SI7 REPORT: High-throughput qPCR for ...

A high-throughput automated qPCR for Phytophthora agathidicida to determine risk of spread by nursery plants

February 2024

Dobbie K, O'Neill R, Simpson S, McLay E, McDougal R. 2024. A high-throughput automated qPCR for Phytophthora agathidicida to determine risk of spread by nursery plants. A report for New Zealand’s Biological Heritage National Science Challenge: Ngā Rākau Taketake. 19 p.

NOTE:

This report is embargoed until 01/03/2025. For further information please contact Kiryn Dobbie at Scion Research: Kirryn.Dobbie@scionresearch.com

ABSTRACT

Phytophthora agathidicida (PA), the causal agent of kauri dieback disease, threatens the long-term survival of kauri (Agathis australis) in Aotearoa. Currently, PA presence can only be confirmed via a soil test, which includes a soil baiting step. The soil baiting protocol is a lengthy, labour-intensive, and costly process. Several factors can affect the accuracy of this test and potentially result in false negative and/or false positive detection. These factors include the presence of other oomycetes, in particular P. cinnamomi, which has been shown to mask the detection of PA. There are several molecular techniques being explored, including a PCR-based technique that can detect PA directly from soil and soil water and qPCR assays for detection of PA and a Loop-Mediated Isothermal Amplification (LAMP) assay that can detect PA from soil baits. These molecular techniques have not yet been assessed for the detection of PA in the presence of another pathogen, such as P. cinnamomi as part of their validation process. Furthermore, automation of PCR-based assays could substantially speed up diagnostics from soil baiting for PA. Slipstream Automation provides both robotic, higher-throughput and improved consistency in DNA extractions and qPCR as well as being an automated service. They provide support for organisations involved in Marker Assisted Selection (MAS) and other large-scale DNA extraction/genetic screening projects. Slipstream Automation costs are significantly lower per sample, and have higher throughput than molecular methods that are currently used for the detection of PA. The aim of this project was to test the possibility of using the high-throughput systems as a viable, cost effective and efficient method for testing of samples for PA. We used two qPCR assays to detect PA in infected soil baits as a model system. In addition, we assessed the effect P. cinnamomi has on the detection of PA using the same qPCR assays and other molecular methods if applicable.

The problem

The current soil baiting protocol for detecting PA is a lengthy, labour-intensive, and costly process. Several factors can affect the accuracy of this test and potentially result in false negative and/or false positive detection. These factors include the presence of other oomycetes, in particular P. cinnamomi, which has been shown to mask the detection of PA.

Client initiatives To investigate the potential of developing a protocol for a high-throughput qPCR assay or assays to detect PA in soil baits and potentially leachates and soils using high-throughput qPCR.

This project

The aim of this project was to test the potential of high-throughput qPCR as a viable cost effective and efficient method for testing of environmental samples for PA. We used two qPCR assays to detect PA in infected soil baits as a model system. In addition, we assessed the effect P. cinnamomi had on the detection of PA using the same qPCR assays and other molecular methods if applicable.

Key results

• Baits were tested using Slipstream Automations, high-throughput qPCR from three different soil baiting experiments which were named experiment 1, 2 and 3.

• For the detection of PA from bait plant material there was a difference in sensitivity between the two qPCR assays. This was due to the gene copy number in the target organism genome.

• Using the Internal Transcribed Spacer (ITS) assay and the ras-related Ypt protein (Ypt1) with DNA of baits from the positive controls (soils baits known to be infected with PA), we found target DNA amplified from 100% and 96% of the baits respectively.

• Using the ITS assay and the Ypt1 assay with DNA of baits from the negative controls (baits known not to be infected with PA) we amplified target DNA from 37% and 2% of the baits respectively

• Target DNA was amplified in all treatments from all studies using both the ITS and Ypt1 including the P. cinnamomi treatment.

• More baits amplified target DNA in experiment 1 than from experiment 2 and 3 which was consistent with what was found from the traditional soil baiting morphological results.

• Optimisation of the two qPCRs should eliminate the false negatives and false positives that are currently occurring.

• This high-throughput automated qPCR method for detecting PA could be a fast and cost-effective option for screening soil baits.

Implications of results for the client

This pilot study has shown promising results although false positive and false negatives were detected. Further optimisation of these qPCRs for PA using Slipstreams high-through automatic methods could speed up diagnostics significantly reduce costs for the detection of PA in soils.

Further work

• Our results should be viewed as a fast succeed/fail pilot study. We conclude that the pilot study succeeded, and results have not ruled out the use of these techniques

• Further trouble shooting to optimise the qPCRs to ensure no false negative or false positive results occur should be carried out, this would include:

• Run a High-Resolution Melting (HRM) assay on negative control samples and P. cinnamomi treatment samples that have amplified target DNA to determine if cross contamination of the PA has occurred in these baits.

• Carry out further serial dilutions for ITS and Ypt1 assay standard curves to reach sensitivity limits of detection and allow an assessment of potential Cq cut-off values for high Cq amplification that may be considered not positive.

• Test the cedar DNA in isolation of PA with the ITS and Ypt1 assays to ensure there are no false positive results and ensure that the cedar DNA is not interfering with the Ypt1 assay resulting in false negative results.

• Test P. cinnamomi DNA in isolation of PA with the ITS and Ypt1 assays to ensure that there are no false positive detections.