CN104975090A - Method for automatically detecting abundance of butane oxidizing bacteria - Google Patents
Method for automatically detecting abundance of butane oxidizing bacteria Download PDFInfo
- Publication number
- CN104975090A CN104975090A CN201510400777.1A CN201510400777A CN104975090A CN 104975090 A CN104975090 A CN 104975090A CN 201510400777 A CN201510400777 A CN 201510400777A CN 104975090 A CN104975090 A CN 104975090A
- Authority
- CN
- China
- Prior art keywords
- pcr
- nucleic acid
- sample
- dna
- workstation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for automatically detecting the abundance of butane oxidizing bacteria. The method comprises the steps: constructing an automatic workstation of an original-state microbial oil gas and hydrate prospecting technique, performing sample processing, completing liquid transfer by adopting an automatic liquid processing device of the workstation, extracting and purifying DNA (deoxyribonucleic acid) by using an automatic nucleic acid extraction device, performing electrophoresis detection with agarose, performing fluorescent quantitive PCR (polymerase chain reaction) detection of the abundance of butane oxidizing bacteria, obtaining results, and the like. Based on an automatic analysis method, automation of the nucleic acid extraction and purification steps of butane oxidizing bacteria is achieved; automation of the preparation of a specific gene PCR reaction system is achieved, errors caused by human factors introduced by manual operation are reduced, the operation steps are carried out in a clean environment, the whole detection process is controlled by computer software, the detection procedure can be traced, and uniform correction of system errors can be realized.
Description
Technical field
The present invention relates to oil gas and hydrate exploration and oil gas and hydrate and hide characterization technique field, more specifically, relate to a kind of automated detection method of butane oxidation bacterium abundance.
Background technology
China is an energy consumption big country.The external dependence degree of China's oil in 2013 is close to 60%.Along with the quickening of industrialization and Development of China's Urbanization, the demand of China to oil gas and the hydrate energy enters sustainable growth period.Although China is implementing energy-saving and emission-reduction and new forms of energy development strategy, in quite over a long time, China still cannot break away from the dependence to hydrocarbon resources, and therefore Chinese energy safety is put on important schedule.For alleviating China energy shortage situation, the exploration and to develop oil gas more efficiently extremely urgent with hydrate of going into overdrive, and efficiently method of exploration and relevant equipment thereof be one of critical path of solution energy demand.
Oil gas and hydrate exploration field comprise the large core technology of geological prospecting, geophysical survey, geochemical prospecting and microbiological prospecting four.Wherein first three kind exploration engineering relative microbes oil gas and hydrate exploration technology have been tending towards ripe.Microbiological prospecting belongs to one of large core technology of oil gas and natural gas hydrate exploration field four, is the important method of carrying out source potential assessment according to microbiological anomaly in surface soil/settling/water.Microbiological prospecting technology because of its fast, economy, result the concern of advantage and extremely educational circles such as easily to explain.The advantage such as microbiological prospecting method has fast, economy and result are easily explained.But because in soil, S & W body, more than 99% microorganism can not be cultivated, therefore the traditional microbiological method of exploration set up based on culture method is difficult to meet exploration requirement in accuracy of detection; Because hydrocarbon gas Micro blazed-grating is dynamically with discontinuous, therefore traditional microbiological method of exploration cannot be in the block discovery hydrocarbon gas Micro blazed-grating of squeezed state at microfissure; Single viable bacteria abnormal index is adopted to be difficult to carry out exploration at the environment such as desert, Gobi desert and ocean deepwater now; Traditional microbiological method of exploration needs double sampling when being used for reservoir characterization, and this not only significantly increases cost, and cannot implement in a lot of mining area, did not carry out microorganism characterize because of the most hydrocarbon-bearing pool of China.
The immature property of microorganism oil gas and hydrate exploration technology is mainly manifested in: (1) in the face of abundance is very low, to have oil gas and hydrate indicative significance, the complex sample of obligate or aerobic-anaerobic microbe, the traditional microbiological oil gas set up based on culture of microorganism and hydrate exploration technology, not only be difficult to survey its bacterial abundance accurate (because in soils and sediments, the microorganism of more than 99% can not be cultivated), data reliability is poor; (2) cannot find that microfissure is in the exploration area oil gas of squeezed state and hydrate is hidden; (3) in the face of complicated and diversified oil gas and hydrate exploration district, particularly desert, Gobi desert, saltings and ocean deepwater district, the traditional microbiological oil gas set up based on culture of microorganism and hydrate exploration technology lost efficacy substantially; Trace it to its cause, mainly traditional microbiological oil gas and hydrate exploration technology only depend on the viable bacteria of single index extremely; (4) based on traditional oil gas of culture method and hydrate exploration technology very low to the processing efficiency of sample, a usual batch sample at least needs 14 days ability to obtain result, and the cycle is long, and efficiency is low, and cost is high; (5) data influence factor is many, fluctuation is large, measures in flow process can introduce a large amount of personal errorss in analysis; Testing process can not be reviewed, and cannot carry out the correction of systematic error.
Wang Jianghai etc. are based on the ortho states detection technique of microbial single-cell and gene, the concept of proposition ortho states microorganism oil gas and the hydrate exploration technology of taking the lead in, principle and technical scheme, and in the fluorescent dye of methane-oxidizing bacteria viable bacteria In-vitro specificity, the Quantitative detection of methane-dead bacterium of butane oxidation bacterium and viable bacteria, setting up of microorganism oil gas and hydrate exploration multiple indexes makes important progress with the aspect such as the PCR strategy of oil gas and the denaturing gradient gel electrophoresis analysis of microflora of hydrate field district, form the viable bacteria with hydrocarbon gas oxidation bacteria and association mushroom thereof, dead bacterium and total bacterium are microorganism oil gas and the hydrate exploration technology of multiple exploration index extremely, but still need to detect bacterial abundance in manual methods at present, essence cannot solve the defect of microorganism oil gas and hydrate exploration technology.
