I am working on DNA barcoding of a marine elasmobranch (guitarfish). Recently, I collected 30 new specimens from Cox’s Bazar. The fish were purchased from fishermen (about 1–2 weeks old, kept in cold storage). I only used muscle tissue, followed sterile handling, changed gloves between samples, and even replaced the ethanol after one day. DNA extraction and PCR were done immediately after preservation.
Still, I got only 7 good sequences of the target species, while 9 samples showed contamination with Shewanella.
I’m not sure whether this is happening because of how the fishermen stored the fish, something in my extraction procedure, or maybe even primer issues. Has anyone working with marine elasmobranchs (or other fish) faced a similar situation, or have ideas on what might be going wrong?
because the tissue was already decomposed before preservation. Shewanella is a common marine bacterium that rapidly invades muscle after death, and by the time the samples were stored in ethanol its DNA had become far more abundant than the degraded host DNA. Standard COI primers then preferentially amplified the bacterial DNA instead of the fish DNA.
Your issue is common when working with marine fish that are not fresh. Shewanella contamination is likely due to the fish being stored for 1–2 weeks, during which these cold-tolerant bacteria can proliferate in the muscle tissue. Universal DNA barcoding primers (e.g., Folmer primers) can amplify both fish and bacterial DNA, and bacterial DNA may dominate the PCR product.
To reduce contamination:
· Use fresh samples: Collect tissue from live or freshly caught fish whenever possible.
· Surface-sterilize tissue: Rinse the area with sterile water or ethanol before sampling.
· Use fish-specific primers: Consider primers like FishF1/FishR1 or Fish-BC/Fish-C.
· Run negative controls: Include extraction blanks and PCR negatives to ensure reagents are clean.
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