Table 2.
Detection of parasites by conventional multiplex PCR (mPCR) assay in hemolymph and whole tissue pepsin-HCl digestion spiking experiments. The lowest level of detection is indicated in bold. Each hemolymph or homogenate replicate is comprised of matrix from one oyster.
| (Oo)cysts/sample | (Oo)cysts/reaction | Amplification/replicates tested |
|||
|---|---|---|---|---|---|
| Cryptosporidium | Giardia | Toxoplasma | |||
| Hemolymph | 10,000 | 1000 | 3/3 | 3/3 | 3/3 |
| 1000 | 100 | 3/3 | 3/3 | 3/3 | |
| 100 | 10 | 2/3 | 1/3 | 3/3 | |
| 10 | 1 | 1/3 | 0/3 | 3/3 | |
| 5 | 0.5 | 2/3 | 0/3 | 3/3 | |
| 0 | 0 | 0/3 | 0/3 | 1/3a | |
| Whole tissue homogenate | 10,000 | 1000 | 3/3 | 1/3 | 3/3 |
| 1000 | 100 | 1/3 | 1/3 | 3/3 | |
| 100 | 10 | 1/3 | 0/3 | 3/3 | |
| 10 | 1 | 0/3 | 0/3 | 2/3 | |
| 5 | 0.5 | 0/3 | 0/3 | 3/3 | |
| 0 | 0 | 0/3 | 0/3 | 0/3 | |
a
T. gondii amplification was found in one of three un-spiked hemolymph samples by mPCR targeting the 18S rRNA gene. A different PCR analysis targeting the B1 gene was attempted to evaluate the polymorphism of T. gondii target bands in spiked and un-spiked samples (Grigg and Boothroyd, 2001); however, T. gondii was not amplified in the B1 assay.