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Detection Methods for SLRSV
Molecular Methods
AmpliDet RNA assay developed by
M. Klerks, Plant Research International BV.
Nucleic acid extraction
100 mg of leaf tissue is ground in liquid nitrogen and dissolved
in RLT-lysisbuffer from the RNeasy plant extraction kit (Westburg,
Quiagen). Extraction was carried out according to the RNeasy
extraction protocol.
Primers / Probes
SLRSV reverse: 5'-AAT TCT AAT ACG ACT CAC TAT AGG GAA GAT CAG CAA CTT CAA AGT TCT CAA-3'
SLRSV forward: 5'-GAC TCT TGC TAA ATA TCC TAG CA-3'
The reverse NASBA primer consistes of a 5'-T7 RNA polymerase
recognition site seqeunce and a 3' target-complementary sequence.
SLRSV probe: Biotin labeled detection probe for specific detection
of amplicons by Northern blotting. 5'-TGG TTT ATG CCT GGT AGG CAT GA-3'
SLRSV MB: Molecular beacon enabling fluorescent real-time detection
of NASBA amplification. On the 5' end a fluorophore, on the 3' end a
quencher (DABCYL) is present. 5'-GCA GCT ATA ATG ACC AGT GAT CAC AAT GAG CTG C-3'
NASBA assay
The NASBA reaction mix consists of 5 µl NASBA-reagents
(160 mM Tris.HCl, pH 8.5, 48 mM MgCl2, 2 mM DTT,
4 mM dNTP, 8 mM ATP, UTP and CTP, 6 mM GTP and 2 mM ITP), 0.7
µl 350 mM KCl, 4 µl 5x primer mix (75 % DMSO and 1 µM
of each primer) and 2.3 µl of RNase free water per reaction.
A volume of 3 µl of sample solution (extracted RNA) is added
to the NASBA reaction mix. The reactions are pre-incubated at
65° C directly followed by 41° C for 5 min. each. The
NASBA reaction starts by adding 5 µl of enzyme mix (375 mM
sorbitol, 2.1 µg BSA, 0.08 U RNase H, 32 U T7 RNA polymerase
and 6.4 U AMV reverse transcriptase) per reaction, incubating
for 5 min. at 41° C, then shortly centrifuging followed by
incubation for 90 min. at 41° C.
Real-time amplification and detection using AmpliDet RNA is
carried out as described above, except that the 2.3 µl
RNase free water is replaced by 1 µl of 10 µM ROX
[5-(and -6)-carboxy-X-rhodamine], 1 µl of molecular beacon
solution (9 ng/µl) and 0.3 µl of RNase free water. The
ROX and molecular beacons must be suspened in RNase free water.
The 90 min. incubation at 41° C is performed in a thermostatic
fluorimeter measuring the emission spectrum of the fluorescent dye
attached to the molecular beacon in real-time for each sample every
2 min.
Northern blotting
NASBA products are analyzed by electrophoresis using a 1 %
pronarose gel with 0.5 µg/ml EtBr. Gels are run at 100 V for
15 min. in buffer containing 40 mM Tris-acetate and 1 mM EDTA,
pH 8.0 (1x TAE). The gel is blotted onto a Z-probe nylon membrane
in 0.3 M NaCl and 30 mM Na-citrate (2x SSC) for 20 min. Nucleic
acids are cross-linked onto the Z-probe by UV-exposure for 2 min.
Hybridization of the biotinylated probe (3 µM) to the NASBA
products is performed at 50° for 30-60 min. in 5x SSC, 7% SDS,
20 mM Na-phosphate, pH 6.7, 10 x Denhart's reagent. The blots are
washed twice with 3x SSC, 1% SDS at 50° for 5 min. and once
with buffer containing 0.1% SDS with 20 mM Na2HPO4,
0.36 M NaCl and 2 mM EDTA (2x SSPE) at room temperature for 10 min.
Subsequent steps are carried out at room temperature. The blots
are incubated for 30 min. with 2 µl streptavidin peroxidase
conjugate in 5x SSPE with 5% SDS, followed by extensive washing,
then incubated in ECL detection reagents (Amersham Pharmacia Biotech,
Freiburg, Germany) and exposed to X-ray films.
NASBA (nucleic acid sequence based amplification) is a
registered trademark in one or more countries. The amplification
technology used in the NASBA system is covered by various
patents and pending patents which are owned by Organon
Teknika BV.
Molecular beacon is a registered trademark in one or more
countries. The technology is covered by various patents and
pending patents which are owned by The Public Health
Research Institute.
AmpliDet RNA is a registered trademark by Plant Research
International BV.
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