Development of primers and a procedure for specific identification of the diatom Thalassiosira weissflogii

Abstract

A recent study showed Thalassiosira weissflogii to be a diatom containing suitable nutrition for larviculture of the black tiger shrimp, Penaeus monodon. Accurate and practical identification of this diatom species is therefore important for commercial hatcheries. The purpose of this study was to establish a DNA-based method of identification to supplement morphological examination, avoiding confusion with other Thalassiosira sp. Primers, 18SF/28SR1, specific for ribosomal DNA genes (3′-end of 18S rDNA through 5′-end of 28S rDNA, covering two internal transcribed spacers), were employed as a first-step polymerase chain reaction, followed by a second nested amplification using specifically designed primers, ITS1-F-D/ITS1-R-D. The nested-PCR result revealed specificity in the detection, distinguishing T. weissflogii from T. pseudonana, Cyclotella meneghiniana, and Chaetoceros sp., and the PCR fragment of the amplified region had a sequence that was 99% identical to the T. weissflogii sequence held by GenBank.