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題名 | 莧科、馬齒莧與蘿蔔白銹菌專一性引子對之敏感度與其偵測應用=Sensitivity and Application of Albugo-specific Primers to Detection of Albugo spp. on Amaranthaceae, Portulaceae and Radish |
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作者 | 蕭茹萍; 王怡靜; 李敏郎; Hsiao, J. P.; Wang, I. C.; Lee, M. L.; |
期刊 | 植物保護學會會刊 |
出版日期 | 20070900 |
卷期 | 49:3 2007.09[民96.09] |
頁次 | 頁197-212 |
分類號 | 433.4 |
語文 | chi |
關鍵詞 | 莧科白銹菌; 馬齒莧白銹菌; 蘿蔔白銹菌; 專一性引子對; 敏感度; 偵測; Albugo bliti; A. portulacae; A. candida; Specific primers; Sensitivity; Detective; |
中文摘要 | 本研究針對莧科、馬齒莧科、旋花科及十字花科等白銹菌 ( Albugo spp. ) 設計專一性引子對,並探討這些引子對之敏感度與其偵測應用。利用序列特徵化增幅區域 ( sequence characterized amplified regions, SCARs ) 篩選白銹菌之專一性片段,設計其特定片段之引子對,並以它種白銹菌株做對照,進行聚合 酶鍊鎖反應( polymerase chain reaction, PCR ) 測試此引子對之專一性,結果顯示Abw260 ( abw260-up: 5'-cgacaagtaccttcctacatc-3'; abw260-dw: 5'- gatgta ggaaggtacttgtcg–3' ) 可專一性偵測白莧及紅莧白銹菌(A. bliti );Abl258 ( abl258-up: 5’-aaggcttgtataggcttatgc-3’; abl258-dw: 5'-gactgtggtcgacatgattta-3' );Abp260 ( abp260-up:5'-gcgttcttgtttgtagcgtta-3';abp260-dw:5'-gacacgagtgcaagtcatcat -3 )則可分別偵測鳥莧白銹菌 ( A. bliti ) 和馬齒莧白銹菌 ( A. portulacae );及Acr250 ( acr250-up: 5'-atgtgaagcggaacgagtgt-3’; acr250-dw: 5'-ttagtggtcatcggctcttc- 3’ ) 可偵測蘿蔔和山東白菜白銹菌 ( A. candida ) 。在白銹菌專一性引子對之敏感度方面,則為每反應液中含有100 pg白銹菌DNA即可測出。至於白銹菌孢囊數量與專一性引子對敏感度之關係,則以玻璃砂 ( Glass beads ) 震盪法萃取孢囊DNA後進行分析,結果顯示含有4顆白銹菌孢囊DNA模版者即可被測出,含17顆孢囊可增幅較大量之專一片段。以白銹菌接種後而尚未產生病徵之白莧、紅莧、鳥莧、馬齒莧及蘿蔔等寄主植物進行測試,結果顯示白銹菌專一性引子對可增幅出接種之白銹菌特定片段,對照組之健康植株則無此特定片段,並進一步偵測不同天數之接種葉,於接種三天後之葉片DNA中,明顯地偵測出白銹菌,因此本研究所設計之白銹菌專一性引子對,可應用於白銹菌輔助鑑定、偵測及監測等用途。 |
英文摘要 | Albugo species were collected from Amaranthaceae, Portulaceae, Convolvulaceae and Brassicaceae in Taiwan. Specificity, sensitivity and application of these Albugo-specific primers were tested in the study. Sequence characterized amplified regions (SCARs) were used to screen the individual specific DNA region of Albugo spp. for design of Albugo-specific primers. Sensitivity of the primers was tested with different Albugo species by polymerase chain reaction. The results showed that Albugo bliti from Amaranthus mangostanus and Am. mangostanus forma ruber were identified specifically by Abw260 primers (abw260-up: 5'-cgacaagtaccttcctacatc-3'; abw260-dw: 5'-gatgtaggaaggtacttgtcg-3'); A. bliti from Am. lividus, and A. portulacae from Portulaca oleracea were identified specifically by Abl258 primers (abl258-up: 5'-aaggcttgtataggcttatgc-3'; abl258-dw: 5'-gactgtggtcgacatgattta-3') and Abp260 primers (abp260-up: 5'-gcgttcttgtttgtagcgtta-3'; abp260-dw: 5'-gacacgagtgcaagtcatcat-3'), respectively; A. candida from Raphanus sativus and Brassica pekinensis were identified specifically by Acr250 primers (acr250-up: 5'-atgtgaagcggaacgagtgt-3'; acr250-dw: 5'-ttagtggtcatcggctcttc-3'). Sensitivity of Albugo-specific primers was tested and 100 pg DNA was the least amount required in each reaction solution. The relationship between the amount of sporangia and sensitivity of these Albugo-specific primers were analyzed. The sporangial DNA was extracted by glass beads method. The result showed that 4 sporangia DNA/reaction could be detected with Albugo-specific primers, but positive PCR band was obtained with 17 sporangia DNA/reaction. In the detection application of these Albugo-specific primers, specific bands of inoculated Albugo spp. were observed distinctly with inoculated but symptomless leaves of Am. mangostanus, Am. mangostanus forma ruber, Am. lividus, P. oleracea and radish. No bands were amplified with control leaves of host plants. DNA was also extracted from leaves of different infected periods after inoculation and analyzed with Albugo-specific primers. The results showed that the specific bands of inoculated Albugo spp. were observed clearly three days after inoculation of host plants. Therefore, the Albugo-specific primers designed in this study could be applied to the identification, detection and monitoring of Albugo spp. |
本系統之摘要資訊系依該期刊論文摘要之資訊為主。