A new approach to apple proliferation detection: a highly sensitive real-time PCR assay

Baric, Sanja; Via, Josef Dalla

doi:10.1016/j.mimet.2003.12.009

Cited by 86 publications

(70 citation statements)

References 25 publications

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“…P. mali'. all isolates were tested positive using a qualitative real-time Pcr approach (Baric and Dalla Via 2004;Baric et al 2006) and quantified applying an absolute standard curve method (Baric et al 2011).…”

Section: Methodsmentioning

confidence: 99%

“…the following cycling conditions were applied on a 7500 Fast real-time Pcr System (applied Biosystems): 2 min at 50 °c, 10 min at 95 °c and 40 cycles of 15 s at 95 °c and 1 min at 60 °c. after termination of amplification reactions, data were analysed using the automatic baseline setting of the 7500 Software Version 2.0.1 (applied Biosystems), while three different approaches were employed to set the threshold: (i) automatic threshold setting as implemented in the 7500 Fast realtime Pcr System analysis software; (ii) threshold fixed qaP-16S-F 5'-cgaacgggtgagtaacacgtaa-3' Baric and Dalla Via (2004) qaP-16S-r 5'-ccagtcttagcagtcgtttcca-3' Baric and Dalla Via (2004) qaP-16S a 5'-FAM-taacctgcctcttagacg-3' Baric and Dalla Via (2004) acO1 (M. domestica) qMd-acO-F 5'-ccagaatgtcgatagcctcgtt-3' Baric et al (2011) qMd-acO-r 5'-ggtgctgggctgatgaatg-3' Baric et al (2011) qMd-acO a 5'-VIC-tacaacccaggcaacg-3' Baric et al (2011) a taqMan-Probes were conjugated with a Minor groove Binder (MgB) and a non-fluorescent quencher dye (nFQ) at the 3'-ends (applied Biosystems) manually at 0.05 for all amplification runs and both targets; and (iii) manual adjustment of the threshold level based on a so-called calibrator sample which was run on each plate. More precisely, the threshold level was set such that the c t values for both target genes of the calibrator sample remained constant over all runs.…”

Section: Methodsmentioning

confidence: 99%

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Quantitative Real-Time PCR Analysis of ‘Candidatus Phytoplasma mali’ Without External Standard Curves

Baric

2012

Erwerbs-Obstbau

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apple proliferation caused by 'Candidatus Phytoplasma mali' is an economically important disease of apple (Malus × domestica). the availability of a simple quantitative approach to assess pathogen load in infected host plants would certainly contribute to a better understanding of pathogenesis and epidemiology. this study proposes a quantification approach not requiring the analysis of external standard curves. it is based on the simultaneous detection of the 16S rrna gene of the pathogen and the 1-aminocyclopropane-1-carboxylate oxidase gene of the host plant in a single-tube reaction using taqMan chemistry. the quantity of the phytoplasma relative to its host plant is determined as the difference between c t values of the two target genes (Δc t ). to assess the agreement of the relative quantification approach with a standard curvebased method, a dataset of 450 Dna samples from infected apple trees was reanalysed. comparison of the Δc t -based relative quantities with the corresponding absolute values revealed high degrees of agreement between the relative and absolute quantification methods. the Δc t procedure can thus be considered adequate for quantification of the phytoplasma in infected host plant tissue. in addition, this approach ensures improved methodological standardisation and increased analysis throughput.Keywords apple proliferation · Malus × domestica · Phytoplasma · relative quantification Quantitative real-time PCR Analyse von 'Candidatus Phytoplasma mali' ohne externe Standardkurven Zusammenfassung Die apfeltriebsucht, verursacht durch 'Candidatus Phytoplasma mali', ist eine wirtschaftlich bedeutende Krankheit des apfelbaumes (Malus × domestica). Die Verfügbarkeit einer einfachen und schnellen analysemethode zur ermittlung der erregermenge in infizierten Wirtspflanzen könnte zu einem besseren Verständnis der Pathogenese und der epidemiologie der Krankheit beitragen. in dieser arbeit wird ein Quantifizierungsverfahren vorgestellt, das ohne die analyse externer Standardkurven auskommt. es beruht auf der Verwendung der taqMan technologie, wobei in einer reaktion gleichzeitig das 16S rrna-gen des erregers und das 1-aminocyclopropan-1-carboxylatoxidase-gen (acO) der Wirtspflanze nachgewiesen werden. Die erregermenge, relativ zur anzahl der genomeinheiten der Wirtspflanze, wird als Differenz der c t Werte (Δc t ) berechnet, die für die beiden genabschnitte ermittelt werden. Zur ermittlung der Übereinstimmung des relativen Quantifizierungsverfahrens mit der konventionellen Standardkurven-Methode wurde ein Datensatz mit 450 Dna-Proben von apfeltriebsucht-infizierten apfelbäumen reanalysiert. Der Vergleich der relativen Δc t -Werte mit den absoluten erregermengen zeigte ein hohes Maß an Übereinstimmung. Das Δc t -Verfahren kann somit als geeignet für die Quantifizierung des apfeltriebsucht-Phytoplasmas in infiziertem Wirtspflanzen-gewebe angesehen werden. Zudem trägt die neue Quantifizierungsmethode zur besseren Standardisierung und durch den geringeren arbeitsaufwand zur erhöhung des analysed...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Quantitative Real-Time PCR Analysis of ‘Candidatus Phytoplasma mali’ Without External Standard Curves

