LNA probes in a real‐time TaqMan PCR assay for genotyping of Giardia duodenalis in wastewaters

Aims: This study describes an approach for genotyping Giardia cysts obtained from wastewater treatment plants (WTPs) in Spain using real‐time PCR (qPCR) in combination with immunomagnetic beads. Methods and Results: A 50‐cycle amplification of a 74‐bp fragment of the Giardia beta‐giardin gene was adopted from a previous qPCR method. Additionally, two locked nucleic acid (LNA) probes were designed (LNA P434 P1 for assemblage A and LNA P434 H3 for assemblage B). All 16 wastewater samples analysed were positive with the immunofluorescence assay (IFA). Assemblage A was detected in all WTP samples using primer–LNA probe P434 P1 set. Giardia duodenalis identification was confirmed by PCR–RFLP analysis and sequencing of the β‐giardin gene in the water samples found positive by IFA and qPCR. Among the 16 assemblage A isolates that were sequenced, two subtypes were identified; 11 corresponded to the A2 subgenotype, whereas three corresponded to the subgenotype A3. A mixture of subgenotypes was found in the remaining two isolates. Conclusions: The newly developed qPCR assays were able to discern G. duodenalis assemblages A and B in wastewater. Significance and Impact of the Study: The real‐time PCR assays provided a rapid method for detection and one‐step genotyping of G. duodenalis from wastewater samples, and its application would contribute to understanding the distribution and abundance of G. duodenalis assemblages A and B in wastewater.