Triplex real-time PCR detection of three quarantine Phytophthora pathogens infecting Malus Miller
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In order to develop a simultaneous and specific molecular detection of the three quarantine Phytophthora pathogens, Phytophthora hibernalis, Phytophthora cambivora and Phytophthora syringae that infect Malus Miller, three pairs of real-time PCR primers (PH-F/PH-R, PC-F/PC-R and PS-F/PS-R) and three probes (PH-Pr, PC-Pr and PS-Pr) labeled with HEX, FAM and ROX, respectively, were designed for P. hibernalis, P. cambivora and P. syringae by alignment analyses of enolase (Enol), ras-like protein Ypt1 and HSP90 gene sequences with other Phytophthora spp. Black Hole Quencher 1 (BHQ1) was used for P. hibernalis and P. cambivora and BHQ2 for P. syringae. Through the optimization of the reaction conditions, a triplex real-time PCR simultaneous detection for the three Phytophthora species infecting Malus Miller was developed. It could achieve simultaneous and specific detection with a sensitivity of 2 × 10−4, 2 × 10−4 and 2 × 10−2 ng/μL genomic DNA, respectively, for P. hibernalis, P. cambivora and P. syringae.
KeywordsMalus Miller Quarantine Phytophthora pathogens P. hibernalis P. cambivora P. syringae Triplex real-time PCR detection
This work was funded by Rapid Identification of Quarantine Fungal Phytophthora Pathogens of Imported Fruits and Seedlings, a Program from The General Administration of Quality Supervision, Inspection and Quarantine of PRC (No: 2012IK286) and Plant Quarantine Pathogen Detection and Application by DNA Barcoding, National “12th Five-Year” Science and Technology Support Program (No: 2012BAK11B02).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interests.
This research did not involve any animal and/or human participants.
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