CN111440847B - High-flux low-cost micro biological sample molecule identification technology - Google Patents
High-flux low-cost micro biological sample molecule identification technology Download PDFInfo
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Abstract
The invention relates to the field of molecular biology, in particular to a high-throughput low-cost micro biological sample molecular identification technology, which comprises two aspects of high-quality genome extraction and sequence detection analysis. The invention aims to reduce the high throughput sequencing cost of molecular identification of trace biological samples. The high-throughput low-cost micro biological sample molecular identification technology comprises the following steps: 1) Collecting a sample; 2) Sample cleavage; 3) Collecting nucleic acid; 4) PCR amplification of the labeled primer; 5) PCR products were mixed sequenced and position reduced. The invention does not need to prepare complex protoplast, has simple receptor source and is suitable for fungal transformation without spores. In addition, the invention simplifies and improves the existing DNA extraction technology, so that researchers can conveniently carry out large-scale high-quality genome extraction by using some simple and small-sized equipment in a laboratory; and meanwhile, common PCR and high-throughput sequencing technology are integrated, and the low-cost PCR sequence amplification and sequencing analysis are realized on the extracted DNA by combining computer analysis.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and relates to a high-throughput low-cost micro biological sample molecular identification technology, which comprises two aspects of high-quality genome extraction and sequence detection analysis of a micro biological sample.
Background
Modern molecular biology techniques have gradually entered the era of high throughput processing in terms of DNA sample extraction, PCR amplification and sequencing, which is essential in many research fields, such as molecular studies of the diversity of certain biological specific genetic loci, which have to be faced with large numbers of samples (including animal and plant cells and microorganisms), which generally rely on expensive automated processing machinery and kits. For laboratory studies under general conditions, if genome extraction, PCR amplification and sequencing are manually performed one by using a kit according to conventional methods, it is not only time consuming and laborious, but also often difficult to withstand in terms of cost. In terms of DNA extraction, it is not difficult to extract high quality DNA of a single sample using a kit, and performing rapid low cost extraction of samples in batches requires improvement and optimization in the method. In recent years, many researchers have proposed many improvements of DNA extraction methods, but often only for one microorganism sample, such as only for gram-negative bacteria, etc., which makes it difficult to extract various samples simultaneously, such as samples of intestinal bacteria that are relatively easy to extract DNA, such as gram-negative bacteria, and gram-positive bacteria that are difficult to extract DNA, such as bifidobacteria; moreover, to meet the need for large-scale extraction, most of the improvements in the past tend to overcomplete the extraction process, severely degrading the quality of the extracted DNA. In terms of sequence detection analysis, PCR and classical sequencing methods, while very mature and reliable, are time-consuming and laborious to sequence thousands of samples, making high throughput sequencing a tremendous amount of sequencing, but the cost of sequencing each sample is not inexpensive and the cost of use is high.
Disclosure of Invention
In order to solve the technical problems, so as to economically and rapidly develop the identification of trace biological sample molecules based on high flux, the invention provides two innovative contents: first, the existing DNA extraction technology is simplified and improved, so that researchers can conveniently, economically and rapidly extract high-quality genomes on a large scale by using some simple and small-sized equipment in a laboratory; integrating common PCR and high-throughput sequencing technology, and combining computer analysis to realize low-cost PCR sequence amplification and sequencing analysis on the extracted DNA;
the invention adopts the following technical proposal to realize the first innovation content:
preferably, the technical scheme comprises the following steps:
