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

https://doi.org/10.1016/j.jviromet.2005.10.004Get rights and content

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 Tm'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 °C/s, compared to 0.4 °C/s, was effective for detecting weak amplicons, and improved resolution of the Tm 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.

Introduction

Real-time PCR with SYBR Green I melting curve analysis is a simple and reliable technique that has been effective for the detection and identification of various pathogens. These include Leishmania species (Nicolas et al., 2002), animal RNA viruses such as Norwalk-like viruses and viruses infecting penaeid shrimp (Beuret, 2004, Mouillesseaux et al., 2003), and plant RNA viruses such as Plum pox virus (PPV; Varga and James, 2005). This approach to real-time PCR can be adapted for quantitative analysis of the target(s) of interest (Papin et al., 2004). SYBR Green I dye binds non-specifically to double-stranded DNA by intercalation and/or minor groove binding (Lekanne Deprez et al., 2002, Mouillesseaux et al., 2003, Zipper et al., 2004). Specific identification may be achieved by melting curve analysis that can be used for identification at the species level (Nicolas et al., 2002), or even identification of strains of a virus pathogen (Varga and James, 2005).

SYBR green I based detection methods are reliable for detecting nucleic acid targets characterized by sequence variability. Papin et al. (2004) found that use of a probe based assay such as TaqMan resulted in failure to detect 47% of possible single nucleotide variants of West Nile virus, whereas a SYBR green I based assay was just as sensitive, and more importantly, it detected 100% of possible variants. Richards et al. (2004) indicated that in the case of Noroviruses, the use of degenerate primers facilitate broad spectrum detection but that probe-type approaches such as TaqMan require high complementarity for probe binding. This may result in failure to detect viruses that have high sequence variability in the probe-binding region. This uncertainty or false negative result is unacceptable in situations where; (a) the result might affect early initiation of treatment which could make the difference between life and death, and (b) where a false negative result might contribute to the release/introduction of plants infected with pathogens, such as aphid-vectored PPV (Avinent et al., 1994), that may be spread to other hosts in the vicinity. Other advantages of SYBR green-based real-time PCR assays include; easy identification of spurious or non-specific amplification, the ability to detect uncharacterized variants, and reduced time for analysis (Papin et al., 2004, Richards et al., 2004, Varga and James, 2005).

PPV is considered the most serious disease affecting stone fruits, members of the Prunus spp. (Nemeth, 1986). There are six recognized strains of PPV including D, M, EA, C, Rec, and W (Wetzel et al., 1991b, Cambra et al., 1994, Nemchinov et al., 1998, Glasa et al., 2004, James and Varga, 2005). These strains vary in aphid transmission, geographic distribution, host range, and pathogenicity, so strain identification is essential for effective control of the virus and improved understanding of the epidemiology of the associated disease.

Varga and James (2005) recently described a real-time multiplex PCR assay using SYBR green I and melt curve analysis for identification of members of the two major strains of PPV, strains D and M. This approach is relatively simple, and more rapid than previously described PPV strain typing methods including RT-PCR with RFLP analysis (Wetzel et al., 1991a), and integrated RT-PCR/nested PCR (Szemes et al., 2001). Varga and James (2005) demonstrated reliable identification of isolates of strain D and M in both herbaceous and woody hosts. Strain specific forward primers amplifying fragments of different sizes were combined with universal PPV primers, and primers targeting the endogenous NADH dehydrogenase gene (Menzel et al., 2002), in a multiplex system. This facilitated simultaneous PPV detection, D or M strain identification, and detection of an endogenous control that reduces false negative results. The 74 bp universal fragment, amplified for isolates of all PPV strains tested (Varga and James, 2005), had melting temperature characteristics with the potential to facilitate identification of isolates of strains other than D and M. In this study, real-time RT-PCR with SYBR green I melting curve analysis of the 74 bp fragment was evaluated for supplementary use in the identification of PPV strains C, EA, and W. Fragment size was assessed for efficacy by comparisons of the 74 bp fragment with a 155 bp fragment. Also, factors such as melt rate and the phenomenon of dye translocation were assessed for their effects on reliable detection and strain identification of PPV.

Section snippets

Virus source

PPV C (a sweet cherry isolate) and PPV EA (El Amar) were obtained as freeze dried tissue samples from A. Myrta, Italy. These isolates were maintained in the herbaceous host Nicotiana benthamiana. PPV 2630 (D-2630) is a Canadian type D isolate mechanically sap-transmitted from peach (Prunus persica var. Redhaven) to N. benthamiana. The virus isolate W3174 is a Canadian isolate of PPV detected in plum (Prunus domestica), and mechanically sap-transmitted to N. benthamiana. This isolate represents

One tube real-time multiplex RT-PCR assay versus two-tube assay; sensivitity comparison

The one-tube real-time multiplex RT-PCR assay, as adapted from the two-tube procedure, was more sensitive with a broader range of detection. Amplification was observed with serially water-diluted total RNA extracts from infected herbaceous hosts, at 10−6 dilution (data not shown). Dilution of cDNA was no longer required with the one tube RT-PCR thus limiting and reducing any chance for contamination and/or technical error. This indicates a more robust reaction. Also, the initial cDNA synthesis

Discussion

Real-time PCR is a powerful diagnostic tool capable of rapidly generating reliable and reproducible results with reduced risks of cross contamination (MacKay, 2004). In this study, single tube real-time RT-PCR with SYBR green I dye and melting curve analysis of a 74 bp amplicon were used for reliable identification of isolates of PPV strains C, EA, and W. When combined with the protocol described by Varga and James (2005) specific strain typing of members of all strains of PPV is possible,

Acknowledgements

We wish to express our gratitude to Arben Myrta for freeze dried samples of PPV C (a sweet cherry isolate) and PPV EA. Also, we would like to thank the CFIA St. Catherines PPV survey crew for providing samples of PPV W3174 and PPV D (2630).

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