Multiplex Real-Time qPCR Assay for Simultaneous and Sensitive Detection of Phytoplasmas in Sesame Plants and Insect Vectors

İkten, Cengiz; Ustun, Rustem; Catal, Mursel; Yol, Engin; Uzun, Bülent

doi:10.1371/journal.pone.0155891

Cited by 27 publications

(10 citation statements)

References 39 publications

(51 reference statements)

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“…When working with phytoplasma, it is important to have reference genes for a broad range of plant or insect host species available. Although several protocols are available for the detection of different phytoplasma species in single-or multiplex assays [14,[16][17][18]24], all of them use internal endogenous controls with a rather narrow host range. Only the 18S rDNA gene has been reported as a universal endogenous qPCR control, working with a broad range of plant species [26].…”

Section: Discussionmentioning

confidence: 99%

“…Nowadays, most tools for phytoplasma detection are based on quantitative PCR (qPCR) techniques, using intercalating DNA dyes (SYBR-Green) or hybridization probes (TaqMan ® ) [13]. Several qPCR protocols are available for the detection of phytoplasmas using different host specific endogenous internal controls [13][14][15][16][17][18][19]. Some of the available host specific internal controls can be used simultaneously with the phytoplasma specific primers in a multiplex qPCR assay [14,15,18,19], for others it is necessary to perform a separate qPCR run [20].…”

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confidence: 99%

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Development of a universal endogenous qPCR control for eukaryotic DNA samples

Mittelberger¹,

Obkircher²,

Oberkofler³

et al. 2020

Plant Methods

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Background: Phytoplasma are obligate intracellular plant-pathogenic bacteria that infect a broad range of plant species and are transmitted by different insect species. Quantitative real-time PCR (qPCR) is one of the most commonly used techniques for pathogen detection, especially for pathogens that cannot be cultivated outside their host like phytoplasma. PCR analysis requires the purification of total DNA from the sample and subsequent amplification of pathogen DNA with specific primers. The purified DNA contains mainly host DNA and only a marginal proportion is of phytoplasmal origin. Therefore, detection of phytoplasma DNA in a host DNA background must be sensitive, specific and reliable and is highly dependent on the quality and concentration of the purified DNA. DNA quality and concentration and the presence of PCR-inhibitors therefore have a direct impact on pathogen detection. Thus, it is indispensable for PCR-based diagnostic tests to validate the DNA preparation and DNA integrity before interpreting diagnostic results, especially in case that no pathogen DNA is detected. The use of an internal control allows to evaluate DNA integrity and the detection of PCR-inhibiting substances. Internal controls are generally host-specific or limited to a defined group of related species. A control suitable for the broad range of phytoplasma hosts comprising different insect and plant species is still missing. Results: We developed a primer and probe combination that allows amplification of a conserved stretch of the eukaryotic 28S rDNA gene. The developed endogenous qPCR control serves as a DNA quality control and allows the analysis of different eukaryotic host species, including plants, insects, fish, fungi, mammals and human with a single primer/probe set in single-or multiplex assays. Conclusions: Quality and performance control is indispensable for pathogen detection by qPCR. Several plant pathogens are transmitted by insects and have a broad range of host species. The newly developed endogenous control can be used with all so far tested eukaryotic species and since multiplexing is possible, the described primer and probe set can be easily combined with other PCR-based pathogen detection systems.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Development of a universal endogenous qPCR control for eukaryotic DNA samples

Mittelberger¹,

Obkircher²,

Oberkofler³

et al. 2020

Plant Methods

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“…A wide variety of PCR primer combinations has been reported in the literature for the detection of phytoplasmas 8 , 49 , and we previously compared the cpn60 -targeted direct PCR 12 to direct PCR using primers P1/Tint 8 . More recently, 16S rRNA-encoding gene-targeted primers have been described that are suitable for both conventional and real-time PCR 50 , but these have not been demonstrated to work with the 16SrXIII group. We therefore chose to compare the cpn60 -targeted direct PCR assay to nested PCR targeting the F2nR2 nested PCR, which is commonly used to detect phytoplasma infections.…”

Section: Discussionmentioning

confidence: 99%

Molecular diagnostic assays based on cpn60 UT sequences reveal the geographic distribution of subgroup 16SrXIII-(A/I)I phytoplasma in Mexico