Ortho states microorganism oil gas and hydrate exploration technology (IsMOST) are oil gas based on modern molecular biology technique and hydrate exploration technology, only has the fast processing in enormous quantities realizing low abundance microbiological specimens, and obtain on the basis of breakthrough of essence in relevant equipment technical field, just likely real by ortho states microbiological prospecting Technology application in oil gas and hydrate exploration aspect.
Summary of the invention
The technical problem that the present invention will solve is the deficiency for existing microorganism oil gas and hydrate exploration technology, especially automatic business processing technical deficiency, a kind of automatic processing method of butane oxidation bacterium abundance is provided, fast processing in enormous quantities and the analysis of low abundance microbiological specimens can be realized based on described method, and ensure to detect analytical results accurately, realize the trackability of testing process, the unified of systematic error can be carried out and correct.
Object of the present invention is achieved by the following technical programs:
A kind of automatic processing method of butane oxidation bacterium abundance is provided, comprises the following steps:
S1. the Automation workstation (isMOST) of ortho states microorganism oil gas and hydrate exploration technology is built; Described workstation comprises automated fluid treatment unit, nucleic acid extraction and purifier apparatus, PCR reaction system configuration-system;
S2. sample preparation, each sample obtains the supernatant liquor being greater than 1.5ml;
S3. ortho states microorganism oil gas of the present invention and hydrate exploration workstation automated fluid treatment unit (isMOST Station 1) is adopted to complete and move liquid; Specifically perform the program of automated fluid treatment unit setting, different reagent is added in corresponding sample panel, board, wash plate and wash-out plate by robotically controlled arm being being moved liquid passage and the operation moving liquid module automatically;
S4. automatic instrument for extracting nucleic acid extraction and purification DNA, after all sample panel are all ready to, perform the program that ortho states microorganism oil gas of the present invention and hydrate exploration workstation (isMOST Station 2) set, the total genomic dna in soil and/or settling.After about 1 hour, nucleic acid extraction and purifying flow process terminate, and move in trace P CR pipe by the nucleic acid extraction liquid of sample, preserve after mark is clear in-20 DEG C of refrigerators;
S5. agarose electrophoresis detects;
Working method is as follows:
1. get 5 μ l DNA extraction liquid electroresis appraisal on 1% agarose, DNA Ladder is DL2000.
If 2. there is >2kbp band, then get 5 μ l DNA extraction liquid and add after aqua sterilisa dilutes 20 times and preserve as pcr template and at 4 DEG C, sample gene group DNA extraction liquid is stored in-20 DEG C.
S6. the fluorescence quantitative PCR detection of butane oxidation bacterium abundance
(1) cultivation of butane oxidation bacterium
The substratum of preparation butane oxidation bacterium; 1% butane oxidation bacterium is inoculated in culturing bottle; Butane is injected after sealing; Cultivate 15 days at suitable temperature, cultivation of transferring thereafter, muddy to the visible nutrient solution of naked eyes; Collect the butane oxidation bacterium thalline cultivating gained, for the nucleic acid extraction of butane oxidation bacterium and the drafting of typical curve.
(2) Auele Specific Primer is designed
According to the CDS sequence of butane oxidation bacterium bmoX gene, design and synthesize the two pairs of Auele Specific Primers that can be used for fluorescence quantitative PCR detection butane oxidation bacterium, primer sequence is as follows:
Pair of primers:
bmoX-F:5'TCGCCCGAGCAGCGGAACGGTTATCT 3'
bmoX-R:5'AGGATTTTCATTGGATCAGGAATAGTAA 3'
The PCR primer length obtained that increases is 1190bp.
Second pair of primer:
bmoX-DF:5'TGGCACCGGTGGGTGTACGAAGACT 3'
bmoX-DR:5'GCGCGATCAG(C:G 3:2)GTCTT(G:C 9:1)CC(G:A 1:9)TC 3'
The PCR primer length obtained that increases is about 400bp.
(3) preparation standard product
Take total genomic dna as template, adopt the target fragment of primer amplified butane oxidation bacterium, and it is connected with carrier T; Connection product is added in competent cell suspension, then extracts plasmid; After order-checking turns out to be butane oxidation bacterium target fragment, then the purity measuring plasmid DNA is also quantitative; Finally be diluted to finite concentration with aseptic double-distilled water ,-20 DEG C of packing are preserved, as the standard substance of quantitative fluorescent PCR.
(4) reaction system and the reaction conditions of real-time fluorescence quantitative PCR is set up
RT-PCR amplification completes in Roche Light Cycler 480 (Roche Diagnostics, Penzberg, Germany).Reaction system is basis
premix Ex Taq
tM(TaKaRa) manufacturer specification preparation.Two couples of primer A189F and mb661R, 955F25_butane and 1517R22, the CDS sequence of amplification butane oxidation bacterium bmoX gene.In settling, total gene DNA adds in each reaction mixture using the level of 1 ~ 10ng as template.By the parameter preparation PCR reaction system listed as follows.
Real-time fluorescence quantitative PCR reaction system:
Real-time fluorescence quantitative PCR reaction conditions
Wherein, being formulated in ortho states microorganism oil gas of the present invention and hydrate exploration workstation (isMOST Station 1) of PCR reaction system completes.Operating process is as follows:
S11. prepare reaction system mixed solution (except 2 μ L DNA profilings) according to table 2, be sub-packed in 2ml sterile centrifugation tube, often pipe is greater than 0.5ml, is placed in the luggage carrier that refrigeration is preserved.
S12. ortho states microorganism oil gas is placed in luggage carrier, as DNA profiling together with the deep-well plates of the band nucleic acid samples of hydrate exploration workstation (isMOST Station 2) Isolation and purification.