(1) And (3) respectively processing according to the dry and wet states of the samples: the microorganism colony and the like are compared with a dry sample, and the sample is directly picked into a centrifuge tube for the next step; if the liquid cell suspension is liquid cell suspension, 10000g of the liquid cell suspension is centrifuged for 1min at 4 ℃, supernatant is removed, sterile water is added for cleaning once, 10000g of the liquid cell suspension is centrifuged for 1min at 4 ℃, and supernatant is removed; the tube can be selected from calandria and 96-hole plate;
(2) According to the volume of the tube, adding glass beads with the diameter of 0.1-0.5 mm to 1/8 of the total volume of the tube, adding protease K suspension with the concentration of 1mg/ml and 1/8 of the total volume of the tube, preserving heat for 5-30 minutes at the temperature of 40-70 ℃, and horizontally or vertically oscillating for 5 minutes at the speed of more than 1000 rpm;
(3) Adding 10% of chelex liquid with the concentration of 1/4 of the total volume of the tube, scattering, and then carrying out ice bath at 95 ℃ for 10-20 min;
(4) Centrifuging at 4deg.C for 1min at 10000g, collecting supernatant as template, and freeze preserving;
the invention adopts the following technical scheme to realize the second innovative content:
preferably, the technical scheme comprises the following steps:
(1) Designing primer marking sites corresponding to different PCR tubes;
(2) PCR amplification and sample mixed sequencing;
(3) Reducing the positions of PCR tubes where different primers are positioned according to the detected primer sequences;
preferably, the step (1) includes the following three steps:
(2) introducing 1-2 mismatched bases between the 5' end of the upstream primer and the middle region of the downstream primer as specific labels, designing 24 mismatched combinations by the upstream primer, and designing 16 mismatched combinations by the downstream primer, so that 384 different primer combinations are formed, and 384 different PCR tube positions can be corresponded;
(3) a 30-35 base joint is added at the 5' end of the primer, and the PCR amplification in the subsequent high-throughput sequencing is linked, so that the length of the primer is increased to about 45 bases, and the amplification stability can be further enhanced;
preferably, the step (2) includes the following three steps:
(3) after PCR reaction, 1-5 μl of the product from each well is mixed and recorded as a sample, and Miseq deep sequencing of Illumina is performed;
preferably, the step (3) includes the following two steps:
(1) identifying the relative marks of the pipe hole positions of a sequence measured by a sample through computer software, classifying, and selecting when 1-2 adjustment bases at the upstream and downstream positions are required to appear during identification so as to remove errors to the greatest extent;
(2) the sequences measured in each well are sequenced, so that the reliability of more sequence readings is higher, fewer sequence readings can be further verified by a classical sequencing method, and the fewer sequences than the set prediction, such as the fewer than 10, are considered errors to be eliminated. Thus, the sequence of each hole can be restored to the greatest extent;
the invention is divided into a microbial genome extraction part and a PCR sequence amplification and sequence analysis part. The extracted part of the microbial genome is characterized in that: the glass beads are utilized to vibrate, enzymolysis of proteinase K, chelate resin and high-temperature and other methods suitable for high-flux operation are utilized to cooperatively play roles, and a large number of microbial samples can be simultaneously released with high-quality DNA in a short time; the PCR sequence amplification and sequence analysis technology is characterized in that different PCR samples are marked by using a primer combination, then the one-time sequencing of the mixture of the different samples is realized by using a deep sequencing technology, and finally the primer mark is sorted out by using a computer technology; the two-part technology can be tightly connected to realize high-flux low-cost operation of the whole process, and each part can also be used as a part of other molecular biology identification technologies;
compared with the existing genome extraction technology and PCR sequence analysis technology, the invention has the following beneficial effects:
(1) Micro DNA extraction can be performed by only a small amount of sample, for example, bacterial colonies are picked from a flat plate, so that the trouble of preparing a large amount of strains is avoided;
