CN101792807A - Method for analyzing microbial community structures - Google Patents
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Abstract
The invention belongs to the technical field of bioanalysis, and particularly relates to a method for analyzing microbial community structures. The method comprises the following steps of: extracting total RNA in an environment sample; separating small-subunit rRNA from the total RNA; carrying out reverse transcription on the small-subunit rRNA by random primer for sequencing; extracting the small-subunit rRNA sequence from the results of sequencing; and confirming category message according to the obtained small-subunit rRNA sequence, and acquiring the characteristics of the microbial community structure. The method does not use specific PCR amplification so as to avoid various deviations in the PCR process; and meanwhile, the method can be used for analyzing the community structure of bacteria, archimycetes and eukaryotic microorganism, thus truly and completely reflecting the characteristics of the microbial community structure in the environment sample.
Description
Technical field
The invention belongs to the bioassay technique field, be specifically related to be a kind of utilization not the rRNA by PCR check order and analyze the method for biological community structure.
Background technology
The analysis of biological community structure is the focus of microbial ecology and Study of Environmental Microbiology always.Therefore, according to the dynamic change that the population structure and the diversity of target sample are resolved its micropopulation, can be for optimizing structure of community, regulate group's function and finding that new critical function microbe groups provides reliable foundation.Along with the development and the widespread use of Protocols in Molecular Biology, from early 1990s, it has also progressively been introduced microbial ecology and environmental microbiology field, for the research in this field provides new method and unprecedented impellent.The advantage of Protocols in Molecular Biology is that it has evaded the puzzlement that most microorganisms in the middle of the environment can not pure culture this problem under condition at present.Along with the molecular biology gene analysis technique of ribosome-RNA(rRNA) (rRNA) gene in microflora's application analytically, the diversity of research environment sample becomes possibility.
These methods mainly based on extract DNA or RNA from environmental sample, are used the specific primer of rRNA that template DNA is increased, or RNA are carried out earlier carrying out specific amplification after the reverse transcription.Provide high-resolution classified information based on the sequencing result of 16S and 18S rRNA amplification and clone library structure for the structure of community diversity analysis of microorganism.Gene fingerprint technology such as DGGE, TGGE, technology such as T-RFLP also change the information fast and accurately that provides for main population in the monitoring group.
Yet based on specific PCR amplification method research biological community structure and multifarious the whole bag of tricks its unavoidable defective is arranged: 1) although the investigator has designed the primer that more and more contains the degeneracy base, at the primer of rRNA gene order can not the matched environment sample in all microorganisms, this can cause certain micro-organisms group to fail to be detected, and the content of certain micro-organisms group is over-evaluated; 2) because PCR method is that template is carried out a plurality of round-robin amplifications, and the amplification efficiency of different microorganisms is not quite similar in the template, and the PCR product that finally obtains can not react the ratio of different microorganisms content in the primary template; 3), thereby can not analyze diversity and the relative content of these microorganisms in group jointly because employed primer is that groups such as bacterium, Archimycetes, fungi are specific.
In recent years, tetra-sodium order-checking high-throughput sequencing technologies such as (pyrosequencing) grows up apace, becomes possibility thereby make low cost obtain high-throughout gene order.By before universal primer, adding the laggard performing PCR amplification of the specific label of sample, the amplified production mixing back order-checking of a plurality of samples can be obtained a large amount of sequences, by label each sample is made a distinction, this method has extensively applied to the biological community structure and the diversity analysis of environmental sample again.But this method still relies on primer, can't avoid the defective of specific PCR amplification.Unit's (grand) genome analysis can be avoided the defective of pcr amplification, but have only result and 16S/18S rRNA gene-correlation about 0.2% in the first genomic data because of gained, so first genome analytical method is not the analytical procedure that is suitable for microbial diversity most.
Among the present invention employed utilization not the check order method of analyzing biological community structure of the rRNA by PCR avoided the defective of the method for above-mentioned PCR-based.
Summary of the invention
The object of the present invention is to provide a kind of energy accurately, structure of community and the method for analyzing diversity of microorganism in the quantitative response environmental sample.
