“Development of point of need diagnostic methods for detection of bonamia ostreae using in-field nucleotide sequencing (7112)”

development of point of need diagnostic methods for detection of bonamia ostreae using in-field nucleotide sequencing

Introduction

Populations of the European flat oyster (Ostrea edulis) have been declining globally due to overexploitation and diseases caused by parasites such as Bonamia ostreae. While technological advances have significantly improved the detection and identification of this pathogen in laboratory settings, there is a pressing need for diagnostic techniques for point-of-need use. Rapid diagnosis of infectious diseases in farms, border controls, and processing units might allow the implementation of measures to mitigate and prevent the spread of diseases. This is particularly crucial for diseases that cannot be eradicated or for which there are limited or no treatments, which is the case for most diseases affecting molluscs. The objective of this study was to develop diagnostic methods for in-field detection and characterization of B. ostreae using in-field nucleotide sequencing.

Methodology

Nine different DNA extraction methods were evaluated to determine their efficiency in obtaining DNA from oyster samples under field conditions. The quantity and quality of the DNA extracted was assessed using fluorescence and spectrophotometric methods. Additionally, PCR amplification of host DNA was performed to assess the presence of inhibitors. For amplification of B. ostreae DNA, fast PCR and recombinase polymerase amplification (RPA) assays were developed. The assays were designed to allow the differentiation of B. ostreae, B. exitosa and B. perspora. Finally, rapid ONT library protocols were implemented to sequence fast PCR and RPA products.

Results and Conclusions

Among the nine DNA extraction methods tested, the Quickextract™ DNA extraction solution was deemed the most suitable based on dna yield (ng/mg), 260/280 and 230/280 ratios, absence of inhibitors, extraction time, and simplicity. The fast PCR approach successfully detected B. ostreae in samples with DNA copy numbers between 245 to 6918 per mg. Using the fast PCR/ONTapproach, a consensus sequence of approximately 500 bp with 100% nucleotide similarity to the reference B. ostreae was obtained. This method enabled the detection and characterization of B. ostreae from an infected oyster in approximately 2 hours (from tissue to sequence). However, this approach tested only a single sample, unlike the RPA with SYBR™ green method, which allowed simultaneous testing of up to 12 samples. RPA products were also successfully sequenced using the ONT rapid barcoding kit. Analysis of naturally infected O. edulis indicated that RPA and fast PCR have similar sensitivity levels.

Keywords

point of need testing, dna extraction, rpa, fast pcr, in-field sequencing, nanopore sequencing, Bonamia ostreae

Funding

FC1216 (DEFRA) and Cefas Seedcorn dp445

1. Batista, Frederico, Cefas, Presenter
2. Kerr, Rose, Cefas, Author
3. Edwards, Matt, Cefas, Author
4. Robert, Hatfield, Cefas, Author
5. Scott, George, Cefas, Author