Development and evaluation of a genus-specific, probe-based, internal-process-controlled real-time PCR assay for sensitive and specific detection of Blastocystis spp

Abstract

Blastocystis is a common intestinal parasite of unsettled clinical significance, which is not easily detected by standard parasitological methods. The genus comprises at least 13 subtypes (STs) (which likely represent separate species), 9 of which have been found in humans. Recent data indicate that at least one of the subtypes is associated with intestinal disease. A quantitative TaqMan 5' nuclease real-time PCR (TaqMan PCR) including an internal process control (IPC) was developed for the detection of Blastocystis and shown to be applicable to genomic DNAs extracted directly from feces. The assay enabled successful amplification of DNAs from all relevant subtypes within the genus (ST1 to ST9). For assay evaluation, 153 samples previously tested by xenic in vitro culture (XIVC) were screened by the TaqMan assay. A total of 49/51 samples positive by XIVC and 13/102 samples negative by XIVC were positive by the TaqMan assay; samples positive by the TaqMan assay and negative by XIVC were subsequently tested by conventional PCR, and amplicons could be identified to the subtype level by sequencing in 69% of the cases. Compared to the TaqMan assay, XIVC had a sensitivity of 79%. This is the first time that a genus-specific, probe-based, internal-process-controlled real-time PCR assay for the detection Blastocystis has been introduced.

Fig 1

Alignment of Blastocystis-specific oligonucleotides (forward, probe, and reverse) and SSU rDNAs from Blastocystis sp. ST1 to ST10, other Blastocystis spp., Proteromonas lacertae, Candida albicans, and Saccharomyces cerevisiae. Polymorphic bases are highlighted in gray. Dashes indicate missing or nonexisting bases.