title: Multilocus amplification of genomic DNA from single cysts of Giardia duodenalis separated using micromanipulation technique.
abstract: Giardia duodenalis is divided into at least eight groups, named assemblages A to H. Assemblages A and B are the only ones able to infect humans and other mammals. The species status for these assemblies is a moot point, but has not gained general acceptance because sexual activity in Giardia is not completely understood. Heterozygosity in G. duodenalis can be detected through simultaneous identification of multiple loci in single cysts or trophozoites. In this paper, we describe a technique that enables simultaneous detection of fragments from four genes from single cysts of G. duodenalis recovered from stool samples. Each cyst from a fecal sample of human origin was separated, the DNA was extracted and amplified by means of multiplex PCR directed to four genes and the multiplex PCR product was further re-amplified using four single PCR (one for each gene). The following loci were detected: beta giardin (bg), GLORF-C4 (orfC4), triose phosphate isomerase (tpi) and glutamate dehydrogenase (gdh). This procedure should make it possible to investigate multiple genes from a single cyst of G. duodenalis assemblage A or B.
authors.affiliation: Departamento de Medicina Veterinária Preventiva e Saúde Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo, SP, Brazil.
authors.forename: Juliana Martins
authors.initials: JM
authors.lastname: Aguiar
authors.affiliation: Departamento de Medicina Veterinária Preventiva e Saúde Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo, SP, Brazil.
authors.forename: Sheila Oliveira da
authors.initials: SO
authors.lastname: Silva
authors.affiliation: Departamento de Farmácia e Laboratório Clínico do Hospital Universitário, Universidade de São Paulo, São Paulo, SP, Brazil.
authors.forename: Valdir Antônio Dos
authors.initials: VA
authors.lastname: Santos
authors.affiliation: Departamento de Medicina Veterinária Preventiva e Saúde Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo, SP, Brazil.
authors.forename: Giselle Ayres Razera
authors.initials: GA
authors.lastname: Rossa
authors.affiliation: Departamento de Medicina Veterinária Preventiva e Saúde Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo, SP, Brazil.
authors.forename: Sueli Akemi
authors.initials: SA
authors.lastname: Taniwaki
authors.affiliation: Departamento de Medicina Veterinária Preventiva e Saúde Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo, SP, Brazil.
authors.forename: Fabio
authors.initials: F
authors.lastname: Gregori
authors.affiliation: Faculdade de Zootecnia e Engenharia de Alimentos, Departamento de Medicina Veterinária, Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo, São Paulo, SP, Brazil.
authors.forename: Lara Borges
authors.initials: LB
authors.lastname: Keid
authors.affiliation: Departamento de Medicina Veterinária Preventiva e Saúde Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo, SP, Brazil. Electronic address: rosoares@usp.br.
authors.forename: Rodrigo Martins
authors.initials: RM
authors.lastname: Soares
mesh_terms: D016054:DNA, Protozoan / Q000235:genetics* / Q000302:isolation & purification*; D005243:Feces / Q000469:parasitology; D016829:Giardia lamblia / Q000235:genetics*; D005873:Giardiasis / Q000469:parasitology; D006579:Heterozygote; D006801:Humans; D008846:Micromanipulation; D058885:Multilocus Sequence Typing; D060885:Multiplex Polymerase Chain Reaction; D016133:Polymerase Chain Reaction
chemical_list: D016054:DNA, Protozoan
keywords: Cysts; Giardia duodenalis; Micromanipulation; Multilocus PCR