Ronit’s Notebook: Identifying Unknown Oyster Sample from Marinelli Shellfish Company (C. gigas vs C. sikamea)

The Marinelli Shellfish Company had an issue with one of their bags of oysters being labelled as Kumamoto oysters (C. sikamea) AND as Pacific Oysters (C. gigas). Obviously, confusion abounded, and ultimately we were tasked with figuring out what the true identity of these mystery oysters were. To do so, DNA was isolated from mantle tissue from the unknown oysters, a sample set of known C. gigas, and a sample set of known C. sikamea. 4 PCR primers were targetting the cytochrome oxidase gene: universal forward and reverse primers (HC02198, LCO1490); reverse primer specific to C. gigas (COCgi269r); and a reverse primer specific to C. sikamea (COCsi546r). Note: this was a multiplex PCR.

Cycling parameters were as follows:

95°C for 10 mins; 30 cycles of 95°C (1 min), 51°C (1 min), 72°C (1 min); 72°C (10 min).

PCR reactions were run on a gel and results are visualized below:

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The first set of 4 samples (offset by ladders) are the unknown samples; the second set of 4 samples are C. gigas; and the third set of 4 samples are C. sikamea.

Using the GeneRuler DNA Ladder as a guide,

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First, we can see that there is a band of approximately 700bp in all samples, indicating that the universal forward and reverse primers did their job (positive primer). Next, we expect to see a band of approximately 260-270 bp in the known C. gigas samples, which we do! Similarly, we also expect to see a band of 550 bp in the known C. sikamea samples, which we also do. (Note: it looks like there is a faint band of 270bp in the C. sikamea samples. Could be a sign of contamination with C. gigas samples?).

In the unknown samples, a prominent band of 270bp is clearly visible, which is what we should see in C. gigas samples, Thus, it seems that these mystery samples are in fact C. gigas. Case closed!