Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Denaturing gradient gel electrophoresis technique for detection of bacterial community structure in rice wine wheat koji

A technology of denaturing gradient gel and technical detection, applied in the direction of biochemical equipment and methods, measuring devices, and the determination/inspection of microorganisms

Inactive Publication Date: 2011-12-14
JIANGNAN UNIV
View PDF3 Cites 5 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to use denaturing gradient gel electrophoresis technology to detect the bacterial microbial community structure in rice wine wheat koji for the problems existing in the traditional separation culture method

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Denaturing gradient gel electrophoresis technique for detection of bacterial community structure in rice wine wheat koji
  • Denaturing gradient gel electrophoresis technique for detection of bacterial community structure in rice wine wheat koji
  • Denaturing gradient gel electrophoresis technique for detection of bacterial community structure in rice wine wheat koji

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0008] Example 1 Genomic DNA Extraction

[0009] Weigh 1.0g wheat koji sample into a 10mL centrifuge tube, add 2.5mL extract solution (100mmol / L Tris-HCl, pH8.0, 40mmol / L EDTA, pH8.0) at the same time, shake and mix well, then add 1mL10% SDS and 2mL benzyl chloride, incubate at 50°C for 1h (mix up and down every 10min), add 1mL of 3mol / L NaAc, ice-bath for 15min, centrifuge at 12,000r / min for 15min, take the supernatant, add an equal volume of isopropanol , Place at -20°C for 2 hours, centrifuge at 12,000 r / min for 15 minutes, discard the supernatant, add 70% ethanol to wash, centrifuge for 15 minutes, discard the supernatant, and add 50 μL TE to dissolve after the ethanol has evaporated.

Embodiment 2

[0010] Embodiment 2PCR-DGGE analysis

[0011] Design a primer pair for amplifying the V3 region of bacterial 16S rDNA, and add a GC clip to the 5' end of the forward primer; the primers are: F1:

[0012] 5'-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGGCCTACGGGAGGCAGCAG-3'

[0013] R1: 5'-ATTACCGCGGCTGCTGG-3'. The 50 μL reaction system includes: 36 μL double distilled water, 5 μL 10×PCR Buffer, 4 μL dNTP, 2 μL primer, 1 μL template, 0.25 μL ExTaq enzyme (5u / μL). Pre-denaturation at 94°C for 4min, denaturation at 94°C for 45S, annealing at 55°C for 45S, extension at 72°C for 45S, 35 cycles, and finally extension at 72°C for 10min. PCR products were separated on denaturing gradient gel electrophoresis; the relevant parameters of electrophoresis were voltage 150V, electrophoresis at 60°C for 4h, acrylamide gel concentration was 8%, and denaturing gradient was 50%-65% (100% denaturant It consists of 7mol / L urea and 40% deionized formamide with a mass fraction of 40%. The electrophore...

Embodiment 3

[0014] Example 3 Bacterial community analysis

[0015] Cut and recover the bands on the DGGE gel: Dilute 10000×SYBR-Green into 1×SYBR-Green for staining. The staining process is divided into 3 times, each staining is 15min; the method of cutting and recovering the gel: use a sterile knife After cutting out the dominant band, put it into a sterilized centrifuge tube, add 200 μL of sterilized double distilled water and refrigerate overnight at 4°C; perform T-cloning after PCR of the cut gel band: PCR amplification primers and amplification The procedure is the same as Example 2. The PCR product obtained in Example 2 was compared with the PCR product corresponding to the positive clone in this example by DGGE. The correct positive clones were sequenced, and the results were compared in Genbank; the structure of the microbial community of wheat koji bacteria in rice wine was obtained. select figure 1 Bands A and B are examples for illustration, the sequencing result of band A i...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a method for detecting the bacterial community structure in rice wine wheat koji by denaturing gradient gel electrophoresis technology. The genomic DNA of the wheat koji sample is extracted by the benzyl chloride method, the bacterial 16S rDNA V3 region is amplified by PCR, and the DGGE electrophoresis band is separated. Secondary PCR and T-cloning after rubber tapping and recovery, selection of positive clones for DGGE band comparison, sequencing after correct comparison, and obtaining relevant microbial information from GenBank. The invention is simple, fast and has good repeatability, and has good guiding significance for analyzing the dominant bacteria and functional microorganisms in rice wine wheat koji.

Description

technical field [0001] The invention relates to a method for detecting the bacterial community structure of wheat koji in rice wine, especially a method for detecting the bacterial community structure of wheat koji in rice wine by using PCR-DGGE (Denaturing gradient gel electrophoresis) technology. The invention belongs to microbial ecological technology field. Background technique [0002] Denaturing gradient gel electrophoresis is an electrophoresis technique for detecting DNA mutations. Muzyer et al. used this technology for the first time in the study of microbial ecology, and confirmed that this technology has obvious advantages in studying microbial diversity. DGGE can separate a mixture of DNA fragments with the same length but different base compositions. Denaturing agents—urea and deionized formamide are added to ordinary acrylamide gels to form a linear gradient from low to high. Under certain temperature conditions, DNA fragments with different base compositions...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12Q1/68C12Q1/04G01N27/447
Inventor 陆健张中华管政兵陈亮亮
Owner JIANGNAN UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com