摘要
传统PCR方法不能诊断柑橘溃疡病菌(Xanthomonas citri subsp.citri Gabriel)的死活状态,往往导致假阳性检测结果。本研究将特异性核酸染料叠氮溴化乙锭(ethidium monoazide bromide,EMA)与PCR技术结合,旨在建立柑橘溃疡病活菌的快速检测技术。根据柑橘溃疡病菌独有的保守蛋白基因设计特异性引物扩增出278bp的靶带,PCR反应的检测下限为25个细胞/25μL或2.75pg/25μL。EMA-PCR结果表明:当卤钨灯曝光时间1min,EMA终浓度为1.0mg/L时,能有效抑制1.0×108 cfu/mL死菌的扩增;当EMA的浓度小于30mg/L时,EMA对上述相同浓度活菌靶基因的扩增没有明显的抑制。EMA-PCR对死活混合菌的扩增表明,活菌数在6.875×101～6.875×105 cfu/PCR范围时,荧光强度与混合体系中活菌的对数值有线性关系。基于以上建立的EMA-PCR活体检测技术,对疑似带病柑橘材料进行检测,结果发现能降低柑橘溃疡病菌检测过程中的假阳性,有望为柑橘溃疡病的检疫检验提供更科学的技术手段。
Conventional PCR method can not distinguish live cells from dead cells of Xanthomonas citri subsp, citri Ga briel, and false positives results can easily be obtained in detection process. Ethidium monoazide bromide （EMA） can se lectively inhibit PCR amplification of DNA from dead cells. Therefore, in this study, EMA was combined with PCR to detect viable cells of X. cirri subsp, cirri. Firstly, primers （Xcc R/ Xcc F） specific to X. cirri subsp, cirri were designed to amplify a 278 bp fragment. The minimum limit of detection was 25 cells/25μL PCR volume or 2.75 pg/25 μL PCR volume. The results of EMA-PCR showed that the optimized light exposure time was at least 1 min, allowing cross- linking of DNA by the EMA in dead cells and photolysing the free EMA in solution. The minimum amount of EMA to completely inhibit the PCR amplification of DNA derived from heat-killed cells was 1.0 mg/L. EMA less than 30 mg/L did not inhibit the PCR amplification of DNA derived from viable cells of X. citri subsp, cirri. A linear relationship was found between the average fluorescent intensity of the DNA bands and the logarithmic val- ue of genomic targets derived from the viable cells in mixtures of viable and dead cells in the range of 6. 875 X 10^1 --6. 875 X l0^5 cfu/PCR. The data of EMA-PCR detection on citrus field samples indicated that 2 mg/L EMA could successfully inhibit PCR amplification of DNA from dead bacteria in filed samples, suggesting a promising and ac curate method to prevent false positive results in detection of X. cirri subsp, citri.
出处
《植物保护》
CAS
CSCD
北大核心
2013年第4期78-84,共7页
Plant Protection
基金
公益性行业(农业)科研专项(201003067)
中央高校基本科研业务费(CDJXS112300)
关键词
柑橘溃疡病菌
EMA-PCR
死菌与活菌
检疫
Xanthomonas citri subsp, citri ~ EMA-PCR~ viable and dead bacteria~ quarantine
