, and with low cost.
Form 1NPK62 test kit (GREDBIO) qPCR system forms
| 2 × PCR damping fluid (containing fluorescence dye and PCR synergistic agent) | 6μL |
| Primer 1 (2 μMs) | 1μL 2 --> |
| Primer 2 (2 μMs) | 1μL |
| Template+H 2O | 3.8μL |
| Taq enzyme (5U/ μ L) | 0.2μL |
| Cumulative volume is 12 μ L |
Embodiment
The present invention is described in further detail by following examples, but is not limited to following embodiment.
Concrete operation method is as follows:
The design of embodiment 1 Auele Specific Primer:
By the amplification of 16SrDNA in certain soil sample, (amplimer is 27F (5 '-AGAGTTTGATCCTGGCTCAG-3 ') and 1492R (5 '-GGTTACCTTGTTACGACTT-3 '), amplification template is certain soil sample sample and TA cloning and sequencing, obtain nearly 800 effectively to clone, except non-perfect fungus accounts for 60.78%, perfect fungus (318) belongs to Bacteroidetes (3.79%) respectively, Firmicutes (31.16%), Proteobacteria (3.55%), Synergistetes (0.63%), the Atopobium (0.13%) of Actinobacteria.By to the Multiple Sequence Alignment of these 318 16S sequences with repeatedly compare, find out more than totally 200, several characteristic SNP (single nucleotide polymorphism) site of respective door.Design primer is attempted in 3 ' the least significant end position using these SNP site as primer.The base principle of design of 3 ' penultimate of primer is, forms a mispairing, also form a mispairing with non-target sequences between it and target sequence.So, primer and target sequence only have the mispairing of a 3 ' penultimate, and have continuous two mispairing of two 3 ' least significant ends with non-target sequences.Amplification length in 100-300 base so that for qPCR.These primer pairs carry out PCR with the TA of the actual 16S sequence of each clone pure plasmid for template and (use NPK02 test kit after the raw work synthesis in Shanghai, the large positive GREDBIO in Shandong) verify and hybrid template checking, and qPCR (uses NPK62 test kit, the large positive GREDBIO in Shandong) (requirement is an amplification out special object band only in checking, the analysis of Tm value only has master tape (not containing primer dimer or little dimer)), determine the specificity of each pair of primer.It is each 1 right that each door selects the primer of best specific amplification.Wherein, Bacteroidetes Auele Specific Primer, its primer pair nucleotide sequence is as follows: ngF (5'-ACGCACGGGTGAGTAACACGTAT-3'), ngR (5'-AGGGGATAAATCCTCTCAGTTCCCCT-3').The Auele Specific Primer of Proteobacteria, its primer pair nucleotide sequence is as follows: bxF (5'-ACCGTAGCTGGTCTGAGAGGATGATAA-3'), bxR (5'-ACACGCTTTACGCCCAGTAATTCCYA-3').The Auele Specific Primer nucleotide sequence of Firmicutes is as follows: hbF (5'-ACAGCAGTGGGGAATCTTCC-3'), hbR (5'-ATAGCCGGGGCTTCCTCCT-3').Syntrophism bacterium door Auele Specific Primer, its primer pair nucleotide sequence is as follows: hyF (5'-AAAACCGCTTTCAGCAGGGAC-3'), hyR (5'-ATAACTCCCGACCAAACAAGC-3').Actinomycetes door Auele Specific Primer, its primer pair nucleotide sequence is as follows: fxF (5'-AAGCGAACGGGATTAGATACCCC-3'), fxR (5'-AAGGTAAGGTTCTTCGGTTTGCA-3').
Embodiment 2 genome sample extraction
Often kind of certain soil sample sample is fresh takes 200 milligrams, and adopt Solarbio soil genomic kit or Shanghai raw work EZUP pillar soil genome extraction agent cassette method to extract genome, last elution volume is 100 μ L.The refrigeration of-80 DEG C, genomic templates sample (10-50ng/ul) is for subsequent use.Soil soil sample can spend Refrigerator store 1-2-80 by plastic bag sealing.
The configuration of embodiment 3qPCR system:
Described qPCR system can use most business archaeal dna polymerase or qPCR test kit.For the NPK62 test kit (containing 2 × buffer) of GREDBIO, the composition of qPCR system is see form 1.
