Identifying Trichuris Species by PCR

Samples testing positive for Trichuris ssp. using the QIMR assay, but negative for Trichuris trichiura using the Smith assay were further analyzed to determine the identity of the infecting species. Using a previously described primer-probe set targeting the coding sequence for the 18S ribosomal subunit [46 (link)], these samples were PCR amplified in 25 μl reactions using the Phusion Hot Start Flex DNA Polymerase Kit (New England Biolabs, Ipswich, MA). PCR reaction conditions were as follows: 16.5 μl PCR-grade water, 400 nM forward primer, 400 nM reverse primer, 0.5 μl dNTPs, 0.75 μl DMSO, 5.0 μl Phusion HF buffer, 0.25 μl Phusion Polymerase, and 1 μl template DNA. Cycling conditions consisted of an initial denaturing step at 98°C for 15 min, followed by 35 cycles of 98°C for 10 sec, 56°C for 15 sec, and 72°C for 15 sec. Following 35 cycles, a final extension step of 72°C for 7 min was performed. PCR products were then sequenced using standard Sanger methodology and resulting sequence data were analyzed using NCBI’s Nucleotide BLAST tool.