CN102329855A - Testing type gene chip for testing archaea communities in oil reservoirs and application thereof - Google Patents
Testing type gene chip for testing archaea communities in oil reservoirs and application thereof Download PDFInfo
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- CN102329855A CN102329855A CN201110071426A CN201110071426A CN102329855A CN 102329855 A CN102329855 A CN 102329855A CN 201110071426 A CN201110071426 A CN 201110071426A CN 201110071426 A CN201110071426 A CN 201110071426A CN 102329855 A CN102329855 A CN 102329855A
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Abstract
The invention discloses a testing type gene chip for testing archaea communities in oil reservoirs and application thereof. The gene chip is arranged on a carrier material, and comprises probes for identifying the categories of archaea in oil reservoirs, probes derived from the probes and a complementary probe and an extension probe therefore of each probe. The specificity probes can identify eighteen categories of archaea in oil reservoirs; and the method adopts the genome DNA (deoxyribonucleic acid) of a sample to be tested as a template, uses a marked primer group to carry out amplification, then hybridizes the obtained amplification product with the probes on the biochip, and determines the category of archaea in an oil reservoir according to a hybridization result. The invention provides a quick, accurate and high-throughput tool for the research on the compositions, structures, functional activities and the like of archaea communities in oil reservoir environments. Meanwhile, the mechanism of increasing the recovery ratio by microorganisms can be known at the genetic level as well, and thereby the invention provides a theoretical basis and technical support for the enhancement of the microbial enhanced oil recovery technology and the on-site implementation effect.
Description
One, technical field:
The present invention relates to a kind of gene chip field, particularly a kind of detection type gene chip that detects oil reservoir bacterial classification in middle ancient times crowd and uses thereof.
Two, background technology:
Along with microbial technique in extensive applications such as petroleum natural gas exploration and environment remediation, the detection evaluation and the colony assay of related microorganisms also become important research contents.Under the oil reservoir extreme environment, abundant microbial population is arranged, wherein, ancient bacterium extensively exists and brings into play important metabolism, but ancient bacterial classification class is various, abundance is lower, and be difficult to utilize pure culture method in laboratory to carry out classification and Detection.
Polymerase chain reaction (PCR), restriction fragment length polymorphism analysis (RFLPs), AFLPs and denaturing gradient gel electrophoresis (DGGE) equimolecular biology techniques have promoted the development of petroleum microorganism research.Yet there are many deficiencies in molecule polymorphum technology commonly used at present: (1) operation is complicated, and the human intervention link is many, poor accuracy; (2) the detection flux is low, provides information few, and time and effort consuming.This regulates and control to have brought inconvenience to timely and effectively ancient flora being dropped into to go.
Three, summary of the invention:
The object of the invention is exactly the above-mentioned defective that exists to prior art; A kind of detection type gene chip that detects oil reservoir bacterial classification in middle ancient times crowd and uses thereof is provided; Can under molecular level, study microflora; Ancient bacterial classification crowd information under the understanding varying environment condition is confirmed dominant population, for microbial technique research level and field conduct effect provide method basic.
Detection type gene chip and the method for use that is used for oil reservoir bacterial classification in middle ancient times crowd provided by the present invention; It is the ancient bacterial classification class that exists in the analytical reagent oil reservoir; Design belongs to specific probe to its 16S ribosome-RNA(rRNA), is template with the genomic dna of detected sample, increases with the primer sets of mark; Then the probe on amplified production that obtains and the biochip is hybridized, confirm the kind of the ancient bacterium of oil reservoir according to results of hybridization.
