Examination of six commonly used laboratory fixatives in HAB monitoring programs for their use in quantitative PCR based on Taqman probe technology

Abstract

Due to the need for more rapid and reliable detection, quantification and enumeration of harmful algal species the use of molecular methods are increasingly being used in monitoring and field studies. However, many studies often require sample fixation to allow for transportation before analyses are conducted. Here, we describe the effects of six fixatives (acidified Lugol's iodine with or without sodium thiosulphate, glutaraldehyde, paraformaldehyde (PFA), formalin and ethanol) on quantitative real-time polymerase chain reaction (qPCR) amplification with Taqman probes. We applied extracted total genomic DNA from four harmful algal species from Danish waters, representing three dinoflagellates (Alexandrium tamarense, Karenia mikimotoi, Karlodinium veneficum and a haptophyte (Prymnesium parvum). The Cq values generated on the qPCR amplification plot were compared to those of an unfixed sample that acted as a control. For all species positive amplifications were achieved from DNA templates from all preserved samples. However, amplification efficiencies between fixatives and species varied. Yet it was found that Lugol's iodine was the most ideal short-term fixative for enumeration of cells by qPCR as well as being the safest to handle. The effect of age on Lugol's iodine fixed samples was also addressed. Samples were fixed and stored at 5°C in the dark and total genomic DNA extracted after 24h, 72h, 1 week, 2 weeks, 1 month and 2 months. Samples remained stable for 1 month for A. tamarense and K. veneficum and 2 months for K. mikimotoi and P. parvum.