应用斑点酶联法检测柑桔溃疡病菌

DETECTION OF Xanthomonas campestris pv.citri BY DOT-ELISA

用浓度为１０８ｃｆｕ／ｍｌＸ．ｃ．ｐｖ．ｃｉｔｒｉ全胞活菌免疫家兔，得到效价为１／２５６０～１／１０２４０的抗血清。应用斑点酶联法（Ｄｏｔ－ＥＬＩＳＡ）检测Ｘ．ｃ．ｐｖ．ｃｉｔｒｉ表明，抗血清经交叉吸收后特异性高，适宜稀释倍数为１∶１０４～１０６，检测灵敏度高可达１０４ｃｆｕ／ｍｌ。加有０．５％Ｔｗｅｅｎ２０或０．５％Ｔｗｅｅｎ８０或０．１％ｔｒｉｔｏｎＸ－１００的ＴＢＳ液为适宜封闭液，酶标抗体以ＨＲＰ－ＳＰＡ较好。在检测无症样品时，与使用酶联板的ＩＭ－ＥＬＩＳＡ、ＤＡＳ－ＥＬＩＳＡ和ＳＰＡ－ＥＬＩＳＡ比较，Ｄｏｔ－ＥＬＩＳＡ快速简便，整个过程只需６小时，结果可靠。

Dot-ELISA was used for the detection of Xanthomonas campestris pv.citri isolates.The antisera with a agglutinative titer of 1/2560 —1/10240 were prepared in rabbits against cultures of X.c.pv. citri isolates.The sensitivity of Dot-ELISA was 10 4 cfu/ml.The antibodies cross-absorbed with other bacteria reacted positively with the 26 isolates of X.c.pv.citri at dilution of 1:10 4-10 6,but negatively with other bacteria.A nitrocellulose membrane with 0 33μm aperture was used as the solid phase and good results were obtained.The use of HRP-SPA resulted in no background color and less nospecific reactions than in the case of using HRP-IgG.The results of detecting 171 symptomless citrus tissue samples by Dot-,SPA-,DAS-and IM-ELISA showed no difference from those obtained from using semiselective medium isolations and pathogenicity tests.However,Dot-ELISA method was even more handy,quicker and more economical for detecting X.c.pv.citri.