(2) The whole operation can be carried out by a plurality of single pipes, and is more suitable for the operation of high-flux equipment such as micro-pore plates and the like;
(3) Is particularly suitable for large-scale strain identification and diversity research of specific gene loci in flora;
(4) The extracted genome has higher quality, and ensures the stability of PCR sequence amplification;
both genome extraction technology and PCR sequence amplification and sequence analysis show the characteristics of high throughput and low cost.
Drawings
FIG. 1 gram-positive bacterium Bifidobacterium longum(Bifidobacterium.longum) The extraction effect in the 8-link PCR tube;
FIG. 2 effect of PCR amplification of intestinal bacteria.
Detailed Description
EXAMPLE 1 DNA extraction of the gram-Positive bacterium Bifidobacterium longum
Among bacteria, generally, the DNA of gram-negative bacteria is relatively easy to extract; gram positive bacteria have relatively thick peptidoglycan cell walls, so that the effects of single heating, freeze thawing and mechanical disruption are generally poor; the method of the invention has better effect;
1. culturing the target strain to an OD value of about 1.0 or more;
2. the bacterial liquid was aspirated into 200. Mu.l of 8-well tubes, centrifuged at 10000g for 1min at 4℃and the supernatant was removed. If the bacterial liquid is thinner, the process can be repeated for 2 to 3 times; then adding sterile water for washing, and centrifuging to remove supernatant;
3. 25 μl glass beads (diameter 0.3 mm) and 25 μl protease K at 1mg/ml concentration were added and mixed well, and incubated at 60deg.C for 10 min;
4. vibrating the microplate mixer at 1200rpm for 6min;
5. adding 50 μl of 10% chelex solution, scattering, heating the cover of the PCR instrument at 95deg.C for 20min, and cooling to 4deg.C;
6. centrifuging 10000g of the mixture at 4 ℃ for 1min, taking supernatant as a template, and freezing and preserving;
7. clear bands can be seen by running electrophoresis of the product (see FIG. 1);
example 2 identification of intestinal flora Using commonly used 16s rRNA primers and PCR amplification
Standard 16s rRNA universal primer forward 515F:5'-GTGCCAGCMGCCGCGG-3', the reverse direction is: 907R:5'-CCGTCAATTCMTTTRAGTTT-3'; the specific amplification is carried out by adopting point mutation cross combination to form a 24 multiplied by 16 primer matrix (table 1) corresponding to 384 sample combinations; the amplification result shows that for the primers with different site mutations, the PCR effect is obvious, the sizes of the primers are all near the target band, and the experimental result is reliable (see figure 2);
TABLE 1 sequences and numbering of the primers
Primer name adaptor (underlined) +universal primer (5 '-3', mutation is identified in lowercase) numbering
1B515F1-A CACTCCATACAGCACGCTCTTCCGATCTTCCCGAaTGCCAGCMGCCGCGG D1
1B515F1-B CACTCCATACAGCACGCTCTTCCGATCTTCCCGAtTGCCAGCMGCCGCGG D2
1B515F1-C CACTCCATACAGCACGCTCTTCCGATCTTCCCGAcTGCCAGCMGCCGCGG D3
1B515F1-D CACTCCATACAGCACGCTCTTCCGATCTTCCCGAGaGCCAGCMGCCGCGG D4
1B515F1-E CACTCCATACAGCACGCTCTTCCGATCTTCCCGAGTaCCAGCMGCCGCGG D5
1B515F1-F CACTCCATACAGCACGCTCTTCCGATCTTCCCGAGTtCCAGCMGCCGCGG D6
1B515F1-G CACTCCATACAGCACGCTCTTCCGATCTTCCCGAGTcCCAGCMGCCGCGG D7
1B515F1-H CACTCCATACAGCACGCTCTTCCGATCTTCCCGAGTGaCAGCMGCCGCGG D8
1B515F1-I CACTCCATACAGCACGCTCTTCCGATCTTCCCGAGTGtCAGCMGCCGCGG E1
1B515F1-J CACTCCATACAGCACGCTCTTCCGATCTTCCCGAGTGgCAGCMGCCGCGG E2
1B515F1-K CACTCCATACAGCACGCTCTTCCGATCTTCCCGAGTGCaAGCMGCCGCGG E3
1B515F1-L CACTCCATACAGCACGCTCTTCCGATCTTCCCGAGTGCtAGCMGCCGCGG E4
1B515F1-M CACTCCATACAGCACGCTCTTCCGATCTTCCCGAGTGCgAGCMGCCGCGG E5
1B515F1-N CACTCCATACAGCACGCTCTTCCGATCTTCCCGAGTGCCtGCMGCCGCGG E6
1B515F1-O CACTCCATACAGCACGCTCTTCCGATCTTCCCGAGTGCCAaCMGCCGCGG E7
1B515F1-P CACTCCATACAGCACGCTCTTCCGATCTTCCCGAGTGCCAtCMGCCGCGG E8
1B907R1-1 CATCATATACAGCACGCTACCTTGATCTTCCCGAaCGTCAATTCMTTTRAGTTT A1
1B907R1-2 CATCATATACAGCACGCTACCTTGATCTTCCCGAtCGTCAATTCMTTTRAGTTT A2
1B907R1-3 CATCATATACAGCACGCTACCTTGATCTTCCCGAgCGTCAATTCMTTTRAGTTT A3
1B907R1-4 CATCATATACAGCACGCTACCTTGATCTTCCCGACaGTCAATTCMTTTRAGTTT A4
1B907R1-5 CATCATATACAGCACGCTACCTTGATCTTCCCGACtGTCAATTCMTTTRAGTTT A5
1B907R1-6 CATCATATACAGCACGCTACCTTGATCTTCCCGACgGTCAATTCMTTTRAGTTT A6
1B907R1-7 CATCATATACAGCACGCTACCTTGATCTTCCCGACCaTCAATTCMTTTRAGTTT A7
1B907R1-8 CATCATATACAGCACGCTACCTTGATCTTCCCGACCtTCAATTCMTTTRAGTTT A8
1B907R1-9 CATCATATACAGCACGCTACCTTGATCTTCCCGACCGaCAATTCMTTTRAGTTT A9
1B907R1-10 CATCATATACAGCACGCTACCTTGATCTTCCCGACCGcCAATTCMTTTRAGTTT A10
1B907R1-11 CATCATATACAGCACGCTACCTTGATCTTCCCGACCGgCAATTCMTTTRAGTTT A11
