A rotor-gene SYBR Green base real time system was developed for the detection and quantification of Betanodavirus. Two primers set were designed to target conserved sequence in the nodavirus capsid protein gene, yielding an amplification product of 122 and 116 nucleotides. A plasmid was cloned and quantify for producing standard curve. The method was then apply to cDNA retrotranscripted from RNA extracted from cell-supernatants and directly from tissues of infected animals. The assay allowed analitycal enumeration of 300 copies of plasmid and 500 target cDNA copies for primer set 1 and 10 copies of plasmid and 20 target cDNA copies for primer set 2. The reproducibility of the assay was evaluated by intra-assay variability of standard control that showed a coefficient of variation (CV) ranging from 1.5 to 4.6 and from 1.5 to 4.9 respectively for the two assaysas. Intra-assay and inter-assay variability were further assessed using high and low amounts of cDNA showing a high reproducibility. The method revealed to be very sensitive and quick, reducing post-PCR processing and suitable for both diagnostic and pathogenesis studies.
Detection and quantification of betanodavirus by real time PCR
GALLETTI, Elena;
2006-01-01
Abstract
A rotor-gene SYBR Green base real time system was developed for the detection and quantification of Betanodavirus. Two primers set were designed to target conserved sequence in the nodavirus capsid protein gene, yielding an amplification product of 122 and 116 nucleotides. A plasmid was cloned and quantify for producing standard curve. The method was then apply to cDNA retrotranscripted from RNA extracted from cell-supernatants and directly from tissues of infected animals. The assay allowed analitycal enumeration of 300 copies of plasmid and 500 target cDNA copies for primer set 1 and 10 copies of plasmid and 20 target cDNA copies for primer set 2. The reproducibility of the assay was evaluated by intra-assay variability of standard control that showed a coefficient of variation (CV) ranging from 1.5 to 4.6 and from 1.5 to 4.9 respectively for the two assaysas. Intra-assay and inter-assay variability were further assessed using high and low amounts of cDNA showing a high reproducibility. The method revealed to be very sensitive and quick, reducing post-PCR processing and suitable for both diagnostic and pathogenesis studies.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
