Challenges with the Applicaion of qPCR Based Tests for Enterococci as a Rapid Beach Mangement Tool in Hawai'i

Abstract

Rapid quantitative PCR based methods (USEPA Method 1609 and 1611) that can evaluate water quality within a few hours are important since urbanization and our changing climate will alter inputs of bacteria into the environment, which can compromise the health of Hawai’i residents and visitors. We identified that assay interference in Hawaiian coastal waters is associated with the DNA extraction process, but not with the PCR amplification procedure. Further we demonstrated that acidification of water samples can alleviate assay interference associated with Hawai’i’s beach water samples. It is speculated that acidification of coastal water samples helped to dissolve calcium carbonate rich coralline particles, common to subtropical and tropical coastal waters, hence eliminating a substratum for DNA to bind. This study envisions, that once the assay can be used to reliably quantify microorganisms (such as enterococci, humanassociated Bacteroides), these tests will be used for beach water quality evaluations at popular beaches in the state of Hawai’i. In this research, water matrixes for PCR inhibitors were tested from water samples gathered at selected beaches on Oahu, by using the EPA Methods 1609 and 1611, Enterococci in Water by TaqMan ®Quantitative Polymerase Chain Reaction (qPCR) with Internal Amplification Control (IAC) Assay. There is a relationship between the sodium chloride and calcium carbonate on Oahu, and the inhibition of the rapid method, used to identify enterococci. The corals in Hawai’i are unique and add in with the water's composition that is interfering with the DNA extraction process, and underestimating the rapid method results.