Real-time RT-PCR and SYBR Green I melting curve analysis for the identification of Plum pox virus strains C, EA, and W: effect of amplicon size, melt rate, and dye translocation.
J Virol Methods
; 132(1-2): 146-53, 2006 Mar.
Article
em En
| MEDLINE
| ID: mdl-16293321
ABSTRACT
Real-time RT-PCR and SYBR green I melt curve analysis of a 74 bp amplicon enabled identification of Plum pox virus strains C, EA, and W, with distinct T(m)'s associated with each strain. This test is a useful supplement to a real-time RT-PCR test described earlier that was used to distinguish PPV strains D and M. A longer fragment of 155 bp was not effective for strain identification. A simplified one-tube protocol, with dithiothreitol eliminated from the reaction, showed similar sensitivity when compared to a two-tube protocol. For melt curve analysis, a slower melt rate of 0.1 degrees C/s, compared to 0.4 degrees C/s, was effective for detecting weak amplicons, and improved resolution of the T(m) of amplicons amplified simultaneously. SYBR green I was useful for duplex melt curve analysis. In repeated melt run treatments (total of 14) of a single sample containing co-amplified targets, complete translocation of SYBR green I was observed, going from a 74 bp fragment to a 114 bp fragment. The duration of the melt run may be a critical factor affecting SYBR green I binding and translocation, and its manipulation may facilitate improved resolution and simultaneous detection of multiple targets. This phenomenon may explain inconsistent SYBR green I fluorescence patterns associated with melt curve analysis of some amplicon complexes.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Compostos Orgânicos
/
Doenças das Plantas
/
Vírus Eruptivo da Ameixa
/
Reação em Cadeia da Polimerase Via Transcriptase Reversa
Tipo de estudo:
Diagnostic_studies
/
Guideline
/
Prognostic_studies
Idioma:
En
Revista:
J Virol Methods
Ano de publicação:
2006
Tipo de documento:
Article