Extracting DNA from whole organism homogenates and the risk of false positives in PCR based diet studies: A case study using spiny lobster larvae
2013
O'Rorke, R. | Jeffs, A.G. | Fitzgibbon, Q. | Chow, S. | Lavery, S.
Better understanding the diet of small marine predators such as the planktonic larvae of spiny lobsters is important for our awareness of interactions within marine assemblages and for species commercialisation. In DNA-based diet studies of small organisms there is a risk that any DNA contaminating the outside of an organism will be detected and falsely assumed to originate from the gut. Experiments with terrestrial predators have overcome the problem of exogenous contamination by treating the exterior of the predator with bleach (sodium hypochlorite). However, the use of bleach is a risky strategy when treating either a rare predator or aquatic predators, which are generally more permeable than terrestrial animals. Many plankton studies have not reported how they dealt with exogenous contamination, or do not use a control during PCR to detect false positives due to exogenous contamination. One approach is to wash the predator with MilliQ filtered water or ethanol and to use the final wash as a PCR template to detect residual DNA. In the present study we report that washing has variable success at removing exogenous contaminants and that using the final wash as a control for exogenous contamination consistently fails. Based on our results we recommend using DNA extracted from a swab of the exterior of the predator as a control for exogenous contamination. We also report on the benefit of using a novel syringe technique to obtain gut content that minimises contact with the predator surface, and therefore the risk of exogenous contamination.
显示更多 [+] 显示较少 [-]