1B907R1-12 CATCATATACAGCACGCTACCTTGATCTTCCCGACCGTaAATTCMTTTRAGTTT A12
1B907R1-13 CATCATATACAGCACGCTACCTTGATCTTCCCGACCGTtAATTCMTTTRAGTTT B1
1B907R1-14 CATCATATACAGCACGCTACCTTGATCTTCCCGACCGTgAATTCMTTTRAGTTT B2
1B907R1-15 CATCATATACAGCACGCTACCTTGATCTTCCCGACCGTCtATTCMTTTRAGTTT B3
1B907R1-16 CATCATATACAGCACGCTACCTTGATCTTCCCGACCGTCcATTCMTTTRAGTTT B4
1B907R1-17 CATCATATACAGCACGCTACCTTGATCTTCCCGACCGTCgATTCMTTTRAGTTT B5
1B907R1-18 CATCATATACAGCACGCTACCTTGATCTTCCCGACCGTCAtTTCMTTTRAGTTT B6
1B907R1-19 CATCATATACAGCACGCTACCTTGATCTTCCCGACCGTCAcTTCMTTTRAGTTT B7
1B907R1-20 CATCATATACAGCACGCTACCTTGATCTTCCCGACCGTCAgTTCMTTTRAGTTT B8
1B907R1-21 CATCATATACAGCACGCTACCTTGATCTTCCCGACCGTCAAcTCMTTTRAGTTT B9
1B907R1-22 CATCATATACAGCACGCTACCTTGATCTTCCCGACCGTCAAgTCMTTTRAGTTT B10
1B907R1-23 CATCATATACAGCACGCTACCTTGATCTTCCCGACCGTCAATaCMTTTRAGTTT B11
1B907R1-24 CATCATATACAGCACGCTACCTTGATCTTCCCGACCGTCAATcCMTTTRAGTTT B12
Note that: the underlined adaptor sequences are based on the primers used for high throughput sequencing, just to name one example;
preparing a PCR reaction solution:
mix adopts the 2X TSINGKE Master Mix of the family of the Optimaceae, the primer is synthesized, the page is purified, and the water is self-contained sterile water; mixing the reaction solution in a 96-deep hole plate;
forward primer (24): water 0.6×24×3.3=48, mix 3.8×24×3.3=301, each primer 0.6×24×3.3=48
Reverse primer (16): water 0.6×16×3.3=32, mix 3.8×16×3.2=201, each primer 0.6×16×3.2=32
Note that: this amount has been increased by 10% of the remaining amount, an amount of 3X 4X 96 wells
The primer corresponds to the template:
adding samples by using a thermo 12-channel (5-50 μl) pipettor, forming 384-well samples by using 4 96-well plates, wherein the corresponding 24×16 primer combinations are 5 μl in each sample adding amount row and column, each well does not comprise a template, and finally 10 μl is added, and finally 5 μl is added;
PCR procedure: 94 ℃ for 5min; 95 ℃ 10s,56 ℃ 50s and 72 ℃ 100s, and the total number of the three times is 35; 72 ℃ for 5min; 30min at 4 ℃;
electrophoresis detection conditions
name description
gel TAE 90ml, 25comb
Agarose(1%) Biowest 0.3g
dye EB(10mg/ml)2μl
buffer TAE,45ML
device DYY-8c
Voltage(V) 120
Current(mA) 150
Time(min) 25
Loading Product 5μl
marker DL 2000 marker
Example 3 identification and reduction of sequence tags to tubes
Sort program was designed using python software:
the program flow is as follows: distributing all sequenced sequence marking sites to corresponding PCR reaction holes > > selecting 20-30 sequences from each hole to integrate into a single fasta file > > performing single BLAST sequence comparison > > classifying and sequencing results in excel > > and outputting homology comparison results to excel; the execution effect is shown in the following table:
the first column in the table is the hole position, the second column is the maximum 20 sequences which are randomly taken out, the third column is the maximum possible strain, and the fourth column is the sequence of the strain;
Cell_position Total_count 1Species 1Hit
515F1-D-907R1-9 20 Veillonella dispar 8
515F1-E-907R1-9 20 Veillonella dispar 7
515F1-H-907R1-11 20 Veillonella dispar 4
515F1-I-907R1-6 20 Veillonella dispar 5
515F1-I-907R1-7 20 Veillonella dispar 7
515F1-J-907R1-8 20 Veillonella dispar 7
515F1-K-907R1-10 20 Veillonella dispar 11
515F1-K-907R1-11 20 Veillonella dispar 8
515F1-K-907R1-12 20 Veillonella dispar 9
515F1-K-907R1-7 20 Veillonella dispar 7
515F1-L-907R1-12 20 Veillonella dispar 8
515F1-L-907R1-9 20 Veillonella dispar 13
515F1-N-907R1-13 5 Veillonella dispar 1
515F1-P-907R1-18 5 Veillonella dispar 1
515F1-O-907R1-11 20 Sphingomonas koreensis 2
515F1-E-907R1-2 20 Microbacterium maritypicum 6
515F1-G-907R1-24 13 Microbacterium maritypicum 4
515F1-A-907R1-1 20 Escherichia fergusonii 16
515F1-A-907R1-10 20 Escherichia fergusonii 17
515F1-A-907R1-11 20 Escherichia fergusonii 15
515F1-A-907R1-12 20 Escherichia fergusonii 15
515F1-A-907R1-13 20 Escherichia fergusonii 19
515F1-A-907R1-14 20 Escherichia fergusonii 14
515F1-A-907R1-15 20 Escherichia fergusonii 13
515F1-A-907R1-16 20 Escherichia fergusonii 10
515F1-A-907R1-17 20 Escherichia fergusonii 10
515F1-A-907R1-18 20 Escherichia fergusonii 15
515F1-A-907R1-19 20 Escherichia fergusonii 13
515F1-A-907R1-20 20 Escherichia fergusonii 14
515F1-A-907R1-22 20 Escherichia fergusonii 13
515F1-A-907R1-23 20 Escherichia fergusonii 12
515F1-A-907R1-3 20 Escherichia fergusonii 17
515F1-A-907R1-4 8 Escherichia fergusonii 6
515F1-A-907R1-5 20 Escherichia fergusonii 14
515F1-A-907R1-6 20 Escherichia fergusonii 12
