1887

Abstract

is the only species that causes amoebiasis in humans. Approximately 50 million people are infected, with 100 000 deaths annually in endemic countries. Molecular diagnosis of is important to differentiate it from the morphologically identical to avoid unnecessary medication. Conventional molecular diagnostic tests require trained personnel, cold-chain transportation and/or are storage-dependent, which make them user-unfriendly. The aim of this study was to develop a thermostabilized, one-step, nested, tetraplex PCR assay for the detection of , and species in cold-chain-free and ready-to-use form. The PCR test was designed based on the small subunit rRNA (SSU-rRNA) gene, which detects the presence of any species, and simultaneously can be used to differentiate from . In addition, a pair of primers was designed to serve as an internal amplification control to help identify inhibitors in the samples. All PCR reagents together with the designed primers were thermostabilized by lyophilization and were stable at 24 °C for at least 6 months. The limit of detection of the tetraplex PCR was found to be 39 pg DNA or 1000 cells for and 78 pg DNA or 1000 cells for , and the specificity was 100 %. In conclusion, this cold-chain-free, thermostabilized, one-step, nested, multiplex PCR assay was found to be efficacious in differentiating from other non-pathogenic species.

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2012-09-01
2020-11-30
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