Human blastocystis subtyping with subtype specific primer developed from unique sequences of SSU rRNA Gene / Nurul 'Izzati Zulkifli

Blastocystis is a single-celled eukaryotic parasite that commonly infects the lower gastrointestinal tract in humans as well as animals. It is genetically diverse whereby 17 subtypes (STs) has been recorded so far. However, ST3 and ST1 are predominant in human stool samples especially in Southeast Asian countries including Malaysia. There is currently a lack of time- and cost-effective detection methods for these STs. Thus, the present study was carried out to develop subtype-specific primers from unique sequences of the SSU rRNA gene of human Blastocystis sp. In this study, 2 sets of primers for both ST1 and ST3 have been developed via PerlPrimer and Oligo Analyser software using sequences acquired from the NCBI database. Next, PCR amplification, followed by gel electrophoresis (1.5% agarose gel, 145 volt, 60 minutes) was performed. The sensitivity of the primers were tested using 10-fold dilutions. The primers were also tested against common enteric bacteria and parasites to ensure its specificity. Each primer pairs successfully amplified the target region of the Blastocystis samples, whereby 138 bp and 233 bp amplicons were acquired for ST1 and ST3, respectively. Furthermore the detection limits of the multiplex PCR were confirmed at 7 ng/μl for the former, and 70 ng/μl for the latter. The primers were also confirmed to be non-specific to the non-Blastocystis samples. Because of the ease of use, specificity and increase in Blastocystis infection in Malaysia, this assay has the potential to greatly improve clinical diagnosis of Blastocystis infection and becoming method of choice.