515F1-A-907R1-7 16 Escherichia fergusonii 7
515F1-A-907R1-8 20 Escherichia fergusonii 12
515F1-A-907R1-9 20 Escherichia fergusonii 13
515F1-B-907R1-1 20 Escherichia fergusonii 12
515F1-B-907R1-10 20 Escherichia fergusonii 11
515F1-B-907R1-11 20 Escherichia fergusonii 14
515F1-B-907R1-12 20 Escherichia fergusonii 13
515F1-B-907R1-13 20 Escherichia fergusonii 14
515F1-B-907R1-14 20 Escherichia fergusonii 14
515F1-B-907R1-18 20 Escherichia fergusonii 9
515F1-B-907R1-19 20 Escherichia fergusonii 10
515F1-B-907R1-2 20 Escherichia fergusonii 6
515F1-B-907R1-20 20 Escherichia fergusonii 14
515F1-B-907R1-22 20 Escherichia fergusonii 12
515F1-B-907R1-23 20 Escherichia fergusonii 10
515F1-B-907R1-24 20 Escherichia fergusonii 16
515F1-B-907R1-3 20 Escherichia fergusonii 10
515F1-B-907R1-4 20 Escherichia fergusonii 14
515F1-B-907R1-5 20 Escherichia fergusonii 9
515F1-B-907R1-6 20 Escherichia fergusonii 12
515F1-B-907R1-7 20 Escherichia fergusonii 10
515F1-B-907R1-8 20 Escherichia fergusonii 8
515F1-B-907R1-9 20 Escherichia fergusonii 9
515F1-C-907R1-1 20 Escherichia fergusonii 15
515F1-C-907R1-10 20 Escherichia fergusonii 13
515F1-C-907R1-11 20 Escherichia fergusonii 15
515F1-C-907R1-13 20 Escherichia fergusonii 8
515F1-C-907R1-15 20 Escherichia fergusonii 12
515F1-C-907R1-16 20 Escherichia fergusonii 9
515F1-C-907R1-17 20 Escherichia fergusonii 10
515F1-C-907R1-18 20 Escherichia fergusonii 11
515F1-C-907R1-2 20 Escherichia fergusonii 15
515F1-C-907R1-22 20 Escherichia fergusonii 10
515F1-C-907R1-24 20 Escherichia fergusonii 10
515F1-C-907R1-3 20 Escherichia fergusonii 15
515F1-C-907R1-4 10 Escherichia fergusonii 6
515F1-C-907R1-5 15 Escherichia fergusonii 7
515F1-C-907R1-6 20 Escherichia fergusonii 9
515F1-C-907R1-7 20 Escherichia fergusonii 17
515F1-C-907R1-8 20 Escherichia fergusonii 15
515F1-C-907R1-9 20 Escherichia fergusonii 10
515F1-D-907R1-1 20 Escherichia fergusonii 13
515F1-D-907R1-10 20 Escherichia fergusonii 6
515F1-D-907R1-11 20 Escherichia fergusonii 13
515F1-D-907R1-12 20 Escherichia fergusonii 17
515F1-D-907R1-13 20 Escherichia fergusonii 12
515F1-D-907R1-14 20 Escherichia fergusonii 9
515F1-D-907R1-18 20 Escherichia fergusonii 11
515F1-D-907R1-2 20 Escherichia fergusonii 15
515F1-D-907R1-20 20 Escherichia fergusonii 16
515F1-D-907R1-22 20 Escherichia fergusonii 12
515F1-D-907R1-23 20 Escherichia fergusonii 12
515F1-D-907R1-24 20 Escherichia fergusonii 10
515F1-D-907R1-3 20 Escherichia fergusonii 12
515F1-D-907R1-4 16 Escherichia fergusonii 10
515F1-D-907R1-5 20 Escherichia fergusonii 7
515F1-D-907R1-6 20 Escherichia fergusonii 8
515F1-D-907R1-7 20 Escherichia fergusonii 9
515F1-D-907R1-8 20 Escherichia fergusonii 7
515F1-E-907R1-1 20 Escherichia fergusonii 12
515F1-E-907R1-10 20 Escherichia fergusonii 12
515F1-E-907R1-11 20 Escherichia fergusonii 11
515F1-E-907R1-12 20 Escherichia fergusonii 13
515F1-E-907R1-13 20 Escherichia fergusonii 9
515F1-E-907R1-14 20 Escherichia fergusonii 13
515F1-E-907R1-15 20 Escherichia fergusonii 10
515F1-E-907R1-16 20 Escherichia fergusonii 7
515F1-E-907R1-17 20 Escherichia fergusonii 10
515F1-E-907R1-23 20 Escherichia fergusonii 10
515F1-E-907R1-24 20 Escherichia fergusonii 10
515F1-E-907R1-3 20 Escherichia fergusonii 11
515F1-E-907R1-4 20 Escherichia fergusonii 14
515F1-E-907R1-5 20 Escherichia fergusonii 7
515F1-E-907R1-6 20 Escherichia fergusonii 10
515F1-E-907R1-7 20 Escherichia fergusonii 7
515F1-E-907R1-8 20 Escherichia fergusonii 10
515F1-F-907R1-1 20 Escherichia fergusonii 11
515F1-F-907R1-10 20 Escherichia fergusonii 12
515F1-F-907R1-11 20 Escherichia fergusonii 13
515F1-F-907R1-12 20 Escherichia fergusonii 13
515F1-F-907R1-13 20 Escherichia fergusonii 10
515F1-F-907R1-16 20 Escherichia fergusonii 8
515F1-F-907R1-17 20 Escherichia fergusonii 12
515F1-F-907R1-19 20 Escherichia fergusonii 6
515F1-F-907R1-2 20 Escherichia fergusonii 11
515F1-F-907R1-20 20 Escherichia fergusonii 12
515F1-F-907R1-23 20 Escherichia fergusonii 11
515F1-F-907R1-24 20 Escherichia fergusonii 12
515F1-F-907R1-3 20 Escherichia fergusonii 15
515F1-F-907R1-4 18 Escherichia fergusonii 8
515F1-F-907R1-5 20 Escherichia fergusonii 9
515F1-F-907R1-6 20 Escherichia fergusonii 10
515F1-F-907R1-7 20 Escherichia fergusonii 12
515F1-F-907R1-8 20 Escherichia fergusonii 10
515F1-F-907R1-9 20 Escherichia fergusonii 12
515F1-G-907R1-1 20 Escherichia fergusonii 14
515F1-G-907R1-10 20 Escherichia fergusonii 13
515F1-G-907R1-11 20 Escherichia fergusonii 12
515F1-G-907R1-12 20 Escherichia fergusonii 11
515F1-G-907R1-13 20 Escherichia fergusonii 14
515F1-G-907R1-14 20 Escherichia fergusonii 11
515F1-G-907R1-19 20 Escherichia fergusonii 5
515F1-G-907R1-2 20 Escherichia fergusonii 9
