การพัฒนา oilgonucleotide probes สำหรับใช้ในการตรวจสอบ methane producing bacteria โดยใช้ 16S rRNA fluorescent hybridization

Development of Oligonucleotide Probes for Detection of Methane Producing Bacteria and Sulfate Reducing Bacteria using 16S rRNA Fluorescent in situ Hybridization

Name: วิวัฒน์ จูประพัทธศรี

Organization : มหาวิทยาลัยเทคโนโลยีพระจอมเกล้าธนบุรี

keyword: Acetate enrich culture

; phylogenetic analysis

; in situ hybridization

; tyramide signal amplification

Abstract: The microbial community structure of an acetate enriched culture, which hasbeen being used in the treatment of cassava starch wastewater, was investigated by16S rDNA analysis. Two 16s rDNA libraries were constructed. Almost-full-lengthor full-length genes coding for the small-subunit rRNA (rDNA) were PCR amplifiedfrom total community genomic DNA using primer specific for Archaea or Bacteriaand subsequently cloned into the pGEM-T vector. Almost of 16s rDNA of Archaeaclone library were affiliated into Methanosaeta sp. and Methanosarcina sp. TheBacteria clones were screened by PCR amplification with a sulfate reducing bacteriaspecific primer set. The group of Bacterial clones revealed into different cluster andrelatedness with sulfate reducing bacteria. However, all of them were clustered intoanaerobic or facultative anaerobic cultures and usually presented into anaerobicwastewater treatment systems. For ensure phylogenetic analysis results, a new 16SrRNA targeted oligonucleotide probe specific for methanogen was developed basedon the retrieved sequences. In situ fluorescent hybridization with the methanogenspecificprobe revealed the majority of long sheathed filament Methanosaeta-likemorphology, with a similar result to the Archaea- specific probe hybridization. Dueto the weak fluorescent signals obtained with Bacteria-specific probe, tyramidesignal amplification (TSA) was used in combination of the conventionalfluorochrome-labeled probe to increased signal for hybridization. TSA system wasconsisted of a fluorochrome labeled-16S rRNA oligonucleotide probe, antifluorochromehorseradish peroxidase (HRP) conjugate, biotin-labeled tyramide, andstreptavidin-fluorescent conjugate. The technique was successfully applied for in situdetection of microbial communities of an acetate enriched culture. Most of bacterialcells were detected in very strong green signal, whereas the case of untreated TSA,most of bacterial cells showed very low signal.

มหาวิทยาลัยเทคโนโลยีพระจอมเกล้าธนบุรี

Address: กรุงเทพมหานคร (Bangkok)

Email: info.lib@mail.kmutt.ac.th

Name: Asst.Prof.Dr. Supapon Cheevadhanarak

Role: Supervisor

Created: 2544

Issued: 2005-10-26

Modified: 2552-11-20

วิทยานิพนธ์/Thesis

ISBN: 9746289594

CallNumber: BIT404

tha

DegreeName: วิทยาศาสตรมหาบัณฑิต

Level: ปริญญาโท

Descipline: เทคโนโลยีชีวภาพ

Grantor: มหาวิทยาลัยเทคโนโลยีพระจอมเกล้าธนบุรี

©copyrights มหาวิทยาลัยเทคโนโลยีพระจอมเกล้าธนบุรี

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3	BIT404ab.txt	2.37 KB	14	2025-11-10 10:07:50

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