Analytical procedure about biological community structure provided by the invention is to after the environmental sample extracting RNA, does not carry out the specific PCR amplification, but the method for after reverse transcription, directly carrying out high-flux sequence and analyzing,
Concrete steps are as follows:
Step 1, total RNA of extraction environmental sample;
Step 2 is separated small subunit (16S/18S) rRNA from total RNA;
Step 3 is carried out reverse transcription by random primer to small subunit rRNA, and reverse transcription product is checked order;
Step 4, extracting goes out small subunit rRNA sequence from sequencing result;
Step 5 according to the small subunit rRNA sequence of gained, obtains the foundation of sequence respective classified information as taxon with the public database contrast, obtains the biological community structure feature thus.
Described separation small subunit rRNA be to separate small subunit rRNA and big subunit (23S/28S) rRNA band by electrophoresis, and small subunit rRNA is reclaimed in rubber tapping.
Described reverse transcription and order-checking by random primer is by the random primer synthetic dsdna, and interrupts at random and/or the length-specific bar of tapping rubber brings and makes and be suitable for order-checking; Perhaps by adding the random primer synthetic dsdna of tag, and with increase this double-stranded DNA and be used for order-checking of the corresponding primer of tag sequence.
The described small subunit rRNA sequence that extracts from sequencing result is to determine in conjunction with check order row and all kinds (16S/18S/23S/28S) the rRNA geneseq database of self-built a plurality of microorganisms and the BLAST result of public nucleotide sequence database whether the row that check order belong to small subunit rRNA sequence.
Described definite classified information according to small subunit rRNA sequence is determined, is to determine classified information with the similarity or the homology of small subunit rRNA sequence and public database, thereby determines biological community structure.
The present invention can carry out quantitative analysis to the bacterium in the environmental sample, Archimycetes, eukaryotic microorganisms simultaneously, it can overcome the research biological community structure of existing PCR-based amplification and the defective of multifarious molecular biology method, because the object that the present invention studied is the RNA of microorganism, therefore the structure of community and the diversity that have active microorganism in can accurately the reaction environment sample, have accurately, remarkable advantages such as quantification, and be suitable for existing high-flux sequence.
The present invention has the following advantages:
(1) the present invention can analyze the biological community structure diversity of various dissimilar environmental samples.Employed method is easy and simple to handle among the present invention, and is with low cost, is applicable to general Molecular Biology Lab's operation.
(2) object that the present invention analyzed is the rRNA of microorganism, and therefore reaction is to have active biological community structure and diversity in the sample.
(3) the present invention is by pcr amplification, therefore evaded in the PCR process because primer specificity and non-specific the deviation that the own inherent skewed popularity of amplification efficiency and PCR is produced.Because the present invention do not need RNA is increased, so the sequencing result ratio of each quasi-microorganism of response sample kind quantitatively.
Description of drawings
The RNA electrophoresis diagram of Fig. 1 for extracting.
Embodiment
Take from the microbial diversity analysis of the active sludge in the sewage disposal plant aeration tank
The active sludge sample is taken from the aeration tank of one tame municipal sewage plant, Shanghai City, and experimental procedure is as follows:
(1) the total RNA extraction agent of liquid nitrogen grinding method and Trizol box (worker, Shanghai are given birth in Shanghai) combines and extracts total RNA of environmental sample.
1) in the centrifuge tube of the 50ml Rnase-free that mud mixture is added, centrifugal 5 minutes of 10000rpm removes liquid.Sample is divided into every part of 0.5g packs in the centrifuge tube of 1.5ml Rnase-free, immerse liquid nitrogen and wait to freeze the back in-80 ℃ of preservations.
2) get in the mortar of granulated glass sphere behind high-temperature sterilization that 0.5g sample and 0.5g diameter are 212-300 μ m.
3) add liquid nitrogen and cover, treat that firmly being ground to sample immediately after liquid nitrogen volatilizees becomes powder.
4) repeat 2) once.
5) in mortar, add 1000 μ l Trizol, treat powder dissolution after, turbid solution is changed in the centrifuge tube of 1.5ml Rnase-free.
