臺灣博碩士論文加值系統

為尋求篩選 E.coli O157:H7 的有效方法, 利用 mEC+novobiocin 培養液
進行增菌培養後, 塗抹平板於 MacConky sorbitol agar(MSA)平板培養基
上, 挑出白色非 sorbitol 發酵型菌落於 LB 培養液 96孔的 micro-
titer plate 上,以轉印的方式, 同時接種在 MacConky (MAC)、EMB 平板
上, 及裝於 96 孔 microtiter plate 中的 SIM, Phenol red-
sorbitol- MUG (PRS-MUG) 培養基。將符合乳糖發酵型、indole正反應、
β-glucuro- nidase 負反應及 EMB 上呈現 E.coli 菌落外觀的菌落挑
出, 進行血清測試。當 novobiocin 為 2μg/ml 時, 增加的 lactose
及 indole 兩個因子可使檢測 E.coli O157 的成功率由 28.2%提高
為77.4%。而novobiocin為 20μg/ml 時, lactose 及indole 兩個因子可
使檢出 E.coli O157 的成功率由 71.2% 提高為 93.7%。且 novobiocin
為 20μg/ml時, 接種量為 3.6 cell/g 時, 對 6件肉品的回收率為
5/6。本方法可節省人力、時間及適合用在大量篩菌的過程。分析16S
rRNA 序列除了有助於了解演化關係,亦可藉16S rRNA 序列的差異性發展
具特異性探針。將 5株 E. coli O157:H7 利用共同性引子進行 PCR 反
應,可放大 16S rDNA的量,再經不對稱 PCR 反應得到單股放大的16S
rDNA 進行定序。獲得的序列與典型 E. coli 的 16S rRNA 進行序列排列
比對,在位置 982 與 1018 bp之間,找到 9 個變異。利用這段變異區,我
們設計了EH1 探針,用來偵測E.coli O157: H7。於特異性試驗中, 12株E.
coli O157:H7 均為正反應, 8株其它E.coli O157:H7 中 1株正反應, 2株
弱正反應而 Escherichia coli, Klebsiella pneumoniae, Shigella
flexner,Salmonella braenderup 均有弱正反應。

An effective screening method was developed for isolate
Escherichia coli O157:H7. After enrichment by mEC+novobicin
broth, appropriate dilutions of the cultures were spread plate
on MSA. Sorbitol negative white colonies were picked and
inoculated in LB broth in 96-well microtiter plate. Muti-
inoculator was used to transfer colonies to MacConky agar plate
、EMB agar plate or SIM, phenol red-sorbitol-MUG (PRS-MUG)in
96-well microtiter plate. The colony is lactose poistive,
indole positive, MUG negative and typical E.coli morphology on
EMB were choosed to do serology test. When the novobiocin is
20μg/ml, the additional factors,lactose and indole, can
increase the isolation on frequence from 70.7% to 93.1%. By
this method E.coli O157:H7 was isolated from 5 of 6 meat sample
inoculated at 3.6 cells/g level.Sequencing of 16S rRNA is a
valuable technique in determining phylogenic relationships.
Comparison of 16S rRNA sequences from closely related bacteria
species can give specific probe to detect 16S rRNA.12 E.coil
O157:H7 16S rDNA was amplified by PCR using universal primers.
The partial 16S rDNA fragment of 5 E.coli O157:H7 were
sequenced. The 16S rDNA sequences of 5 E.coli O157:H7 and
typical E.coli were analyzed by gene There are 9 base variants
during position 982 to 1018. EH1 was designed as E.coli O157:H7
16S rDNA probe. In the specific test,12 E.coli O157:H7 are
positive; 3 of 8 other E.coli are posotive(1 strain) or weak
posotive (2 strain), Escherichia hermannii,Klebseilla
pneumoniae,Shigella flexneri and Salmonella braenderup are weak
positive.