台灣十字花科黑腐病菌之酵素連結抗體檢側法

Identification and Detection of Xanthomonas Campestris pv. Campestris in Taiwan byEnzyme-Linked Immunosorbent Assay

黃㯖昌(Tze-Chung Huang)

《臺東區農業改良場研究彙報》 2輯 (1988/06) Pp. 107-125

https://doi.org/10.6959/RBTDAIS.198806.0107

摘要

以黑腐病菌（Xanthomonas campestris pv. campestris）XC38菌株之抗血清為材料，比較雙層夾心式及間接式酵素連結抗體檢測法檢測黑腐病菌之專一性與靈敏度，顯示二者靈敏度相近，但以雙層夾心式酵連抗體法之專一性較高，供試之31株其他病原細菌及腐生菌中，僅X. campestris pv. vesicatoria出現強干擾反應，但供試之62個黑腐病菌株中，有3個呈現負反應。抗原細菌以添加0.1% Tween 80及0.125% glutaraldehyde之磷酸緩衝生理食鹽水當作萃取液萃取後，在雙層夾心式酵連抗體法中反應最強，而背景呈色亦低，偵測同源抗原細菌之靈敏度可達3.3 × 10^4 CFU/ml，肉眼可判別之反應則約在1.3×10^5～2.6×10^5之間，此一方法同時亦適用於病組織中細菌之檢測。以雙層夾心式酵連抗體法偵測種帶或土帶黑腐病菌，一般而言，靈敏度不及以選擇性培養基SMA之直接分離法，但其反應不受雜菌多寡之影響，對大量或污染嚴重之種子樣品，仍不失為可行之病菌偵測法，同時亦可輔助培養基直接分離法確認可疑之菌落，也可應用於田間作物或雜草可疑病徵之快速鑑定。

關鍵字

十字花科黑腐病菌 ； 酵素連結抗體驗測法

並列摘要

Double sandwich (DAS), and indirect (ID) ELISA were compared for the assay of Xanthomonas campestris pv. campestris (abbreviated to Xcc) by using an antiserum against strain XC38 of Xcc Sensitivities of DAS-ELISA and ID-ELISA were similar, but DAS-ELISA was more specific than ID-ELISA. ”When 32 strains of bacteria other than Xcc were tested in DAS-ELISA, only X. campestris pv. vesicatoria reacted positively, however, 3 out of 62 Xcc strains tested, then failed to react with anti-XC38 r-globulin in DAS-ELISA. An extraction buffer consisting of 0.01 M-phosphate buffer (pH 7.2), 0.1% Tween 80 and 0.125% glutaraldehyde for antigens was found that it increased specific reaction and decreased nonspecific background. Using this procedure, the sensitivity of DAS-ELISA was at 3.3×10^4 CFU/ml for pure culture of Xcc when monitored photometrically and was at 1.3×10^5-2.6×10^5 CFU/ml when determined visibly. This procedure was also suitable for the detection of Xcc in diseased leaf tissues. Generally, the sensitivity of DAS-ELISA was laver than that of direct isolation with selective medium, but the reaction in DAS-ELISA seldom intefered by other microorganisms existing in samples. It may be used for assay of large-scale seed samples, confirmation of suspected colonies on selective medium, or rapid identification of suspected diseases on cruciferous crops or weeds.