Detection of DNA and RNA plant viruses by PCR and RT-PCR using a rapid virus release protocol without tissue homogenization

Thomson, Darelle; Dietzgen, Ralf G.

doi:10.1016/0166-0934(95)00022-m

Cited by 102 publications

(34 citation statements)

References 19 publications

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“…Using crude preparation of tissues, apple stem grooving virus, potato virus Y and banana streak virus were detected by RT-PCR or real-time PCR (Marinho et al, 1998;Chandelier et al, 2001;Delanoy et al, 2003). Besides, simple and rapid methods for nucleic acid liberation towards suitable and sensitive detection of some viruses and viroids by PCR have been described by Thomson and Dietzgen (1995) and Nakahara et al (1999), respectively.…”

Section: Discussionmentioning

confidence: 99%

“…The DNA extraction efficiency of this crude preparation method was further assessed by semi-quantitative evaluation of phytoplasma DNA using real-time PCR. The application of crude preparation of infected plants has been previously tested for virus detection by PCR amplification (Marinho et al, 1998;Marbot et al, 2002;Delanoy et al, 2003); furthermore a simple plant tissue preparation without homogenization suitable for detection of viruses by PCR was described by Thomson and Dietzgen (1995).…”

Section: Introductionmentioning

confidence: 99%

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A simple and rapid protocol of crude DNA extraction from apple trees for PCR and real-time PCR detection of ‘Candidatus Phytoplasma mali’

Aldaghi

Massart

Dutrecq

et al. 2009

Journal of Virological Methods

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a b s t r a c tDifferent PCR protocols have been established for detection of European fruit trees phytoplasmas; however the majority of the procedures for extracting phytoplasma DNA are complex, time consuming, and expensive, with a risk of contamination or loss of target DNA. In present study, a crude extract preparation method previously used to detect other plant pathogens was adapted to samples from apple trees infected by 'Candidatus Phytoplasma mali'. End-point and real-time PCR detection of 'Ca. P. mali' were used to compare this extraction procedure with an established method for efficient extraction of purified DNA. The crude extract proved fully adequate for phytoplasma detection in samples from 86 in vitro and 35 in vivo apple shoots or plants and 10 periwinkle plants. High inter-and intra-run reproducibility was obtained for phytoplasma detection with different TaqMan MGB-or SYBR Green-based real-time PCR protocols applied to the crude extracts. Real-time PCR applied to serially diluted crude and purified extracts revealed the same phytoplasma detection limit (dilution up to 10 5 ). All results confirm the suitability of this simple, quick, efficient extraction technique for accurate detection of 'Ca. P. mali' in different types of apple and periwinkle samples.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A simple and rapid protocol of crude DNA extraction from apple trees for PCR and real-time PCR detection of ‘Candidatus Phytoplasma mali’

Aldaghi

Massart

Dutrecq

et al. 2009

Journal of Virological Methods

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“…PCR amplifications were carried out using diluted extracts (1:100 in distilled water). The PCR amplification was performed using specific primer pairs previously described for BBTV CP and DNA-R fragments (Harding et al, 1993;Thomson and Dietzgen, 1995;Amin et al, 2008). All 79 samples were first subjected to the PCR amplification of a 349 bp fragment of the putative BBTV replicase gene using primer pairs BBT1 forward (5'-CTCGTCATGTGCAAGGTTATGTCG-3') and BBT2 reverse (5'-GAAGTTCTCCAGCTATTCATCGCC-3') for detection of the virus in different samples (Harding et al, 1993;Thomson and Dietzgen, 1995).…”

Section: S1mentioning

confidence: 99%

“…These samples were collected from diverse local banana genotypes namely AAA-EA, ABB, AAB and AABB types cultivated at different altitudes across the three countries. Diagnostic tests confirming the viral status of samples were performed using previously described PCR analysis (Harding et al, 1993;Thomson and Dietzgen, 1995).…”

Section: Samplingmentioning

confidence: 99%

Towards understanding the diversity of banana bunchy top virus in the Great Lakes region of Africa

Niyongere¹,

Lepoint²,

Losenge³

et al. 2015

Afr. J. Agric. Res.