6) be that the granulated glass sphere of 212-300 μ m adds in the centrifuge tube of the 1.5ml Rnase-free contain the 0.5g sample with the 0.5g diameter, add 300 μ l DEPC-ddH
2O.
7) centrifuge tube is placed on the vibrator (Labnet, the U.S.), use maximum speed vibration 10 minutes.
8) in centrifuge tube, add 500 μ l Trizol.
Annotate: step 2)-5) can change step 6)-8 according to different samples into), step 2)-5) be applicable to that content of microorganisms is more in the sample, the sample that impurity is more, step 6)-8) be applicable to that content of microorganisms is less, the sample that impurity is less.
9) add 100 μ l chloroforms, thermal agitation 30 seconds, centrifugal 5 minutes of 120000rpm room temperature
10) water is changed in the centrifuge tube of new 1.5ml Rnase-free, add 150 μ l dehydrated alcohols, pipe is put upside down for several times repeatedly behind the mixing liquid is moved on in the RNA column, the RNA column is placed on the 2ml collection tube.
11) room temperature was placed 2 minutes, centrifugal 1 minute of 8000rpm.
12) liquid in the collection tube is outwelled, added 450 μ l RPE solution, centrifugal 30 seconds of 10000rpm.
13) repeating step 12) once.
14) liquid in the collection tube is outwelled centrifugal 1 minute of 10000rpm.
15) the RNA column is positioned on the centrifuge tube of new 1.5ml Rnase-free, adds 50 μ l DEPC-ddH
2O placed 2 minutes for 75 ℃.
16) 10000rpm is centrifugal 1 minute.
(2) from total RNA, separate small subunit rRNA.
1) the above-mentioned RNA that obtains is carried out 1% agarose electrophoresis, damping fluid 1 * TAE, voltage 100V, 30 minutes.
2) the TAE solution of agarose gel being put into 300ml 2 μ g/ml ethidium bromides soaked 10 minutes.
3) with blob of viscose as under the 312nm UV-light, the result cuts off the blob of viscose of 16S/18S rRNA position as shown in Figure 1.
4) (article No.: specification sheets operation R1011) obtains the RNA purified product to reclaim test kit according to the RNA glue of Zymoclean (U.S.).
5) use nanodrop 3300 (U.S.) that the RNA that reclaims is carried out quantitatively, dyestuff uses Ribogreen (Sigma, the U.S.).
(3) to RNA reverse transcription product random sequencing.
1) the double-stranded cDNA synthetic agent box that uses Invitrogen (U.S.) synthesizes the RNA synthetic dsdna that reclaims to use the random hexamer primer.
2) the synthetic double-stranded DNA is broken into the fragment of 400-1000bp at random.
3) use Luo Shi GS-FLX tetra-sodium sequencing system to carry out high-flux sequence to these fragments.
(4) extracting from a large amount of random sequencing sequences of 16S/18S rRNA sequence is gone out.
1) all sequence operation ncbi databases (http://www.ncbi.nlm.nih.gov/) is carried out local BLAST.
2) choose various bacteria respectively, Archimycetes, fungi, protozoic 16S, 23S, or 18S, the full length sequence of 28S rRNA gene carry out BLAST from database construction with check order row.
3) in conjunction with 1) and 2) result that obtains, all sequences is divided into 16S, 18S, 23S, 28S rRNA gene and other.Bad differentiation type is manual to be analyzed.
(5) foundation of using the classified information of sequence correspondence to determine to the 16S/18S rRNA sequence of a large amount of random sequencings as taxon.
1) for 16S rRNA sequence, the Classifier instrument of use RDP database (
Http:// rdp.cme.msu.edu/classifier/classifier.jsp) classify.
2) for 18S rRNA sequence, the local BLAST of operation ncbi database (http://www.ncbi.nlm.nih.gov/), database date is up-to-date, and use MEGAN software (http://www-ab.informatik.uni-tuebingen.de/software/megan) is analyzed BLAST result and checked.
Obtain 33017 of original series altogether by above-mentioned experimental procedure, wherein greater than 20783 of the sequences of 400bp, 16S, the shared ratio of 18S rRNA sequence is respectively 32.3% (6721) and 25.8% (5361).
