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, internalprocess-controlled real-time PCR assay for the detection Blastocystis has been introduced.

Recent Developments in Blastocystis Research

“…The latter has the added advantages of automatically assigning allele types to the SSU-rDNA as well as using the consensus subtype nomenclature (unlike GenBank where the subtype is included only if one was part of the accession submission and no attempt to impose a standard nomenclature is made). Because of the occasional problem of nonspecific amplification, it is recommended that samples be screened first for positivity, where possible, using Real-Time PCR (Stensvold et al, 2012b) An example of how necessary this approach can be exists in the case of ST13. The subtype was found in a Quokka (a marsupial) and named by Parkar et al (2010) in Australia, who showed it to cluster near ST5 in phylogenetic trees.…”

“…The rationale for this is unexplained -why would having 4 vs. 6 organisms per field be a significant difference? The recent descriptions of Real-Time PCR diagnostic tools for Blastocystis Stensvold et al, 2012b) will make detection of the organisms easier as well as allowing exploration of any role for infection intensity in symptomatology.…”

Molecular Identification and Subtype Analysis of Blastocystis

“…The latter two samples might represent negative samples, depending on the Blastocystis DNA concentration and stability of the positive controls. Hence, controls will need to be run to determine the exact sensitivity and specificity (for more information, please look up Stensvold et al, 2012a). Figure. 1.…”

“…The PCR will not give information on subtype. An internal process control (IPC) can be included (for IPC development, primers and probe, please see Stensvold et al, 2012a).…”

“…This is also one of the reasons why this PCR should not be used diagnostically. Hence, it is recommended that barcoding be used only for molecular characterization of already known positive samples-not for screening; state-of-the-art screening uses real-time PCR as described by Stensvold et al, 2012a. Used on DNA extracted from Blastocystis-positive cultures, the primers are usually very specific.…”