515F1-G-907R1-20 20 Escherichia fergusonii 11
515F1-G-907R1-3 20 Escherichia fergusonii 13
515F1-G-907R1-4 20 Escherichia fergusonii 13
515F1-G-907R1-5 20 Escherichia fergusonii 14
515F1-G-907R1-6 20 Escherichia fergusonii 12
515F1-G-907R1-7 20 Escherichia fergusonii 10
515F1-G-907R1-8 20 Escherichia fergusonii 13
515F1-G-907R1-9 20 Escherichia fergusonii 14
515F1-H-907R1-1 20 Escherichia fergusonii 13
515F1-H-907R1-10 10 Escherichia fergusonii 3
515F1-H-907R1-13 20 Escherichia fergusonii 13
515F1-H-907R1-16 20 Escherichia fergusonii 10
515F1-H-907R1-18 20 Escherichia fergusonii 12
515F1-H-907R1-19 20 Escherichia fergusonii 12
515F1-H-907R1-2 20 Escherichia fergusonii 13
515F1-H-907R1-20 20 Escherichia fergusonii 12
515F1-H-907R1-23 10 Escherichia fergusonii 6
515F1-H-907R1-24 15 Escherichia fergusonii 6
515F1-H-907R1-3 20 Escherichia fergusonii 10
515F1-H-907R1-4 20 Escherichia fergusonii 12
515F1-H-907R1-5 20 Escherichia fergusonii 12
515F1-H-907R1-6 20 Escherichia fergusonii 7
515F1-H-907R1-7 20 Escherichia fergusonii 8
515F1-H-907R1-8 20 Escherichia fergusonii 5
515F1-H-907R1-9 18 Escherichia fergusonii 8
515F1-I-907R1-1 20 Escherichia fergusonii 10
515F1-I-907R1-10 20 Escherichia fergusonii 13
515F1-I-907R1-11 20 Escherichia fergusonii 12
515F1-I-907R1-12 20 Escherichia fergusonii 8
515F1-I-907R1-13 6 Escherichia fergusonii 4
515F1-I-907R1-15 7 Escherichia fergusonii 3
515F1-I-907R1-19 9 Escherichia fergusonii 3
515F1-I-907R1-2 20 Escherichia fergusonii 14
515F1-I-907R1-21 4 Escherichia fergusonii 2
515F1-I-907R1-22 9 Escherichia fergusonii 3
515F1-I-907R1-23 6 Escherichia fergusonii 3
515F1-I-907R1-24 5 Escherichia fergusonii 2
515F1-I-907R1-3 20 Escherichia fergusonii 12
515F1-I-907R1-4 20 Escherichia fergusonii 11
515F1-I-907R1-5 20 Escherichia fergusonii 9
515F1-I-907R1-8 20 Escherichia fergusonii 9
515F1-I-907R1-9 20 Escherichia fergusonii 7
515F1-J-907R1-1 20 Escherichia fergusonii 13
515F1-J-907R1-10 3 Escherichia fergusonii 2
515F1-J-907R1-11 20 Escherichia fergusonii 13
515F1-J-907R1-12 20 Escherichia fergusonii 13
515F1-J-907R1-13 7 Escherichia fergusonii 5
515F1-J-907R1-14 5 Escherichia fergusonii 3
515F1-J-907R1-15 7 Escherichia fergusonii 4
515F1-J-907R1-16 8 Escherichia fergusonii 2
515F1-J-907R1-17 20 Escherichia fergusonii 15
515F1-J-907R1-18 6 Escherichia fergusonii 3
515F1-J-907R1-2 20 Escherichia fergusonii 14
515F1-J-907R1-20 20 Escherichia fergusonii 12
515F1-J-907R1-21 9 Escherichia fergusonii 5
515F1-J-907R1-22 9 Escherichia fergusonii 5
515F1-J-907R1-23 3 Escherichia fergusonii 2
515F1-J-907R1-24 6 Escherichia fergusonii 4
515F1-J-907R1-3 20 Escherichia fergusonii 6
515F1-J-907R1-4 20 Escherichia fergusonii 11
515F1-J-907R1-5 20 Escherichia fergusonii 12
515F1-J-907R1-6 20 Escherichia fergusonii 13
515F1-J-907R1-7 20 Escherichia fergusonii 12
515F1-J-907R1-9 13 Escherichia fergusonii 5
515F1-K-907R1-1 20 Escherichia fergusonii 15
515F1-K-907R1-13 12 Escherichia fergusonii 7
515F1-K-907R1-14 20 Escherichia fergusonii 13
515F1-K-907R1-17 20 Escherichia fergusonii 11
515F1-K-907R1-2 20 Escherichia fergusonii 11
515F1-K-907R1-21 20 Escherichia fergusonii 12
515F1-K-907R1-23 20 Escherichia fergusonii 14
515F1-K-907R1-3 20 Escherichia fergusonii 13
515F1-K-907R1-4 20 Escherichia fergusonii 12
515F1-K-907R1-5 20 Escherichia fergusonii 14
515F1-K-907R1-6 20 Escherichia fergusonii 13
515F1-K-907R1-8 20 Escherichia fergusonii 9
515F1-K-907R1-9 20 Escherichia fergusonii 7
515F1-L-907R1-1 20 Escherichia fergusonii 10
515F1-L-907R1-10 20 Escherichia fergusonii 13
515F1-L-907R1-11 20 Escherichia fergusonii 14
515F1-L-907R1-13 11 Escherichia fergusonii 5
515F1-L-907R1-14 20 Escherichia fergusonii 11
515F1-L-907R1-17 20 Escherichia fergusonii 8
515F1-L-907R1-18 20 Escherichia fergusonii 15
515F1-L-907R1-2 20 Escherichia fergusonii 17
515F1-L-907R1-20 20 Escherichia fergusonii 12
515F1-L-907R1-21 20 Escherichia fergusonii 10
515F1-L-907R1-22 20 Escherichia fergusonii 9
515F1-L-907R1-23 20 Escherichia fergusonii 15
515F1-L-907R1-24 9 Escherichia fergusonii 6
515F1-L-907R1-3 20 Escherichia fergusonii 15
515F1-L-907R1-4 20 Escherichia fergusonii 12
515F1-L-907R1-5 20 Escherichia fergusonii 7
515F1-L-907R1-6 20 Escherichia fergusonii 7
515F1-L-907R1-7 20 Escherichia fergusonii 13
515F1-L-907R1-8 20 Escherichia fergusonii 11
515F1-M-907R1-1 20 Escherichia fergusonii 8
515F1-M-907R1-10 20 Escherichia fergusonii 7
515F1-M-907R1-11 20 Escherichia fergusonii 13
515F1-M-907R1-12 20 Escherichia fergusonii 9
515F1-M-907R1-13 14 Escherichia fergusonii 7
515F1-M-907R1-15 20 Escherichia fergusonii 11
515F1-M-907R1-19 20 Escherichia fergusonii 10
515F1-M-907R1-2 20 Escherichia fergusonii 10