S13. the program that ortho states microorganism oil gas and hydrate exploration workstation (isMOST Station 1) set is performed, the mix reagent set up needed for PCR reaction system and 2 μ L DNA profilings are added successively in corresponding, new deep-well plates, namely complete the configuration of PCR reaction system.
Subsequent step reference literature (Shao Mingrui etc., 2013, the foundation of three kinds of oil and gas indication bacterium quantifying PCR methods and the Preliminary Applications in oil-gas field soil thereof, biotechnology is circulated a notice of, 4th phase, 172-178) operate, after experiment terminates, with LightCycler 480Software 1.5.0 software, experimental result is analyzed, the abundance of butane oxidation bacterium in sample can be obtained.
Beneficial effect of the present invention:
Based on the present invention, following beneficial effect can be obtained:
(1) nucleic acid extraction of hydrocarbon gas oxidation bacteria and association mushroom thereof and purification step is made to realize automatization;
Based on scientific analysis in conjunction with a large amount of optimization experiment result of study, set up optimum extraction and the purifying flow process of hydrocarbon gas oxidation bacteria and association mushroom nucleic acid thereof, according to its best nucleic acid extraction and purifying flow process, accurately moved the technology such as liquid, anti-hanging drop, mechanical arm and software control by hyperchannel, realize the automatization of soils/sediments amplifying nucleic acid Isolation and purification step.
(2) preparation of hydrocarbon gas oxidation bacteria and association mushroom specific gene PCR system thereof is made to realize automatization;
The technology such as liquid, anti-hanging drop, mechanical arm and software control are accurately moved by hyperchannel, accurate control completes the precise amounts of each reagent needed for quantitative fluorescent PCR system of hydrocarbon gas oxidation bacteria and association mushroom specific gene thereof, realize the automatization of PCR system preparation, the error that human factor when reducing manual operations causes.
(3) further, the design of laminar flow hood ensures that the operation stepss such as nucleic acid extraction are run in clean environment, guarantee on the one hand the accuracy of impact without outside contamination and detected result, on the other hand volatilizable reagent is reclaimed, ensure the safety of operator and the clean and tidy of laboratory environment.
In summary, outstanding beneficial effect is: (1) solve abundance very low, to have oil gas and hydrate indicative significance, the abundance of obligate or aerobic-anaerobic microbe complex sample detects, data reliability is high; (2) the present invention is based on the detection of many indexes oil gas and hydrate to the hydrocarbon gas oxidation bacteria of indicative significance, can successfully be applicable to carry out oil gas and hydrate exploration in complicated and diversified exploration area; (3) processing efficiency of sample is very high, and the cycle is short, and cost falls; (4) automatic business processing, eliminates various factors, reduces data fluctuations, effectively avoids analyzing the personal errors measuring and introduce in flow process; (5) whole testing process is by computer software control, and testing process can be reviewed, and the unified of feasible system error corrects.
Accompanying drawing explanation
The structural representation of Fig. 1 mechanical arm of the present invention.
Fig. 2 is of the present invention moves liquid schematic diagram.
Fig. 3 is of the present invention move liquid passage is arranged move liquid module pictorial diagram.
Fig. 4 analytical procedure of the present invention detects the pcr amplification electrophorogram of bacterium in oceanic sediment Duplicate Samples.
The electrophorogram of the pcr amplification of bacterium, wherein a:CTAB-phenol chloroform method in Fig. 5 manual process terrestrial soil sample; B: RNA isolation kit.
1-mechanical arm, 2-moves liquid passage, and 3-moves plate mechanical manipulator, and 4-receives and rises point sample and 96 or 384 passages, 5-piston, 6-sleeve, 7-pressure transmitter, 8-air column, 9-liquid.
Embodiment
The present invention is further illustrated below in conjunction with the drawings and specific embodiments.In apparatus of the present invention and system, maximum inventive point is the correlation technique of the other field of the existing routine of optimization and conformity, solves the drawback existing for manual operations when the sample analysis carrying out microorganism oil gas and hydrate exploration detects.Annexation between some parts and parts can be achieved with reference to association area proven technique, does not therefore repeat one by one at this, or therefore limits the scope of the invention.
Embodiment 1
The present embodiment provides the automatic processing device of a kind of ortho states microorganism oil gas and hydrate exploration technology, comprises automated fluid treatment unit and nucleic acid extraction and purifier apparatus;
Whole experimentation to the automatization application of sample of sample, reaction reagent, preparation and process, and to be placed in fully automatic operation under operatorless environment by integrating various terminal analysis test set and to provide result by described automated fluid treatment unit.Preferably, described automated fluid treatment unit is made up of the structure of four parts: service platform, mechanical arm, the carrier moving liquid passage and experimental article and utility appliance; Described service platform is provided with some positioning tracks (T), for the carrier of the various experimental article of fixed placement.Service platform is the framework of whole workstation, is semi-enclosed structure, and platform front has can the pneumatic control door of manual open and close.Platform is designed with a lot of positioning track, different experimental articles, different discharge orders, the carrier size required for it is also different, can occupy the position of different quantities T.Service platform can be divided into broad variety by the capacity of water of processing sample.Main Types has three kinds: compact type service platform: 30 T, can place at most 25 SBS international standard plates (as 96 hole microwell plates); Universal service platform: 54 T, can place at most 45 SBS international standard plates (as 96 hole microwell plates); Extended service platform: 71 T, can place at most 55 SBS international standard plates (as 96 hole microwell plates).
The structural representation of described mechanical arm is shown in shown in accompanying drawing 1, and mechanical arm 1 loads the liquid instrument and move plate mechanical manipulator 3 of moving, described in move liquid instrument comprise single or multiple independent control move liquid passage, described mechanical arm 1 drives and moves liquid passage 2 in X-direction accurately movement; Move on liquid passage 2 and some micro-sampling modules 4 of moving liquid module and receiving upgrading are set.Figure 3 provides a kind of pictorial diagram of moving liquid module.
