PDplus™ Technical specifications 

PDplus™ is a brand new service for the molecular characterisation of a target pathogen identified using a Pocket Diagnostic™ lateral flow device (LFD).  The licensed technique for collecting DNA using an LFD, followed by real-time PCR, means precise diagnosis of pathogens is now possible and more cost-effective than ever before.  The first target pathogen for PDplus™ is Phytophthora.

Phytophthora diagnosis
PDplus™ is limited initially to the confirmation of five plant pathogenic species of Phytophthora from a wide range of host plants:

P. cinnamomi

P. ramorum
P. kernoviae
P. fragariae
P. cactorum

Host species from which Phytophthora has been detected and diagnosed using PDplus™ include:

Banksia

Rhododendron
Camellia
Viburnum
Strawberry
Calluna

How PDplus™ works
PDplus™ is a two stage process:
1. You carry out a Pocket Diagnostic™ LFD test to confirm a positive result for the target pathogen, then send the LFD to Arborcura
2. The DNA is analysed from the positive LFD to confirm the exact identity of the target pathogen.  Results are sent to you within 1 week of Arborcura receiving the LFD.

Stage 1 – the lateral flow device test
PDplus™ uses the highly-regarded Phytophthora Pocket Diagnostic™ test kit as the method of collecting and cleaning DNA from the sample.  The Pocket Diagnostic Phytophthora LFD is used widely by the industry for the detection of the pathogen.  The monoclonal antibody at the core of the LFD recognises a mycelial protein found in all species of Phytophthora, and hence gives a very effective method of screening plant samples for the presence of the pathogen.

Stage 2 – the PCR analysis
An ultra-sensitive molecular detection method known as real-time PCR (polymerase chain reaction) is used to confirm the identity of the target pathogen on the LFD.  A range of pathogen specific tests is available, as mentioned above.  In order to rule out false negative results, internal control tests are performed to assess the quality of the DNA.  Two targets are used, one for plant DNA and the other for fungal DNA.  A positive result for one or both of these targets indicates the presence of amplifiable DNA.

Stability of samples
Samples taken from plant material should be run on the LFD test according to the standard instructions as soon as possible after disrupting in the extraction buffer.  Once the LFD test has been run, the bound DNA is stable.  Tests have shown that storage at room temperature in dry conditions for several months has no affect on the detection of pathogen DNA in subsequent PCR analysis. 

Protected technology
Arborcura has an exclusive license to provide the PDplus™ service in Australasia.   The service is available to customers worldwide, apart from those in the UK and USA.

Validation
Validation of the technique has been carried out alongside conventional laboratory methods of Phytophthora identification.  Samples of plant material from the field were diagnosed using current practice, standard laboratory PCR methods and a combination of LFD and TaqMan PCR.
Of 114 samples tested, 97% showed agreement on Phytophthora speciation between the standard laboratory PCR and the LFD/PCR method. 

Limitations
PDplus™ analysis is not quantitative.  PDplus™ cannot identify species of Phytophthora other than those listed above.  For best results, a good clean LFD result is required, although successful diagnosis has been carried out on LFDs where high levels of contaminants have been present in the sample, for example, soil and compost.  PDplus has been validated extensively with foliage and 'soft' samples.  Use of the technique with samples of bark and wood is being validated.

As with any diagnostic technique, it is advisable to obtain confirmatory results from another diagnostic method before making decisions. If P. cinnamomi, ramorum or kernoviae are identified in the sample, the customer is obliged to inform their local relevant plant health authorities.