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PCRs were monitored in real time by acquiring data with the SYBR Green channel (Ex 488?nm and Em 522?nm). The threshold cycles (Ct) were always lower than 30. The amplicons were processed immediately after the qPCR. Samples were heated to 95��C for 15?s, followed by 1?min at 60��C. They were melted from 60 to 95��C, and the fluorescence was recorded 3��3 times per degree with a temperature ramp rate of 2��2��C?s?1. The Tm was directly measured by the internal software (StepOne Software v2.1; Applied Biosystems). Normalization with ROX dye was not used. The efficiencies of PCRs (ranging from 1��85 to 19��7) were calculated according to Pfaffl��s method (Pfaffl 2001). For comparative purposes, the amplicons of the reference strains (Table?1) and those of a subset http://www.selleckchem.com/products/PD-0325901.html (29 out of 70) of Prototheca isolates were also sequenced using ABI Prism 3100 Avant Sequencer (Applied Biosystems). The sequences were analysed using the SeqMan Module of Lasergene Package (DNA Star, Madison, UK). To check the robustness of the method and to explore the existence of possible differences in the Tm change, we performed serial dilutions of DNA (by using the same elution buffer used during the DNA extraction), from 101 to 10?4?ng?��l?1, for three representative strains (P.?zopfii gen. 1 SAG 2063, P.?zopfii gen. 2 IZSLER 219509/2 and P.?blaschkeae SAG 2064). In addition, to avoid the effect of small amount of PCR product due by low starting DNA concentrations, that can lead to misleading identification of the appropriate curve, we prolonged the cycles until 40. http://www.selleck.cn/products/JNJ-26481585.html Melting temperature of different Prototheca spp. strains was expressed http://www.selleckchem.com/products/epz-6438.html as mean?��?standard deviation (SD). The significance of the differences was assessed by one-way anova. In agreement with the current literature (Sharma 1996), significance was accepted at the P?