Multilocus amplification of genomic DNA from single cysts of Giardia duodenalis separated using micromanipulation technique.

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.