Ecole Nationale Supérieure Agronomique de Montpellier ... - CIAM

2.4. Semi-quantitative real-time PCR
The specific primer pair designed for conventional PCR (ESFYf/r) could also be used
in semi-quantitative PCR. The reactions were performed in 20 µl with Fullvelocity SYBR
Green QPCR Master mix (Stratagene), 5 µl of a 10-fold dilution of the total DNA extract
(from plant or insect), and 0.30 µM of each primer. Thermal cycle parameters were as
follows: 5 min at 95°C (1 cycle) followed by 10 s at 95°C, 30 s at 62°C (40 cycles) and 1 min
at 95°C, 30 s at 55°C, 30s at 95°C (1 cycle). Analyses were performed using the MX 3000P
real-time PCR system and software (v.2 Stratagene).
2.5. Quantitative real-time PCR
The sequence delimited by ESFYf/r was not suited to design primers (ECAQf/r) and
probe ECAQp. There (Table 1) were designed only in the 16S rDNA region of the
phytoplasma genome using the Primer Express (version 1.0) software (Applied Biosystems).
The probe (Applied Biosystems) was labeled at its 5’-end with the fluorescent reporter dye
VIC and at the 3’-end with the fluorescent quencher TAMRA (6-carboxy-tetramethylrhodamine).
Sequence alignment using GenBank database showed the specificity of the designed
primers and probes in relation to other phytoplasmas belonging to the 16SrX group or
occurring in stone fruit trees (fig.3).
To take account of any variability in the yield of DNA extraction, an internal standard (an
housekeeping gene from C. pruni) was used. As no sequence was available for C. pruni, we
designed a primer pair and a probe on a highly conserved portion of the 18S rDNA gene. The
search for this gene was done with 7 C. pruni caught in different French areas to avoid
possible intraspecific differences in the C. pruni populations. Total DNA from a single insect
was extracted according to the method described above. Primers were designed on the highly
conserved 18S rDNA gene. First, a 500 bp segment was amplified by standard PCR
techniques using the primers previously designed for this region in the Rotifera-
Acanthocephala clades : 5’-CCACATCCAAGGAAGGCAGCAGGC-3’ (forward) and 5’-
CCCGTGTTGAGTCAAATTAA-3’ (reverse) and according to the thermal cycle parameters
described by Miquelis et al. (2000).
The PCR products were directly sequenced using an automated sequencer (Megabace,
Amersham), and the 7 C. pruni sequences were deposited in GenBank (accession numbers:
??). A BLAST search on the NBCI database was used to detect any possible homology with
known prokaryote sequences. We obtained the first DNA sequence of C. pruni (286 bp),
which revealed some intraspecific genetic diversity within the 18S rDNA (outside the
fragment chosen to design the primers). CPf/q/r were designed in a slowly evolving zone of
this gene, as confirmed by the complete homology with the corresponding sequence from
Trioza eugeniae (Psyllidae). Thus, CPf/q/r are expected to amplify any C. pruni, and probably
any Cacopsylla. However, this fragment is different from the homologous regions in the rest
of living organisms. The probe was labeled at its 5’-end with the fluorescent reporter dye
FAM (6-carboxy-fluorescein) and at the 3’-end with the fluorescent quencher dye, TAMRA
(6-carboxy-tetramethyl-rhodamine). All the sequences of primers and TaqMan probes are
listed in Table 1.
Reactions were performed in 25 µl with 2X TaqMan Universal PCR Master mix (Applied
Biosystems), on 5µl of total DNA. Psyllid DNA and ‘Ca. P. prunorum’ DNA quantification
required an optimization of the primers and probes concentrations. Optimized concentrations
were respectively 200 nM of CPf, 200 nM of CPr, 200 nM of probe and 600 nM of ECAQf,
600 nM of ECAQr, 250 nM of probe. Thermal cycle parameters were as follows: 2 min at
50°C (1 cycle) and 10 min at 95°C (1 cycle) followed by 15 s at 95°C and 1 min at 60°C (40
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