三重PCR检测黄瓜靶斑病菌、炭疽病菌和细菌性角斑病菌

doi:10.3864/j.issn.0578-1752.2016.16.006

Abstract

Abstract: 【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 andC. orbiculare and 16S 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/CC4R and CC5F/CC5R primers could specifically amplify ITS sequence of C. cassiicola; CL1F/CL1R, CL2F/CL2R, CL3F/CL3R, CL3F/CL4R, CL3F/CL5R, CL3F/CL6R and CL3F/CL7R could specifically amplify ITS sequence of C. orbiculare; PS3F/PS4R and PS4F/PS4R primers could specifically amplify 16S rDNA sequence of P. syringae pv. lachrymans. Fragments in lengths of 370, 275 and 698 bp were amplified by CC5F/CC5R, 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/CC5R, 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/CC5R, 0.4 μmol·L^-1 of CL3F/CL5R and 0.16 μmol·L^-1 of PS3F/PS4R. 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.

Key words: Corynespora cassiicola;Colletotrichum orbiculare; Pseudomonas syringae pv. lachrymans, molecular detection, triplex PCR

GAO Shi-gang, ZENG Rong, XU Li-hui, LUO Jin-yan, CHEN Lei, DAI Fu-ming. Triplex PCR Detection for Corynespora cassiicola,Colletotrichum orbiculare and Pseudomonas syringae pv. lachrymans[J].Scientia Agricultura Sinica, 2016, 49(16): 3119-3129.

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