Objective: To establish a multiplex PCR assay for detecting Angiostrongylus cantonensis larvae in Pomacea canaliculata.
Methods: A pair of specific primers was designed based on the sequences of the small subunit rDNA of A. cantonensis (GenBank jAY295804), in combination with 16s rDNA specific primers of P. canaliculata, a multiplex PCR was developed. The PCR was performed on positive and negative snails, and the amplified products were analyzed by agarose gel electrophoresis and DNA sequencing. DNA template of 200 III stage larvae of A. cantonensis was diluted by negative snail DNA (1200 ng/microl, 120 ng/microl, 12 ng/microl, 1200 pg/microl, 120 pg/microl and 12 pg/microl), to find the minimum detectable level. Single blind method was used to evaluate the accuracy. After being detected by lung microscopy, 172 snails from field were tested by the multiplex PCR to assess the sensitivity and specificity.
Results: Agarose gel electrophoresis and DNA sequencing analysis indicated that the target sequences were efficiently amplified by the PCR assay (550 bp for P. canaliculata, 405 bp for A. cantonensis). The minimum detectable level was 120 pg/microl. The coincidence between the two methods stood for 84.3% (145/172), including 45 positives and 100 negatives. 24 snails were PCR positive and microscopy negative, 3 snails were PCR negative and microscopy positive. The sensitivity and specificity of multiplex PCR was 93.8% and 80.6%, respectively. Its positive rate (40.1%, 69/172) was significant higher than that of lung-microscopy (27.9%, 48/172)(chi2 = 14.8, P < 0.01).
Conclusion: A multiplex PCR method has been developed for the detection of A. cantonensis larvae in P. canaliculata.