Preferably, each liquid passage 2 that moves on described mechanical arm 1 has independently dynamic positioning system, when hyperchannel operates simultaneously, each passage can carry out non-unification, asymmetrical movement at Y, Z axis and move liquid simultaneously, channel pitch is from adjusting, and the multiple tracks of the sample hose or porous plate that meet all size operates simultaneously.Passage can directly automatic loading and unloading disposable tip or reusable pin type suction nozzle.
Alternatively, described mechanical arm can be 2 ~ 6, and different mechanical arm performs simultaneously and different moves liquid task.
Further, mechanical arm drives and moves liquid passage in X-direction accurately movement, is accurately located by suction nozzle.The displacement accuracy of mechanical arm is ± 0.1mm.Mechanical arm can load and variously move liquid instrument, as single or multiple independent control move liquid passage, and 1,2,4,8,12 or 16 difference configuration moving liquid passage can be provided; For more high-throughput, what also can arrange 96 passages or 384 passages moves liquid module, and receives the micro-sampling module of upgrading.Move plate mechanical manipulator being mounted with the mechanical arm moving liquid passage also can install simultaneously, and do not need extra mechanical arm to control.
The liquid technology of moving of described fluid treatment appts is based on air displacement principle, is similar to normally used Manual liquid transfering device, promotes air carry out imbibition and tapping by the movement of passage inner carrier 5.Adopt this principle to move liquid and do not need the syringe pump of step motor control, various conduit and System Solution, not easily produce the systemic contamination of workstation inside, avoid the crossed contamination that sample room produces due to systemic contamination.See shown in accompanying drawing 2, in accompanying drawing 2,5 is piston, and 6 is sleeve, and 7 is pressure transmitter, and 8 is air column, and 9 is liquid.
Described utility appliance comprises sample oscillation device, incubating device, sample stowage unit, sample number into spectrum scanning and recording unit, the transfer device of experiment consumptive material, the connecting device of upstream and downstream equipment.Based on described utility appliance except performing except pipetting, other experimental duties a lot of can also be performed, as sample vibration, hatch, the transfer of the sample of automatic loading and recording strip numbering, experiment consumptive material, the function such as to be connected with upstream and downstream experimental installation.
Described nucleic acid extraction and purifier apparatus are full-automatic nucleic acid magnetic bead extraction and purification system, and principle of work is first by concussion sample, makes nucleic acid free out, then is held by the nucleic acid in sample with large volume extraction magnetic head, is transferred in new centrifuge tube; Such repetitive operation 3 times, just can obtain very pure nucleic acid, detects and other molecular biology researches provide highly purified nucleic acid samples for the automatization configuration of the follow-up PCR of development reaction system and real-time fluorescence quantitative PCR.In sample handling processes, the nucleic acid purity not only obtained is high, and its extraction efficiency also very high (for the marine sediment samples that bacterial abundance is very low, still can obtain enough nucleic acid for follow-up work).Adopt existing conventional computer technology, in described nucleic acid extraction and purifier apparatus, built-in multiple nucleic acid purification program, is controlled by computer software and realizes automated operation.
Embodiment 2
Based on the automatization sample processing device of ortho states microorganism oil gas of the present invention and hydrate exploration technology, the present embodiment provides the Automation workstation of a kind of ortho states microorganism oil gas and hydrate exploration technology, described workstation comprises the automatic business processing sampling device of ortho states microorganism oil gas and hydrate exploration technology, also comprises laminar flow hood and PCR reaction system configuration-system.
Ultra-clean laminar flow hood is set above described automated fluid treatment unit and/or nucleic acid extraction and purifier apparatus, ensures aseptic Working environment; Also vent line can be connected, by the discharge of poisonous waste that produces or recovery in experiment.Such setting can protect the health and safety of operator, also can ensure the security of sample and experiment flow.
Based on automated installation of the present invention and workstation, the automated analysis to hydrocarbon gas oxidation bacteria and association mushroom abundance thereof can be realized, comprise the following steps:
S1. building platform, workplatform arranges positioning track, for the carrier of the various experimental article of fixed placement;
S2. in automated fluid process and PCR reaction system inking device (isMOST Station-1) and nucleic acid extraction and purifier apparatus (isMOST Station-2), the supernatant of sample be dispensed into sample deep-well plates from sample hose through automatic sampling and add magnetic bead and damping fluid sample deep-well plates, washings and elutriant being sub-packed in washings deep-well plates simultaneously;
S3. the deep-well plates that supernatant is housed is placed on the carrier dish of automated fluid treatment unit with the deep-well plates that washings is housed respectively;
S4., in nucleic acid extraction and purifier apparatus, the extraction and purification of nucleic acid is completed;
To sample deep-well plates and washings deep-well plates, reagent needed for sample liquid or nucleic acid extraction and purifying is added respectively from reagent trough particular by moving liquid passage, by liquid-transfering device, the washings deep-well plates of adding sample liquid or described reagent is moved to suction magnetic potential, through Electrical heads probe into deep-well plates stir and hold magnetic bead carry out suction magnetic and suction waste liquid, complete DNA wash-out;
S5. in PCR reaction system configuration-system, PCR reaction is carried out;
S6. PCR reaction result is obtained.
Wherein, sample described in S2 step is multiple, is sub-packed in multiple sample hose, continuous automatic sampling, and to sample hose or deep-well plates scanning bar code one by one, record sample number into spectrum, realizes sample and follow the tracks of, simplify experimental implementation, and ensure the safety of experimenter.
Embodiment 3 application experiment
Extract soil and/or settling amplifying nucleic acid with ortho states microorganism oil gas and the automatization of hydrate exploration technology (isMOST) workstation and carry out the abundance measurement of butane oxidation bacterium in conjunction with real-time fluorescence quantitative PCR instrument.