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The genetic variability of banana bunchy top virus (BBTV) isolates from the Great Lakes region of Africa (GLRA) spanning Burundi, the Democratic Republic of the Congo and Rwanda was assessed to better understand BBTV diversity and its epidemiology for improved disease management. DNA-R and DNA-S fragments of the virus genome were amplified and sequenced in this study. These two BBTV fragments were previously used to classify isolates into the South Pacific and the Asian groups. Phylogenetic analyses based on nucleotide sequences involving GLRA isolates and those obtained from the GenBank database were carried out. Sequence similarity for both DNA-R and DNA-S fragments ranged between 99.1 to 100.0% among the GLRA isolates, 96.2 to 100.0% and 89.7 to 94.3% between the GLRA isolates and those previously clustering in the South Pacific and the Asian groups, respectively. These results showed that GLRA isolates belong to the South Pacific group and are phylogenetically close to the reference Indian isolate. The similar banana cultivars and BBTV isolates across the GLRA implied that the disease may have mainly spread through exchange of planting material (suckers) between farmers. Thus, farmers' awareness and quarantine measures should be implemented to reduce BBTV spread in the GLRA.

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“…To mitigate or prevent a viral disease from occurring, the conventional RT-PCR has become the one routine technique to detect the plant RNA viruses due to its sensitivity and ready availability (Thomson and Dietzgen, 1995). So far, however, little attention was paid to the primer-independent cDNA synthesis during plant RNA virus identification or diagnosis, probably because this event has no or little effect on the final result of common RT-PCR used for these purposes.…”

mentioning

confidence: 99%

Self-priming on the plant viral RNAs during reverse transcription-PCR

Zhang¹,

Wu²,

Zhang³

et al. 2015

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Summary. -The occurrence of the primer-independent cDNA synthesis during RT-PCR analysis of human and animal RNA viruses has been well documented. Conversely, there is scant knowledge about this event in plant RNA viruses. Here we show that the primer-independent cDNA synthesis occurs in all eight different plant RNA viruses tested in this study, suggesting a common phenomenon for RT-PCR analysis of plant RNA viruses. Additional experiments indicate that the event is likely contributed to by RNA self-priming, and can be effectively reduced or eliminated through increasing temperature of the RT reaction.Keywords: RT-PCR; primer-independent cDNA synthesis; self-priming; plant RNA virus; RT temperature * Corresponding author. E-mail: liweimin01@caas.cn; phone: +86-10-82106117. C. Zhang. and H.N. Wu contributed equally to this work. Abbreviations: CGMMV = cucumber green mottle mosaic virus; CMV = cucumber mosaic virus; gRNA = genomic RNA; ORSV = odontoglossum ring-spot virus; PVX = potato virus X; TCV = turnip crinkle virus; TMV U1 = tobacco mosaic virus U1 strain; TNV = tobacco necrosis virus; TRV = tobacco rattle virus RT-PCR is one of the most common methods of molecular biology used to detect and quantify RNA expression levels in both cells and small quantities of tissues. Within this process, RT is the first and fundamental step, in which the RNA template is converted into its cDNA by reverse transcriptase in the presence of exogenous oligonucleotide referred to as primer, providing template DNA for subsequent PCR amplification. The primer-dependent mechanism for RT reaction has been generally accepted over the years. However, during RT-PCR analysis of human and animal RNA viruses, bacterial operons, as well as eukaryotic cellular RNAs, it has been observed that cDNA could be synthesized by reverse transcriptase without the addition of exogenous oligonucleotide (Gunji et al

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Detection of DNA and RNA plant viruses by PCR and RT-PCR using a rapid virus release protocol without tissue homogenization

Cited by 102 publications

References 19 publications

A simple and rapid protocol of crude DNA extraction from apple trees for PCR and real-time PCR detection of ‘Candidatus Phytoplasma mali’

A simple and rapid protocol of crude DNA extraction from apple trees for PCR and real-time PCR detection of ‘Candidatus Phytoplasma mali’

Towards understanding the diversity of banana bunchy top virus in the Great Lakes region of Africa

Self-priming on the plant viral RNAs during reverse transcription-PCR

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