18S rRNA sequence can be divided into 11 Eukaryotic doors, is mainly: podophyll door, Ciliophora, wheel animalcule door, vertebrate, Annelita, the multiple door in top etc.16S rRNA sequence is mainly bacterium and a spot of Archimycetes, and bacterium can be divided into 19 doors, is mainly Proteobacteria, the actinomycetes door, Bacteroidetes, wart germ door, green curved bacterium door, Firmicutes, floating mould doors etc., it is the sequence of Archimycetes 16S rRNA that 34 sequences are arranged, and is mainly methane germ guiding principle.In addition, also have a large amount of sequences that can be defined as 16S or 18S rRNA in existing public database, not have corresponding classified information, this is because existing P CR primer is to mate all microorganisms, and the present invention has avoided the step of specific PCR amplification, the skewed popularity and the limitation of primer have been avoided, thereby can obtain more the sequence information of the microorganism of in the past not finding.
In order to verify and result's contrast of the present invention, use the high-flux sequence method of PCR-based to analyze equally to this sample, concrete steps are as follows:
(1) sample extraction RNA, 16S rRNA are tapped rubber recycling step with of the present invention identical.
(2) carry out the reverse transcription of primer specificity for the 16S rRNA that obtains in () and obtain the regional pcr amplification product of 16S rRNA V3 (the hypermutation zone on the bacterial 16 S rRNA gene is commonly used to analyze bacterial flora structure and diversity).
1) the double-stranded cDNA synthetic agent box that uses Invitrogen (U.S.) is with the synthesizing single-stranded DNA of RNA that reclaims, and the primer that uses is 519R (5 '-GTATTACCG CGG CTG CTG G-3 ').
2) with 1) in the cDNA that obtains as template, use the MasterMix PCR test kit of Tiangen (Beijing) to increase, the primer that uses is 338F (5 '-ACT CCT ACG GRA GGC AGC AG-3 ') and 519R, reaction conditions is 95 ℃ of pre-sex change 5 minutes, 94 ℃ 30 seconds, 53 ℃ 30 seconds, 72 ℃ 45 seconds, 30 circulations, last 72 ℃ were extended 7 minutes.
3) the PCR product is run 1% agarose electrophoresis, (about 200bp) cuts off with corresponding band.
4) use Axygen (U.S.) DNA glue to reclaim test kit and carry out purifying.
(3) use Luo Shi GS-FLX tetra-sodium sequencing system to carry out high-flux sequence to 16S rRNA V3 zone pcr amplification product.
To not screening among the present invention, obtain containing 2428 sequences in complete V3 district by the sequence that contains the V3 zone in the resulting 16S rRNA of the pcr amplification sequence.This is organized sequence and compares discovery (as shown in table 1) by the resulting sequence of pcr amplification: 1) result of two groups of resulting classified informations of sequence roughly is similar.2) dominant bacteria difference is little in two groups of resulting classified informations of sequence, but less wart germ door, green curved bacterium door and the floating mould Men Ze of content has bigger difference.In order further to analyze the reason that this difference produces, the sequence that primer and the present invention of two groups of sequences obtains has been carried out comparative analysis (referring to table 1), in the sequence of these 3 doors, the unmatched ratio of sequence that existing primer and the present invention obtain is higher, and this is also just explaining the less reason of sequence of these 3 doors in the sequence that obtains by PCR method.Also have a certain proportion of sequence and existing primer not to mate in other, this has illustrated that also method of the present invention avoided use PCR, has avoided the interference of primer, can obtain microflora's diversity widely.
The V3 sequence pairing bacterium diversity comparison that the amplification of table 1 gained V3 of the present invention regional sequence and PCR-based obtains and to the analysis of the universal primer sequence used in the pcr amplification.
Diversity to the V3 sequence that obtains of V3 sequence and PCR-based amplification in the sequence of the present invention is carried out statistical analysis, 400 sequences of picked at random in the sequence that two kinds of methods obtain, when the sequence difference of employing 0.03 to 0.3 comes artificially but sequence to be divided into activity classification unit (OTU), rarefaction, ACE, statistical result such as Chao1 is as shown in table 2.The bacterial flora diversity that the present invention embodied all is far longer than the PCR-based resultant sequence that increases.This illustrates that again the inventive method is applied to the advantage in the microbial diversity analysis.
