DNA molecular analysis was carried out using published PCR-relevant protocols [15 (link), 19 (link)]. In brief, the genotyping analysis was performed using assemblages-specific primers that amplify, in the case of the PCR-tpi, a 148-bp fragment of the assemblage A and 81-bp fragment of assemblage B [19 (link)]. The other PCR (PCR-E1-HP) amplifies a 165-bp amplicon for assemblage A and 272-bp fragment for assemblage B [15 (link)].
The PCR reaction mixture was done using a AmpliTaq DNA Polymerase with GeneAmp 10x PCR buffer Kit (Applied Biosystems, USA) in a total volume of 25 μL and comprised 10 μL of 10x PCR buffer (Applied Biosystems, USA), 0.2 mM of each deoxynucleoside triphosphate (dNTP) (Applied Biosystems, USA), 1 U of Taq polymerase (Applied Biosystems, USA), 0.4 μM of each primer, and 5 μL of DNA template, with ultrapure water used as a negative control.
The DNA was amplified using a thermocycler (Gene Amp PCR System 9700, Applied Biosystems, USA). The PCR products were analyzed by 2% agarose gel electrophoresis, stained with 0.5 μg/mL of ethidium bromide, and then visualized on a UV transilluminator (Syngene, U:Genius, Belgium).
DNA from axenic cultures of G. duodenalis strains WB-C6 (assemblage A) and GS (assemblage B) was used as positive controls, while ultrapure water was included in negative controls.
The PCR reaction mixture was done using a AmpliTaq DNA Polymerase with GeneAmp 10x PCR buffer Kit (Applied Biosystems, USA) in a total volume of 25 μL and comprised 10 μL of 10x PCR buffer (Applied Biosystems, USA), 0.2 mM of each deoxynucleoside triphosphate (dNTP) (Applied Biosystems, USA), 1 U of Taq polymerase (Applied Biosystems, USA), 0.4 μM of each primer, and 5 μL of DNA template, with ultrapure water used as a negative control.
The DNA was amplified using a thermocycler (Gene Amp PCR System 9700, Applied Biosystems, USA). The PCR products were analyzed by 2% agarose gel electrophoresis, stained with 0.5 μg/mL of ethidium bromide, and then visualized on a UV transilluminator (Syngene, U:Genius, Belgium).
DNA from axenic cultures of G. duodenalis strains WB-C6 (assemblage A) and GS (assemblage B) was used as positive controls, while ultrapure water was included in negative controls.
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