Detecting Aphanomyces Invadans in Pure Cultures and EUS-infected Fish Lesions by Applying PCR
DOI:
https://doi.org/10.18034/mjmbr.v3i2.410Keywords:
PCR, EUS, Aphanomyces invadans, DNA extractionAbstract
Aphanomyces invadans is an oomycete fungus which causes Epizootic Ulcerative Syndrome (EUS) disease in wide range of fresh and brackish water fish worldwide imposing serious economic losses. A diagnostic procedure, based on a polymerase chain reaction method (PCR) was developed to detect infection of fish with the A.invadans. A set of primers (1APM 1F and 1APM 6R) was used to specifically amplify A. invadans DNA. The PCR amplifies a 400 bp amplicon. A protocol for the extraction of A. invadans DNA from infected fish tissue and pure fungal cultures was developed. The method was tested on seven EUS-susceptible fish species (snakehead, snakeskin gourami, moonlight gourami, koi carp, catfish, gold fish, climbing perch) and one EUS-resistant fish (tilapia), artificially infected with A. invadans and pure cultures of Aphanomyces spp., Saprolegnia spp., Achlya spp., and Allomyces sp. Detection of A. invadans was possible at the early stage of sampling, which was 24 hours post injection in both EUS-susceptible and resistant fish. Resistant fish was found to be PCR-negative after 6 days of inoculation but in susceptible fish PCR-positive results obtained even after day 28 or in dead fish. Therefore PCR may be a useful method for detection EUS infection in fish from early stage of disease onset.
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