A kind of detection type gene chip that detects oil reservoir bacterial classification in middle ancient times crowd, its preparation method is:
(1) the ancient bacterium specific probe of design oil reservoir
(1) oil reservoir sample collecting;
(2) the ancient bacterium 16S of oil reservoir ribosome-RNA(rRNA) sequence is collected: with universal primer, ancient bacterium 16S ribosome-RNA(rRNA) fragment is increased, and order-checking;
(3) ancient bacterium specific probe: according to above-mentioned sequencing result, the ancient Pseudomonas specific probe of design oil reservoir, the bar code sequence probe sequence general structure on the chip is: NH
2-TTTTTTTTTTTTTTT-bar code sequence, promptly 5 of probe ' end is amido modified, and amino next door connects poly-T15;
(2) preparation gene chip:
Use point sample instrument with probe with the fixed order set to the slide of aldehyde radical modification, the array that gene chip is made up of the positive quality control PC to the negative Quality Control NC of the negative Quality Control BC of the positive quality control QC of 83 probes of 18 ancient Pseudomonas, point sample, point sample, hybridization and hybridization;
Probe sequence such as following table:
QC is that an end has the Hex mark, and the oligonucleotide probe that the other end is amido modified is used to observe chip point sample and fixed efficient, and its sequence is NH2-TTTTTTTTTTTTTTTAGAGTGCTTGGTGCCATAAC-HEX; BC is 50% methyl-sulphoxide DMSO, and being used for Quality Control point sample process has the n.s. residual contamination; NC is one section amido modified oligonucleotide probe, can not hybridize with all sequences to be detected in the hybridization solution, is used for observing that specific hybridization nothing but, its sequence are arranged is NH2-TTTTTTTTTTTTTTTGCAACCACCACCGGAGG; PC is one section amido modified oligonucleotide probe, can with the fluorescently-labeled complementary sequence hybridization that adds in the hybridization solution, be used for the Quality Control of crossover process, its sequence is NH2-TTTTTTTTTTTTTTTGGTATCGCGACCGCATCCCAATCT.
And detection oil reservoir bacterial classification in middle ancient times crowd's of the present invention detection type gene chip, its method of use is:
(a) nucleic acid amplification
Be template with oil reservoir sample gene group DNA respectively, with upstream primer and downstream primer pcr amplification;
Amplification system is following, comprises 0.2mM dNTPs, and 1 * Qiagen PCR buffer adds MgCl2 to 2mM, pH 8.7,1 HotStarTaq DNA Polymerase of unit and 0.1ng masterplate DNA, upstream and downstream primer; In the 25 μ l amplification systems, the concentration of primer is 0.02 μ M;
The amplification parameter is:
Earlier 95 ℃ 15 minutes; Then 94 ℃ 30 seconds, 55 ℃ 1 minute, 72 ℃ 90 seconds, 35 circulations;
Last 72 ℃ 10 minutes; 4 ℃ of preservations;
(b) gene chip hybridization
Get 10 μ l PCR products and be added in the 20 μ l hybridization buffers, 98 ℃ of sex change 5 minutes, ice bath, hybridization mixture join in the dot matrix and react.Then chip is placed in 42 ℃ of water-baths and hybridized 1 hour; Take out slide respectively in two kinds of washing lotions 42 ℃ respectively wash 2min, washing lotion I:0.3 * SSC/0.1%SDS, washing lotion II:0.06 * SSC; At last, slide centrifuge dripping slightly;
(c) data analysis
Use chip scanner scanning chip, according to fluorescence signal intensity and the ancient flora of the scan image analysis purposes oil reservoir structure that falls.
Detection oil reservoir bacterial classification in middle ancient times crowd's of the present invention detection type gene chip, its purposes is: gene chip is the oligonucleotide arrays that is used to detect Archaeoglobus, Geoglobus, Methanobacterium, Methanocalculus, Methanococcoides, Methanoculleus, Methanoplanus, Methanothermococcus, Methanothrix/Methanosaeta, Pyrodictium, Thepmococcus, Thermofilum, Ferroglobus, Methanocella, Methanosarcina, Methanothermobacter, Methermicoccus or Picrophilus.
The invention has the beneficial effects as follows: the present invention have high specificity, highly sensitive, the result is reliable and stable and be suitable for the in batches characteristics of pattern detection, is applicable to ancient bacterial classification cluster analysis natural, simulating oil deposit; In addition, the present invention is as a kind of revolutionary analytical technology, and biochip is integrated so that it was had, the performance of microminiaturization and robotization has been brought into play more and more important effect analyzing biological technical field.The advantage of biochip high-throughput, the parallel detection of many indexs; Exactly can satisfy the research demand of oil pool microorganisms group; Use ancient bacterial classification crowd detection chip and carry out the identification and analysis of group, can under molecular level, study, ancient bacterial classification crowd information under the understanding varying environment condition microflora; Confirm dominant population, for microbial technique research level and field conduct effect provide the method basis.
Four, description of drawings:
Accompanying drawing 1 is the hybridization dot matrix collection of illustrative plates of embodiments of the invention.
Five, embodiment:
Embodiment 1: following is that example is explained the present invention to detect the Shengli Oil Field sample, should be understood that these embodiment only are used to explain the present invention, limits the scope of application of the present invention and be not used in.Following experimental technique if no special instructions, is ordinary method.
