Multiplex PCR was performed with three pairs of primers, and three different sizes were obtained: 597 bp E. encrasicolus, 598 bp E. japonicus, 380 bp E. mordax, and 250 bp E. ringens.  For the species E. encrasicolus and E. japonicus, PCR-RFLP was used as an additional technique to distinguish between them because their NTS sequences showed considerable similarity.
PCR
Multiplex PCR was performed with three pairs of primers, and three different sizes were obtained: 597 bp E. encrasicolus, 598 bp E. japonicus, 380 bp E. mordax, and 250 bp E. ringens. 
Multiplex PCR
For the species E. encrasicolus and E. japonicus, PCR-RFLP was used as an additional technique to distinguish between them because their NTS sequences showed considerable similarity.
PCR-RFLP