Pérez‐López

Rodríguez-Martínez

Olivier

et al. 2017

Sci Rep

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Geographically diverse samples from strawberry exhibiting symptoms of Strawberry Green Petal (SbGP), periwinkle plants with virescence, and blackberry, blueberry, and raspberry plants displaying yellowing and inedible fruits, were assayed for the presence of phytoplasma DNA. PCR targeting the 16S rRNA-encoding gene and chaperonin-60 (cpn60) showed that the plants were infected with phytoplasma subgroup16SrXIII-(A/I)I (SbGP/MPV). To examine the geographic distribution of this pathogen in Mexico, we designed an array of cpn60-targeted molecular diagnostic assays for SbGP/MPV phytoplasma. A fluorescent microsphere hybridization assay was designed that was capable of detecting SbGP/MPV phytoplasma in infected plant tissues, successfully differentiating it from other known phytoplasma cpn60 UT sequences, while identifying a double infection with SbGP/MPV and aster yellows (16SrI) phytoplasma. Two quantitative assays, quantitative real-time PCR (qRT-PCR) and droplet digital PCR (ddPCR), gave similar results in infected samples. Finally, a loop-mediated isothermal amplification (LAMP) assay provided rapid detection of SbGP/MPV phytoplasma DNA. Application of these assays revealed that SbGP/MPV phytoplasma is widely distributed in Central Mexico, with positive samples identified from eleven localities within three states separated by hundreds of kilometres. These results also provide tools for determining the presence and geographic distribution of this pathogen in plant and insect samples in other localities.

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“…P. aurantifolia' in tissues of Mexican lime plants was designed on the 16Sr RNA gene (Mazraie et al, 2019). Pest and plant (sesame) specific reagents with different fluorescent dyes were used for simultaneous multiple detection and quantification of 16SrII and 16SrIX phytoplasmas in sesame plant and insect vector samples (Ikten et al, 2016). A rapid real-time group-specific LAMP assay was developed to detect 16SrII phytoplasmas (Bekele et al, 2011) Specificity of ribosomal primers and probes was not assessed on the different 16SrII strains (Ikten et al, 2016;Mazraie et al, 2019 The pest can be detected by conventional nested PCR, and distinguished from 'Ca.…”

Section: Intraspecific Diversitymentioning

confidence: 99%

Pest categorisation of the non‐EU phytoplasmas of tuber‐forming Solanum spp.

Bragard¹,

Dehnen‐Schmutz²,

Gonthier³

et al. 2020

EFS2

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Following a request from the European Commission, the EFSA Panel on Plant Health performed a pest categorisation of four phytoplasmas of tuber‐forming Solanum spp. known to occur only outside the EU or having a limited presence in the EU . The only tuber‐forming species of Solanum reported to be phytoplasma infected is S. tuberosum . This opinion covers ‘ Candidatus Phytoplasma americanum’, ‘ Ca . P. aurantifolia’‐related strains ( GD 32; St_ JO _10, 14, 17; PPT ‐ SA ; Rus‐343F; PPT ‐ GTO 29, ‐ GTO 30, ‐ SINTV ; Potato Huayao Survey 2; Potato hair sprouts), ‘ Ca . P. fragariae’‐related strains ( YN ‐169, YN ‐10G) and ‘ Ca . P. pruni’‐related strains (Clover yellow edge; Potato purple top AK pot7, MT 117, AK pot6; PPT ‐ COAHP , ‐ GTOP ). Phytoplasmas can be detected by molecular methods and are efficiently transmitted by vegetative propagation. Phytoplasmas are also transmitted in a persistent and propagative manner by some insects belonging to families within Cicadomorpha, Fulgoromorpha and Sternorrhyncha (order Hemiptera). No transovarial, pollen or seed transmission has been reported. The reported natural host range of the phytoplasmas categorised here varies from restricted (‘ Ca . P. americanum’, and ‘ Ca . P. fragariae’‐related strains) to wide (‘ Ca . P. aurantifolia’‐related strains and ‘ Ca . P. pruni’‐related strains), thus increasing the possible entry pathways in the latter case. S. tuberosum is widely cultivated in the EU . All the categorised phytoplasmas can enter and spread through the trade of host plants for planting, and by vectors. Establishment of these phytoplasmas is not expected to be limited by EU environmental conditions. The introduction of these phytoplasmas in the EU would have an economic impact. There are measures to reduce the risk of entry, establishment, spread and impact. Uncertainties result from limited information on distribution, biology and epidemiology. All the phytoplasmas categorised here meet the criteria evaluated by EFSA to qualify as potential Union quarantine pests, and they do not meet all the criteria to qualify as potential regulated non‐quarantine pests, because they do not occur or are not known to be widespread in the EU . ...

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Multiplex Real-Time qPCR Assay for Simultaneous and Sensitive Detection of Phytoplasmas in Sesame Plants and Insect Vectors

Cited by 27 publications

References 39 publications

Development of a universal endogenous qPCR control for eukaryotic DNA samples

Development of a universal endogenous qPCR control for eukaryotic DNA samples

Molecular diagnostic assays based on cpn60 UT sequences reveal the geographic distribution of subgroup 16SrXIII-(A/I)I phytoplasma in Mexico

Pest categorisation of the non‐EU phytoplasmas of tuber‐forming Solanum spp.

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