2.4. PCR for genotyping

A 530 bp fragment of the triosephosphate isomerase (tpi) gene for Giardia was amplified by nested PCR [³⁹]. The cycling protocol for both reactions included an initial cycle of 94 °C for 2 min, followed by 35 cycles of 94 °C for 45 s, 50 °C for 45 s, 72 °C for 60 s and a final extension of 72 °C for 10 min. The reaction volume was 50 μl containing 1 μl of genomic DNA template, standard PCR buffer (5xGreen GoTaq^® Reaction Buffer, Promega USA), 10 mM of each dNTP, 50 mM MgCl₂, 1.25 U of Taq polymerase (GoTaq^® G2 DNA Polymerase, Promega, USA) and 25 pmol of each oligonucleotide primer (Table 1 ).

Primers utilized in nested PCR reactions amplifying tpi, bg and SSU rRNA of Giardia and gp60 of Cryptosporidium parvum from faecal samples.

A region within the β-giardin (bg) gene of Giardia was amplified using an established nested PCR protocol (by Lalle et al., 2005) [⁴⁰]. Briefly, the cycling protocol for the first reaction included one cycle of 95 °C for 5 min, followed by 35 cycles of 95 °C for 30 s, 65 °C for 30 s, 72 °C for 60 s and a final extension of 72 °C for 7 min. The annealing temperature of the secondary reaction was reduced to 55 °C for 30 s. Five microliters of genomic DNA was used for both reactions in a final volume of 25 μl containing standard PCR buffer (5xGreen GoTaq^® Reaction Buffer, Promega USA), 1.25 U of Taq polymerase (GoTaq^® G2 DNA Polymerase, Promega, USA), 25 mM dNTPs and 10 pmol of each primer (Table 1).

In cases when PCR results were negative or inconclusive, PCR was repeated with native faecal material (tpi: n = 16; bg: n = 17).

Tpi negative Giardia samples were further analysed amplifying a fragment of the small subunit rRNA (SSU rRNA) using nested PCR [^41,42]. In a 20 μl reaction volume 5 μl of genomic DNA, standard PCR buffer (5xGreen GoTaq^® Reaction Buffer, Promega USA), 25 mM dNTPs, 1.25 U Taq polymerase (GoTaq^® G2 DNA Polymerase, Promega, USA) and 25 pmol of each primer was used (Table 1). The cycling protocol for both reactions included one cycle of 95 °C for 2 min, followed by 35 cycles of 96 °C for 20 s, 59 °C for 20 s, 72 °C for 30 s and a final extension of 72 °C for 7 min.

For genotype analysis of C. parvum, a 60-kD glycoprotein (gp60) gene fragment was amplified [⁴³]. One microliter of genomic DNA was used in a 20 μl reaction volume with standard PCR buffer, 25 mM dNTPs, 25 mM MgCl₂, 1.25 U Taq (GoTaq^® G2 DNA Polymerase, Promega, USA) and 20 pmol of each primer. The cycling protocol included one cycle of 94 °C for 2 min, followed by 30 cycles of 95 °C for 50 s, 56 °C for 50 s, 65 °C for 60 s, and a final extension of 65 °C for 5 min. For nested PCR annealing temperature was increased to 60 °C and 0.5 μl DNA template from the previous PCR round was used.

PCR products were subjected to electrophoresis in a 2.0% agarose gel and visualized with ultraviolet light (LumiBIS 1.4, DNR Bio-Imaging Systems Ltd., Israel)