Baric

2012

Erwerbs-Obstbau

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“…Phytoplasma pyri' (pear decline, PD), 'Ca. Phytoplasma pruni' (European stone fruit yellows, ESFY) important pathogens of fruit trees (12,76,48,156,3,4,113,23,128,41). Most of the primer/probe systems are targeting 16S rDNA gene though some others genes or even randomly cloned DNA fragments to which no specific function is assigned have been used (Table 3).…”

Section: Real-time Pcrmentioning

confidence: 99%

“…Therefore, to avoid false positives specific probe can be included. So far, several sequence-specific detection tools are available: the chloroplast chaperonin 21 gene (8); cytochrome oxidase gene (71); the chloroplast gene for tRNA leucine (12); and the 18S rDNA gene (30, 118, 113, www.intechopen.com 128) addressed as targets to control the quality of total DNA extracted. SYBR Green I is one of the cheapest chemistry for real-time PCR detection, but the specificity of the reaction is extremely low, and needs to be checked.…”

Section: Target Gene Referencesmentioning

confidence: 99%

“…Phytoplasma detection is now routinely done by different nucleic acid techniques based on polymerase chain reaction (PCR) (144,12,52,165). The procedures developed in the last 20 www.intechopen.com years are now used routinely and are adequate for detecting phytoplasma infection in plant propagation material and identifying insect vectors, thus helping in preventing the spread of the diseases and their economical impact.…”

Section: Introductionmentioning

confidence: 99%

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Polymerase Chain Reaction for Phytoplasmas Detection

Delić¹

2012

Polymerase Chain Reaction

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“ Candidatus Phytoplasma ”

Harrison¹,

Gundersen‐Rindal²,

Davis³

2015

Bergey's Manual of Systematics of Archaea and Bacteria

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Phy.to.plas'ma. Gr. masc. n. phytos a plant; Gr. neut. n. plasma something formed or molded, a form. Tenericutes / Mollicutes / Acholeplasmatales / incertae Sedis ‐ Family II / “Candidatus Phytoplasma” Phytoplasmas (Sears and Kirkpatrick, 1994) are wall‐less, nutritionally fastidious, phytopathogenic prokaryotes 0.2–0.8 µm in diameter that morphologically resemble members of the Mollicutes . Sequencing of nearly full‐length PCR‐amplified 16S rRNA genes (Gundersen et al., 1994; Namba et al., 1993; Seemüller et al., 1994), combined with earlier studies (Kuske and Kirkpatrick, 1992b; Lim and Sears, 1989), provided the first comprehensive phylogeny of the organisms and showed that they constitute a unique, monophyletic clade within the Mollicutes . These organisms are most closely related to members of the genus Acholeplasma within the Anaeroplasma clade as defined by Weisburg et al. (1989). Sustained culture in cell‐free media has not yet been demonstrated for any phytoplasma. Their genome sizes have been estimated to range from 530 to 1350 kb, and the G+C content of phytoplasma DNA is about 23–30 mol%. The presence of a characteristic oligonucleotide sequence in the 16S rRNA gene, CAA GAY BAT KAT GTK TAG CYG GDC T, and standard codon usage indicate that phytoplasmas represent a distinct taxon for which the name “ Candidatus Phytoplasma” has been adopted by specialists in the molecular biology and pathogenicity of these and similar phytopathogenic organisms (IRPCM Phytoplasma/Spiroplasma Working Team – Phytoplasma Taxonomy Group, 2004). At present, the designation “ Candidatus ” must still be used for new types.

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A new approach to apple proliferation detection: a highly sensitive real-time PCR assay

Cited by 86 publications

References 25 publications

Quantitative Real-Time PCR Analysis of ‘Candidatus Phytoplasma mali’ Without External Standard Curves

Quantitative Real-Time PCR Analysis of ‘Candidatus Phytoplasma mali’ Without External Standard Curves

Polymerase Chain Reaction for Phytoplasmas Detection

“ Candidatus Phytoplasma ”

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