Development of a hybridisation-based detection assay for PVdI

The main objective of this study was to develop a robust hybridization assay for the detection of PlVd-I to reduce the per tree cost and allow for more extensive screening of plant material.

The RNA transcripts and labelled probes were successfully constructed for the detection of PlVd-I. Initial evaluations indicated that the colorigenic detection strategy will most likely not be sensitive enough for a routine assay and we subsequently converted to chemiluminescent detection exclusively. However after numerous optimization attempts, the hybridisation assay sensitivity was not high enough to compare to RT-PCR effectivity to detect PlVd-I. An alternative strategy was selected and a LAMP assay was designed and tested on PlVd-I infected material.

Probes transcribed from the constructed plasmids were evaluated for their ability to detect control pDNA constructs and RNA transcripts. These probes performed with similar sensitivity. Probes were able to detect controls to single digit picogram levels but were unable to detect the viroid in RNA extracts or crude plant extracts. Additional optimisation made no difference in sensitivity. The alternative LAMP assay was able to detect PlVd-I from crude plant extracts eliminating the need for an RNA extraction and reducing the associated cost and labour of the assay significantly.

The development of a hybridization assay to reduce the cost of per tree testing is an essential part of the marbling management strategy. All efforts were made to improve the efficiency of the hybridisation assay, however only an alternative approach yielded encouraging results. This project delivered a LAMP assay that is able to detect PlVd-I in crude extracts. Additional validations are required for the assay to be rolled out to plant improvement organisations.

A LAMP assay for the detection of PlVd-I from crude plant extracts was designed. This assay needs additional validations, but has the potential to be cost effective for the screening of a large number of samples.