烟草五种土传病原菌的多重PCR快速检测

  目的  建立一种可同时检测烟草黑胫病菌（Phytophthora parasitica）、青枯病菌（Ralstonia solanacearum）、立枯病菌（Rhizoctonia solani）、根腐病菌（Fusarium oxysporum）和根黑腐病菌（Thielaviopsis basicola）5种烟草重要土传病原菌的五重PCR快速检测方法。  方法  筛选5种病原菌的特异性引物组合，通过不同的引物浓度和退火温度对多重PCR体系进行优化，检测体系的灵敏度，并对土壤和植株样品进行测试，验证其实用性。  结果  根据青枯病菌fliC基因、立枯病菌RPB2基因、根腐病菌COI基因以及根黑腐病菌TEF1基因设计特异性引物，并结合已报道的黑胫病菌parA1基因的特异性引物，成功建立5种烟草土传病害的多重PCR检测方法。反应体系（25 μL）:Sf1/Sr1、Ff1/Fr1、Pf1/Pr1每条引物分别0.3 μL，Rf1/Rr1每条引物各1.8 μL，TBf1/TBr1每条引物各1 μL，2×PCR Mix 12.5 μL，退火温度为58℃，灵敏度可达到100 pg/μL。  结论  本研究建立的多重PCR体系能够同时快速检测烟草黑胫病菌、青枯病菌、立枯病菌、根腐病菌和根黑腐病菌以及田间带菌烟草病株和土壤，为田间烟草土传病害的早期诊断提供依据。 

Rapid detection of five soil-borne pathogens in tobacco by multiplex PCR

  Objective  The aim of this study was to develop a multiplex PCR assay for simultaneous detection of five important soil-borne pathogens in tobacco, including Phytophthora parasitica, Ralstonia solanacearum, Rhizoctonia solani, Fusarium oxysporum and Thielaviopsis basicola.  Methods  Five pairs of specific primers were selected to analyze the influencing factors of five-plex PCR. The primer concentration and annealing temperature were optimized, and the sensitivity of the system was detected. Then the soil and plant samples were tested to verify the utility of the five-plex PCR detection system.  Results  The primers were designed based on the fliC gene of R. solanacearum, the RPB2 gene of R. solani, the COI gene of F. oxysporum and the TEF1 gene of T. basicola. Combined with reported specific primer for parA1 gene of P. parasitica, a multiplex PCR detection methods of five soil-borne pathogens of tobacco were successfully established. The reaction system with total volume of 25 μL contained 0.3 μL of Sf1/Sr1, Ff1/Fr1, Pf1/Pr1, 1.8 μL of RF1/Rr1, 1 μL of TBf1/TBr1 respectively, 2×PCR Mix at 12.5 μL. The annealing temperature was 58℃, and the detection limitation was 100 pg/μL.  Conclusion  The five-plex PCR detection system established in this study can quickly detect P. parasitica, R. solanacearum, R. solani, F. oxysporum, T. basicola in the infected plant and soil, which provides a basis for early diagnosis and effective control of tobacco soil-borne diseases.