Estimating Giardia cyst viability using RT-PCR

Abstract

The aim of this work was to evaluate the use of molecular techniques for the detection of viable Giardia cysts in the environment to assess public health issues. Three target genes were selected: the heat shock protein gene, HSP70, which is expressed in response to stress; the giardin gene, which encodes a structural protein; and, alcohol dehydrogenase E (ADHE), a novel gene encoding an enzyme involved in the metabolism of energy. We tested the efficiency of five protocols for the extraction of either genomic DNA or total RNA from Giardia cysts: two of these protocols were previously cited in the literature and three consisted of commercial DNA extraction kits. The brands of enzyme were determined according to the primers chosen and the amplification conditions were optimised: 2.5 mM MgCl2, 0.5 mM primers and 60°C for annealing temperature. A semi-nested PCR method and an RT semi-nested PCR procedure were developed to detect mRNA from these three genes and to estimate the viability of Giardia cysts.