CN102978292B - DGGE/TGGE (Denaturing Gradient Gel Electrophoresis/Temperature Gradient Gel Electrophoresis) analysis method of specific 18S rDNA (Deoxyribose Nucleic Acid) fragment without being based on GC clamp strategy - Google Patents
DGGE/TGGE (Denaturing Gradient Gel Electrophoresis/Temperature Gradient Gel Electrophoresis) analysis method of specific 18S rDNA (Deoxyribose Nucleic Acid) fragment without being based on GC clamp strategy Download PDFInfo
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Abstract
The invention provides a DGGE/TGGE (Denaturing Gradient Gel Electrophoresis/Temperature Gradient Gel Electrophoresis) analysis method of specific 18S rDNA (Deoxyribose Nucleic Acid) fragment without being based on a GC clamp strategy. The specific 18S rDNA fragment is a fragment containing a terminal region of 18S rDNA, which is far away from an ITS (Internal Transcript Space) sequence; and with a 18S rDNA sequence of a Saccharomyces cerevisiae standard strain as a reference, the terminal region of the 18S rDNA, which is far away from the ITS sequence, is equivalent to a fragment which starts from any nucleotide within a range of the first to the 30th and ends with the 291th-locus nucleotide. According to the invention, a result consistent with that obtained by the use of the GC clamp can be obtained under the same condition by adopting the DGGE/TGGE analysis of the specific 18S rDNA without adopting the GC clamp, therefore, the experiment cost can be reduced and the experiment operation steps are reduced.
Description
Technical field
The present invention relates to a kind of DGGE/TGGE analytical procedure of specific 18S rDNA fragment, refer in particular to a kind of not DGGE/TGGE analytical procedure of the specific 18S rDNA fragment based on GC clamping plate strategy.
Background technology
DGGE(denaturing gradient gel electrophoresis) and TGGE(temperature gradient gel elec-trophoresis (TGGE)) be two kinds of electrophoretic techniques based on sequence dna fragment difference, be usually used in early days effective detection of transgenation, become at present the important means of microorganism species Study on Diversity.Adopt these two technology can separation length identical but DNA fragmentation that sequence is different, the separation of fragment is to rely on double-stranded DNA to have the different behaviors of unwinding during swimming in the middle of the gel that contains chemical denaturant gradient (DGGE) or thermograde (TGGE) to realize.In order as far as possible effectively to detect all sudden changes in the middle of sequence, conventionally in one end of target fragment, add the high GC fragment of the preceding paragraph 30bp-50bp---by synthetic, be added in 5 ' end of primer, through its product end of pcr amplification, just contain this fragment; When carrying out DGGE or TGGE analysis, this high GC region will make target fragment be unlikely to unwind completely in denatured gradient environment, just can realize all possible single base mutation is detected, and this is GC clamping plate strategies.For flora structural research, effective identification of the various single base differences of sequence, will contribute to the multifarious accurate parsing of flora.
Correlative study shows, if adopt native sequences for the fragment within the scope of 50-1000bp, DGGE can only detect the 50%[V.C. Sheffield of all possible single base mutation, D.R. Cox, L.S. Lerman, R.M. Myers. Attachment of a 40-base-pair G+C-rich sequence (GC-clamp) to genomic DNA fragments by the polymerase chain reaction results in improved detection of single-base changes[J].
proc.Natl.Acad. Sci.USA.1989,86:232-236.], and employing GC clamping plate strategy, can greatly improve the sudden change recall rate of DGGE/TGGE, almost can detect any possible single base mutation [R.M. Myers, S.G. Fischer, T. Maniatis, et al. Nearly all single base substitutions in DNA fragnents joined to a GC-clamp can be detected by denaturing gradient gel electrophoresis [J] .Nucleic Acids Res. 1985, 13 (9): 3131-3145.], therefore this strategy is adopted by nearly all DGGE or TGGE correlative study (comprising flora structural research) at present.Yet when carrying out flora structural research, need to synthesize simultaneously and comprise the long primer that contains GC clamping plate and not containing the general primer two cover primers of GC clamping plate, especially the long primer that contains GC clamping plate needs higher cost conventionally more than 60bp, occurs sometimes in addition the situation that GC long primer amplification efficiency reduces.In fact, when adopting some specific DNA fragmentation to carry out DGGE or TGGE analysis, having or not of GC clamping plate can't have influence on experimental result.
Summary of the invention
The invention provides a kind of not DGGE/TGGE analytical procedure of the specific 18S rDNA fragment based on GC clamping plate strategy, the DGGE/TGGE that the specific 18S rDNA of all employings fragment is carried out analyzes without GC clamping plate, just can obtain with the consistent result of GC clamping plate, can save experimental cost, reduce experimental implementation step.
It is as follows that the present invention solves the problems of the technologies described above adopted technical scheme:
A DGGE/TGGE analytical procedure for the specific 18S rDNA fragment based on GC clamping plate strategy not, described specific 18S rDNA fragment refers to and comprises 18S rDNA away from the fragment of the stub area of ITS sequence, with
saccharomyces cerevisiaethe 18S rDNA sequence of reference culture NCYC 505 is reference, and described 18S rDNA is equivalent to it away from the stub area of ITS sequence and arises from the fragment that arbitrary Nucleotide within the scope of the 1st to the 30th site terminates in the 291st site Nucleotide.
Described DGGE/TGGE analytical procedure comprises following steps:
The genomic dna of a, extraction institute study sample;
B, specific 18S rDNA fragment increases;
C, the specific 18S rDNA fragment that step b amplification is obtained are carried out DGGE or TGGE analysis;
D, rubber tapping are reclaimed object band and adopt same primer pair fragment again to increase; Described same primer is for the primer of the specific 18S rDNA fragment that increases in step b;
The fragment of e, counterweight amplification is carried out sequencing, the sequence of mensuration be submitted on NCBI, utilize Blast in GenBank database, carry out similarity searching and according to result judge sequence the kind of corresponding bacterial strain.
When the present invention adopts specific 18S rDNA fragment to carry out DGGE analysis, the denaturing agent gradient upper limit is less than 40%.
Remarkable advantage of the present invention: the invention provides a kind of not DGGE/TGGE analytical procedure of the specific 18S rDNA fragment based on GC clamping plate strategy, the DGGE/TGGE that the specific 18S rDNA of all employings fragment is carried out analyzes without GC clamping plate, just can obtain with the consistent result of GC clamping plate, can save experimental cost, reduce experimental implementation step.
Accompanying drawing explanation
Fig. 1 is that 18S rDNA is away from the stub area schematic diagram of ITS sequence; The ITS fragment that only shows 18S rDNA fragment in figure and be adjacent, what wherein black region band portion represented is 18S rDNA fragment, what white area band portion represented is ITS fragment, double-headed arrow region representation be that 18S rDNA is away from the stub area of ITS sequence.
Fig. 2 is the sequence alignment collection of illustrative plates of the 18S rDNA stub area of 16 kinds of fungies; In figure, 1-16 represents the numbering of 16 kinds of bacterium, and the kind of representative refers to table 1, and in figure, NS1 represents primer; In figure, the 1-160 region of ruler sign is low GC content fragment, and three high GC content fragments are contained in 160-280 region, roughly mark respectively with three square frames.The target site of primer NS1 is arranged in 18S rDNA away from the stub area of ITS sequence.Although the 18S rDNA sequence of each fungi of collecting differs in the initial situation of the end away from ITS sequence, but at least originate in the 20th site, for easy analysis, the 18S rDNA sequence end of part fungi, with primer NS1 sequence polishing, can't have influence on the sequence characteristic of its low GC content like this.
Fig. 3 is that the agarose gel electrophoresis of primer pair NS1-fung and NS1-GCfung amplified fragments detects collection of illustrative plates.
Fig. 4 is that the DGGE of primer pair NS1-fung and NS1-GCfung amplified fragments analyzes collection of illustrative plates.
Fig. 5 is that the agarose gel electrophoresis of the heavy amplified fragments of primer pair NS1-fung detects collection of illustrative plates; Heavy amplification template is that liquid is reclaimed in the rubber tapping of 20 DGGE bands in Fig. 4, and the letter in figure represents the sign of template in Fig. 4.
Embodiment
One, the present invention is based on the DGGE/TGGE analysis principle of specific 18S rDNA fragment:
18S rDNA sequence through each the main fungi kind at present known is analyzed discovery, and 18S rDNA has special sequence characteristic away from the stub area (referring to Fig. 1) of ITS sequence: the low GC region that 180bp left and right is contained in its one end, is equivalent to
saccharomyces cerevisiaethe 1-183 site of the 18S rDNA sequence of reference culture NCYC 505 (Accession Number is Z75578), its average GC content only has 35% left and right, and has high mutability; The other end that is close to low GC region contains three arranges compact high GC region, is equivalent to respectively
saccharomyces cerevisiae184-202 site, 223-244 site and the 268-291 site of the 18S rDNA sequence of reference culture NCYC 505 (Accession Number is Z75578), average GC content is more than 65%, and has good conservative property.Just cause 18S rDNA to have low melting district and high melting district away from the stub area of ITS sequence, and melting nature difference between the two will be very greatly different like this.Therefore the fragment that comprises this stub area can utilize low melting district difference to carry out separation under the condition of low denatured gradient, and remaining part is equivalent to the high district that melts and can unwind and can serve as GC clamping plate.
Described specific 18S rDNA fragment refers to and comprises 18S rDNA away from the fragment of the stub area of ITS sequence, with
saccharomyces cerevisiaethe 18S rDNA sequence of reference culture NCYC 505 is reference, and described 18S rDNA is equivalent to its 1st fragment to the 291st nucleotide site scope away from the stub area of ITS sequence.(based on existing fungi 18S rDNA primer, as the amplified fragments of primer pair NS1-fung or primer pair NS1m-NS2+10 belong to as described in specific 18S rDNA fragment, primer sequence is as follows:
NS1（SEQ ID NO.1）：5'-GTAGTCATATGCTTGTCTC-3'；
fung（SEQ ID NO.2）：5'-ATTCCCCGTTACCCGTTG-3'；
GCfung（SEQ ID NO.3）：5'-CGCCCGCCGCGCCCCGCGCCCGGCCCGCCGCCCCC GCCCCATTCCCCGTTACCCGTTG-3'；
NS1m（SEQ ID NO.4）：5'-CCAGTAGTCATATGCTTGTC-3'；
NS2+10（SEQ ID NO.5）：5'-GAATTACCGCGGCTGCTGGC-3'）。
The low GC region that described 18S rDNA contains away from one end of the stub area of ITS sequence, approximately 180 Nucleotide of its total length, it should be noted that specific 18S rDNA fragment might not comprise this low GC region total length, with
saccharomyces cerevisiaethe 18S rDNA sequence of reference culture NCYC 505 is reference, and described low GC region can arise from and be equivalent to arbitrary Nucleotide within the scope of its 1st to the 30th site.
Described low GC region has high mutability and refers to that, for most of fungi kinds, this region fragment sequence is different.Described three high GC regions have good conservative property and refer to for most of fungi kinds, all have the fragment of such three high GC contents in corresponding region.
Wherein, for specific 18S rDNA fragment, its separation in DGGE/TGGE depends primarily on low GC region, therefore can adopt low denaturing agent gradient scope.In view of low GC zone leveling GC content only has 35% left and right, so the denaturing agent gradient upper limit is less than 40%.
Wherein for specific 18S rDNA fragment, the primer corresponding zone GC content of relative with its a low melting district end other end can not be lower than 40%, to guarantee that this end can not unwind under low denatured gradient condition.This point is very easily reached.(as the amplified fragments of primer pair NS1-fung or primer pair NS1m-NS2+10 meets this point).
Two, the present invention's DGGE/TGGE analytical procedure of the specific 18S rDNA fragment based on GC clamping plate strategy not:
The genomic dna of a, extraction institute study sample;
B, specific 18S rDNA fragment increases;
C, the specific 18S rDNA fragment that step b amplification is obtained are carried out DGGE or TGGE analysis;
D, rubber tapping are reclaimed object band and adopt same primer pair fragment again to increase; Described same primer is for the primer of the specific 18S rDNA fragment that increases in step b;
The fragment of e, counterweight amplification is carried out sequencing, the sequence of mensuration be submitted on NCBI, utilize Blast in GenBank database, carry out similarity searching and according to result judge sequence the kind of corresponding bacterial strain.
Below in conjunction with specific embodiment, the present invention will be further described, but the present invention is not limited to this.
Before the present embodiment carries out, it is example that the Fujian red rice yellow wine of take is brewageed by the 18S rDNA sequence of the fungi flora in song, and the sequence characteristic to it away from the stub area of ITS sequence is analyzed.Brewage with isolating the pure bacterial strain of 23 fungal strain song from four kinds of typical Fujian red rice yellow wines early stage in laboratory, through identifying, has 16 kinds.In Silva database, collect the 18S rDNA sequence of these fungi kinds, utilize clustalx software (clustalx 1.8) to compare, the comparison result in intercepting primer NS1 respective ends region as shown in Figure 2; The sequence in primer NS1 respective ends region is melted to characteristic and carry out statistical study, result is as shown in table 1.The low GC region known in conjunction with Fig. 2 and table 1,160bp left and right is contained in collected 18S rDNA sequence one end, is equivalent to
saccharomyces cerevisiaethe 20-183 site of the 18S rDNA sequence of reference culture NCYC 505 (Accession Number is Z75578), its average GC content only has 35% left and right, and has high mutability; The other end that is close to low GC region contains three arranges compact high GC region (Fig. 2 square frame part), is equivalent to respectively
saccharomyces cerevisiae184-202 site, 223-244 site and the 268-291 site of the 18S rDNA sequence of reference culture NCYC 505 (Accession Number is Z75578), average GC content is more than 65%, and has good conservative property.Just cause 18S rDNA to have low melting district and high melting district away from the stub area of ITS sequence, and melting nature difference between the two will be very greatly different like this.Therefore the fragment that comprises this stub area can utilize low melting district difference to carry out separation under the condition of low denatured gradient, and remaining part is equivalent to the high district that melts and can unwind and can serve as GC clamping plate.By analysis, the 18S rDNA sequence of each at present known main fungi kind also has identical characteristic.
With
saccharomyces cerevisiaethe 18S rDNA sequence of reference culture NCYC 505 is reference, the target site of primer fung is 351-368, the expanding fragment length that is to say primer pair NS1-fung is about 350bp, comprises 18S rDNA away from the stub area of ITS sequence, therefore belongs to described specific 18S rDNA fragment.
The sequence characteristic information of the 18S rDNA stub area of 16 kinds of fungies of table 1
Note: GaoGC district 1,2,3 corresponds respectively in Fig. 2 three boxed area from left to right.Wherein 8 and 12 two kind of its 18S rDNA sequence of fungi from GenBank database, the 18S rDNA sequence of all the other 14 kinds of fungies all derives from Silva database.
embodiment 1:it is research object that the present embodiment be take the red colouring agent for food, also used as a Chinese medicine that is purchased from Yongchun area and Youxi area, adopts the sequence of primer pair NS1-fung and NS1-GCfung(primer NS1, fung and GCfung to refer to sequence table simultaneously) amplified fragments carry out DGGE in order to analyze the fungi flora in distiller's yeast.
The present embodiment concrete steps are as follows:
1, the extraction of genomic dna
Reference literature (Heng Zhu, Feng Qu and Li-Huang Zhu. Isolation of genomic DNAs from plants, fungi and bacteria using benzyl chloride. Nucleic Acids Research. 1993,21(2): 5279-5280.) adopt Benzyl chloride method to extract the total genomic dna of microorganism in distiller's yeast.
2, the amplification of specific 18S rDNA fragment
The genomic dna extracting in step 1 of take is template, adopts respectively primer pair NS1-fung and primer pair NS1-GCfung to carry out the amplification of specific 18S rDNA fragment, and PCR condition is: 95 ℃ of denaturation 7min; 94 ℃ of sex change 1min, 50 ℃ of annealing 1min, 72 ℃ are extended 2min, circulate 36 times; 72 ℃ are extended 10min eventually.Amplified fragments carries out agarose gel electrophoresis detection, and collection of illustrative plates as shown in Figure 3.In Fig. 3, with M, represent that DNA Marker is in the middle of the 1st swimming lane; With I, represent Yongchun red colouring agent for food, also used as a Chinese medicine sample, comprise that the 2nd and the 3rd swimming lane is respectively the amplified production of primer pair NS1-GCfung and NS1-fung; With II, represent Youxi red colouring agent for food, also used as a Chinese medicine sample, comprise that the 4th and the 5th swimming lane is respectively the amplified production of primer pair NS1-GCfung and NS1-fung.As shown in Figure 3, institute's amplified fragments band is single, and expanding effect is good.Known with reference to DNA Marker, in figure, band is about respectively 350bp and 390bp, meets respectively the length range of NS1-fung and NS1-GCfung amplified fragments.
3, the DGGE of amplified fragments analyzes
DGGE analytical procedure reference literature (Muyzer G, Waal EC, Uitterlinden AG.Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl.Environ.Microbiol, 1993,59:695-700.) carry out.Deposition condition has the optimization through early stage, and top condition is: gum concentration is 8%, and denaturing agent gradient is 20%-40%, and temperature control is 60 ℃, and voltage is 75V, and the time is 12h.DGGE collection of illustrative plates as shown in Figure 4, represents Yongchun red colouring agent for food, also used as a Chinese medicine sample with I, comprises that the 1st and the 2nd swimming lane is respectively the amplified production of primer pair NS1-GCfung and NS1-fung; With II, represent Youxi red colouring agent for food, also used as a Chinese medicine sample, comprise that the 3rd and the 4th swimming lane is respectively the amplified production of primer pair NS1-GCfung and NS1-fung.
For Yongchun red colouring agent for food, also used as a Chinese medicine, the DGGE banding pattern of primer pair NS1-GCfung and NS1-fung amplified fragments (swimming lane 1 and swimming lane 2) is consistent as can be known from Fig. 4, all there are six clearly demarcated bands identified and position basically identical, in figure, use respectively a, b, c, d, e, f and a ', b ', c ', d ', e ', f ' in corresponding position, to represent the DGGE band of primer pair NS1-fung and primer pair NS1-GCfung amplified production; For Youxi red colouring agent for food, also used as a Chinese medicine, the DGGE banding pattern of primer pair NS1-GCfung and NS1-fung amplified fragments (swimming lane 3 and swimming lane 4) is consistent as can be known from Fig. 4, all there are four clearly demarcated bands identified and position basically identical, in figure, use respectively g, h, i, j and g ', h ', i ', j ' in corresponding position, to represent the DGGE band of primer pair NS1-fung and primer pair NS1-GCfung amplified production.Hence one can see that in the present embodiment, and the flora diversity analysis result that adopts primer pair NS1-fung to obtain with primer pair NS1-GCfung amplified fragments is consistent.In addition, because the amplified fragments of primer pair NS1-GCfung has had more the GC clip portion of 40bp than the amplified fragments of primer pair NS1-fung, so the former band can pull up lame a bit.
4, rubber tapping is reclaimed and heavily increases
To the recovery of tapping rubber of 20 DGGE bands in Fig. 4, add frozen the spending the night of 50ul sterilizing deionized water, after thawing, get 1ul and reclaim liquid as template, adopt primer pair NS1-fung again to increase, condition is with original.Heavy amplified production carries out agarose gel electrophoresis detection, and collection of illustrative plates respectively as shown in Figure 5.As seen from Figure 5, heavily the fragment pillar location of amplification is consistent, and banding pattern is single, and expanding effect is good.
5, carry out sequencing and Molecular Identification
The fragment that counterweight amplification obtains check order (sequencing result refers to sequence table), through compare of analysis, find: band a and a ', b and b ', c and c ' (sequence is as shown in SEQ ID NO.8), d and d ' (sequence is as shown in SEQ ID NO.9), e and e ', f and f ', g and g ', h and h ', i and i ', the sequencing result of j and j ' be consistent (in addition, band a, a ', g, the sequencing result of g ' is consistent (sequence is as shown in SEQ ID NO.6), band b, b ', h, the sequencing result of h ' is consistent (sequence is as shown in SEQ ID NO.7), band e, e ', the sequencing result of i and i ' is consistent (sequence is as SEQ ID NO.10), band f, f ', the sequencing result of j and j ' is consistent (sequence is as shown in SEQ ID NO.11)).This result shows that the amplification of two pairs of primers has obtained each group on all four fragment of sequence except GC clamping plate, and the phenomenon consistent with the DGGE banding pattern of two pairs of primer amplification fragments conforms to.Each corresponding band sequence is identical, illustrates that the flora results of structural analysis that adopts in the present embodiment NS1-fung primer to obtain with NS1-GCfung primer amplification fragment will be consistent.The sequence of measuring is submitted on NCBI utilizes Blast in GenBank database, to carry out similarity searching, the results detailed in Table 2.
Table 2 kind qualification result
By the present embodiment, proved that the DGGE/TGGE of specific 18S rDNA fragment analyzes, can without GC clamping plate in the situation that, obtain with the consistent result of GC clamping plate, therefore can save experimental cost, reduce experimental implementation.
<110> University of Fuzhou
Mono-kind of <120> is the DGGE/TGGE analytical procedure of the specific 18S rDNA fragment based on GC clamping plate strategy not
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<213> artificial sequence
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<211> 58
<212> DNA
<213> artificial sequence
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<211> 20
<212> DNA
<213> artificial sequence
<400> 4
ccagtagtca tatgcttgtc 20
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<213> monascus (
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The two spore droughts of <213> mould (
xeromyces bisporus)
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ataaattgct tatacttaga catgcatggc ttaatctttg 280
<210> 9
<211> 280
<212> DNA
<213> wood-sugar fermentation yeast (
scheffersomyces stipitis)
<400> 9
aagttgatag ggcagaaatt tgaatgaacc atcgccggca caggccatgc gattcgaaaa 60
gttattatga atcaccaagg actcccgaaa gggattgatt ttttatctaa taaatacatc 120
ccttccaaaa atccgggttt tttagcatgt attacttcta aaattacaac ggtatatatg 180
tagtaaggta ctctatctat aaacgataag agatttgtga gccattcgca gtttcactga 240
ataaattgct tatacttcaa catgcatggc ttcttctttg 280
<210>10
<211> 280
<212> DNA
<213> monascus (
monascus sp.)
<400> 10
ttgatagggc agaatttgaa tgaaccatcg ccggcgcaag gccatgcgat tcgtttagtt 60
attatgattc accaaggagc cccgaagggc gttggttttt tatctaataa atacacccct 120
tccgaagtcg gggtttttag catgtattag ctctagaatt accacaggta tccatgtagt 180
aaggtactat caaataaacg ataactgatt taatgagcca ttcgcagttt cacagtataa 240
attgcttaca cttagacatg catggcttaa tctttgagac 280
<210>11
<211> 280
<212> DNA
The two spore droughts of <213> mould (
xeromyces bisporus)
<400> 11
gttgataggg cagaatttga atgaaccatc gccggcgcaa ggccatgcga ttcgctaagt 60
tattatgact caccaaggag ccccgaaggg cattggtttt ttatctaata aatacacccc 120
ttccgaagtc ggggttttca gcatgtatta gctctagaat taccacaggt atccatgtag 180
taaggtacta tcaaataaac gataactgat ttaatgagcc attcgcagtt tcacagtata 240
aagtgcttac acttagacat gcatggctta atctttgaga 280
Claims (1)
1. a DGGE/TGGE analytical procedure for the specific 18S rDNA fragment based on GC clamping plate strategy not, is characterized in that: described specific 18S rDNA fragment refers to and comprise 18S rDNA away from the fragment of the stub area of ITS sequence, with
saccharomyces cerevisiaethe 18S rDNA sequence of reference culture NCYC 505 is reference, and described 18S rDNA is equivalent to it away from the stub area of ITS sequence and arises from the fragment that arbitrary Nucleotide within the scope of the 1st to the 30th site terminates in the 291st site Nucleotide; Described method comprises following steps:
The genomic dna of a, extraction institute study sample;
B, specific 18S rDNA fragment increases; Amplimer is NS1 and fung, and sequence is respectively as shown in SEQ ID NO.1 and SEQ ID NO.2;
C, the specific 18S rDNA fragment that step b amplification is obtained are carried out DGGE or TGGE analysis;
D, rubber tapping are reclaimed object band and adopt same primer pair fragment again to increase; Described same primer is for primer NS1 and the fung of the specific 18S rDNA fragment that increases in step b;
The fragment of e, counterweight amplification is carried out sequencing, and it is upper that the sequence of mensuration is submitted to NCBI, utilizes Blast in GenBank database, to carry out similarity searching, and according to result judge sequence the kind of corresponding bacterial strain;
Described
saccharomyces cerevisiaethe 18S rDNA sequence of reference culture NCYC 505, its NCBI number of logging in is Z75578.
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| CN102071249A (en) * | 2009-11-25 | 2011-05-25 | 贵州仁怀茅台镇金士酒业有限公司 | Method for identifying Maotai-flavor Daqu liquor |
| CN102242193A (en) * | 2011-05-09 | 2011-11-16 | 南昌大学 | Application of DGGE (Denaturing Gradient Gel Electrophoresis) method to microorganism quick sort |
| CN102559862A (en) * | 2010-12-24 | 2012-07-11 | 云南天士力帝泊洱生物茶集团有限公司 | Dominant fungi in microbial community of puer tea and spectrum thereof |
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| CN102071249A (en) * | 2009-11-25 | 2011-05-25 | 贵州仁怀茅台镇金士酒业有限公司 | Method for identifying Maotai-flavor Daqu liquor |
| CN102559862A (en) * | 2010-12-24 | 2012-07-11 | 云南天士力帝泊洱生物茶集团有限公司 | Dominant fungi in microbial community of puer tea and spectrum thereof |
| CN102242193A (en) * | 2011-05-09 | 2011-11-16 | 南昌大学 | Application of DGGE (Denaturing Gradient Gel Electrophoresis) method to microorganism quick sort |
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