OBJECTIVE: To establish a PCR assay for detecting the third-stage larvae of Angiostrongylus cantonensis in Pomacea canaliculata. METHODS: Polymerase chain reaction primers were designed by the software Lasergene, based on the specific cDNA of the third-stage larvae of A.cantonensis in Genbank. The total RNA was prepared from the third-stage larvae of A.cantonensis and of the snails by TRIzol one-step protocol. Amplification by RT-PCR was carried out following the kit protocol. RESULTS: RT-PCR assay revealed a clear differentiation between infected and negative snails. When a mixture of the total RNA from the negative snails and the third-stage larvae of A.cantonensis was tested by the PCR assay, the detectable level was 128 pg RNA, a concentration close to one third-stage larva of A.cantonensis, minimum concentration that could be found by naked eyes. The minimum detected total RNA concentration of the third-stage larvae of A.cantonensis was 105 pg by PCR assay. CONCLUSION: A PCR assay has been developed for detecting A.cantonensis larva in Pomacea canaliculata.
OBJECTIVE: To establish a PCR assay for detecting the third-stage larvae of Angiostrongylus cantonensis in Pomacea canaliculata. METHODS: Polymerase chain reaction primers were designed by the software Lasergene, based on the specific cDNA of the third-stage larvae of A.cantonensis in Genbank. The total RNA was prepared from the third-stage larvae of A.cantonensis and of the snails by TRIzol one-step protocol. Amplification by RT-PCR was carried out following the kit protocol. RESULTS: RT-PCR assay revealed a clear differentiation between infected and negative snails. When a mixture of the total RNA from the negative snails and the third-stage larvae of A.cantonensis was tested by the PCR assay, the detectable level was 128 pg RNA, a concentration close to one third-stage larva of A.cantonensis, minimum concentration that could be found by naked eyes. The minimum detected total RNA concentration of the third-stage larvae of A.cantonensis was 105 pg by PCR assay. CONCLUSION: A PCR assay has been developed for detecting A.cantonensis larva in Pomacea canaliculata.