1. oil reservoir sample collecting: select certain recovery well, take a sample according to ordinary method.
2. oil reservoir sample nucleic acids for preparation: use the ordinary method genomic dna.
3. nucleic acid amplification: be template with oil reservoir sample gene group DNA respectively, with upstream primer and downstream primer pcr amplification; PCR carries out in 1 pipe.
Amplification system is following, comprises 0.2mM dNTPs, and 1 * Qiagen PCR buffer adds MgCl2 to 2mM, and pH 8.7, the 1 HotStarTaq DNA Polymerase of unit (Qiagen, Hilden, Germany) and the 0.1ng DNA, the upstream and downstream primer.In the 25 μ l amplification systems, the concentration of primer is 0.02 μ M.
The amplification parameter is:
Earlier 95 ℃ 15 minutes; Then 94 ℃ 30 seconds, 55 ℃ 1 minute, 72 ℃ 90 seconds, 35 circulations;
Last 72 ℃ 10 minutes; 4 ℃ of preservations.
4. preparation gene chip
The array that chip is made up of 83 probes, QC (positive quality control of point sample), BC (the negative Quality Control of point sample), NC (the negative Quality Control of hybridization) and PC (positive quality control of hybridization) to 18 ancient Pseudomonas.
Use point sample instrument with probe with the fixed order set to the slide of aldehyde radical modification.The sealing chip on not with oligonucleotide bonded aldehyde radical.Treat after the chip seasoning subsequent use.
4. gene chip hybridization
Get 10 μ l PCR products and be added to (6 * SSC, 5 * Denhardt ' s reagent, 25% methane amide, 0.1%SDS, 5nM c-PC (with chip PC complementary sequence, 5 ' end TAMRA mark) in the 20 μ l hybridization buffers.98 ℃ of sex change 5 minutes, ice bath, hybridization mixture join in the dot matrix and react.Then chip is placed in 42 ℃ of water-baths and hybridized 1 hour.Take out slide respectively in two kinds of washing lotions 42 ℃ respectively wash 2min, washing lotion I:0.3 * SSC/0.1%SDS, washing lotion II:0.06 * SSC.At last, slide centrifuge dripping slightly.
5. data analysis
Use chip scanner scanning chip.According to the fluorescence signal intensity analysis, totally seven kinds on its main ancient mushroom type is respectively Archaeoglobus sp.; Methanocalculu sp.; Methanobacterium sp., Methanoplanus sp., Thermococcus sp.; Methanothermococcus sp., Methanothermobacter sp..This is consistent with the result who the PCR product is built the clone library sequencing analysis.
Sequence table
<160>12
<210>1
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>1
TCCCAGGTK?AGACCGGGTTT?20
<210>2
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>2
CGATCCCAGG?TKAGACCGGG 20
<210>3
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>3
CCTCGATCCC?AGGTKAGACC?20
<210>4
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>4
TGGACCCBTG?TCTCAGGGTC?20
<210>5
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>5
CAGGTKAGAC?CGGGTTTAGG?20
<210>6
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>6
TTCCAGCACC?CATCACCTAT?20
<210>7
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>7
AGTCTGGTAG?GGTCTTCAGC?20
<210>8
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>8
GCATTCCAGC?ACCCATCACC?20
<210>9
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>9
ATCCCAGGTT?AGACCGGGTT?20
<210>10
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>10
TCGATCCCAG?GTTAGACCGG?20
<210>11
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>11
ACCGCGCGNT?TATGACACGC?20
<210>12
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>12
GCGCGNTTAT?GACACGCGAT?20
<210>13
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>13
TTCACCGCGC?GNTTATGACA?20
<210>14
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>14
CCNAAGGATC?CGCTGGTAAC?20
<210>15
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>15
AGTGTGCCCG?GCATCCCNAA?20
<210>16
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>16
CCTTTCRGAG?ACAGAGCATT?20
<210>17
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>17
CACCCGTCTC?CTATGAGGNA?20
<210>18
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>18
CCGTCTCCTA?TGAGGNATTA?20
<210>19
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>19
GGACCTTTCR?GAGACAGAGC?20
<210>20
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>20
GGCGGACCTT?TCRGAGACAG?20
<210>21
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>21
TCCCAGGTGG?TTCGCTTCAC?20
<210>22
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>22
GCGATTCAGG?TAARGTCTTT?20
<210>23
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>23
CACAGAGTAC?CCATCATCCC?20
<210>24
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>24
AGAGTACCCA?TCATCCCGAA?20
<210>25
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>25
ATCCCGAAG?GACATGCTGGC?20
<210>26
