Rapid and reliable detection of phytoplasma by loop-mediated isothermal amplification targeting a housekeeping gene

Sugawara, Kazuki; Himeno, Misako; Keima, Takuya; Kitazawa, Yugo; Maejima, Kensaku; Oshima, Kenro; Namba, Susumu

doi:10.1007/s10327-012-0403-9

Cited by 42 publications

(24 citation statements)

References 43 publications

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“…The groEL gene has been used to differentiate 'Ca.P. asteris'-related strains (Mitrović et al 2011;Alvarez et al 2013), and as target to develop diagnostic methods (Sugawara et al 2012). In our study, 16S and cpn60 UT sequence analysis resulted in the identification of four consensus sequences representing two strains, MBS-Puebla and MBS-Veracruz, both belonging to the subgroup 16SrI-B.…”

Section: Discussionmentioning

confidence: 49%

“…Recently, other ribosomal and protein-encoding genes have been used to improve phytoplasma strain differentiation and/or to develop diagnostic methods (Lee et al 2010;Davis et al 2013;Dumonceaux et al 2014). The Cpn60-encoding gene has been successfully used as a marker for phytoplasma detection and differentiation (Mitrović et al 2011;Sugawara et al 2012;Alvarez et al 2014), and an approximately 550 bp portion of this gene, known as the chaperonin 60 universal target (cpn60UT) (Goh et al 1996) can be universally accessed and used to differentiate phytoplasmas (Pérez-López et al 2014;Dumonceaux et al 2014). …”

Section: Introductionmentioning

confidence: 99%

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Maize bushy stunt phytoplasma affects native corn at high elevations in Southeast Mexico

et al. 2016

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In the 2013-2014 growing season, field surveys were conducted in native corn fields located in high altitude agricultural communities in the 'Sierra Norte de Puebla' in Mexico. Symptoms typical of maize bushy stunt (MBS) disease were observed and DNA extracted from symptomatic native corn plants was used as template to confirm the presence of phytoplasmas.Amplification and sequencing of 16S rRNA-encoding sequences and chaperonin 60 universal target (cpn60 UT) sequences followed by in vitro restriction fragment length polymorphism and phylogenetic analyses revealed that the phytoplasma detected belongs to the subgroup 16SrI-B, 'Candidatus Phytoplasma asteris'. Based on 16S rRNA-encoding gene sequence analysis and on a single nucleotide polymorphism within the cpn60 UT sequence, two MBS strains, MBS-Puebla and MBS-Veracruz, were identified. This is the first detection of MBS phytoplasma (MBSP) affecting native corn and the first molecular survey made in corn fields in Mexico to detect and characterize MBSP. We discuss these results in light of the potential evolutionary relationship between corn and MBSP.

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Section: Discussionmentioning

confidence: 49%

Section: Introductionmentioning

confidence: 99%

Maize bushy stunt phytoplasma affects native corn at high elevations in Southeast Mexico

et al. 2016

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“…El genoma publicado de "Ca. Phytoplasma onion yellows" (OY-M) (Oshima et al, 2004), fue utilizado por Sugawara et al (2012) para el diseño de oligonucleótidos a partir del gen conservado groEL. La sensibilidad de la técnica, el espectro y la robustez de su estudio fueron probados a partir del diseño de diecinueve juegos de oligonucleótidos analizados en distintos grupos de fitoplasmas.…”

Section: Amplificación Isotérmica (Lamp)unclassified

Compilación de oligonucleótidos para la detección y clasificación de fitoplasmas

et al. 2018

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Phytoplasmas are phytopathogenic prokaryotes of great importance because they have been linked to numerous diseases around the world. The aim of these research was to present the general characteristics, the size and composition of the genome and the genes and/or regions used as molecular markers for the identification and characterization of phytoplasmas. Among its main hosts are fruit and wood trees, vegetables, cut flowers and weeds, which generate alterations in the hormonal balance producing symptoms such as philodias and virescence. Because its isolation in vitro has not been possible, the detection and characterization has been carried out, mainly, with molecular techniques. In addition, the use of New Generation Sequencing (NGS) has allowed to know the complete genome of some phytoplasmas, as well as the regions and genes that constitute it. In the present bibliographic review, the information generated from the molecular techniques and NGS sequencing is compiled, as well as the primers reported to identify some groups of phytoplasmas.

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“…The LAMP method has also been adapted to detect RNA through the addition of a reverse transcriptase and is referred to as reverse transcription LAMP (RT-LAMP) (Notomi et al 2000). Due to its simple operation and costeffectiveness, LAMP and RT-LAMP have been used increasingly to diagnose plant diseases caused by viruses and virus-like pathogens (Boubourakas et al 2009;Fukuta et al 2003Fukuta et al , 2004Okuda et al 2005;Sugawara et al 2012).…”

Section: Introductionmentioning

confidence: 99%

Rapid detection of fig mosaic virus using reverse transcription loop-mediated isothermal amplification

et al. 2015

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Fig mosaic virus (FMV), a negative-strand RNA virus, is a causal agent of fig mosaic disease, which occurs in almost all countries where figs are grown and severely affects worldwide fig production.Reverse transcription-polymerase chain reaction (RT-PCR) using FMV-specific primers has typically been used to detect FMV, but faster and easier detection methods for FMV are required because RT-PCR requires multiple steps and special equipment. In this study, we established a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method to detect FMV. We designed LAMP primer sets based on sequence conservation among FMV isolates. The RT-LAMP reaction using a primer set targeting RNA3 in total RNA extracted from FMV-infected fig leaves resulted in rapid detection within 15 min. In addition, we established a fast and simple method of direct sampling from fig leaves using a wooden toothpick. The RT-LAMP reaction specificity and reactivity when using the direct sampling method were almost comparable to those obtained using isolated total RNA. Moreover, geographically and phylogenetically distinct FMV isolates were detectable using the FMV RT-LAMP assay. The assay presented here provides a practical method to detect FMV.

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Rapid and reliable detection of phytoplasma by loop-mediated isothermal amplification targeting a housekeeping gene

Cited by 42 publications

References 43 publications

Maize bushy stunt phytoplasma affects native corn at high elevations in Southeast Mexico

Maize bushy stunt phytoplasma affects native corn at high elevations in Southeast Mexico

Compilación de oligonucleótidos para la detección y clasificación de fitoplasmas

Rapid detection of fig mosaic virus using reverse transcription loop-mediated isothermal amplification

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