Abstract. To accurately discriminate between individual metacercariae of Paragonimus heterotremus and P. westermani occurring in Thailand, polymerase chain reaction (PCR)-based molecular methods were established and subjected to an evaluation. We first amplified and sequenced the second internal transcribed spacer (ITS2) region of the nuclear ribosomal DNA of the two species. Based on their nucleotide differences, P. heterotremus and P. westermani were unequivocally discriminated from each other. These nucleotide differences were further utilized to select the ApaL1 endonuclease site for PCR-restriction fragment length polymorphism (PCR-RFLP) analyses and to design species-specific primers for multiplex PCR reactions. Both PCR-RFLP and multiplex PCR methods allowed a more rapid and labor-effective species discrimination. Furthermore, the multiplex PCR method enabled the most efficient discrimination because species identification involved a single round of PCR in a single tube. In Thailand, P. heterotremus is the only species affecting humans. Thus, the methods established in the present study can be used as reliable tools to identify the lung fluke metacercariae that cause human disease. primers. All of these methods utilize nucleotide differences in the second internal transcribed spacer (ITS2) of the nuclear ribosomal DNA (rDNA) for dicrimination between the two species. In the present study, we focused on the lung flukes occurring in Thailand and applied the methods for species discrimination between individual metacercariae of P. heterotremus and P. westermani. MATERIALS AND METHODS Parasite material and DNA isolation The metacercariae of P. heterotremus and P. westermani DNA amplification, restriction digestion and sequencing The rDNA region spanning the ITS2 from individual metacercariae of the two species was amplified by PCR using the primers, 3S (forward, 5'-GGTACCGGTGGATCACTCGGCTCGTG-3') and A28 (reverse, 5'-GGGATCCTGGTTAGTTTCTTTT CCTCCGC-3'). These primers were designed on the basis of the conserved rDNA sequences of the Schistosoma specie
