Development of real-time quantitative PCR (QPCR) for the detection and enumeration of Pseudo-Nitzschia (Bacillariophyceae)

Chui, Siew Jin. (2014) Development of real-time quantitative PCR (QPCR) for the detection and enumeration of Pseudo-Nitzschia (Bacillariophyceae). [Final Year Project Report] (Unpublished)

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Abstract

Amnesic Shellfish Poisoning (ASP) has been known to be a life-threatening phenomenon upon consumption of contaminated shellfish that contains domoic acid (DA), a neurotoxin produced by the diatom Pseudonitzschia. One third of the species of Pseudo-nitzschia have been known to produce DA. Thus, the aim of this study is to develop a rapid and sensitive detection tool for identification of the morphologically closely similar species of Pseudo-nitzschia. In this study, a real-time quantitative polymerase chain reaction (qPCR) assay was used to identify and quantify Pseudo-nitzschia in the natural samples. Samples were collected along the coast of Sarawak. Isolation of single-chain cells frqm the field was carried out by micropipetting technique. Clonal cultures were established and grown in sterile F/2 medium with a pH of 7.8-7.9, 25 ± 0.5 oc, under a 12:12 h L:D photoperiod. A total of three strains of Pseudo-nitzschia were identified under transmission electron microscope (TEM). They are Pseudo-nitzschia pungens. A pair of species-specific primers targeting the internal transcribed spacer (ITS) region of ribosomal RNA gene was designed in silico. Genomic DNA of Pseudo-nitschia and dinoflagellates was extracted and amplified with the primers to check for the primer specificity. A total of four Pseudo-nitzschia strains were successfully amplified with a length of approximately 150 bp. The amplicons were then purified and sequenced. Sequence analysis showed that high similarity to P. pungens. The primer specificity was confirmed and verified using qPCR approach. The primers specifically targeted samples of Pseudo-nitzschia but no amplification on non-targeted products based on the results of melting and quantification curve. qPCR is believed to be a rapid, cost-effective method for detecting the species of Pseudo-nitzschia in accordance to the incidence of ASP.

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