In soil and/or sediment sample the total genomic dna of butane oxidation bacterium extract and total count quantitative analysis method and specific experiment step as follows:
One, reagent preparation
1, the EDTA of Buffer TE:10mM Tris-HCl, 1mM, adjusts pH to be 8.0;
2,20mg/ml lysozyme soln preparation: 20mg N,O-Diacetylmuramidase is dissolved in 1ml Buffer TE;
3, DNA extract: 10mM Tris-Hcl, 1M EDTA, 0.5%SDS;
4,20mg/ml Proteinase K;
5, Binding Buffer: buy from Omega company;
6, Buffer PHB: buy from Omega company;
7, the aqueous ethanolic solution (now with the current) of SPMWash Buffer:78% volume ratio.
8, bead suspension (Magpure particles): magnetic microsphere (diameter is 60nm) (the profit micro-nano novel material Science and Technology Ltd. difficult to understand) 50ng comprising thin layer nano-silicon parcel in every milliliter of bead suspension, all the other are water.
Two, sample preparation
1, from drying, fine ground soil and/or sediment sample, take 2.5g, be placed in 10ml sterile centrifugation tube.
2, in centrifuge tube, add 1.5ml lysozyme soln (20mg/ml), vibration suspends, 50 DEG C of water-baths 1 hour;
3, add 400 μ l DNA extracts successively, 25 μ l Proteinase K Solution (20mg/ml), place 2h in 55 DEG C of water-baths.
4, the centrifugal 5min of 6000 × g, obtains supernatant liquor; Be loaded on by supernatant liquor in new 10ml sterile centrifugation tube, again the centrifugal 5min of 6000 × g, each sample obtains the supernatant liquor being greater than 1.5ml.
Three, complete move liquid with ortho states microorganism oil gas of the present invention and hydrate exploration workstation (isMOST Station 1)
1,10ml centrifuge tube is placed in luggage carrier.
2, reagent is ready to by according to the form below 1.
3, perform the program that ortho states microorganism oil gas and hydrate exploration workstation (isMOST Station 1) set, different reagent is added in the sample panel of correspondence, board, wash plate and wash-out plate automatically by operation robotically controlled arm being being moved liquid passage and move liquid module.
Table 1: reagent inventory
Four, nucleic acid extraction and purifier apparatus extract DNA automatically
After all sample panel being all ready to, perform the program that ortho states microorganism oil gas of the present invention and hydrate exploration workstation (isMOST Station 2) set, the total genomic dna in settling.After about 1 hour, nucleic acid extraction and purifying flow process terminate, and move in trace P CR pipe by the nucleic acid extraction liquid of sample, preserve after mark is clear in-20 DEG C of refrigerators.
Five, agarose electrophoresis detects
1. get 5 μ l DNA extraction liquid electroresis appraisal on 1% agarose, DNA Ladder is DL2000.
If 2. there is >2kbp band, then get 5 μ l DNA extraction liquid and add after aqua sterilisa dilutes 20 times and preserve as pcr template and at 4 DEG C, sample gene group DNA extraction liquid is stored in-20 DEG C.
Six, nucleic acid concentration and purity detecting
Trace dna determinator is used to measure its concentration and purity to the sample gene group DNA extracted.As OD260=1, double-stranded DNA (dsDNA) concentration is about 50 μ g/ml
Single stranded DNA (ssDNA) concentration is about 37 μ g/ml
Oligonucleotide concentration is about 30 μ g/ml
Pure dna: OD260/OD280 ≈ 1.8 (if be greater than 1.9, shows have RNA to pollute; If be less than 1.6, show there is the pollution such as protein and phenol).
Seven, the fluorescence quantitative PCR detection of butane oxidation bacterium quantity
(1) cultivation of butane oxidation bacterium
The substratum of preparation butane oxidation bacterium; 1% butane oxidation bacterium is inoculated in culturing bottle; Butane/butane is injected after sealing; Cultivate 15 days at suitable temperature, cultivation of transferring thereafter, muddy to the visible nutrient solution of naked eyes; Collect the butane oxidation bacterium thalline cultivating gained, for the nucleic acid extraction of butane oxidation bacterium and the drafting of typical curve.
(2) Auele Specific Primer is designed
According to the CDS sequence of butane oxidation bacterium bmoX gene, design and synthesize the Auele Specific Primer that can be used for fluorescence quantitative PCR detection butane oxidation bacterium.
Two pairs of primer sequences are as follows:
Pair of primers:
bmoX-F:5'TCGCCCGAGCAGCGGAACGGTTATCT 3'
bmoX-R:5'AGGATTTTCATTGGATCAGGAATAGTAA 3'
The PCR primer length obtained that increases is 1,190bp.
Second pair of primer:
bmoX-DF:5'TGGCACCGGTGGGTGTACGAAGACT 3'
bmoX-DR:5'GCGCGATCAG(C:G 3:2)GTCTT(G:C 9:1)CC(G:A 1:9)TC 3'
The PCR primer length obtained that increases is about 400bp.
(3) preparation standard product;
The microbial total genomic dna that obtains is extracted for template with S4, adopt the object sheet degree of following primer pair amplifies bmoX, product is separated object band through 2% agarose gel electrophoresis, and be connected with pMD18-T Vector carrier, connect product to add in competent cell suspension, then extract plasmid, turn out to be after methane-oxidizing bacteria object fragment through PCR and order-checking, use this plasmid DNA purity of spectrophotometric determination and quantitatively, be finally diluted to 1 × 10 with aseptic double-distilled water
9copy/μ L ,-20 DEG C of packing are preserved, for the standard substance of quantitative fluorescent PCR.