Embodiment 4qPCR quantitative criterion: the quantitative criterion material of each is the pure plasmid stoste sample that 16SrDNA obtains through F27-R1492 amplified production TA clone (using TaKaRa based on the TA cloning system of pMD-19T), measure its actual concentrations, and be diluted to 10 by 10 times
-10secondary.Because the sized molecules amount of above-mentioned plasmid is unique, mass concentration and volumetric molar concentration can Accurate Measurements, and for single qPCR product, functional quality concentration and volumetric molar concentration can; And in qPCR reaction in this technology, what standard model increased is single product, and sample amplification is the mix products of the many kinds of certain, so the concentration of standard model must use volumetric molar concentration, the result that sample measures out is also volumetric molar concentration.The pure plasmid sample of 5 doors extracts according to the requirement of raw work SanPrep pillar extraction of plasmid DNA test kit SK8191, and elution volume 100 microlitre, loading 10 microlitre runs agarose electrophoresis and checks on the quality and its concentration of quantitative assay.Plasmid measures concentration through OD260, OD280 and is converted into copy/L (often liter of how many molecules). and plasmid stock carries out doubling dilution (each dilution 10 times) as the quantitative standard of each qPCR.Specific amplification ensures that the melt curve analysis of qPCR product only has one group of peak of a unimodal or melting temp closely (difference is generally no more than 5 degree); According to the door specific primer design of the art of this patent, by seeing that amplified production is all the kind of target door to the cloning and sequencing of use door primer amplified product.The specificity of amplified production and the configuration of door specific criteria sample can ensure the validity that qPCR is quantitative.The quantitative plasmid by each door oneself of each is as quantitative criterion, and door Auele Specific Primer ensures that amplified production is the kind sequence of target door, so just can ensure the validity that qPCR is quantitative.The above-mentioned of 5 doors quantitatively doing together with in a qPCR, just can once complete the quantitative of 5 doors, then obtains the useful information of Bacterial community composition.
Embodiment 5 utilizes Bacteroidetes Auele Specific Primer to carry out the quantitative comparison of Bacteroidetes to certain enterprise four kinds of soil samples (be labeled as 1. 2. 3. 4., following instance is same).Bacteroidetes primer: ngF (5'-ACGCACGGGTGAGTAACACGTAT-3'), ngR (5'-AGGGGATAAATCCTCTCAGTTCCCCT-3') specific primers amplify length is 185 base pairs.According to the qPCRC of each concentration standard sample and sample
tvalue can be calculated the concentration of target product of four samples is as follows:
Standard plasmid mother liquor DNA molecular concentration is 75649000 × 10
8copy/L, dilution gradient is by 10
-3to 10
-8as follows:
| Gradient dilution | -3 | -4 | -5 | -6 | -7 | -8 |
| Concentration (10 8Copy/L) | 75649 | 7564 | 756 | 75.6 | 7.56 | 0.75 |
The absolute concentration that sample calculates:
| Sample | ① | ② | ③ | ④ |
| Concentration (10 8Copy/L) | 2 | 744 | 14163 | 851 |
Embodiment 6 utilizes syntrophism bacterium door Auele Specific Primer to carry out the quantitative comparison of syntrophism bacterium door to certain enterprise four kinds of soil samples.Syntrophism bacterium door primer: hyF (5'-AAAACCGCTTTCAGCAGGGAC-3'), hyR (5'-ATAACTCCCGACCAAACAAGC-3'), specific primers amplify length is 208 base pairs.According to the qPCRC of each concentration standard sample and sample
tvalue can be calculated the concentration of target product of four samples is as follows:
Standard plasmid mother liquor DNA molecular concentration is 67323000 × 10
8copy/L, dilution gradient is by 10
-3to 10
-8as follows:
| Gradient dilution | -3 | -4 | -5 | -6 | -7 | -8 |
| Concentration (10 8Copy/L) | 67323 | 6732 | 673 | 67 | 6.7 | 0.67 |
The absolute concentration that sample calculates:
| Sample | ① | ② | ③ | ④ 4 --> |
| Concentration (10 8Copy/L) | 0 | 140 | 385 | 4 |
Embodiment 7 utilizes actinomycetes door Auele Specific Primer to carry out the quantitative comparison of actinomycetes door to certain enterprise four kinds of soil samples.Actinomycetes door primer: fxF (5'-AAGCGAACGGGATTAGATACCCC-3'), fxR (5'-AAGGTAAGGTTCTTCGGTTTGCA-3'), specific primers amplify length is 157 base pairs.According to the qPCRC of each concentration standard sample and sample
tvalue can be calculated the concentration of target product of four samples is as follows:
Standard plasmid mother liquor DNA molecular concentration is 1134140 × 10
8copy/L, dilution gradient is by 10
-1to 10
