(RT)-qPCR

(RT-)qPCR reagent kits are available for detecting HLVd, various viruses and fungi, and Russet mites, as well as for sexing seedlings.  RT-qPCR is the only reliable method of detecting HLVd.  Even tiny amounts of viroid RNA can be detected.  Each assay generates an exponential fluorescence curve where the fluorescence is plotted against the number of amplification cycles.  The number of cycles at which the fluorescence crosses a baseline set by the operator (quantification cycles, qC) is usually used to score a sample as positive or negative.  However, results can be false positive or false negative, depending on where the baseline is set.  Positive samples are more reliably scored by looking at the fluorescence curve shapes.  Quantifying HLVd numbers in samples collected in the greenhouse is not practical because the signal strength is affected by the amount of tissue in the sample, any contamination, and the sampling sites.  Usually, several leaf punches or root clippings are combined in each assay, making quantification even less dependable.

We strongly recommend setting up in-house qPCR testing for any larger-scale commercial grow.  The cost of used qPCR equipment has dropped significantly with the declining demand for COVID-19 testing.  In-house testing reduces the cost per sample and turnaround time significantly.  Questionable samples can be re-run immediately.  All steps from sampling to scoring are under the grower’s control.  When compared to the potential losses HLVd causes, in-house qPCR is an excellent investment.