三重PCR检测黄瓜靶斑病菌、炭疽病菌和细菌性角斑病菌

Triplex PCR Detection for Corynespora cassiicola, Colletotrichum orbiculare and Pseudomonas syringae pv. lachrymans

【目的】开发一种可同时检测黄瓜靶斑病菌(Corynespora cassiicola)、黄瓜炭疽病菌(Colletotrichum orbiculare)和黄瓜细菌性角斑病菌(Pseudomonas syringae pv.lachrymans)的三重PCR快速检测方法。【方法】根据ITS或16S r DNA序列分别设计黄瓜靶斑病原菌、黄瓜炭疽病菌和黄瓜细菌性角斑病菌特异性检测引物;通过鉴定引物的特异性,筛选可在目标菌株中扩增出特异片段的特异引物;选择可以组合的3种病原菌特异性引物进行三重PCR,对引物浓度、退火温度、延伸时间和循环数分别进行优化,优化三重PCR体系。【结果】针对黄瓜靶斑病菌、黄瓜炭疽病菌和黄瓜细菌性角斑病菌分别设计出5对、7对和6对特异性引物,其中引物CC4F/CC4R和CC5F/CC5R可特异扩增黄瓜靶斑病菌ITS,引物CL1F/CL1R、CL2F/CL2R、CL3F/CL3R、CL3F/CL4R、CL3F/CL5R、CL3F/CL6R和CL3F/CL7R可特异扩增黄瓜炭疽病菌ITS,引物PS3F/PS4R和PS4F/PS4R可特异扩增黄瓜细菌性角斑病菌16S rDNA。引物CC5F/CC5R、CL3F/5R和PS3F/4R扩增片段长度分别为370、275和698 bp,其混合扩增产物可在3%琼脂糖凝胶上得到充分分离,这3对引物选择作为三重PCR引物。在三重PCR反应体系中,当引物CC5F/CC5R、CL3F/5R和PS3F/4R的终浓度分别为0.16、0.4和0.16μmol·L^(-1)时,3个目的片段能同时得到有效扩增;当退火温度大于65℃时,部分目的片段不能有效扩增。最终建立并验证了适合上述3种黄瓜主要病原菌的三重PCR检测体系,即25μL PCR反应体系中含有12.5μL 2×Hiff^(TM) PCR Master Mix(With Dye)、0.16μmol·L^(-1) CC5F/CC5R、0.4μmol·L^(-1) CL3F/CL5R、0.16μmol·L^(-1) PS3F/PS4R。反应程序:95℃预变性3 min;95℃变性30 s,65℃退火30s,72℃延伸2 min,35个循环;最后72℃延伸10 min。【结论】建立的三重PCR方法能够快速检测田间采集的黄瓜发病叶片中的黄瓜靶斑病菌、黄瓜炭疽病菌和黄瓜细菌性角斑病菌,灵敏度均可达到0.4 pg·μL^(-1)。

【Objective】The objective of this study is to develop a rapid triplex PCR detection method of Corynespora cassiicola, Colletotrichum orbiculare and Pseudomonas syringae pv. lachrymans. 【Method】 Specific primers for the three pathogens were designed based on ITS sequences of C. cassiicola and C. orbiculare and 16 S rDNA sequence of P. syringae pv. lachrymans, which were confirmed by amplifying specific fragments. Three pairs of primers specific to C. cassiicola, C. orbiculare or P. syringae pv. lachrymans were suitable for triplex PCR and selected for triplex PCR. Primer concentration, annealing temperature, extension time and cycle number were optimized to develop the triplex PCR detection system. 【Result】Five, seven and six pairs of primers were designed specific to C. cassiicola, C. orbiculare and P. syringae pv. lachrymans, respectively. CC4F/CC4 R and CC5F/CC5 R primers could specifically amplify ITS sequence of C. cassiicola; CL1F/CL1 R, CL2F/CL2 R, CL3F/CL3 R, CL3F/CL4 R, CL3F/CL5 R, CL3F/CL6 R and CL3F/CL7 R could specifically amplify ITS sequence of C. orbiculare; PS3F/PS4 R and PS4F/PS4 R primers could specifically amplify 16 S r DNA sequence of P. syringae pv. lachrymans. Fragments in lengths of 370, 275 and 698 bp were amplified by CC5F/CC5 R, CL3F/5R and PS3F/4R, respectively, which were separated fully by 3% agarose gel electrophoresis, and the three pairs of primers were used for triplex PCR. Three target fragments were amplified effectively with 0.16 μmol·L^-1 of CC5F/CC5 R, 0.4 μmol·L^-1 of CL3F/5R and 0.16 μmol·L^-1 of PS3F/4R in 25 μL of triplex PCR system. Part target fragments were not amplified effectively at 65℃ of annealing temperature. Finally, the triplex PCR system suitable for the detection of C. cassiicola, C. orbiculare and P. syringae pv. lachrymans were developed and confirmed. This PCR system contained 12.5 μL of 2×Hiff^TM PCR Master Mix（With Dye）, 0.16 μmol·L^-1 of CC5F/CC5 R, 0.4 μmol·L^-1 of CL3F/CL5 R and 0.16 μmol·L^-1 of PS3F/PS4 R. The PCR program was as follows： an initial denaturation at 95℃ for 3 min followed by 35 cycles of denaturation（95℃for 30 s）, annealing（65℃ for 30 s） and extension（72℃ for 2 min）, and a final extension at 72℃ for 10 min. 【Conclusion】The detection method could rapidly detect C. cassiicola, C. orbiculare and P. syringae pv. lachrymans from leaves infected by the three pathogens, the sensitivity of which was 0.4 pg·μL^-1.