The V3 sequence statistical analysis that the amplification of V3 sequence and PCR-based obtains in table 2 sequence of the present invention
OTU number when the value in the bracket is 2428 for sequence V3 sequence number of the present invention.
Claims (5)
1. the analytical procedure of biological community structure is characterized in that concrete steps are as follows:
Step 1, total RNA of rapid extraction sample;
Step 2 is separated small subunit (16S/18S) rRNA from total RNA;
Step 3 is carried out reverse transcription by random primer to small subunit rRNA, and reverse transcription product is checked order;
Step 4, extracting goes out small subunit rRNA sequence from sequencing result;
Step 5 is determined classified information according to the small subunit rRNA sequence of gained, and obtains the biological community structure feature.
2. method according to claim 1 is characterized in that described separation small subunit rRNA, be to separate small subunit rRNA and big subunit (23S/28S) rRNA band by electrophoresis, and small subunit rRNA is reclaimed in rubber tapping.
3. method according to claim 1 is characterized in that described reverse transcription and order-checking by random primer, is by the random primer synthetic dsdna, and interrupts at random and/or the length-specific bar of tapping rubber brings and makes and be suitable for order-checking; Perhaps by adding the random primer synthetic dsdna of tag, and with increase this double-stranded DNA and be used for order-checking of the corresponding primer of tag sequence.
4. method according to claim 1, it is characterized in that the described small subunit rRNA sequence that extracts from sequencing result, is to determine in conjunction with check order row and all kinds rRNA geneseq database of self-built a plurality of microorganisms and the BLAST result of public nucleotide sequence database whether the row that check order belong to small subunit rRNA sequence.
5. method according to claim 1 is characterized in that described definite classified information according to small subunit rRNA sequence, is to determine classified information with the similarity or the homology of small subunit rRNA sequence and public database, thereby determines biological community structure.
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| CN105407728A (en) * | 2013-07-21 | 2016-03-16 | 霍勒拜欧姆公司 | Methods and systems for microbiome characterization, monitoring and treatment |
| GB2531741A (en) * | 2014-10-28 | 2016-05-04 | Bisn Laboratory Services Ltd | Molecular and bioinformatics methods for direct sequencing |
| CN104568680A (en) * | 2015-01-14 | 2015-04-29 | 浙江大学 | Community monitoring method of microorganism carried by air particulate matters with multiple particle sizes |
| CN105238867A (en) * | 2015-11-05 | 2016-01-13 | 复旦大学 | Methane/ ammoxidation bacterial community structure analysis method based on two-step method |
| CN105238867B (en) * | 2015-11-05 | 2019-10-15 | 复旦大学 | Methane/ammonia oxidizing bacteria COMMUNITY STRUCTURE method based on two-step method PCR |
| CN107805658A (en) * | 2017-09-19 | 2018-03-16 | 华南师范大学 | A kind of screening technique of the eucaryon miniature organism T RFLP fragment sensitive to water quality |
| CN109187922A (en) * | 2018-09-10 | 2019-01-11 | 西北农林科技大学 | The research method of biological community structure and organic carbon response relation in revegetation |
| WO2020088596A1 (en) * | 2018-10-31 | 2020-05-07 | Coyote Diagnostics Lab (Beijing) Co., Ltd. | Methods, systems and kits for predicting premature birth condition |
| CN111187850A (en) * | 2018-11-14 | 2020-05-22 | 中国科学院青岛生物能源与过程研究所 | Method for analyzing succession of microbial communities in tobacco mellowing process |
| CN111996245A (en) * | 2020-08-27 | 2020-11-27 | 复旦大学 | Method for analyzing microbial community structure based on reverse transcription of full-length small subunit ribosomal RNA |
| CN111996245B (en) * | 2020-08-27 | 2022-12-16 | 复旦大学 | Method for analyzing microbial community structure based on reverse transcription of full-length small subunit ribosomal RNA |
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