-6as follows:
| Gradient dilution | -1 | -2 | -3 | -4 | -5 | -6 |
| Concentration (10 8Copy/L) | 113414 | 11341 | 1134 | 113 | 11.3 | 1.13 |
The absolute concentration that sample calculates:
| Sample | ① | ② | ③ | ④ |
| Concentration (10 8Copy/L) | 0 | 298986 | 13 | 0 |
Embodiment 8 utilizes Firmicutes Auele Specific Primer to carry out the quantitative comparison of Firmicutes to certain enterprise four kinds of soil samples.The Auele Specific Primer of Firmicutes is as follows: hbF (5'-ACAGCAGTGGGGAATCTTCC-3'), hbR (5'-ATAGCCGGGGCTTCCTCCT-3').Specific primers amplify length is 135 base pairs.According to the qPCRC of each concentration standard sample and sample
tvalue can be calculated the concentration of target product of four samples is as follows:
Standard plasmid mother liquor DNA molecular concentration is 195071744 × 10
8copy/L, dilution gradient is by 10
0to 10
-5as follows:
| Gradient dilution | 0 | -1 | -2 | -3 | -4 | -5 |
| Concentration (10 8Copy/L) | 195071744 | 19507174 | 1950717 | 195071 | 19507 | 1950 |
The absolute concentration that sample calculates:
| Sample | ① | ② | ③ | ④ |
| Concentration (10 8Copy/L) | 122505 | 37972420 | 597973824 | 6704090 |
Embodiment 9 utilizes Proteobacteria Auele Specific Primer to carry out the quantitative comparison of Proteobacteria to certain enterprise four kinds of soil samples.Auele Specific Primer following institute bxF (5'-ACCGTAGCTGGTCTGAGAGGATGATAA-3') of Proteobacteria, bxR (5'-ACACGCTTTACGCCCAGTAATTCCYA-3').Specific primers amplify length is 264 base pairs.According to the qPCRC of each concentration standard sample and sample
tvalue can be calculated the concentration of target product of four samples is as follows:
Standard plasmid mother liquor DNA molecular concentration is 3666710 × 10
10copy/L, dilution gradient is by 10
-1to 10
-6as follows:
| Gradient dilution | -1 | -2 | -3 | -4 | -5 | -6 |
| Concentration (10 10Copy/L) | 366671 | 36667 | 3666 | 366 | 36.6 | 3.66 |
The absolute concentration that sample calculates:
| Sample | ① | ② | ③ | ④ |
| Concentration (10 10Copy/L) | 673 | 2818 | 1946 | 9 |
Claims (3)
1. measure a quantivative approach for flora composition structure based on door Auele Specific Primer, the method comprises the following steps:
(1). the design of door Auele Specific Primer;
(2) the configuration of .qPCR system;
(3) the setting of .qPCR condition and operation;
It is characterized in that: the door Auele Specific Primer that the primer used in described qPCR system designs for step (1).
2. one kind measures the quantivative approach of flora composition structure based on door Auele Specific Primer, it is characterized in that, door specific primer design described in claim 1 is the quantivative approach of total copy number of ribosomal gene based on each, but not the detailed number of each each Species Cell comprised.
3. one kind measures the quantivative approach of flora composition structure based on door Auele Specific Primer, it is characterized in that, the door specific primer design described in claim 1 is door specificity SNP (single nucleotide polymorphism) site based on each ribosomal gene.This site uses as 3 ' least significant end position of primer.The base principle of design of 3 ' penultimate of primer is, does not have mispairing or form a mispairing (weak mispairing: A-C, T-G between it and target sequence; Or the mispairing of medium tenacity: A-A, C-C, G-G), also form a mispairing (strong mispairing: T-T, T-C, A-G with non-target sequences; Or the mispairing of medium tenacity: A-A, C-C, G-G).So, primer and target sequence match completely or only have the mispairing of a 3 ' penultimate, generally do not affect expanding effect, and have continuous two mispairing of two 3 ' least significant ends with non-target sequences, generally cause increasing unsuccessfully.So can obtain an Auele Specific Primer.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106222249A (en) * | 2016-07-14 | 2016-12-14 | 哈尔滨工业大学（威海） | The method for designing measuring the species-specific primer of known group information species in microbiologic population and the method measuring strain content |
| CN109251989A (en) * | 2018-11-23 | 2019-01-22 | 哈尔滨工业大学（威海） | A kind of method of methane bacterial content in quantitative detection pit mud |
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2015
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106222249A (en) * | 2016-07-14 | 2016-12-14 | 哈尔滨工业大学（威海） | The method for designing measuring the species-specific primer of known group information species in microbiologic population and the method measuring strain content |
| CN109251989A (en) * | 2018-11-23 | 2019-01-22 | 哈尔滨工业大学（威海） | A kind of method of methane bacterial content in quantitative detection pit mud |
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