515F1-M-907R1-20 20 Escherichia fergusonii 6
515F1-M-907R1-22 20 Escherichia fergusonii 13
515F1-M-907R1-23 12 Escherichia fergusonii 7
515F1-M-907R1-24 10 Escherichia fergusonii 4
515F1-M-907R1-3 20 Escherichia fergusonii 13
515F1-M-907R1-4 20 Escherichia fergusonii 14
515F1-M-907R1-5 20 Escherichia fergusonii 6
515F1-M-907R1-6 20 Escherichia fergusonii 8
515F1-M-907R1-7 20 Escherichia fergusonii 13
515F1-M-907R1-8 20 Escherichia fergusonii 8
515F1-M-907R1-9 20 Escherichia fergusonii 9
515F1-N-907R1-1 13 Escherichia fergusonii 5
515F1-N-907R1-10 20 Escherichia fergusonii 10
515F1-N-907R1-11 20 Escherichia fergusonii 14
515F1-N-907R1-12 20 Escherichia fergusonii 10
515F1-N-907R1-15 11 Escherichia fergusonii 4
515F1-N-907R1-17 9 Escherichia fergusonii 4
515F1-N-907R1-18 9 Escherichia fergusonii 5
515F1-N-907R1-2 5 Escherichia fergusonii 2
515F1-N-907R1-20 9 Escherichia fergusonii 5
515F1-N-907R1-21 7 Escherichia fergusonii 4
515F1-N-907R1-22 10 Escherichia fergusonii 4
515F1-N-907R1-23 5 Escherichia fergusonii 3
515F1-N-907R1-24 5 Escherichia fergusonii 4
515F1-N-907R1-3 20 Escherichia fergusonii 13
515F1-N-907R1-4 20 Escherichia fergusonii 9
515F1-N-907R1-5 20 Escherichia fergusonii 16
515F1-N-907R1-6 20 Escherichia fergusonii 11
515F1-N-907R1-7 20 Escherichia fergusonii 8
515F1-N-907R1-8 20 Escherichia fergusonii 13
515F1-N-907R1-9 20 Escherichia fergusonii 7
515F1-O-907R1-1 18 Escherichia fergusonii 13
515F1-O-907R1-10 20 Escherichia fergusonii 5
515F1-O-907R1-2 20 Escherichia fergusonii 12
515F1-O-907R1-22 20 Escherichia fergusonii 10
515F1-O-907R1-3 20 Escherichia fergusonii 14
515F1-O-907R1-4 20 Escherichia fergusonii 12
515F1-O-907R1-5 20 Escherichia fergusonii 12
515F1-O-907R1-6 20 Escherichia fergusonii 11
515F1-O-907R1-7 20 Escherichia fergusonii 8
515F1-O-907R1-8 20 Escherichia fergusonii 10
515F1-O-907R1-9 20 Escherichia fergusonii 9
515F1-P-907R1-1 10 Escherichia fergusonii 4
515F1-P-907R1-10 20 Escherichia fergusonii 11
515F1-P-907R1-12 11 Escherichia fergusonii 2
515F1-P-907R1-13 6 Escherichia fergusonii 3
515F1-P-907R1-14 8 Escherichia fergusonii 3
515F1-P-907R1-17 20 Escherichia fergusonii 11
515F1-P-907R1-19 8 Escherichia fergusonii 3
515F1-P-907R1-20 8 Escherichia fergusonii 5
515F1-P-907R1-21 5 Escherichia fergusonii 4
515F1-P-907R1-22 5 Escherichia fergusonii 2
515F1-P-907R1-23 4 Escherichia fergusonii 3
515F1-P-907R1-24 3 Escherichia fergusonii 2
515F1-P-907R1-3 20 Escherichia fergusonii 13
515F1-P-907R1-4 20 Escherichia fergusonii 10
515F1-P-907R1-5 20 Escherichia fergusonii 10
515F1-P-907R1-6 20 Escherichia fergusonii 10
515F1-P-907R1-7 20 Escherichia fergusonii 7
515F1-P-907R1-8 20 Escherichia fergusonii 10
515F1-P-907R1-9 20 Escherichia fergusonii 8
515F1-A-907R1-21 20 Enterococcus hirae 11
515F1-B-907R1-15 20 Enterococcus hirae 10
515F1-B-907R1-16 20 Enterococcus hirae 9
515F1-B-907R1-17 20 Enterococcus hirae 7
515F1-B-907R1-21 20 Enterococcus hirae 10
515F1-C-907R1-12 20 Enterococcus hirae 5
515F1-C-907R1-14 20 Enterococcus hirae 9
515F1-C-907R1-19 20 Enterococcus hirae 10
515F1-C-907R1-20 20 Enterococcus hirae 11
515F1-C-907R1-21 20 Enterococcus hirae 10
515F1-C-907R1-23 20 Enterococcus hirae 11
515F1-D-907R1-16 20 Enterococcus hirae 14
515F1-D-907R1-17 20 Enterococcus hirae 11
515F1-D-907R1-19 20 Enterococcus hirae 10
515F1-D-907R1-21 20 Enterococcus hirae 9
515F1-E-907R1-18 20 Enterococcus hirae 8
515F1-E-907R1-19 20 Enterococcus hirae 9
515F1-E-907R1-20 20 Enterococcus hirae 12
515F1-E-907R1-21 20 Enterococcus hirae 13
515F1-E-907R1-22 20 Enterococcus hirae 10
515F1-F-907R1-14 20 Enterococcus hirae 10
515F1-F-907R1-15 20 Enterococcus hirae 9
515F1-F-907R1-18 20 Enterococcus hirae 10
515F1-F-907R1-21 20 Enterococcus hirae 13
515F1-F-907R1-22 20 Enterococcus hirae 9
515F1-G-907R1-15 20 Enterococcus hirae 12
515F1-G-907R1-16 20 Enterococcus hirae 9
515F1-G-907R1-17 20 Enterococcus hirae 8
515F1-G-907R1-18 20 Enterococcus hirae 9
515F1-G-907R1-21 20 Enterococcus hirae 11
515F1-G-907R1-22 20 Enterococcus hirae 11
515F1-G-907R1-23 5 Enterococcus hirae 2
515F1-H-907R1-12 17 Enterococcus hirae 5
515F1-H-907R1-14 20 Enterococcus hirae 14
515F1-H-907R1-15 20 Enterococcus hirae 12
515F1-H-907R1-17 20 Enterococcus hirae 10
515F1-H-907R1-21 20 Enterococcus hirae 11
515F1-H-907R1-22 20 Enterococcus hirae 9
515F1-I-907R1-14 2 Enterococcus hirae 1
515F1-I-907R1-17 10 Enterococcus hirae 5
515F1-I-907R1-18 3 Enterococcus hirae 1
515F1-I-907R1-20 6 Enterococcus hirae 2
515F1-J-907R1-19 5 Enterococcus hirae 2
515F1-K-907R1-15 20 Enterococcus hirae 14
515F1-K-907R1-22 20 Enterococcus hirae 10
515F1-K-907R1-24 5 Enterococcus hirae 2
