Presentation give an idea on how to analyse bivalve hemolymph for pathogenic DNA

1. PROCESSING AND STORAGE OF
MOLLUSC HEMOLYMPH FOR ANALYSIS
• Hemolymph collected was diluted with an equal volume of
Alsever’s solution (20.8 g glucose, 8.0 g sodium citrate, 22.5 g
sodium chloride, 1 L distilled water, pH 7.2) (Sarocha et al.,2018)
and used for further analysis and storage
• DNA was extracted from the collected hemolymph and analysed
for Viral infections using primers for Ostreid herpes virus,
Iridovirus and Abalone herpes virus and used for detecting
pathogenic bacteria in the haemolymph.

2. CONT…
• Briefly, 500µl of hemolymph diluted in equal volume of
Alsever’s solution was centrifuged and cells pelletized.
Pellet was washed in 400µl STE buffer.(100mM NaCl, 10mM
Tris HCl, 1mM EDTA, pH 8.0)
• Cells were centrifuged at 10,000xg for 2 min, supernatant
was discarded and pellet resuspended in 200µl milliQ water.
Aliquot of 100µl Tris saturated phenol was added to the cell
suspension followed by 60S of vortexing.

3. CONT…
• The solution was centrifuged at 13,000g for 5 min at
4°C . Aliquot of 160µl of supernatant was transferred
to a clean tube and extracted twice using
chloroform. Supernatant was transferred to a clean
tube. An aliquot of 5µl RNase was added and
incubated at 37°C for 10 min.
• An aliquot of 100µl chloroform was again added and
the solution was further centrifuged at 13,000g for
5min. Aqueous phase containing purified DNA was
pipetted out and stored at -20°C.