Absolute and relative Q-PCR quantification of cryptic species of the Rhabditis (Pellioditis) marina species complex
Tezerji, R.S. (2010). Absolute and relative Q-PCR quantification of cryptic species of the Rhabditis (Pellioditis) marina species complex. MSc Thesis. Universiteit Gent. Faculteit Wetenschappen. Vakgroep Biologie: Gent. 41 pp.
Absolute and relative Q-PCR quantification of cryptic species of the Rhabditis (Pellioditis) marina species complex’’ Raheleh Sheibani Tezerji Abstract A quantitative real-time polymerase chain reaction (qPCR) assay using SYBR Green (double-stranded DNA binding dye) was developed for rapid detection and quantification of cryptic species of the Rhabditis (Pellioditis) marina species complex. Previously published sequences of ribosomal internal transcribed spacer region (ITS1, 5.8S and ITS2) of R. (P.) marina were used to design primers to target unique, conserved regions for each of the four dominant cryptic species occurring in Belgium and The Netherlands. The qPCR assay showed to be efficient and specific for each species. In this study two compatible methods based on absolute and relative analyses were tested. Absolute quantification determines the exact copy number of ITS by relating the cycle at which a signal is observed (= Ct value) to a standard curve which contains a serial dilution of a known concentration of ITS. The data were calculated by Second Derivative Maximum analysis method and ddCt method. For each species, the copy number of ITS was assessed by applying the qPCR assay on one nematode specimen. The obtained range of ITS copy number was then used to calculate the absolute number of specimens from each species. For the relative quantification, a mixture of DNA from the four species was used. Species were present in the following proportions: 25:25:25:25, 50:16:16:16 or 99:1. Relative quantification was able to detect these differences. The methods introduced in this study are convenient to detect and quantify cryptic species of R. (P). marina. Keywords: Rhabditis (Pellioditis) marina, Real-time quantitative PCR (QPCR); Absolute quantification; Relative quantification.
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