Development of real-time PCR based assays for simultaneous and improved detection of citrus viruses

Loconsole, Giuliana; Saponari, María; Savino, V.

doi:10.1007/s10658-010-9653-6

Cited by 28 publications

(18 citation statements)

References 34 publications

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“…; Loconsole et al. ), but no comparison has been done with TAS‐ELISA or biological indexing. Here, we report for the first time the use of a RT‐qPCR as a diagnostic tool in Argentina, applying a new and less‐expensive protocol using SYBR Green.…”

Section: Discussionmentioning

confidence: 99%

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Improved Detection of Citrus psorosis virus and Coat Protein‐Derived Transgenes in Citrus Plants: Comparison Between RT‐qPCR and TAS‐ELISA

Francesco

Costa

Plata

et al. 2015

Journal of Phytopathology

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Citrus is one of the most economically important fruit crops in the world. Citrus psorosis is a serious disease affecting mainly oranges and mandarins in Argentina and Uruguay. The causal agent is Citrus psorosis virus (CPsV), an ophiovirus with a tripartite ssRNA genome of negative polarity. The coat protein (CP), the most abundant viral protein in infected plants, has been used to detect CPsV by TAS‐ELISA, but only biological indexing, requiring 1 year, is the current and validated technique for diagnosis of citrus psorosis. In this study, a SYBR Green RT‐qPCR protocol was developed, with primers designed to the most conserved region of the cp gene. We tested their specificity and sensitivity in comparison with TAS‐ELISA. This RT‐qPCR was applied successfully to field samples from Argentina, to a variety of isolates from different countries maintained in the greenhouse, to young seedlings and old trees from a psorosis natural transmission plot, and to transgenic citrus expressing the cp gene of CPsV or a fragment thereof. This method allowed accurate quantification of viral titer and cp gene expression in transgenic plants, which could not be detected previously. The sensitivity and reliability of quantitative CPsV detection were improved with greater speed using commercial reagents, and the sensitivity was three orders of magnitude higher than that of TAS‐ELISA. All these data encourage its validation.

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Section: Discussionmentioning

confidence: 99%

“…Recently, many studies have shown the reliability of this tool even for the simultaneous detection of several viruses in citrus (Loconsole et al. ; Lin et al. ; Saponari et al.…”

Section: Introductionmentioning

confidence: 99%

Improved Detection of Citrus psorosis virus and Coat Protein‐Derived Transgenes in Citrus Plants: Comparison Between RT‐qPCR and TAS‐ELISA

Francesco

Costa

Plata

et al. 2015

Journal of Phytopathology

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“…Rapid and accurate multiple detection of CTV, CPsV and CVV was achieved using the triplex one step real-time RT-PCR protocol. These assays may prove useful as a tool to support quarantine, eradication and certification programs, resulting faster, more sensitive, relatively easier to perform and less cost effective than the conventional detection procedures [29].…”

Section: Real Time Rt-pcrmentioning

confidence: 99%

Recent advances in Citrus psorosis virus

2014

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“…In addition, these tests require specific indicator plants for each pathogen (Roistacher, 1991;Durán et al, 1993), which must be combined with the technique of sequential polyacrylamide gel electrophoresis (sPAGE). The most recent method for the detection of pathogens is PCR real time; however, this technique is still quite expensive, which is the principal problem for implementing its routine use in laboratories (López et al, 2003), though for detecting viral diseases in citrus, the results have been shown to be fast and reliable (Rizza et al, 2009;Loconsole et al, 2010).…”

Section: Multiple Rt-pcrmentioning

confidence: 99%

Simultaneous detection of CTV, CEVd and HSVd using Arizona 861 S1 Citron and RT-PCR

2014

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The simultaneous use of a rapid diagnostic method with the effective cleaning of infected budwood is a powerful preventative method for controlling viral diseases. The objective of this study was the multiple detection of CTV, CEVd, and HSVd, through bio-amplification in Arizona 861-S1 citron plants, combined with two incubation temperatures and multiple RT-PCR in two steps. Citron indicator plants were inoculated with the three pathogens, both individually and simultaneously, with four un-inoculated plants as controls. After four months of incubation at the two different temperatures, both the multiple and individually inoculated plants showed symptoms associated with these pathogens, which were subsequently corroborated through the use of multiple RT-PCR in two steps. This diagnostic method could be employed as an efficient, cost-effective alternative to the methods that are currently in use for detecting CTV, CEVd and HSVd, nevertheless its effectiveness should also be evaluated with tests using different isolates of these pathogens.

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Development of real-time PCR based assays for simultaneous and improved detection of citrus viruses

Cited by 28 publications

References 34 publications

Improved Detection of Citrus psorosis virus and Coat Protein‐Derived Transgenes in Citrus Plants: Comparison Between RT‐qPCR and TAS‐ELISA

Improved Detection of Citrus psorosis virus and Coat Protein‐Derived Transgenes in Citrus Plants: Comparison Between RT‐qPCR and TAS‐ELISA

Recent advances in Citrus psorosis virus

Simultaneous detection of CTV, CEVd and HSVd using Arizona 861 S1 Citron and RT-PCR

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