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>26
ACAGCCTGCA?TNTACAGGCA?20
<210>27
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>27
GCCTGCATNT?ACAGGCACTC?20
<210>28
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>28
ATNTACAGGC?ACTCGAGGTT?20
<210>29
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>29
TGCATNTACA?GGCACTCGAG?20
<210>30
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>30
TCAGAATCCA?ACTCCGGGCT?20
<210>31
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>31
TCAGTGCCGT?TTCYCATTGT?20
<210>32
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>32
GTGCCGTTTC?YCATTGTCCT?20
<210>33
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>33
AGTGTCCYCA?TCATTCCGGA?20
<210>34
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>34
GTCCYCATCA?TTCCGGAGAA?20
<210>35
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>35
ATTCCGGAGA?ACATGCTRGC?20
<210>36
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>36
AGTVGCAACA?TAGGGCACGG?20
<210>37
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>37
ATCYCTGCCA?GCCCTACGGT?20
<210>38
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>38
YCTGCCAGCC?CTACGGTTAA?20
<210>39
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>39
CCGTAGGATT?TAAACAGRGA?20
<210>40
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>40
CCTCTCAGCG?CTTCAGGYAA?20
<210>41
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>41
CACTCCBGAG?AGTACGCTGG?20
<210>42
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>42
TCCBGAGAGT?ACGCTGGCAA?20
<210>43
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>43
CAGTGTACCC?GTCACTCCBG?20
<210>44
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>44
TGTACCCGTC?ACTCCBGAGA?20
<210>45
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>45
ACCCGTCACT?CCBGAGAGTA?20
<210>46
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>46
AGTAAKCATC?CCGACCGCTC?20
<210>47
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>47
AAKCATCCCG?ACCGCTCGCG?20
<210>48
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>48
TTTCGGCGAG?GGACCCGTTC?20
<210>49
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>49
CCCAGTAAKC?ATCCCGACCG?20
<210>50
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>50
AGGCCCAGTA?AKCATCCCGA?20
<210>51
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>51
GACACGCGGG?TACTAGGGAT?20
<210>52
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>52
GGGRAGGACT?GGCAACTGGG?20
<210>53
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>53
TCTCGGCGTG?TCCGGCAAGA?20
<210>54
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>54
AACTGRGGGC?GGGTTTAGGG?20
<210>55
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>55
CTTTCGGCCT?GAGGACCTTC?20
<210>56
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>56
TCTCAGCTCG?TCTGGCAAGA?20
<210>57
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>57
CNGCCATTGC?AGCTCGCGTG?20
<210>58
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>58
TCTTCAGCCC?GACCTTCATC?20
<210>59
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>59
CAGTGGAHAT?GGGTCTCGCT?20
<210>60
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>60
CAACAGTGGA?HATGGGTCTC?20
<210>61
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>61
TAGCAACAGT?GGAHATGGGT?20
<210>62
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>62
CATACAACTG?TCGATCCCGG?20
<210>63
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>63
CCAGCGGTCT?CCACMGTGTA?20
<210>64
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>64
GAACTACGRA?TGGGTTTGTG?20
<210>65
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>65
CTACGRATGG?GTTTGTGAGA?20
<210>66
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>66
RTCCCGGAGG?ACATGCTGGT?20
<210>67
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>67
GGACCCATTG?TCCCATYCAT?20
<210>68
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>68
AGAGTACCCA?TCRTCCCGGA?20
<210>69
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>69
CCCATAAGGG?TTCCGCTGGT?20
<210>70
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>70
ATAAGGGTTC?CGCTGGTAAC?20
<210>71
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>71
ACCCGRAGGT?GTTTCGGTGA?20
<210>72
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>72
ACCTCGAGTC?ATGATAGTAT?20
<210>73
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>73
CCGACCTCGA?GTCATGATAG?20
<210>74
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>74
GACCTTCATC?TCG?CTGTCKC?20
<210>75
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>75
CCTGACCTTC?ATCTCGCTGT?20
<210>76
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>76
CAGCCTGACC?TTCATCTCGC?20
<210>77
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>77
CTAATCCGGT?AAGGTCTTCA?20
<210>78
