番茄细菌性斑点病菌、溃疡病菌、青枯病菌和疮痂病菌的四重PCR检测方法

Quadruple PCR Detection of Pseudomonas syringae pv. tomato,Clavibacter michiganensis subsp. michiganensis,Ralstonia solanacearum and Xanthomonas campestris pv.vesicatoria in Infected Tomato Tissues

针对引起番茄细菌性病害的细菌性斑点病菌(Pseudomonassyringae pv. tomato,Pst)、溃疡病菌(Clavibacter michiganensis subsp. michiganensis,Cmm)、青枯病菌(Ralstonia solanacearum,Rs)以及疮痂病菌(Xanthomonas campestris pv. vesicatoria,Xcv),建立了四重PCR检测技术,为病害的快速、准确诊断提供技术支持。根据gap1基因设计Pst特异性引物BW-F/BW-R,经PCR条件优化,扩增出了375bp的特异性片段。将设计的引物与已报道的3种细菌特异性引物组合,通过设定不同的退火温度、引物浓度、循环次数以及延伸时间,探索影响四重PCR扩增的因素,优化了其反应体系。四重PCR反应体系中的引物对BW-F/BW-R、Fan1/Fan2、RS-1-F/RS-3-R和XCVF/XCVR可分别扩增出细菌性斑点病菌、溃疡病菌、青枯病菌和疮痂病菌长度为375、146、716和517bp的特异性目的片段,反应体系退火温度为57.1℃,4对引物的终浓度分别为0.24、0.16、0.16和0.08μmol·L^(-1),延伸时间45 s,35个循环。该四重PCR反应体系可快速检测田间番茄发病植株中的细菌性斑点病菌、溃疡病菌、青枯病菌和疮痂病菌,灵敏度达到10-1 ng·μL^(-1)。

:In order to establish a rapid and specific detection method for tomato diseases,a quadruple PCR assay was designed for identifing the pathogens including Pseudomonas syringae pv. tomato(Pst), Clavibacter michiganensis subsp. michiganensis(Cmm),Ralstonia solanacearum(Rs)and Xanthomonas campestris pv. vesicatoria(Xcv). The specific primers(BW-F/BW-R)of Pst were designed based on the gap1 gene sequence,which can amplify 375 bp specific fragment by optimizing the PCR conditions. Besides,three pairs of specific primers of Cmm(Fan1/Fan2),Rs(RS-1-F/RS-3-R),and Xcv(XCVF/XCVR)used in this study were referenced previous studies. The primer concentration,the annealing temperature,amplification cycles,and the extension time were optimized to obtain the best ratio of primers and amplification conditions. Then,the rapid quadruple PCR detection of pathogens in tomato was established. The annealing temperature was 57.1 ℃,the extension time was 45 s and the number of cycles was 35. The final concentrations of BW-F/BW-R primers,Fan1/Fan2 primers,RS-1-F/RS-3-R primers and XCVF/XCVR primers were 0.24,0.16,0.16 and 0.08 μmol · L^-1 ,respectively. The expected sizes of amplification bands were 375,146,716 and 517 bp for BW-F/BW-R,Fan1/Fan2,RS-1-F/RS-3-R and XCVF/XCVR,respectively. The quadruple PCR can simultaneously detect Pst,Cmm,Rs and Xcv in infected plants tissue,with the detection limits of 0.1 ng · μL^-1 gDNA. This study indicated that quadruple PCR might be a useful tool for rapid and sensitive diagnosis and provided an effective technical support for pathogenic identification.