免疫捕捉PCR和经典PCR方法检测水稻细菌性谷枯病菌灵敏性比较

Sensitivity comparison of the ISE method and classical PCR to detect Burkholderia glumae

将免疫吸附富集(ISE)和经典PCR结合以提高水稻细菌性谷枯病的检测效率,并且利用多克隆抗体技术成功地获得了4种抗血清,即抗Burkholderia glumae全菌体血清ASBg14,ASBg32,ASBg26和ASBg04,ODD法测定该4种抗血清的效价均达到1∶32以上,利用凝聚法测定的所有抗血清的效价也均达到了1∶5 120以上,通过两种方法结合测定效价均显示为合格抗体。通过对4种抗血清的专化性进行研究,结果显示ASBg14和ASBg32专化性较高,抗血清ASBg26和ASBg04专化性不是很理想。用直接PCR技术和免疫捕捉PCR技术检测谷枯病菌,结果表明,所有谷枯病菌都能产生500 bp左右的特异性片段,非谷枯病菌的8个菌株均无特异性片段产生。两种检测方法的灵敏度比较发现,直接PCR技术能检测到1×105cfu·mL-1左右的悬浮液,免疫捕捉PCR技术能检测到103cfu·mL-1左右悬浮液,免疫捕捉PCR比直接PCR灵敏性提高102倍。检测人工接种的病稻种实验中,直接PCR技术能检测到2粒及以上带菌种子制得浸悬液,而免疫捕捉PCR技术仅1粒带菌种子即可。

The present study combined the fluoride ion-selective electrode （ ISE） method with classical PCR to increase the detection efficiency.The 4 antisera of rabbit anti whole cell of B.glumae have been successfully obtained by common serological method , which were ASBg14, ASBg32, ASBg26 and ASBg04.The antibody of 4 antisera could be effective when diluted by 5 120 times and reached 1∶32 by ODD test.The specificity of antibody of ASBg14 and ASBg32 was very high; however, the specificity of ASBg26 and ASBg04 was not very satisfying.The results showed that all the strains of B.glumae tested produced 500 bp specific fragments by the ISE and the direct PCR method, while others showed negative PCR result .Comparing the sensitivity of these 2 detecting methods, about 1 ＆#215;105 cfu·mL-1 of the bacterial suspension were detected by direct PCR and below 1 ×103 cfu·mL-1 can only be detected by immunocapture PCR method .In the detecting experiments of the artificial inoculated seeds , at least 2 seeds should be required by direct PCR;however, only 1 seed was enough for detection by immunocapture PCR .