A rapid diagnostic test and mobile "lab in a suitcase" platform for detecting Ceratocystis spp. responsible for Rapid ‘Ōhi‘a Death

Abstract

We describe a field compatible molecular diagnostic test for two new species of Ceratocystis that infect `ōhi`a (Metrosideros polymorpha) and cause the disease commonly known as Rapid `Ōhi`a Death. The diagnostic is based on amplification of a DNA locus within the internal transcribed spacer region that separates fungal 5.8S ribosomal genes. The assay uses forward and reverse primers, recombinase polymerase, and a fluorescent probe that allows isothermal (40oC) amplification and simultaneous quantification of a 115 base pair product with a battery operated fluorometer. DNA extractions are field compatible and can be done by heating wood drill shavings to 100oC in Instagene® solution containing Chelex® resin to bind potential amplification inhibitors. The initial heat treatment is followed by a short bead beating step with steel ball bearings and zirconium beads to release DNA. DNA is subsequently purified with a magnetic bead based extraction method that does not require silica columns or centrifugation. The assay is designed around a portable “lab-in-a-suitcase” platform that includes a portable fluorometer, miniature centrifuge, and heat block that operate off either 120V AC power sources or a 12 volt battery with a portable inverter, a magnetic rack designed for 1.5 ml tubes and magnetic bead DNA purification, pipettes and consumable reagents and tubes. The entire assay from DNA extraction to results can be performed in less than 90 minutes on up to six independent samples plus a positive and negative control. Sensitivity based on suspensions of Ceratocystis endoconidia (spores) that were added to wood shavings and processed under field conditions by Instagene® magnetic bead DNA extraction was up to 163 spores/mg wood for Species A and 55 spores/mg wood for Species B in 95% of replicates as determined by probit analysis. Sensitivity increased 5–10 fold to 19 spores/mg wood for Species A and 9 spores/mg wood for Species B when extractions were performed with a commercial, silica column based DNA purification kit. The test did not cross react with other common fungi that have been isolated from `ōhi`a.