- 期刊
火鶴花細菌性葉枯病病原菌PCR引子之研發與應用
Development and Application of PCR Primer for Xanthomonas axonopodis pv. dieffenhachiae, the Causal Agent of Anthurium Blight
摘要
本實驗利用聚合酵素連鎖反應(polymerase chain reaction，簡稱PCR)技術偵測火鶴花細菌性葉枯 病病原菌(Xanthomonas axonopodis pv. Dieffenbachiae，原名X. campestris pv. Dieffenbachiae，簡稱XCD)。實驗中使用之引子對是依據葉枯病菌16S-23S rDNA基因區間(spacer region)之核?酸序列而設計。藉由16S-23S rRNA基因的保守特性，在16S rDNA序列之3'端和23S rDNA序列之5'端設計廣效性引子對Bfl/Brl，以增幅出包含有基因區間之585 bp PCR產物，並選殖入質體pCRII完成核?酸解序，XCD之16S-23S rDNA基因區間核?酸序列顯示包含了tRNAAla和tRNAIle的基因序列。利用XCD及其他Xanthomonas spp.細菌之16S-23S rDNA基因區間核?酸序列比對後，依據其序列差異設計出PCR引子對Difl/Dirl，其可從XCD DNA模版中專一地擴增出一大小為409 bp之PCR產物，而不會由其他測試之Xanthomonas spp.細菌、其他種類細菌及火鶴花葉表細菌之DNA模版中擴增出任何產物。PCR引子對Difl/Dirl的偵測極限為1.5 pg之純化DNA或3-4個XCD細胞。
並列摘要
One pair of primers for the polymerase chain reaction-based assay were devel oped for the detection of the causal agent of anthurium blight, Xanthomonas axonopodis pv. dieffenbachiae (XCD). Universal primers targeting conserved sequences flanking the 3' end of the 16S and the 5'end of the 23S rRNA genes were synthesized and used to amplify the 16S-23S rDNA internal transcribed spacer of XCD. A 585 bpPCR product containing spacer region thus amplified was cloned in pCRII for nucleotide sequence determination. Sequences for tRNAAla and tRNAIle genes were revealed in the 16S-23S rDNA spacer region. Primer pair Difl/Dirl, synthesized according to the sequence of the spacer region, could effectively amplify a 409 bp DNA fragment from the DNA template prepared from XCD, but not from any other tested Xanthomonas spp., other bacterial species or epiphytic bacteria isolated from anthurium. A minimum of 1.5 pg DNA or 3-4 XCD cells was sufficient to amplify the specific XCD PCR fragments using the primer pair Difl/Dirl. Rapid detection of XCD from anthurium samples by PCR was also evaluated.
延伸閱讀
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