Detection of apple chlorotic leaf spot virus using a 5′ nuclease assay with a fluorescent 3′ minor groove binder-DNA probe

Abstract

The development of a real-time 5% nuclease RT-PCR assay for the detection of apple chlorotic leaf spot virus (ACLSV) from infected plant material is described. A short fluorogenic 3% minor groove binder-DNA hydrolysis probe was used to circumvent genome variability between isolates and target a short conserved sequence. The covalent attachment of the minor groove binder moiety at the 3% end of the probe increased the probe/target duplex stability and raised the melting temperature to a range suitable for real-time analysis. The method is rapid, sensitive and takes place within a single tube without post-PCR handling of the amplification products.

Host‐range studies, genetic diversity and evolutionary relationships of ACLSV isolates from ornamental, wild and cultivated Rosaceous species

“…1) and resulted in ACLSV detection in 154 samples from ornamental, wild and cultivated species of the Rosaceae family. It should be noted that most of the isolates detected by molecular methods did not react positively with the ELISA assays, probably due to low viral titres Menzel et al, 2002;Salmon et al, 2002). Therefore, for reliable ACLSV detection in certification and quarantine programmes, it is recommended that both ELISA and nested RT-PCR assays should be used.…”

Evaluation of Reference Genes for the Relative Quantification of Apple stem grooving virus and Apple mosaic virus in Apple Trees

“…We propose employing GAPDH and S19 as housekeeping genes for accurate quantification of ASGV and ApMV in apple leaf samples. The detection limit for both viruses was found around 70 copies of viral genome by one-step RT-qPCR.The fluorescence-based quantitative real-time PCR (qPCR) has been used to detect and measure nucleic acids of a wide range of plant viruses with RNA and DNA genome [13,16,17]. Two main qPCR based techniques: relative and absolute quantification have emerged from various studies on virus detection and quantification of virus titer.…”

“…The fluorescence-based quantitative real-time PCR (qPCR) has been used to detect and measure nucleic acids of a wide range of plant viruses with RNA and DNA genome [13,16,17]. Two main qPCR based techniques: relative and absolute quantification have emerged from various studies on virus detection and quantification of virus titer.…”

Development of Real-Time PCR for the Rapid Detection of Episomal Banana streak virus (BSV)

“…To date, applications of real-time PCR and particularly 5 nuclease assay in plant virology are increasing, but the genomic variability of the virus can impair the fluorogenic process with a single TaqMan probe. Recently, Salmon et al (23,24) described a 5 nuclease assay based on 3 MGB probes for the real-time detection of plant viruses (Apple chlorotic spot virus and Apple stem pitting virus). The chemical modification of the probe allows the use of suitable melting temperatures with shorter probes; this property is critical to target-conserved sequences in viruses with high genomic variability.…”