J L Alonso1, I Amorós, G Cuesta. 1. Instituto de Ingeniería del Agua y Medio Ambiente, Universidad Politécnica, Valencia, Spain. jalonso@ihdr.upv.es
Abstract
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 beta-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.
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 beta-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.
Authors: Deiviane A Calegar; Beatriz C Nunes; Kerla J L Monteiro; Polyanna A A Bacelar; Brenda B C Evangelista; Mayron M Almeida; Jurecir Silva; Jéssica P Santos; Márcio N Boia; Lauren H Jaeger; Filipe A Carvalho-Costa Journal: Microorganisms Date: 2022-04-30
Authors: Felix Weinreich; Andreas Hahn; Kirsten Alexandra Eberhardt; Simone Kann; Torsten Feldt; Fred Stephen Sarfo; Veronica Di Cristanziano; Hagen Frickmann; Ulrike Loderstädt Journal: Microorganisms Date: 2022-06-28