Development and validation of a multiplex RT-PCR method for the simultaneous detection of five grapevine viroids

Hajizadeh, Mohammad Reza; Navarro, Beatriz; Bashir, Nemat Sokhandan; Torchetti, Enza Maria; Serio, Francesco Di

doi:10.1016/j.jviromet.2011.09.022

Cited by 54 publications

(21 citation statements)

References 27 publications

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“…(Sano 2003) could therefore have resulted in the widespread occurrence of HSVd in these three cultivars in China. Furthermore, we found that the ubiquitous infection of hops by HLVd opens up the possibility of using the HLVd viroid as an internal control in multiplex RT-PCR, as has been previously proposed for HSVd in grapevine (Hajizadeh et al 2012).…”

Section: Resultssupporting

confidence: 67%

“…2007), tomato (Matsushita et al. 2010) and grapevine (Hajizadeh et al. 2012), no protocols have been developed for detecting concurrent viroid infections in hop.…”

Section: Introductionmentioning

confidence: 99%

“…Thus, reliable protocols that can be used for quick identification of diseased plants from hop yards or cultured materials need to be established. Although multiplex RT-PCR, a method for detecting simultaneous infections by more than one viroid species in the same host, has been reported for apple (Di Serio et al 2002), citrus (Ito et al 2002;Wang et al 2009), chrysanthemum (Hosokawa et al 2007), tomato (Matsushita et al 2010) and grapevine (Hajizadeh et al 2012), no protocols have been developed for detecting concurrent viroid infections in hop. Therefore, we developed a multiplex RT-PCR for the simultaneous detection of the three viroid species known to naturally infect hops in China.…”

Section: Introductionmentioning

confidence: 99%

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Simultaneous Detection of Three Viroid Species Infecting Hops in China by Multiplex RT‐PCR

Lu¹,

Zhang²,

Xu³

et al. 2012

Journal of Phytopathology

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We have developed a multiplex RT‐PCR protocol for the simultaneous detection of three viroids in three different genera that infect hops: Hop latent viroid (HLVd; Cocadviroid), Hop stunt viroid (HSVd; Hostuviroid) and Apple fruit crinkle viroid (AFCVd; Apscaviroid). The method was validated by testing 175 hop samples collected from the Xinjiang autonomous region of China. All samples were found to be positive for HLVd but negative for AFCVd, confirming the widespread or even ubiquitous infection of HLVd and the low incidence of AFCVd in hops in China. In addition, HSVd was detected in 22.86% of the samples tested. This rapid and reliable multiplex RT‐PCR assay provides an effective method for detection of three important viroid species in large‐scale surveys for disease management in hops.

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Section: Resultssupporting

confidence: 67%

“…2007), tomato (Matsushita et al. 2010) and grapevine (Hajizadeh et al. 2012), no protocols have been developed for detecting concurrent viroid infections in hop.…”

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Simultaneous Detection of Three Viroid Species Infecting Hops in China by Multiplex RT‐PCR

Lu¹,

Zhang²,

Xu³

et al. 2012

Journal of Phytopathology

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“…Developing reliable, cost‐effective methods for detecting viruses is imperative for controlling and preventing plant diseases. mRT‐PCR methods have been successfully applied in the past for the detection of viruses (Gambino and Gribaudo ; Jarosova and Kundu ; Hajizadeh et al ; Yu et al ). We previously showed that co‐infections with viruses are quite common in peach plants in China (Yu et al ; Lu et al ), with PaLV (Zhou et al ), NSPaV (Lu et al ) and PeVD (unpublished data) co‐infecting peach.…”

Section: Resultsmentioning

confidence: 99%

Multiplex RT‐PCR to simultaneously detect three viruses that infect peach

Yang

Zhou

et al. 2019

Lett Appl Microbiol

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Significance and Impact of the Study: In this study, we developed a multiplex reverse transcriptase PCR (mRT-PCR) assay for simultaneously detecting three viruses that infect peach: peach-associated luteovirus (PaLV), peach virus D (PeVD) and nectarine stem-pitting-associated virus (NSPaV). This assay is simple, easy to perform, reliable and cost-effective, and can thus be applied for large-scale surveys of PaLV, PeVD and NSPaV. AbstractPeach is a major crop in China, and like any other stone fruit, virus and viruslike diseases can reduce the yield and quality of the fruit. Herein, we developed a multiplex RT-PCR (mRT-PCR) assay for simultaneously detecting three viruses known to infect peach: peach-associated luteovirus (PaLV), peach virus D (PeVD) and nectarine stem-pitting-associated virus (NSPaV). Plant nad5 mRNA was used as the internal control. Field samples that were co-infected with PaLV, PeVD and NSPaV were used; we identified three primer pairs to be the most specific for detecting these viruses, followed by determining the ideal concentration of each primer pair and optimizing the annealing temperature for mRT-PCR. We also assessed the detection limit using serial dilutions of RNA and cDNA. The newly developed mRT-PCR assay could simultaneously detect PaLV, PeVD and NSPaV. To validate the reliability of mRT-PCR for virus detection, mRT-PCR was used to detect viruses in the leaves of 21 peach plants collected in Liaoning Province, China. The obtained results revealed the presence of single and co-infections. To conclude, the mRT-PCR assay developed herein is sensitive, reliable and economical, and we believe that it can thus be used for large-scale surveys of PaLV, PeVD and NSPaV.Letters in Applied Microbiology ISSN 0266-8254

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“…Simultaneous detection lessens costs, labour and time but, at the same time, the amount of amplified DNA is lower than with single RT-PCR (Ito et al, 2002a;Narayanasamy, 2011a). Nevertheless, multiplex RT-PCR reactions, such as fluorescent mRT-PCR (Di Serio et al, 2002), mRT-PCR-ELISA (Shamloul et al, 2002) and regular mRT-PCR (Nie & Singh, 2000;Ito et al, 2002a;Ragozzino et al, 2004;Hataya, 2009;Wang et al, 2009;Matsushita et al, 2010;Hajizadeh et al, 2012;Lin et al, 2012;Lu et al, 2012;Song et al, 2013;Gambino et al, 2014;Kumar et al, 2014;Olivier et al, 2014) are increasingly being used.…”

Section: Rt-pcrmentioning

confidence: 99%

Diagnostic techniques for viroids

et al. 2016

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The fast growth of the human population forces us to produce more food, but higher crop production also leads to the fast spread of diseases. Plant pathology deploys a wide range of methods that do not provide an adequate solution to all disease losses. In the case of viroids, therapeutic means of control are not available; therefore control strategies are more focused on the development of reliable detection methods to quickly exclude the infected plant material. Although viroids are the smallest and simplest plant pathogens, their identification and detection is not straightforward. Each viroid–host combination is specific, and for reliable identification, all steps from sampling to final detection must be performed accurately. In this review, several methods for viroid detection in various host plants are discussed, including their advantages and disadvantages. Even though relatively new molecular methods enable fast and sensitive detection of viroids, a combination of different methods gives the most reliable identification. Techniques based on nucleic acids may be the future for viroid detection but they still cannot replace biological indexing, which is usually essential in epidemiological and aetiological studies.

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Development and validation of a multiplex RT-PCR method for the simultaneous detection of five grapevine viroids

Cited by 54 publications

References 27 publications

Simultaneous Detection of Three Viroid Species Infecting Hops in China by Multiplex RT‐PCR

Simultaneous Detection of Three Viroid Species Infecting Hops in China by Multiplex RT‐PCR

Multiplex RT‐PCR to simultaneously detect three viruses that infect peach

Diagnostic techniques for viroids

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