A multiplex TaqMan qPCR assay for sensitive and rapid detection of phytoplasmas infecting Rubus species

Linck, Holger; Krüger, Erika; Reineke, Annette

doi:10.1371/journal.pone.0177808

Cited by 18 publications

(9 citation statements)

References 32 publications

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“…There are a number of assays one can employ for SNP genotyping to facilitate species identification, e.g. TaqMan real-time PCR (Dhami et al 2016;Zhang et al 2016;Linck et al 2017), High-Resolution Melt (HRM) real-time PCR (Dhami et al 2014;Ajamma et al 2016), species-specific PCR (Sint et al 2016), KASP genotyping (Middlesex, UK), and SNP microarrays. We chose to adopt the Agena MassARRAY platform in combination with iPLEX chemistry (Gabriel et al 2009) to maximize the number of SNP markers we can multiplex in a single assay to reduce false positive rate and increase rigor of species identification.…”

Section: Discussionmentioning

confidence: 99%

Sequencing of Tuta absoluta genome to develop SNP genotyping assays for species identification

et al. 2019

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Tuta absoluta is one of the most devastating pests of fresh market and processing tomatoes. Native to South America, its detection was confined to that continent until 2006 when it was identified in Spain. It has now spread to almost every continent, threatening countries whose economies rely heavily on tomatoes. This insect causes damage to all developmental stages of its host plant, leading to crop losses as high as 80 to 100%. Although T. absoluta has yet to be found in the U.S. and China, which makes up a large portion of the tomato production in the world, computer models project a high likelihood of invasion. To halt the continued spread of T. absoluta and limit economic loss associated with tomato supply chain, it is necessary to develop accurate and efficient methods to identify T. absoluta and strengthen surveillance programs. Current identification of T. absoluta relies on examination of morphology and assessment of host plant damage, which are difficult to differentiate from that of native tomato pests. To address this need, we sequenced the genomes of T. absoluta and two closely related Gelechiidae, Keiferia lycopersicella, and Phthorimaea operculella, and developed a bioinformatic pipeline to design a panel of 21 SNP markers for species identification. The accuracy of the SNP panel was validated in a multiplex format using the iPLEX chemistry of Agena MassARRAY system. Finally, the new T. absoluta genomic resources we generated can be leveraged to study T. absoluta biology and develop species-specific management strategies. Key Message  Surveillance and identification of Tuta absoluta are challenging because it is morphologically similar to closely related species, e.g. Keiferia lycopersicella and Phthorimaea operculella.  We generated new genomic sequences for these three species and identified single nucleotide polymorphisms (SNPs) to facilitate species identification.  We validated a multiplex genotyping panel of 21 SNPs using the iPLEX MassARRAY platform and confirmed its accuracy for species identification.  We generated new molecular tools and genome resources to aid Tuta absoluta management. leaves, where the larvae will emerge from eggs and begin mining host tissues. Larvae can also enter the stems through the buds, and feed within the tomato fruit, leaving them unmarketable. Its distribution was largely confined to South America until it was first detected in Spain in 2006 (Desneux et al. 2010; Guillemaud et al. 2015). Since then T. absoluta has spread rapidly and is now

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Section: Discussionmentioning

confidence: 99%

Sequencing of Tuta absoluta genome to develop SNP genotyping assays for species identification

et al. 2019

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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%

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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“…After centrifugation, the pellet was washed with 70% ethanol and dissolved in 20 µl of deionized sterile water. DNA samples were tested for phytoplasma DNA with the Multiplex TaqMan qPCR assay for insect samples as described in Linck, Krüger, and Reineke ().…”

Section: Methodsmentioning

confidence: 99%

Preliminary survey on putative insect vectors for Rubus stunt phytoplasmas

Linck

Reineke

2019

J Applied Entomology

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Phytoplasmas are cell wall‐less phytopathogenic bacteria which are associated with a disease in Rubus species known as Rubus stunt. Symptoms range from stunting, witches’ broom, small leaves, short internodes, enlarged sepals, phyllody and flower proliferation to fruit malformations. Phytoplasmas can be spread by vegetative propagation and by phloem‐feeding insect vectors. However, little is known about the spectrum and distribution of putative Rubus stunt insect vectors. In this study, a screening of putative insect vectors of Rubus stunt in raspberry plantations in southern and northern Germany was carried out during two successive years (2014 and 2015) with multiple sampling dates throughout the growing seasons. A total of 2,891 hemipteran insects were sorted, identified to family, genus or species level when possible, and a subset of 319 DNA samples containing a sum of 932 selected individuals representing all identified species, sampling locations and sampling dates were tested for phytoplasma DNA using qPCR. Altogether, eight DNA samples were positive for phytoplasma DNA, among them species from the genera Euscelidius, Macrosteles, Euscelis, Anaceratagalliaand Psammotettix. These data will form the basis for choosing and timing appropriate control measures against Rubus stunt and also for potential insect vector transmission experiments.

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A multiplex TaqMan qPCR assay for sensitive and rapid detection of phytoplasmas infecting Rubus species

Cited by 18 publications

References 32 publications

Sequencing of Tuta absoluta genome to develop SNP genotyping assays for species identification

Sequencing of Tuta absoluta genome to develop SNP genotyping assays for species identification

Development of a universal endogenous qPCR control for eukaryotic DNA samples

Preliminary survey on putative insect vectors for Rubus stunt phytoplasmas

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