Nested-PCR检测种传番茄细菌性溃疡病菌

摘要

利用番茄溃疡病菌特异性引物对纯菌液、模拟带菌种子及自然感染种子提取液分别进行Direct-PCR和Nested-PCR检测.结果在纯菌液中,Direct-PCR的检测灵敏度为105 cfu/mL,Nested-PCR为102 cfu/mL;在模拟带菌种子提取液中,Direct-PCR的检测灵敏度为107 cfu/mL,Nested-PCR为104 cfu/mL；在自然感染种子提取液中,稀释104倍后Nested-PCR仍然可以检出.建立的番茄种子Nested-PCR检测技术,无需经过核酸提取步骤,可以在8h内完成整个检测过程,方便快捷,成本低廉,可作为番茄种子带菌的常规检测方法.%Tomato bacterial canker is the most important bacterial diseases which caused substantial economic losses in the worldwide. Seeds were the main pathway of long-distance spread and seed-borne Clavibacter michiganensis subsp. Michiganensis (Cmm) were difficult to detect by current methods because of they were low rate of infected. Direct-PCR and Nested-PCR were performed for the detection of Cmm in bacterial suspensions, artificial contaminated seed extracts and naturally infected seed extracts, respectively. The detection sensitivity of Direct-PCR is 105 cfu/mL and 107 cfu/mL in bacterial suspensions and artificial contaminated seed extracts, respectively. But the sensitivity of Nested-PCR was 102 cfu/mL and 104 cfu/mL respectively. And Nested-PCR could provide positive result in 104 fold dilution in naturally infected seed extracts. The Nested-PCR developed in our study was convenient and low-cost, which could be used a routine method for the detection of tomato bacterial canker.