Use of dried high-phenolic laden host leaves for virus and viroid preservation and detection by PCR methods

Sipahioğlu, Hikmet Murat; Usta, Mustafa; Ocak, M.

doi:10.1016/j.jviromet.2006.06.009

Cited by 36 publications

(20 citation statements)

References 22 publications

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“…While a portion of the same sample was stored at −80°C another part dried by a previously reported method and stored at 4°C (Sipahioğlu et al, 2006). The results reported that leaf samples can effectively be used after they are dried.…”

Section: Discussionmentioning

confidence: 99%

Genetic Diversity in the Coat Protein Genes of Prune dwarf virus Isolates from Sweet Cherry Growing in Turkey

Öztürk

Çevik

2015

The Plant Pathology Journal

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Sweet cherry is an important fruit crop with increasing economical value in Turkey and the world. A number of viruses cause diseases and economical losses in sweet cherry. Prune dwarf virus (PDV), is one of the most common viruses of stone fruits including sweet cherry in the world. In this study, PDV was detected from 316 of 521 sweet cherry samples collected from 142 orchards in 10 districts of Isparta province of Turkey by double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA). The presence of PDV in ELISA positive samples was confirmed in 37 isolates by reverse transcription- polymerase chain reaction (RT-PCR) method. A genomic region of 862 bp containing the coat protein (CP) gene of PDV was re-amplified from 21 selected isolates by RT-PCR. Amplified DNA fragments of these isolates were purified and sequenced for molecular characterization and determining genetic diversity of PDV. Sequence comparisons showed 84–99% to 81–100% sequence identity at nucleotide and amino acid level, respectively, of the CP genes of PDV isolates from Isparta and other parts of the world. Phylogenetic analyses of the CP genes of PDV isolates from different geographical origins and diverse hosts revealed that PDV isolates formed different phylogenetic groups. While isolates were not grouped solely based on their geographical origins or hosts, some association between phylogenetic groups and geographical origins or hosts were observed.

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Section: Discussionmentioning

confidence: 99%

Genetic Diversity in the Coat Protein Genes of Prune dwarf virus Isolates from Sweet Cherry Growing in Turkey

Öztürk

Çevik

2015

The Plant Pathology Journal

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“…In addition, the existence of polysaccharides may reduce the capacity to resuspend precipitated RNA (Wilkins and Smart ; Sipahioglu et al . ). Reverse transcription may be inhibited, for example, by direct interaction of the enzyme with melanin (Eckhart et al .…”

Section: Mechanisms Of Action Of Pcr Inhibitorsmentioning

confidence: 97%

PCR inhibitors - occurrence, properties and removal

et al. 2012

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Summary The polymerase chain reaction (PCR) is increasingly used as the standard method for detection and characterization of microorganisms and genetic markers in a variety of sample types. However, the method is prone to inhibiting substances, which may be present in the analysed sample and which may affect the sensitivity of the assay or even lead to false‐negative results. The PCR inhibitors represent a diverse group of substances with different properties and mechanisms of action. Some of them are predominantly found in specific types of samples thus necessitating matrix‐specific protocols for preparation of nucleic acids before PCR. A variety of protocols have been developed to remove the PCR inhibitors. This review focuses on the general properties of PCR inhibitors and their occurrence in specific matrices. Strategies for their removal from the sample and for quality control by assessing their influence on the individual PCR test are presented and discussed.

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“…Phenol levels may be reduced with the use of polyvinyl-pyrrolidone or the addition of 467 borate (Wilkins and Smart, 1996). Drying plant samples at 65°C for 2 days, prior to DNA extraction, 468 could also help to cancel out the effect of phenolic inhibitors (Sipahioglu et al, 2006). 469…”

Section: Identification Of Xf Subspecies In Environmental Plant Samplmentioning

confidence: 99%

Novel tetraplex qPCR assays for simultaneous detection and identification ofXylella fastidiosasubspecies in plant tissues

Dupas

Briand

Jacques

et al. 2019

Preprint

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AbstractXylella fastidiosa is an insect-borne bacterium confined to the xylem vessels of plants. This plant pathogen has a broad host range estimated to 560 plant species. Five subspecies of the pathogen with different but overlapping host ranges have been described, but only three subspecies are widely accepted, namely subspecies fastidiosa, multiplex and pauca. Initially limited to the Americas, Xf has been detected in Europe since 2013. As management of X. fastidiosa outbreaks in Europe depends on the identification of the subspecies, accurate determination of the subspecies in infected plants as early as possible is of major interest. Thus, we developed various tetraplex and triplex qPCR assays for Xylella fastidiosa detection and subspecies identification in planta in a single reaction. We designed primers and probes using SkIf, a bioinformatics tool based on k-mers, to detect specific signatures of the species and subspecies from a dataset of 58 genome sequences representative of X. fastidiosa diversity. We tested the qPCR assays on 39 target and 30 non-target strains, as well as on 13 different plant species spiked with strains of the different subspecies of X. fastidiosa, and on samples from various environmental and inoculated host plants. Sensitivity of simplex assays was equal or slightly better than the reference protocol on purified DNA. Tetraplex qPCR assays had the same sensitivity than the reference protocol and allowed X. fastidiosa detection in all spiked matrices up to 103 cells.mL−1. Moreover, mix infections of two to three subspecies could be detected in the same sample with tetraplex assays. In environmental plant samples, the tetraplex qPCR assays allowed subspecies identification when the current method based on multilocus sequence typing failed. The qPCR assays described here are robust and modular tools that are efficient for differentiating X. fastidiosa subspecies directly in plant samples.

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Use of dried high-phenolic laden host leaves for virus and viroid preservation and detection by PCR methods

Cited by 36 publications

References 22 publications

Genetic Diversity in the Coat Protein Genes of Prune dwarf virus Isolates from Sweet Cherry Growing in Turkey

Genetic Diversity in the Coat Protein Genes of Prune dwarf virus Isolates from Sweet Cherry Growing in Turkey

PCR inhibitors - occurrence, properties and removal

Novel tetraplex qPCR assays for simultaneous detection and identification ofXylella fastidiosasubspecies in plant tissues

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