515F1-L-907R1-15 20 Enterococcus hirae 20
515F1-L-907R1-16 20 Enterococcus hirae 17
515F1-L-907R1-19 20 Enterococcus hirae 13
515F1-M-907R1-14 17 Enterococcus hirae 6
515F1-M-907R1-16 20 Enterococcus hirae 12
515F1-M-907R1-17 20 Enterococcus hirae 16
515F1-M-907R1-18 20 Enterococcus hirae 18
515F1-M-907R1-21 20 Enterococcus hirae 13
515F1-N-907R1-14 3 Enterococcus hirae 1
515F1-N-907R1-16 10 Enterococcus hirae 2
515F1-N-907R1-19 5 Enterococcus hirae 2
515F1-O-907R1-13 5 Enterococcus hirae 2
515F1-O-907R1-14 5 Enterococcus hirae 3
515F1-O-907R1-15 20 Enterococcus hirae 8
515F1-O-907R1-19 20 Enterococcus hirae 12
515F1-O-907R1-20 20 Enterococcus hirae 14
515F1-O-907R1-23 6 Enterococcus hirae 3
515F1-O-907R1-24 4 Enterococcus hirae 1
515F1-P-907R1-16 20 Enterococcus hirae 16
515F1-A-907R1-24 20 Enterococcus gallinarum 13
515F1-D-907R1-15 20 Enterococcus gallinarum 11
515F1-K-907R1-18 20 Enterococcus gallinarum 10
515F1-K-907R1-19 20 Enterococcus gallinarum 14
515F1-K-907R1-20 20 Enterococcus gallinarum 12
515F1-O-907R1-17 20 Enterococcus gallinarum 9
515F1-O-907R1-18 20 Enterococcus gallinarum 10
515F1-O-907R1-21 20 Enterococcus gallinarum 14
515F1-P-907R1-15 20 Enterococcus gallinarum 11
515F1-O-907R1-16 20 Clostridiumparaputrificum 4
515F1-A-907R1-2 20 Clostridiumbutyricum 14
515F1-O-907R1-12 9 Clostridiumbutyricum 3
515F1-P-907R1-2 20 Clostridiumbutyricum 14
515F1-P-907R1-11 20 Acinetobacter johnsonii 6
515F1-K-907R1-16 20 [Clostridium] innocuum 10
515F1-I-907R1-16 2 [Clostridium] bolteae 1
Claims (4)
1. a high-throughput low-cost micro biological sample molecule identification technology, which is characterized by comprising the following steps:
(1) Sample collection: collecting a trace microorganism sample in a small centrifuge tube, centrifuging the sample containing liquid at a high speed for a short time, and removing a liquid part;
(2) Sample lysis: adding glass beads with the diameter of 0.1-0.5 mm to 1/8 of the total volume of the tube, adding protease K suspension with the concentration of 1mg/ml of the total volume of the 1/8 tube, preserving the temperature at 40-70 ℃ for 5-30 minutes, and immediately performing horizontal or vertical oscillation at a speed of more than 1000 rpm;
(3) Nucleic acid collection: adding 5-10% of chelex solution with concentration of 1/4 of the total volume of the tube, maintaining the temperature at 80-95 ℃ for 10-30min, refrigerating and centrifuging at a high speed for a short time, and taking supernatant for refrigerating and preserving;
(4) PCR amplification of labeled primers:
(1) designing universal upstream and downstream primers according to a conserved region of a gene sequence to be amplified;
(2) introducing 1-2 mismatched bases between the 5' end of the upstream primer and the middle region of the downstream primer as specific labels, designing 24 mismatched combinations by the upstream primer, and designing 16 mismatched combinations by the downstream primer, so that 384 different primer combinations are formed, and 384 different PCR tube positions can be corresponded;
(3) adding a 30-35 base joint at the 5' end of the primer to connect with the subsequent PCR amplification in high-throughput sequencing, and further enhancing the amplification stability;
(4) amplifying each sample by using primers with different marking sites;
(5) PCR product mix sequencing and positional reduction: and mixing the PCR products of each tube, performing high-throughput sequencing, and reducing each sequence into each position sample tube through the difference of the primer mark combinations of each tube.
2. The high throughput low cost micro-biological sample molecular characterization technique of claim 1, wherein: the microphotosamples herein generally refer to various biological samples that facilitate micropipette manipulation.
3. The high throughput low cost micro-biological sample molecular characterization technique of claim 1, wherein: the extraction container can be operated in a plurality of single-tube operation, 12-connected tube, 96-well plate or 384-well plate devices at the same time, so that high throughput and low cost are realized.
4. The high throughput low cost micro-biological sample molecular characterization technique of claim 1, wherein the specific contents of step (5) are:
(1) mixing 1-5 mu l of each tube of amplified products, and taking the mixed products as one sample for high-throughput sequencing;
(2) hundreds of thousands of sequences obtained by sequencing are identified by using computer software, and the sequences are distributed into each sample tube again.
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