TITLE: Non-invasive Detection Method for Bonamia ostreae in Ostrea edulis using the Franklin qPCR machine
AUTHORS: Lavanya M Vythalingam, Tim Regan, Tim Bean

[DESCRIPTION]
This is a comprehensive protocol for using the Franklin qPCR (Biomeme Inc.) machine to detect the presence of Bonamia ostreae parasite in European flat oyster (Ostrea edulis). Previous methods relied on sacrificially dissecting animals to perform histology. While sensitive, these methods were destructive and untested animals carrying Bonamia ostreae are not detected. Here, we present a non-invasive alternative method to detect Bonamia ostreae from Ostrea edulis pseudofaeces/faeces using a portable qPCR machine.

[STEPS]
SECTION: Sample Collection
2. Separate collected animals and cover with seawater (approx. 1 L for every 200 g of live animal). 
 

SECTION: Sample Collection
1. Collect oysters and wash to remove any excess mud/sediment on their shells.
SECTION: Sample Collection
3. Aerate buckets overnight for 960 min at ambient temperature on sampling location (see Figure 1). 

  
SECTION: Sample Collection
4. Collect the sediment remaining in the bottom of each bucket (faeces and pseudofaeces) using a  Pasteur pipette into a 1.5 mL Eppendorf tube. 
 

SECTION: Sample Collection
5. OPTIONAL: Store samples collected at -20 °C until further use.
SECTION: DNA Extraction
7. Extract DNA from the sediment samples by physical beating in 2 mL Lysing matrix A tubes and filter through a 1 uM barb column filter. 

SECTION: DNA Extraction
10. Process filtered sediment through the Biomeme M1 extraction cartridge (see Figure 2), according to the manufacturer’s protocol (refer to link below).

 M1 Sample Prep Cartridge Techniques on Vimeo


  
SECTION: On-site Franklin qPCR assay
15. Run a qPCR assay using the Franklin qPCR machine against the three probe-based assays. Primers and probes used in this assay is given below (Table 1).
 

SECTION: On-site Franklin qPCR assay
12. Transfer 20 µL  of extracted DNA to Biomeme Go-Strip assay tube (see Figure 3). 

  
 

 

SECTION: On-site Franklin qPCR assay
16. The Franklin qPCR protocol consisted of 1 min minute heat activation and 45 cycles of 95 °C for 1 second and 60 °C for 20 s. 

SECTION: Sample Collection
6. Disinfect all equipment with working strength bleach according to biosecurity SOPs. 
SECTION: On-site Franklin qPCR assay
11. Store DNA at -4 °C until further use.
SECTION: DNA Extraction
8. Use a syringe with a 1 uM pre-filter to remove the supernatant without debris. 
SECTION: DNA Extraction
9. Make two holes in the first M1 chamber and transfer sample into the Biomeme M1 cartridge.
SECTION: On-site Franklin qPCR assay
18.  
  
SECTION: On-site Franklin qPCR assay
13.  Follow the instructions on connecting the qPCR machine to the phone using the Biomeme app. Choose the LyoDNA test  (Figure 4) and follow the on screen instructions. 

   
SECTION: On-site Franklin qPCR assay
14. Place the Biomeme Go-Strip assay tubes into the Franklin device in the correct orientation (see Figure 5). 

   
SECTION: On-site Franklin qPCR assay
17. Use the Biomeme app on the phone to analyse the qPCR results to determine the presence of Bonamia ostreae. Refer to Figure 6 for an example of positive Bonamia ostreae presence.