PCR、DIA与致病性测定法检测柑桔溃疡病菌的比较

Comparison of PCR,DIA and Pathogenicity Assay for Detection of Xanthomonas axonopodis pv. citri,the Causal Agent of Citrus Bacterial Canker Disease

依据柑桔溃疡病菌全基因组序列的独有保守区域设计筛选出的特异性引物对JYF5/JYR5,用于柑桔溃疡病菌的PCR检测,具有很好的检测特异性、灵敏度和稳定性。同时比较了PCR与DIA(斑点免疫结合技术)及传统的致病性测定法在检测灵敏度、稳定性及田间样品检出率等方面的差异。结果表明,PCR的检测灵敏度可达103～104cfu·ml-1(每个反应体积约10个细菌),明显高于DIA(104～105cfu·ml-1,每个样点约300个细菌);PCR、DIA和致病性测定法检测田间显症样品的检出率均可达到100%,而检测柑桔无症样品的检出率依次降低。此外,通过排除PCR抑制物质和在PCR反应体系中加入终浓度为15%的甘油,有效地降低了检测中存在的假阴性。

Polymerase chain reaction (PCR) and newly designed primers, JYF5/JYR5, were tested for selective detection of Xanthomonas axonopodis pv. citri (Xac). The efficiency and reliability of PCR detection method were compared with dot immunobinding assay (DIA) and classical pathogenicity test techniques for detecting Xac in suspensions of pure cells and extracts of citrus samples. Detection limit of PCR was 10 cells or 1.56pg of target DNA per reaction, the sensitivity of PCR assay is higher than DIA (300 cells or more per sample dot). The detection rate of the three techniques (Pathogenicity test, DIA and PCR assay) for Xac reach 100% for symptomatic citrus sample. Differences among the three techniques were clearly evident when using citrus tissues without symptoms, the detection frequency from the least to the most was pathogenicity test, DIA and PCR. Add 15% glycerol in PCR reaction mix can reduce the effect of inhibiting compounds in sample and avoid false negative of PCR.