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植物菌質體廣效型PCR引子之評估

Evaluation of universal PCR primers for the detection of phyloplasmas

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摘要


本研究乃利用植物病原菌質體之核糖體RNA基因序列,設計出多組之聚合酶連鎖反應引子序列,並加以評估此類廣效型(universal)聚合酶連鎖反應引子(primer pairs)偵測十一種植物病原菌質體病害與本省所發生之疑似梨衰弱病病株中植物菌質體之效果。所評估之f_1/ r_1、R_(16)F_2/R_(16)R_2、R_(16)F_2/R_(16)rP_1、 R_(16)F_2/ L_1、R_(16)F_2/ rH_3、fD_1/ L_1等六組廣效型聚合酶連鎖反應引子對,各有不同之廣效程度,而聚合酶連鎖反應產物大小分別爲0.63 kb 、1.25 kb、1.35 kb 1.6 kb、1.7 kb及1.7 kb。另以十三種細菌菌株全DNA爲模板DNA進行PCR反應,結果引子對f_1/ r_1及R_(16)F_2/R_(16)R_2具最佳之專一性。在疑似梨衰弱病病株植物菌質體之檢測時,此六對引子對均曾在一些梨樹病組織採樣中增幅出與以植物菌質體DNA爲模板時之PCR產物大小相同之DNA片段,但以國外報告之梨樹衰弱病菌質體專一性引子對fPD/rPDS進行測試時,卻無任何産物被增幅出。在以R_(16)F_2/R_(16)R_2進行偵測敏感度試驗時,對疑似梨衰弱病之病株全DNA,其可偵測到100 ng,對絲瓜簇葉病、甘藷簇葉病大陸品系、甘藷簇葉病、翠菊黃化病西岸品系、翠菊黃化病紐澤西品系、花生簇葉病及楡樹黃化病之罹病日日春全DNA,依序分別可偵測到100ng 、100ng 丶 3.1 ng 、3.1ng、50 ng、6.25 ng及12.5 ng;而在f_1/ r_1的偵測敏感度方面,對於疑似梨衰弱病之罹病梨樹全DNA可測到6.25 ng,對絲瓜簇葉病、甘藷簇葉病大陸品系、甘藷簇葉病、翠菊黃化病西岸品系、翠菊黃化病紐澤西品系、花生簇葉病及楡樹黃化病之罹病日日春全DNA則分別可測到4 ng , 1.5 ng 、12.5 ng 、25 ng 、0.16 ng 、6.25 ng 及0.8 ng,而對甘蔗白葉病罹病甘蔗與水稻黃萎病罹病水稻之全DNA之偵測敏感度則分別爲 0.8 ng及32 pg。

並列摘要


six universal PCR primer pairs, f_1/ r_1, R_(16)F_2/ R_(16)R_2, R_(16)F_2/ R_(16)rP_1, R_(16)F_2/L_1, R_(16)F_2/ rH_3 and fD_1/ L_1 for the detection of phytoplasmas were evaluated in this study. All the primer pairs tested can amplify the phytoplasma-specific PCR products of 0.63 kb, 1.25 kb. 1.35 kb. 1.6 kb, 1,7 kb. and 1.7 kb in length from phytoplasma infected plants. respectively. Besides total DNA of 13 bacteria species were also used to evaluated the specificity of these six universal primer pairs applied in the PCR amplification. Among these, primer pairs f_1/ r_1, and R_(16)F_2/ R_(16)R_2 have the best specificity. PCR products of the size similar to that of phytoplasma specific fragment had ever been amplified with all of the six universal PCR primer pairs using DNA templates prepared from pear-decline-like pear tree in Taiwan. On the other hand, the PCR primers specific for pear decline phytolasma, fPD/ rPDS, had never amplified any fragment from diseased pear tissue collected. The DNA templates prepared from diseased pear trees that gave PCR products were collected in October and December in 1994 and March August and October in 1995. The sensitivity for primer pair R_(16)F_2/R_(16)R_2 to amplify the specific fragment from the total DNA of the pear-decline-like plants is 100 ng, and that for primer pair f_1/r_1 is 6.25 ng

被引用紀錄


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