A novel fluorescence-based multiplex real-time PCR assay for rapid and simultaneous detection of leafminers

Anuradha Sooda *1, Disna Gunawardana 1, Lalith Kumarasinghe 1


1 Plant Health & Environment Laboratory, Ministry of Agriculture and Forestry, PO box 2095, Auckland

Liriomyza spp. are leafminer pests associated with imported fresh produce, plants and other commodities. Rapid and precise identification of juvenile individuals of Liriomyza spp. is of crucial importance to enable appropriate biosecurity decisions to be made at the border or post-border. We have developed a multiplex TaqMan real-time PCR assay that can simultaneously detect Liriomyza huidobrensis, L. sativae and L. trifolii in a single test. Species-specific primers and probes were designed against existing genomic sequences within the mitochondrial cytochrome oxidase I gene. The real-time assay was specific for L. trifolii, L. huidobrensis or L. sativae, both in simplex and multiplex formats. Serial dilution results showed reliable amplification at a 10-4 dilution (1 pg of DNA) and generally even at 10-5 dilutions (0.1 pg), which allows the possibility of using only a small amount of tissue for DNA extraction.


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