(4) reaction system and the reaction conditions of quantitative fluorescent PCR is set up;
Real-time fluorescence quantitative PCR amplification completes in Roche Light Cycler 480 (Roche Diagnostics, Penzberg, Germany).Reaction system is basis
premix Ex Taq
tM(TaKaRa) manufacturer specification preparation.By the CDS sequence of specific primers amplify butane oxidation bacterium bmoX gene.In soil and/or settling, total gene DNA adds in each reaction mixture using the level of 1 ~ 10ng as template.
Set up quantitative fluorescent PCR reaction system as listed in table 2 and reaction conditions:
Table 2
Wherein, being formulated in ortho states microorganism oil gas of the present invention and hydrate exploration workstation (isMOST Station 1) of PCR reaction system completes.Operating process is as follows:
S1. prepare reaction system mixed solution (except 2 μ L DNA profilings) according to table 2, be sub-packed in 2ml sterile centrifugation tube, often pipe is greater than 0.5ml, is placed in the luggage carrier that refrigeration is preserved.
S2. ortho states microorganism oil gas is placed in luggage carrier, as DNA profiling together with the deep-well plates of the band nucleic acid samples of hydrate exploration workstation (isMOST Station 2) Isolation and purification.
S3. the program that ortho states microorganism oil gas and hydrate exploration workstation (isMOST Station 1) set is performed, the mix reagent set up needed for PCR reaction system and 2 μ L DNA profilings are added successively in corresponding, new deep-well plates, namely complete the configuration of PCR reaction system.
Subsequent step reference literature (Shao Mingrui etc., 2013, the foundation of three kinds of oil and gas indication bacterium quantifying PCR methods and the Preliminary Applications in oil-gas field soil thereof, biotechnology is circulated a notice of, 4th phase, 172-178) operate, after experiment terminates, with LightCycler 480Software 1.5.0 software, experimental result is analyzed, the abundance of butane oxidation bacterium in sample can be obtained.
Embodiment 4 Automation workstation serviceability test of the present invention
1. sample preparation ability
Sum up from detection system operating records of the present invention, the initial time of process 96 oceanic sediment Duplicate Samples is 11: 06: 26, and the termination time is 12: 46: 03, about consuming time 1 hour 40 points (namely 100 minutes); Detection system of the present invention is by running 8 hours every day, then every day can processing sample number=(8 hours × 60 points/hour) × 96/100 points]=461; Annual working time, then detection system of the present invention every year can processing sample 13.83 ten thousand by 300 days.
2. extract the effect of nucleic acid
From above-mentioned 96 nucleic acid samples obtained, choose arbitrarily 9 samples carry out pcr amplification, its electrophorogram as shown in Figure 4.In order to carry out the contrast of nucleic acid extraction effect, the electrophorogram of bacterium in manual process terrestrial soil sample is showed in Fig. 5.Comparison diagram 4 and Fig. 5 can obviously find out:
(1) Nucleic acid quality extracted by detection system of the present invention is very high, and highly stable and have repeatability (1 to the 9 canescence band see in Fig. 4);
(2) detection system nucleic acid purification of the present invention obtains very good (in Fig. 4, the brightness of canescence band is basically identical), this and obvious different (Fig. 5 a: nucleic acid does not extract in Fig. 5; Fig. 5 b: although nucleic acid extracts, has from very bright to banded variation trend dimmed gradually, reflects that nucleic acid purification obtains bad);
(3) distance between the bacterium band amplified as can be seen from sample and M band (mark Marker), the bacterial nucleic acid extracted by hand, its molecular weight is roughly at about 20,000 (Fig. 5); And with the bacterial nucleic acid that detection system of the present invention is extracted, its molecular weight is roughly at about 200,000 (Fig. 5), reflect that extracting nucleic acid by detection system of the present invention is not easy hydrolysis, this provides better sample for the follow-up abundance to bacterium carries out fluorescent quantitation, and quantitative accuracy can be made higher, more accurate.
(4) detection system of the present invention can provide the true record of process of the test simultaneously, makes obtained data not only have trackability, and has same error because of it, therefore can carry out unified error correction to data.
3. the contrast experiment of same sample
According to the inventive method and adopt the method for the manual extraction of existing routine to extract comparative result that same marine sediment samples gained extracts nucleic acid effect please be shown in Table 3:
Table 3
* the sedimental degree of depth under seawater.
Find out from upper table 3: the efficiency that (1) extracts nucleic acid by the inventive method is obviously high than the efficiency of manual extraction, on average exceeds about 14%; (2) obviously good than the stability (error is about 7.6%) of manual extraction nucleic acid by the stability (error is about 0.3%) of the inventive method extraction nucleic acid.
SEQUENCE LISTING
<110> Guangzhou Enenta Chemical Science & Technology Co., Ltd.
The automated detection method of a <120> butane oxidation bacterium abundance
<130>
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 26
<212> DNA
<213> artificial sequence primer bmoX-F
<400> 1
tcgcccgagc agcggaacgg ttatct 26
<210> 2
<211> 28
<212> DNA
<213> artificial sequence primer bmoX-R
<400> 2
aggattttca ttggatcagg aatagtaa 28
<210> 3
<211> 25
<212> DNA
<213> artificial sequence primer bmoX-DF
<400> 3
tggcaccggt gggtgtacga agact 25
<210> 4
<211> 19
<212> DNA
<213> artificial sequence primer bmoX-DR
<400> 4
gcgcgatcag gtcttcctc 19
Claims (6)
1. an automated detection method for butane oxidation bacterium abundance, is characterized in that, comprises the following steps:
S1. the Automation workstation of ortho states microorganism oil gas and hydrate exploration technology is built; Described workstation comprises automated fluid treatment unit, nucleic acid purification equipment, PCR reaction system configuration-system;
S2. sample preparation, each sample obtains the supernatant liquor being greater than 1.5 ml;
S3. adopt ortho states microorganism oil gas of the present invention and hydrate exploration workstation automated fluid treatment unit to complete and move liquid; Specifically perform the program of automated fluid treatment unit setting, different reagent is added in corresponding sample panel, board, wash plate and wash-out plate by robotically controlled arm being being moved liquid passage and the operation moving liquid module automatically;
S4. automatic instrument for extracting nucleic acid extraction and purification DNA, after all sample panel being ready to, performing the setting program of ortho states microorganism oil gas of the present invention and hydrate exploration workstation, obtains the total genomic dna in soil and/or settling; Through automated fluid treatment unit, the nucleic acid extraction liquid of sample is moved in trace P CR pipe after nucleic acid extraction and purifying flow process terminate, preserve in-20 DEG C of refrigerators after mark is clear;
S5. agarose electrophoresis detects;
S6. the fluorescence quantitative PCR detection of butane oxidation bacterium abundance, obtains its abundance result.
