Development of a Specific Primer-Based Method for Detecting Bithynia siamensis siamensis, an Intermediate Host of the Liver Fluke Opisthorchis viverrini

Abstract

Background: The liver fluke Opisthorchis viverrini is a major public health concern in Southeast Asia and a leading cause of cholangiocarcinoma. Its transmission relies on freshwater snails of the genus Bithynia as the first intermediate hosts. However, accurately identifying these morphologically similar snails remains challenging. Objectives: This study aimed to develop species-specific primers for the accurate molecular identification of Bithynia siamensis siamensis, which serves as an important first intermediate host of O. viverrini. Methods: Random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) was employed to isolate a unique DNA fragment specific to B. siamensis siamensis. A distinct 820 bp RAPD-derived fragment was obtained, sequenced, and analyzed. From this fragment, a conserved internal region was selected and used to design a species-specific primer pair (BSS2F/BSS2R), which produces a 280 bp diagnostic amplicon. The specificity and sensitivity of the newly developed assay were subsequently evaluated against other freshwater snail species. Results: The designed primers consistently amplified a 280 bp fragment exclusive to B. siamensis siamensis, with no cross-reactivity to B. siamensis goniomphalos, B. funiculata, or other cohabiting snail species. The assay detected as little as 5 ng of genomic DNA and achieved 90% amplification success across samples from four provinces. Conclusion: The RAPD-derived primer pair provides a rapid, reliable, and cost-effective molecular tool for the specific detection of B. siamensis siamensis. This approach enhances epidemiological surveillance of O. viverrini transmission and supports integrated One Health strategies for controlling opisthorchiasis in endemic regions.