PCR法快速检测柑桔溃疡病菌的简易制样技术

A RAPID TECHNIQUE FOR SAMPLE PREPARATION FOR XANTHOMONAS AXONOPODIS PV. CITRI DETECTION WITH PCR

采用依据柑桔溃疡病菌独有蛋白基因的保守区段设计的特异性引物及其扩增体系进行测试。4种浸泡液中,浸泡液B的浸提效果最好,最佳浸泡时间为10～15min。浸提液进行加热灭活与不加热灭活处理,PCR扩增结果无明显差异。对于无症柑桔样品,浸泡液中加入终浓度为0.3%的Na2SO3可以降低植物色素、多元酚和铜制剂残留的Cu2+等抑制PCR反应物质的干扰作用。样品浸提液经过离心处理,不仅可以有效浓缩靶细菌,提高检测灵敏度,而且可以除去PCR反应抑制物质,大大降低了检测的假阴性。

A pair of specific primers designed from published nucleotide sequences encoding a conserved hypothetical protein in Xanthomonas axonopodis pv. citri (Xac) genome and its amplification was employed. Of the 4 soaking liquids used, soaking liquid B gave the best effect in extraction and the best soaking time was 10 to 15 minutes at room temperature. No obvious difference was noticed in the results of PCR amplification whether the extract liquid was heat-deactivated or not. Presence of PCR reaction-inhibiting compounds in plant tissues such as pigments and polyphenols was one of the major problems. Addition of sodium sulphite to the soaking liquid to a final concentration of 0.3% reduced the interference by those compounds. For symptomless citrus materials, centrifugation of the extract liquids effectively increased the concentration of the target bacteria and substantially eliminated the inhibiting compounds in the plant tissues.