EMA-qPCR方法快速检测番茄溃疡病菌活菌研究  

Study on Rapid Detecting of Live Clavibacter michiganensis subsp. michiganensis by EMA-q PCR

作　　者：周大祥[1,2] 熊书[3] 

机构地区：[1]重庆三峡学院生命科学与工程学院,重庆404100 [2]重庆大学生命科学学院,重庆400300 [3]重庆三峡医药高等专科学校基础医学部,重庆404120

出　　处：《西南农业学报》2017年第1期99-104,共6页Southwest China Journal of Agricultural Sciences

基　　金：重庆市自然科学基金(cstc2016jcyj A2132);重庆市教委科学技术研究项目(KJ1502601);重庆三峡医药高等专科学校科研苗圃工程项目(2014mpxz4)

摘　　要：将叠氮溴乙锭(EMA)与实时荧光定量PCR技术相结合(EMA-q PCR),建立一种有效快速检测番茄溃疡病活菌的方法。以番茄溃疡病菌Pat-1基因为检测靶标,菌体经EMA渗透处理,再进行q PCR特异性扩增。结果显示,q PCR检测灵敏度为1.0×101CFU/m L;当EMA的浓度为2.0μg/m L时,能有效抑制1.0×107CFU/m L灭活死菌的扩增,对活菌的扩增没有影响。当活菌数在1.0×101~1.0×105CFU内,每个q PCR反应体系中活菌CFU数与Ct值呈线性相关(R2=0.987)。不同温度处理后EMA-q PCR检测番茄溃疡病菌的存活情况并与平板计数法进行比较,表明待检样品可在4和20℃短期保存。对疑似带病番茄种子样品进行EMA-q PCR检测,发现EMA-q PCR方法能减少番茄溃疡病菌PCR检测的假阳性结果。本研究建立的EMA-q PCR方法是一种能有效检测番茄溃疡病活菌的方法,并能有效避免PCR检测实际样品可能造成的假阳性结果。A novel method to rapidly detect only viable ceils of Clavibacter michiganensis subsp, michiganensis（CMM） was established by using the EMA-qPCR. The Pat-1 was used as the target gene for qPCR detection of CMM. Samples were treated with EMA prior to DNA extraction. DNA was then amplified by qPCR to detect only viable CMM ceils. The sensitivity of qPCR detection was 1.0 ×10t CFU/mL. 2. 0 μg/mL EMA could completely inhibit the PCR amplification of DNA derived from dead cells with the concentration of 1.0 × 107 CFU/mL, but without inhibition to viable cells. A standard curve was generated relating the number of genomic targets per PCR to the Ct values of the EMA-qPCR. A linear range of DNA amplification was observed from 1.0 ×101 -1.0 ×105 genomic targets per PCR. EMA-qPCR method was used to evaluate the survival rate of CMM treated with different temperatures for a short time, compared with the method of plate count. The results indicate that samples can be stored for a short time under 4 and 20 ℃The data of EMA-qPCR detection on tomato seed samples indicated that 4.0μg/mL EMA could successfully inhibit PCR amplification of DNA from dead bacteria in the samples. The EMA-qPCR method established in this work can effectively avoid false positive results of CMM detection.

关 键 词：番茄溃疡病菌 叠氮溴乙锭(Ethidium monoazide bromide EMA) 实时荧光定量PCR(q PCR) 

分 类 号：Q93-33[生物学—微生物学]