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>78
CAGCTAATCC?GGTAAGGTCT?20
<210>79
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>79
AAGCGCTAGA?ATTTACCTGG?20
<210>80
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>80
ACCCGCGTAG?GTAATTGCAA?20
<210>81
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>81
CYTTCACCAT?CCAAACATTC?20
<210>82
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>82
TCTGAATGGC?TATAGGGAAT?20
<210>83
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>83
AGTTCTGAAT?GGCTATAGGG?20
Claims (3)
1. detection type gene chip that detects oil reservoir bacterial classification in middle ancient times crowd is characterized in that its preparation method is:
(1) the ancient bacterium specific probe of design oil reservoir
(1) oil reservoir sample collecting;
(2) the ancient bacterium 16S of oil reservoir ribosome-RNA(rRNA) sequence is collected: with universal primer, ancient bacterium 16S ribosome-RNA(rRNA) fragment is increased, and order-checking;
(3) ancient bacterium specific probe: according to above-mentioned sequencing result, the ancient Pseudomonas specific probe of design oil reservoir, the bar code sequence probe sequence general structure on the chip is: NH
2-TTTTTTTTTTTTTTT-bar code sequence, promptly 5 of probe ' end is amido modified, and amino next door connects poly-T15;
(2) preparation gene chip:
Use point sample instrument with probe with the fixed order set to the slide of aldehyde radical modification, the array that gene chip is made up of the positive quality control PC to the negative Quality Control NC of the negative Quality Control BC of the positive quality control QC of 83 probes of 18 ancient Pseudomonas, point sample, point sample, hybridization and hybridization;
QC is that an end has the Hex mark, and the oligonucleotide probe that the other end is amido modified is used to observe chip point sample and fixed efficient, and its sequence is NH2-TTTTTTTTTTTTTTTAGAGTGCTTGGTGCCATAAC-HEX; BC is 50% methyl-sulphoxide DMSO, and being used for Quality Control point sample process has the n.s. residual contamination; NC is one section amido modified oligonucleotide probe, can not hybridize with all sequences to be detected in the hybridization solution, is used for observing that specific hybridization nothing but, its sequence are arranged is NH2-TTTTTTTTTTTTTTTGCAACCACCACCGGAGG; PC is one section amido modified oligonucleotide probe, can with the fluorescently-labeled complementary sequence hybridization that adds in the hybridization solution, be used for the Quality Control of crossover process, its sequence is NH2-TTTTTTTTTTTTTTTGGTATCGCGACCGCATCCCAATCT.
2. detection oil reservoir bacterial classification in middle ancient times crowd's according to claim 1 detection type gene chip is characterized in that its method of use is:
(a) nucleic acid amplification
Be template with oil reservoir sample gene group DNA respectively, with upstream primer and downstream primer pcr amplification;
Amplification system is following, comprises 0.2mM dNTPs, and 1 * Qiagen PCR buffer adds MgCl2 to 2mM, pH 8.7,1 HotStarTaq DNA Polymerase of unit and 0.1ng masterplate DNA, upstream and downstream primer; In the 25 μ l amplification systems, the concentration of primer is 0.02 μ M;
The amplification parameter is:
Earlier 95 ℃ 15 minutes; Then 94 ℃ 30 seconds, 55 ℃ 1 minute, 72 ℃ 90 seconds, 35 circulations;
Last 72 ℃ 10 minutes; 4 ℃ of preservations;
(b) gene chip hybridization
Get 10 μ l PCR products and be added in the 20 μ l hybridization buffers, 98 ℃ of sex change 5 minutes, ice bath, hybridization mixture join in the dot matrix and react.Then chip is placed in 42 ℃ of water-baths and hybridized 1 hour; Take out slide respectively in two kinds of washing lotions 42 ℃ respectively wash 2min, washing lotion I:0.3 * SSC/0.1%SDS, washing lotion II:0.06 * SSC; At last, slide centrifuge dripping slightly;
(c) data analysis
Use chip scanner scanning chip, according to fluorescence signal intensity and the ancient flora of the scan image analysis purposes oil reservoir structure that falls.
3. detection type gene chip that detects oil reservoir bacterial classification in middle ancient times crowd, it is characterized in that its purposes is: gene chip is the oligonucleotide arrays that is used to detect Archaeoglobus, Geoglobus, Methanobacterium, Methanocalculus, Methanococcoides, Methanoculleus, Methanoplanus, Methanothermococcus, Methanothrix/Methanosaeta, Pyrodictium, Thermococcus, Thermofilum, Ferroglobus, Methanocella, Methanosarcina, Methanothermobacter, Methermicoccus or Picrophilus.
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