臺灣博碩士論文加值系統

本研究目的是進行耐磺胺劑好氣性堆肥嗜高溫菌之分類並分析其dhps gene。研究中應用PCR技術增幅耐sulfamethoxazole好氣性堆肥嗜高溫菌之16S rDNA及dhps gene，並將PCR產物定序分析。將堆肥處理中心分離之菌株共計200株，培養於含sulfamethoxazole 512μg/ml之TSB broth中，判定其中具耐藥性之菌株共有105株（52.5%），由其中選取17株進行16S rDNA分類及dhps gene 序列分析。以16S rDNA 5’及3’端之保留區設計引子對所增幅PCR產物約為1465 bp，直接將產物進行序列分析，並依據Biolog自動鑑定系統及16S rDNA序列所做出親源分類樹狀圖（phylogenetic tree）進行細菌分類。結果顯示菌株編號B39為Brevibacillus agri，編號B77、B78、B86、B87、B88、B89及B113等七株為Geobacillus stearothermophilus，編號A73、A74、A78、A79、A86 、B105、B108及B110等八株為Ureibacillus thermosphaericus。參考文獻B. subtilis dhps gene序列設計之引子對以PCR技術偵測17株耐sulfamethoxazole嗜高溫菌，將其PCR產物純化，經重組質體及定序分析，僅編號B108菌株產物經比對確認為dhps gene之片段。再以B108菌株及Bacillus subtilis之dhps gene序列設計引子對，其中B77、B86、B87、B88、B89、B108及B110等7株，其PCR產物大小407 bp之片段，與原先設計大小一致。選取B110菌株產物純化，經重組質體及定序分析，確認為dhps gene之片段。與ATCC 6633S及GenBank登錄編號M34053 B. subtilis相比較，菌株編號B108 dhps gene第185個胺基酸（Asn變為Ser）及菌株編號B110第244個胺基酸（Gly變為Arg）發生改變，這些改變與磺胺劑抗藥性之相關性，需再進一步研究。

The study is aimed to classify sulfamethoxazole-tolerant thermophilic bacteria isolated from aerobic compost, and to analyze the dhps gene sequence among these bacteria. Polymerase chain reaction（PCR）was performed to amplify part of the dhps gene and 16S rDNA. PCR products were subcloned, and sequence. Total 200 bacteria isolates from compost treatment centers were tested in TSB broth with sulfamethoxazole 512μg/ml. There were 105 isolates (52.5%) growing well (regard as sulfamethoxazole—tolerant). Among 105 isolates, 17 isolates were selected for 16S rDNA-based classification and dhps gene sequence analysis. Using primers emcompassing the 5’ and 3’ conserved region of 16S rDNA, the expected PCR product is about 1465bp. PCR products resulted from different bacterial isolates were sequence. Classification of these bacteria were based on the biochemical characteristics analyzed by Biolog MicroStation system as well as the phylogenetic trees derived from 16S rDNA sequence. Isolate B39 was classified as Brevibacillus spp. Seven isolates including B77, B78, B86, B87, B88, B89 and B113 were identified as Geobacillus stearothermophilus. Eight isolates including A73, A74, A78, A79, A86, B105, B108 and B110 were identified as Ureibacillus thermosphaericus.
According to Bacillus subtilis dhps gene sequence from literature, primers were designed and PCR was performe for 17 sulfamethoxazole-tolerant isolates. The expected fragment of PCR product was purified, subcloned and sequenced. There was only product of B108 isolate confirmed to be fragment of dhps gene. Based on the dhps gene sequence of isolate B108 and Bacillus subtilis, we designed PCR primers and amplified part of the dhps gene among 17 sulfamethoxazole-tolerant isolates. The expected fragment of PCR product is about 407bp. This PCR product was shown in seven isolates including B77, B86, B87, B88, B89, B108 and B110. The expected fragment from B110 was subcloned, sequenced and confirmed to be fragment of dhps gene. Comparing to B. subtilis ATCC 6633S and GenBank #M34053 dhps gene, B108 isolate 185th amino acid（Asn to Ser）and B110 isolate 244th amino acid（Gly to Arg）were changed. There is a relationship between these changes and the sulfa-resistance . However, the further study is required for understanding.

目 錄
頁次
中文摘要………………………………………………………………..Ⅰ
英文摘要………………………………………………………………..Ⅲ
目錄……………………………………………………………………..Ⅴ
表次……………………………………………………………………..Ⅷ
圖次……………………………………………………………………..Ⅸ
第一章 緒 言…..……………………………………………………...1
第二章 文獻探討....…………………………………………………...3
第一節 堆 肥....……………………………………………………3
1.1 堆肥之處理方式及其特點 ………………………………….3
1.2 堆肥嗜高溫菌分類及其作用 …………………………...…..5
1.3 堆肥中藥物殘留及抗藥性細菌對公共衛生之影響 ..…….6
第二節 磺胺劑及其抗藥性 ………………………………….…8
2.1 磺胺劑之一般性質及其作用機制 …………………….……8
2.2 磺胺劑抗藥性菌之產生機制 ..……………………….…..10
2.3 Sulfamethoxazole（SMX）在臨床上之使用.…………….12
第三節 細菌分類法……………………………………………...14
3.1 表現型分類法（phenotypic methods） …………….….….15
3.2 基因型分類法（genotypic methods） …………….………17
3.3 16S rDNA（rRNA）之類緣分析 ……………….…….….18
3.4 Bacillus 現今分類狀況..…………………………………..19
第四節 磺胺劑抗藥性與dhps gene .………………….…..…..20
4.1 dhps gene之位置與功能…………………… ……..……...20
4.2 dhps gene變異與磺胺劑抗藥性產生之關聯……………..21
4.3 利用dhps gene檢出磺胺劑抗藥菌之探討….…………….25
4.4 dhps gene應用在細菌分類之探討………………………..28
第三章 材料與方法…………………………………………………..30
第一節 細菌之來源、培養、鑑定及保存……………………...30
1.1 細菌之來源及培養………………………………………...30
1.2 細菌之鑑定………………………………………………...30
1.3 菌種之保存………………………………………………...32
第二節 細菌DNA之萃取………………………………………32
第三節 16S rDNA……………………………………………….33
3.1 16S rDNA增幅………………………………………….…33
3.2 16S rDNA定序…………………………………………….35
第四節 dhps gene... ……………………………………………..36
4.1 細菌dhps gene增幅及產物純化………………………….36
4.2 構築含有dhps gene之重組質體………………………….37
4.3 dhps gene重組質體核酸定序……………………………..39
4.4 細菌dhps gene初步試驗及引子設計…………………….40
第五節 序列分析方法…………………………………………..41
第四章 結 果………………………………………………………..42
第一節 耐sulfamethoxazole細菌之培養及鑑…....…….……….42
1.1 耐sulfamethoxazole細菌之培養………………………….42
1.2 耐sulfamethoxazole細菌之鑑定………………………….42
第二節 16S rDNA序列比對….……………………………..…...43
第三節 建立菌種演化親緣關係…………………………………44
第四節 dhps gene之偵測與分析………………………………..44
第五章 討 論…………………………………………………….…..47
參考文獻………………………………………………………………..88

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