SI3 & SI4 REPORT: Lateral flow ImmunoStrips ...

Efficacy of commercially available Phytophthora (sp) lateral flow ImmunoStrips in the sensitive detection of Phytophthora agathidicida

June 2024

Wood M, Pushparajah S, Milner D. 2024. Efficacy of commercially available Phytophthora (sp) lateral flow ImmunoStrips in the sensitive detection of Phytophthora agathidicida. A report for New Zealand’s Biological Heritage National Science Challenge: Ngā Rākau Taketake. 16 p.

NOTE

This report is embargoed until 31/05/2025. For further information please contact Marion Wood: Marion.Wood@plantandfood.co.nz

ABSTRACT

Background:

The interest in, and uptake of readily available, commercially attractive lateral flow devices (ImmunoStrips) has recently undergone a significant phase-change due to their extensive use associated with the Covid-19 pandemic. The use of similar ImmunoStrips designed to specifically detect plant pathogens is also currently feasible due the availability of similar, commercially convenient devices. However, such plant pathogen directed devices are prone to issues with sensitivity and the possibility of delivering false positive and negative results. This research has set out to evaluate the commercially available Phytophthora spp. lateral flow device (LFD) (Pocket Diagnostic Abingdon Health PLC) rapid tests and explore their potential when coupled to an additional downstream qPCR-based amplification step to identify Phytophthora agathidicida (PA) quickly and simply in plant and/or soil samples. Such lateral flow devices are routinely used to detect a multitude of pathogens, but their reliability can be severely restricted due to their cross reactivity with other related Phytophthora species or even with non-related pathogens such as Pythium (sp). Using these serological diagnostic strips as means of enriching for Phytophthora in a biological sample, DNA can be purified and eluted from a positive test strip and subsequently analysed specifically for PA using either qPCR (as recently reported by McDougal et al., 2021) or by using digital droplet PCR (ddPCR). This second technique is reported to require less stringent sample preparation and so may lend itself to the laboratory-based diagnostic analyses of soil-based samples.

Project aims:

To address this this research proposal has four main themes: (i) using a positive test strip as the basis for downstream DNA isolation and sequential qPCR analysis to detect Phythophthora agathidicida; (ii) using a positive test strip as the basis for downstream DNA isolation and sequential ddPCR analysis to detect PA (iii) evaluate the ease of use of the LFDs in the field and their potential role for pathogen surveillance both as originally intended as a point of use detection test kit and as an alternative to the need for soil sampling for diagnostic purposes from a mana whenua perspective and (iv) using LFD strips as means of enriching for Phytophthora (sp) in a biological sample, viable pathogens may be cultivated from a positive test strip and subsequently analysed specifically for PA (and other potential co-immobilised pathogens) using either visual characteristics and/or baiting assay and diagnosed using a supplemental molecular assay such as LAMP (as recently reported by Winkworth et al., 2020), qPCR (McDougal et al., 2021) or potentially ddPCR.

Project results:

The use of molecular strategies, such as qPCR and ddPCR to identify specific plant pathogens remains attractive but is limited by their lack of ability to be translated into a robust field-based tool. Overcoming the need for costly transportation of soil samples to laboratories for downstream diagnostic testing remains a desirable goal. This research indicates that the use of Whatman® 1 cm filter paper discs are a cheap, readily available matrix that may have the potential to be used in the field to sequester PA (or other plant pathogens suspended in a soil slurry) for sending to laboratories for downstream testing. This is an attractive alternative and is being evaluated further. ddPCR has been demonstrated to be a robust alternative to the standard qPCR-probe strategy and is relatively easy to use but is reliant on costly hardware. The specificity demonstrated using the ddPCR strategy, without the need for a costly probe, further suggests that ddPCR is a strong contender as an alternative molecular technique for the diagnostic identification of PA, but unlike qPCR it is highly unlikely to be available as a portable tool. Further work to assess the specificity of the assays and their available primer/probe combinations is on-going.

Overcoming the dependency of sending soil samples back to laboratories, in terms of time and cost is highly desirable. Our initial findings suggest that the use of the sample pad of the Agdia Phytophthora ImmunoStrip® provides a useful matrix for sequestering PA, potentially providing significant savings to field users who no longer have to harvest and transport weighty soil samples to the laboratory for downstream molecular validation. However, this cost could be further significantly reduced with the alternative use of Whatman® filter paper discs that are much easier to handle and cheaper than the ImmunoStrips or soil samples themselves. This early-stage research requires additional development to ascertain the false positive / false negative rates and whether the sequestered PA can be also used to plate on selective medium for visual confirmation of the PA pathogen but looks to be promising and warrants further development. Unfortunately, the direct application of the Agdia Phytophthora ImmunoStrip® to detect PA in soil baiting scenarios failed, irrespective of whether the soil sample was previously assessed as being PA positive following plating on selective media and/or molecular validation using qPCR. This strongly suggests that these ImmunoStrips, in their current format, are unsuitable as a robust in-field diagnostic/surveillance tool, and it is not recommended that they are used in this manner for the detection of PA.