2. the automated detection method of butane oxidation bacterium abundance according to claim 1, is characterized in that, it is as follows that agarose electrophoresis described in S5 step detects working method:
(1) get 5 μ l DNA extraction liquid electroresis appraisal on 1% agarose, DNA Ladder is DL2000;
(2) if there is > 2 kbp band, then get 5 μ l DNA extraction liquid and add after aqua sterilisa dilutes 20 times and preserve as pcr template and at 4 DEG C, sample gene group DNA extraction liquid is stored in-20 DEG C.
3. the automated detection method of butane oxidation bacterium abundance according to claim 1, it is characterized in that, described in S6 step, the fluorescence quantitative PCR detection of butane oxidation bacterium abundance comprises the following steps:
(1) cultivation of butane oxidation bacterium;
(2) Auele Specific Primer is designed;
(3) preparation standard product;
Extract the microbial total genomic dna that obtains for template with S4, adopt following primer pair amplifies
bmoXobject sheet degree, product is separated object band through 2% agarose gel electrophoresis, and be connected with pMD 18-T Vector carrier, connecting product adds in competent cell suspension, extract plasmid again, turn out to be after methane-oxidizing bacteria object fragment through PCR and order-checking, use this plasmid DNA purity of spectrophotometric determination and quantitatively, be finally diluted to 1 × 10 with aseptic double-distilled water
9copy/μ L ,-20 DEG C of packing are preserved, for the standard substance of quantitative fluorescent PCR;
(4) reaction system and the reaction conditions of quantitative fluorescent PCR is set up;
Wherein, being formulated in ortho states microorganism oil gas of the present invention and hydrate exploration workstation of PCR reaction system completes;
Operating process is as follows:
S61. will prepare PCR reaction system mixed solution, and be sub-packed in sterile centrifugation tube through automated fluid treatment unit, often pipe is greater than 0.5 ml, is placed in the luggage carrier that refrigeration is preserved;
S62. ortho states microorganism oil gas is placed in luggage carrier, as DNA profiling together with the deep-well plates of the band nucleic acid samples of hydrate exploration workstation Isolation and purification;
S63. perform the program that ortho states microorganism oil gas and hydrate exploration workstation set, in that the mix reagent set up needed for PCR reaction system and DNA profiling are added successively correspondence, new deep-well plates, namely complete the configuration of PCR reaction system;
(5) routine carries out operation and the data processing of real-time fluorescence quantitative PCR instrument, obtains the abundance result of butane oxidation bacterium in sample.
4. the automated detection method of butane oxidation bacterium abundance according to claim 3, is characterized in that, step (2) described primer is two right, and pair of primers is
bmoX-F: 5' TCGCCCGAGCAGCGGAACGGTTATCT 3'；
bmoX-R: 5' AGGATTTTCATTGGATCAGGAATAGTAA 3'；
Second pair of primer is:
bmoX-DF: 5' TGGCACCGGTGGGTGTACGAAGACT 3'；
bmoX-DR: 5' GCGCGATCAG(C:G 3:2)GTCTT(G:C 9:1)CC(G:A 1:9)TC 3'。
5. the automated detection method of butane oxidation bacterium abundance according to claim 3, it is characterized in that, the described quantitative fluorescent PCR reaction system of step (4) is:
SYBR
? Premix Ex Taq
TM(2Х) 10.0 μL；
PCR Forward Primer (10 μM) 0.4 μL；
PCR Reverse Primer (10 μM) 0.4 μL；
Microbial total genomic dna 2.0 μ L;
DdH
2o (sterile purified water) 7.2 μ L;
Total 20.0 μL。
6. the automated detection method of butane oxidation bacterium abundance according to claim 3, it is characterized in that, the described quantitative fluorescent PCR reaction conditions of step (4) is:
(a) Pre-incubation 1 cycle(s) None ；
(b) Amplication 45 cycle(s) Quantification ；
(C) Melting 1 cycle(s) Melting Curves ；
(d) Cooling 1 cycle(s) None 。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510400777.1A CN104975090B (en) | 2015-07-09 | 2015-07-09 | A kind of automated detection method of butane oxidation bacterium abundance |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510400777.1A CN104975090B (en) | 2015-07-09 | 2015-07-09 | A kind of automated detection method of butane oxidation bacterium abundance |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104975090A true CN104975090A (en) | 2015-10-14 |
| CN104975090B CN104975090B (en) | 2018-07-20 |
Family
ID=54272053
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510400777.1A Active CN104975090B (en) | 2015-07-09 | 2015-07-09 | A kind of automated detection method of butane oxidation bacterium abundance |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104975090B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107460109A (en) * | 2017-07-23 | 2017-12-12 | 新疆昆泰锐生物技术有限公司 | A kind of reaction system for bacterium colony PCR is prepared and sampling device and PCR instrument |
| CN109423458A (en) * | 2017-08-30 | 2019-03-05 | 中国石油化工股份有限公司 | A kind of Rhodococcus sp and its identification method and application |
