Establishment of a Quantitative Real-Time PCR Assay for the Quantification of Apple Proliferation Phytoplasmas in Plants and Insects

Abstract

A quantitative PCR (qPCR) assay was established for a sensitive and specific quantification of apple proliferation (AP) phytoplasmas in plants and in insect vectors. Different AP phytoplasma-specific primer pairs previously selected in a non-ribosomal DNA fragment of AP phytoplasma were tested. Among these, primer pair AP3/AP4 has been chosen for the qPCR assay because it amplifies a small sized 162 bp fragment of AP phytoplasma and produces no artefact bands. Thus, with these primers the SYBR™ Green technology could be used to monitor the amplification of the PCR products in real-time. The absolute quantification of the phytoplasmas in the samples was done by using the standard curve quantification method. The plasmid pUCI196 containing the chromosomal fragment of AP phytoplasmas from which the specific primers were derived was used as standard. Serial dilutions of the plasmid were done in total DNA extracts of healthy plants and healthy psyllids, respectively. For insects, total DNA of single individuals was extracted and subjected to PCR. Thus, AP phytoplasmas could be quantified in single individuals. For plant material, quantification of AP phytoplasmas was done with reference to a defined fresh weight of the material prior to DNA extraction. The inter-assay and intra-assay reproducibility of the method was analysed by comparing the Ct-values for given samples. The reproducibility was high both with plant and insect samples. Great differences in phytoplasma load could be found in different insect vector individuals whereas the analysed plant material was more homogenously infected. The established method is now suitable for the study of the infectivity of the insect vectors as well as for the evaluation of the resistance in plant material

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

“…P. mali' detection in both in vitro and in vivo plant samples by three different real-time PCR protocols (Jarausch et al, 2004;Torres et al, 2005;Aldaghi et al, 2007) and with high inter-and intrarun reproducibility. For detection of phytoplasma, the two SYBR Green real-time PCR protocols (protocols 2 and 3) used in present study yielded different C t values possibly due to different levels of primer specificity and/or to the different master mixes used (protocol 3 was not applicable with the master mix used in protocol 2).…”

“…Three established real-time PCR protocols, a TaqMan MGB method (protocol 1) (Aldaghi et al, 2007(Aldaghi et al, , 2008 and two SYBR green methods (protocol 2 and 3) (respectively, Jarausch et al, 2004;Torres et al, 2005), were also used for phytoplasma detection from both DNA extract preparation methods. Reactions were performed in a total volume of 25 l containing 1× qPCR MasterMix for TaqMan or SYBR Green from Eurogentec (respectively for protocols 1 and 2), or Power SYBR Green ® PCR MasterMix from Applied Biosystems, Warrington, UK (for protocol 3), 400 nM of each primer (used in original protocols), 200 nM TaqMan MGB probe (if applicable), and 5 l purified or crude DNA extract.…”

Polymerase Chain Reaction for Phytoplasmas Detection

“…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).…”

“…This property allows the use of shorter probes, with higher specificity than conventional TaqMan ones and the discrimination of even single nucleotide www.intechopen.com mismatched (83,128). Furthermore, applying the same protocols, phytoplasmas DNA could be also detected in insect samples (113,76,48,71) what is also decisive in the search for other potential vectors. A well-optimized reaction is essential for accurate results, which must be further analysed.…”

“…This approach was complex, several steps, such as electrophoresis, image analysis of gel, compensating for differences in intensity due to the different sizes of the product from the pathogen target and the internal standard, were required before the band intensities could be plotted for linear regression analysis. However, nowadays absolute quantification of phytoplasma DNA was achieved per gram of extracted tissue (161,23) or per insect vector (76). Possibility of the method to quantify amount of phytoplasma DNA in plant tissue and insect vectors gave opportunity to better understand biology and epidemiology of the pathogens, to allow examination of different multiplication rates and to calculate the concentration in their plant and vector host (161,142,23) as well as to study interactions of different phytoplasma species or strains present in mixed infection (100,19).…”