Abstract:

In 2022, a total of 34 and 27 brook crabs were collected from Xingshan and Baokang Counties, respectively, in Hubei Province, a region where Paragonimus westermani is endemic. The DNA was extracted by NaOH digestion method after crushing from crab. Based on the 5.8S ribosomal RNA sequence of P. westermani, Primer Explorer V5 software was used to design specific primers for loop-mediated isothermal amplification (LAMP) of P. westermani, and a LAMP detection method for P. westermani was established. The specificity, sensitivity, amplification efficiency, and field application effect of the method were evaluated, and compared with precipitation microscopy. The results indicated that the LAMP method displayed a detection limit of a single P. westermani metacercaria without any cross-reactivity with five non-target species, including Schistosoma japonicum, Clonorchis sinensis, Fasciola hepatica, Capillaria hepatica, and hookworm. The assay’s performance was quantified using the average turbidity time (Tt value) of 29.0 min and the peak turbidity rate (Df) of 0.237 min for four metacercariae samples. The LAMP method revealed 24 and 0 positive stream crabs infceted with P. westermani in Xingshan County and Baokang County, with positive rates of 70.6% (24/34) and 0 (0/27), respectively; the precipitation microscopy method revealed 23 and 0 positive stream crabsin Xingshan County and Baokang County, with positive rates of 67.6% (23/34) and 0 (0/27), respectively. There is no statistically significant difference in the results between the two detection methods (χ² = 0.2, P > 0.05). Conclusively, the LAMP method developed in this study is a straightforward, specific, and highly sensitive diagnostic tool for the detection of P. westermani in freshwater crabs. Its proven efficacy suggests that it is well-suited for in-field surveys or as a screening mechanism in endemic regions.

Key words: Paragonimus westermani, Lop mediated isothermal amplification, Creek crab, Detection effect