| CN113202455A (en) * | 2021-06-02 | 2021-08-03 | 中国石油天然气股份有限公司西南油气田分公司川中油气矿 | Oil exploration method and system based on Internet of things |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102174645A (en) * | 2011-03-01 | 2011-09-07 | 广州安能特化学科技有限公司 | Representation method of oil-gas exploration and oil-gas reservoir by taking vital bacterium abnormality and dead bacterium abnormality of methane-oxidizing bacteria as indicators |
| CN102492603A (en) * | 2011-11-22 | 2012-06-13 | 马庆伟 | Automated instrument for nucleic acid extraction and mass spectrum sample application |
| CN202830011U (en) * | 2012-08-29 | 2013-03-27 | 北京万泰生物药业股份有限公司 | Automated nucleic acid extraction platform |
| CN104313178A (en) * | 2014-11-27 | 2015-01-28 | 中国石油化工股份有限公司 | Fluorogenic quantitative PCR detecting method of surfactant-producing viable bacteria |
-
2015
- 2015-07-09 CN CN201510400777.1A patent/CN104975090B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102174645A (en) * | 2011-03-01 | 2011-09-07 | 广州安能特化学科技有限公司 | Representation method of oil-gas exploration and oil-gas reservoir by taking vital bacterium abnormality and dead bacterium abnormality of methane-oxidizing bacteria as indicators |
| CN102492603A (en) * | 2011-11-22 | 2012-06-13 | 马庆伟 | Automated instrument for nucleic acid extraction and mass spectrum sample application |
| CN202830011U (en) * | 2012-08-29 | 2013-03-27 | 北京万泰生物药业股份有限公司 | Automated nucleic acid extraction platform |
| CN104313178A (en) * | 2014-11-27 | 2015-01-28 | 中国石油化工股份有限公司 | Fluorogenic quantitative PCR detecting method of surfactant-producing viable bacteria |
Non-Patent Citations (3)
| Title |
|---|
| 王皖蒙: "油气资源勘探的环境指示微生物技术研究", 《中国优秀硕士学位论文全文数据库 基础科学辑》 * |
| 邵明瑞 等: "三种油气指示菌定量PCR方法的建立及其在油气田土壤中的初步应用", 《生物技术通报》 * |
| 邵明瑞 等: "油气田土壤DNA提取方法及油气指示菌基因定量结果的比较", 《工业微生物》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107460109A (en) * | 2017-07-23 | 2017-12-12 | 新疆昆泰锐生物技术有限公司 | A kind of reaction system for bacterium colony PCR is prepared and sampling device and PCR instrument |
| CN109423458A (en) * | 2017-08-30 | 2019-03-05 | 中国石油化工股份有限公司 | A kind of Rhodococcus sp and its identification method and application |
| CN109423458B (en) * | 2017-08-30 | 2022-08-19 | 中国石油化工股份有限公司 | Rhodococcus and identification method and application thereof |
| CN113202455A (en) * | 2021-06-02 | 2021-08-03 | 中国石油天然气股份有限公司西南油气田分公司川中油气矿 | Oil exploration method and system based on Internet of things |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104975090B (en) | 2018-07-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103993007B (en) | The simple and easy method of a kind of high efficiency extraction DNA from pedotheque | |
| AU2013329615A1 (en) | Field-based qPCR microbial monitoring | |
| CN104651350A (en) | Extraction method of total DNA of microorganisms from soil sample | |
| CN105018609B (en) | A kind of automated detection method of methane-oxidizing bacteria abundance | |
| CN104975090B (en) | A kind of automated detection method of butane oxidation bacterium abundance | |
| CN105018343B (en) | Ortho states microorganism oil gas and the automation sample processing device and Automation workstation of hydrate exploration technology | |
| CN103882105B (en) | Method for measuring content of saturated hydrocarbon-degrading gene AlkB in petroleum-contaminated soil | |
| CN205152235U (en) | A micro -fluidic chip , detecting system and device for gene somatotype detects | |
| KR20170124728A (en) | Reagent for detecting cross contamination of PDX-model related with human and mouse, the kit comprising the same, and the method for the cross contamination detection | |
| CN102337347A (en) | Characteristic nucleotide sequence for identifying ophiocordyceps crinalis, as well as probes and method thereof | |
| Peng et al. | Methods for genomic characterization and maintenance of anaerobic fungi | |
| CN113528614B (en) | Plant phyllosphere surface microorganism metagenome detection method | |
| CN109762916A (en) | The method and primer special of quantitative detection sulfate reducing bacteria | |
| CN112592965B (en) | E.coli host DNA residue detection kit adopting TaqMan probe method | |
| CN106480167B (en) | Method for exploring oil gas by using microorganisms | |
| CN103602697A (en) | High-flux carrier construction method | |
| Hall et al. | 5 Methods to Study the Bacterial Ecology of Freshwater Environments | |
| KR101288419B1 (en) | Primer composition and kit for isothermal amplification reaction for detecting Pectobacterium carotovorum subsp. comprising specific primer set, and method for detecting Pectobacterium carotovorum subsp. using the primer set | |
| CN101691613B (en) | Primer group for detecting vibrio coralliilyticus by using LAMP, quick diagnosis kit and detecting method | |
| CN113444823B (en) | Primer group and method for identifying streptomyces albidoflavus W68 by using primer group | |
| CN113151425B (en) | Single cell sequencing method for improving accuracy based on key indexes | |
| Blaby-Haas et al. | Using YFP as a reporter of gene expression in the green alga Chlamydomonas reinhardtii | |
| CN103088134B (en) | Method for detecting microorganism with hydrocarbon degradation function under anaerobic conditions | |
| Zhou | Fungal screening and phylogenetic tree construction | |
| CN103361410B (en) | Plasmid standard molecule for detecting transgenic rice Ke-Ming-Dao No.2 and construction of plasma standard molecule |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |