CN102277432A

CN102277432A - Detection method of bacterial community structure in yellow rice wine wheat starter by denatured gradient gel electrophoresis technology

Info

Publication number: CN102277432A
Application number: CN2011102228108A
Authority: CN
Inventors: 陆健; 张中华; 管政兵; 陈亮亮
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2011-08-04
Filing date: 2011-08-04
Publication date: 2011-12-14

Abstract

The invention discloses a detection method of a bacterial community structure in yellow rice wine wheat starter by a denatured gradient gel electrophoresis technology. The method comprises the following steps: extracting DNA (deoxyribonucleic acid) of a genome of a wheat starter sample by adopting a benzyl chloride method; amplifying 16S rDNA (ribosomal deoxyribonucleic acid) V3 region of a bacterium by PCR (polymerase chain reaction); separating by DGGE (denatured gradient gel electrophoresis) strip; tapping and reclaiming, and performing secondary PCR and T-cloning; selecting positive cloning to perform DGGE strip comparison; and sequencing after correct comparison, and acquiring related information of microorganisms from GenBank. The detection method is simple and quick and has good repeatability, and has good instructional significance in analyzing dominant bacteria and functional microorganisms in the yellow rice wine wheat starter.

Description

Denaturing gradient gel electrophoresis technique detects the method for bacterial flora structure in the yellow rice wine wheat starter

Technical field

The present invention relates to a kind of method that detects yellow rice wine wheat starter bacterial flora structure, especially a kind of PCR-DGGE (Denaturing gradient gel electrophoresis that utilizes, denaturing gradient gel electrophoresis) technology for detection yellow rice wine wheat starter bacterial flora structure the invention belongs to the microbial ecological technical field.

Background technology

Denaturing gradient gel electrophoresis technique is a kind of electrophoretic technique that detects dna mutation.Muzyer etc. have adopted this technology first in microbial ecological research, and have confirmed that this technology has obvious superiority aspect the research microbial diversity.But DGGE can the identical based composition of separation length dna fragmentation mixture inequality, thereby denaturing agent---urea and deionized formamide add the linear gradient that forms in the common acrylamide gel from low to high to.Under certain temperature condition, the dna fragmentation melting temperature(Tm) of different based compositions is inequality, thereby can cause different electrophoretic mobilities, so when these dna fragmentations are carried out electrophoresis in the gel that adds denaturing agent, can move to the different positions of gel, the identical but dna fragmentations that based composition is different of length are separated the most at last.PCR and DGGE are joined together, under suitable denatured gradient condition, can tell the difference of a base pair, the resolving power height.Gel pattern after the dyeing by what of band number to a certain extent can response sample in the difference formed of microorganism, what of microorganism in the sample brightness of band can reflect to a certain extent.

The method of traditional research microbial diversity mainly depends on culture method, promptly separation and purification, form and the microscopic examination by bacterial strain, physiological and biochemical property relatively after carry out bacterial strain classification identify, this method steps complexity, workload are big, and are difficult to carry out correct evaluation and classification for the similar bacterial strain of physiological and biochemical property.Yellow rice wine is one of traditional brewing wine of China, and " with the wheat koji, with bent wine brewing " is the characteristic of Chinese rice wine.Wheat koji plays an important role in the brewing process of yellow rice wine, for brewageing of yellow rice wine provides multiple prozyme, flavour substances.Research for the yellow rice wine wheat starter biological community structure at present mainly concentrates on traditional separation and Culture and preliminary evaluation.Use the composition of bacterial micro-organism structure of community in the PCR-DGGE technical Analysis yellow rice wine wheat starter, to judging and identifying that superior microorganism, functional microorganism in the wheat koji have important theory and practice significance.

Summary of the invention

The objective of the invention is to problem, utilize denaturing gradient gel electrophoresis technique to detect bacterial micro-organism structure of community in the yellow rice wine wheat starter at traditional isolated culture existence.The present invention does not rely on traditional separation and Culture, utilizes to be fit to DGGE isolated bacterial universal primer, in conjunction with predominant bacteria and the functional microorganism in the PCR-DGGE technical Analysis yellow rice wine wheat starter.Characteristics such as that present method has is easy, quick, good reproducibility are formed in conjunction with the bacterial micro-organism structure of community that traditional separation method can more comprehensively react in the wheat koji, for the functional microorganism of research yellow rice wine wheat starter provides strong proof.

Technical scheme of the present invention: utilize the Benzyl Chloride method to extract the genomic dna of yellow rice wine wheat starter sample, the 16S rDNA V3 district of pcr amplification bacterium, DGGE electrophoretic separation dna fragmentation, cut the band that glue reclaims the microorganism correspondence, secondary PCR amplification and T-clone and select positive colony, the comparison of DGGE electrophoretic band, order-checking are identified and are cut the microorganism of adhesive tape band correspondence.

The present invention is by bacterial micro-organism structure of community in the PCR-DGGE technology for detection yellow rice wine wheat starter, can be widely used in the yellow rice wine wheat starter sample of analyzing different process, different areas and the rule that changes with bacterial micro-organism structure of community in a kind of yellow rice wine wheat starter sample making process, for the isolation identification of functional microorganism provides reference.

Specific implementation method

Embodiment 1 extracting genome DNA

Take by weighing 1.0g wheat koji sample and put into the 10mL centrifuge tube, add 2.5mL extracting solution (100mmol/L Tris-HCl, pH8.0 simultaneously, 40mmol/L EDTA, pH8.0) behind the vibration mixing, add 1mL10%SDS and 2mL Benzyl Chloride again, 50 ℃ of insulation 1h (every 10min turn upside down mixing), the NaAc that adds 1mL 3mol/L, ice bath 15min, 12, the centrifugal 15min of 000r/min, get supernatant, add isopyknic Virahol, place 2h, 12 for-20 ℃, the centrifugal 15min of 000r/min, abandon supernatant, add 70% washing with alcohol, centrifugal 15min, abandon supernatant, treat that ethanol is evaporated completely the back and adds 50 μ L TE dissolving.

Embodiment 2PCR-DGGE analyzes

The primer in design amplification bacterial 16 S rDNA V3 district is right, and adds the GC folder at 5 ' end of forward primer; Primer is: F1:

5’-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGGCCTACGGGAGGCAGCAG-3’

R1：5’-ATTACCGCGGCTGCTGG-3’。50 μ L reaction systems comprise: 36 μ L distilled waters, 5 μ L10 * PCR Buffer, 4 μ L dNTP, the primer of 2 μ L, the template of 1 μ L, the ExTaq enzyme of 0.25 μ L (5u/ μ L).94 ℃ of pre-sex change 4min, 94 ℃ of sex change 45S, 55 ℃ of annealing 45S, 72 ℃ are extended 45S, 35 circulations, last 72 ℃ are extended 10min.The PCR product is separated on denaturing gradient gel electrophoresis; Electrophoretic correlation parameter is voltage 150V, 60 ℃ of following electrophoresis 4h, acrylamide gel concentration is 8%, denatured gradient is that (100% denaturing agent is that 40% deionized formamide is formed by 7mol/L urea and massfraction to 50%-65%, electrophoresis result as shown in Figure 1, three repetitions of three swimming lanes for being provided with.

Embodiment 3 bacterial flora analyses

Band on the DGGE glue is cut glue to be reclaimed: 10000 * SYBR-Green is diluted to 1 * SYBR-Green dyes, its dyeing course divides 3 times, and 15min dyes at every turn; Cut the method that glue reclaims: put into the centrifuge tube of the bacterium of going out after with aseptic pocket knife the advantage band being downcut, add 4 ℃ of refrigerator overnight of sterilization distilled water of 200 μ L; Carry out the T-clone after cutting adhesive tape band PCR: primer that pcr amplification adopts and amplification program embodiment 2 are the same.The PCR product PCR product corresponding with positive colony in the present embodiment that obtains among the embodiment 2 carried out the DGGE comparison.Correct positive colony of comparison is checked order, carry out result's comparison at Genbank; Obtain the structure of yellow rice wine wheat starter bacterial micro-organism group.Choose A among Fig. 1, the B band is that example describes, A band sequencing result is shown in SEQ ID NO.3, B band sequencing result is shown in SEQ ID NO.4.The Genbank part comparison result of A, B is respectively as Fig. 2, shown in Figure 3.Final definite most probable microorganism of A, B is shown in following table (table 1).

Table 1 microorganism similarity is identified

Though the present invention with preferred embodiment openly as above; but it is not in order to qualification the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; all can do various changes and modification, so protection scope of the present invention should be with being as the criterion that claims were defined.

Claims

1. denaturing gradient gel electrophoresis technique detects the method for bacterial flora structure in the yellow rice wine wheat starter, after it is characterized in that extracting wheat koji sample gene group DNA, carrying out band by specific primer pcr amplification bacterial 16 S rDNA V3 district, DGGE electrophoresis separates, after reclaiming, band carries out secondary PCR and T-clone, select the sub-DGGE band comparison of positive colony, compare the relevant information that checks order and obtain microorganism to GenBank in correct back.

2. the described method of claim 1 is characterized in that described sample is any yellow rice wine wheat starter sample to be detected, and sample adopts the sampling of division method, and the weight of final sample is 1g.

3. the described method of claim 1 is characterized in that described specific primer is F1:

5’-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGGCCTACGGGAGGCAGCAG-3’，R1：5’-ATTACCGCGGCTGCTGG-3’。

4. the arbitrary described method of claim 1-3 is characterized in that described pcr amplification program is: 94 ℃ of pre-sex change 4min, 94 ℃ of sex change 45S, 55 ℃ of annealing 45S, 72 ℃ of extension 45S, 35 circulations, last 72 ℃ of extension 10min.

5. the described method of claim 1-2, the correlation parameter that it is characterized in that described denaturing gradient gel electrophoresis is: voltage 150V, 60 ℃ of following electrophoresis 4h, acrylamide gel concentration is 8%, denatured gradient is 50%-65%, and 100% denaturing agent is that 40% deionized formamide is formed by 7mol/L urea and massfraction.

6. the described method of claim 1-2, it is characterized in that describedly to the band removal process on the DGGE glue being: 10000 * SYBR-Green is diluted to 1 * SYBR-Green dyes, dyeing course divides 3 times, and 15min dyes at every turn; Put into the centrifuge tube of the bacterium of going out after with aseptic pocket knife the advantage band being downcut, add 4 ℃ of refrigerator overnight of sterilization distilled water of 200 μ L.