PCR Detection of BegomoViruses Using Degenerate Primers
Sample Preparation
It is necessary to extract DNA from samples. Although some studies report that
clarification of sap by centrifugation is sufficient, amplification has not been obtained in
our hands with the method described by Wyatt and Brown (1996).
We have used only fresh tissues or frozen (-20C), selecting a mix of immature and
mature leaves, but avoiding necrotic tissues; however, various experts have reported good
results with dried leaves. If using dried leaves, it is recommended to dry them at ambient
temperature between sheets of paper changed daily for three days. For extraction, a
commercial DNA extraction kit (Qiagen’s DNeasy Plant Mini Kit; Cat. No. 69104) is
effective.
Master Mix: per 25 µL rxn (using ABI GeneAmp 9700)
Sterile NPW
7.35 µL
1.5 mM dNTP
2.5
5 µM primer prV324 (+)
4.0
5 µM primer prC889 (-)
4.0
10X Titanium Taq buffer
2.5
Titanium Taq DNA polymerase
0.65
Aliquot 21 µL per rxn tube
Add: 4.0 µL sample DNA extract (or controls as noted below).
(The protocol of Dr. Brown requires addition of 1.0 g (=1000 ng) of total sample DNA
extract (plant plus viral) per reaction. While this is recommended if available, in
Vincelli’s sabbatical experiments, successful amplification was achieved with less, as
little as 10 ng total DNA/reaction or less, although the band on the gel may be faint.)
Controls
Negative control:
Positive control #1:
Positive control #2:
4 l Nanopure water
4 µl of a PCR-+ sample DNA OR 4 µl of a 1/500 dilution
amplicon of same (05-1576, tomato).
4 l sample DNA extract plus 1µl of Positive control #1 amplicon
at 1/100 dilution. If results show evidence of PCR inhibition,
retest sample extract at 10-fold dilution.
Cycling Protocol (GeneAmp 9700): Protocol listed as ba-bmo
1. 94ºC for 120 sec
2. 35 cycles of: 92ºC for 60 sec
3.
60ºC for 20 sec
4.
72ºC for 30 sec
Expected product size: Size products in 1.5% agarose. Expected size of the amplicon is
576-579 bp. Amplicon can be sequenced (by direct sequencing or cloning) to assign a
provisional species name, but conclusive identification requires sequencing of the entire
DNA-A component.
Duration: 1 hr 20 min.
SyBr Green Master Mix: per 25 µL rxn (using Cepheid’s SmartCycler)
Sterile NPW
SCAR
1.5 mM dNTP
5 µM primer prV324 (+)
5 µM primer prC889 (-)
10X Titanium Taq buffer
Titanium Taq DNA polymerase
5X SyBr Green
Add: 4.0 µL sample DNA extract
2.35 µL
2.5
2.5
4.0
4.0
2.5
0.65
2.5
Aliquot 21 µL per rxn tube
Cycling Protocol:
1. 94ºC for 120 sec
optics off
2. 35 cycles of: 92ºC for 60 sec
off
3.
60ºC for 20 sec
on
4. 72ºC for 30 sec
5. Melt Curve
Expected product Tm: 83.8 – 86.2. Template-free controls in this assay have a Tm of
76.4-78.0.
Duration: 1 hr 22 min.
Primers1
Prepare to a concentration of 5 μM (5 pmol/l). Store at -20°C.
prV324 (+): 5’ GCC-YAT-RTA-YAG-RAA-GCC-MAG 3’
prC889(-): 5’ GGR-TTD-GAR-GCA-TGH-GTA-CAT-G 3’
References
Brown, J. K., A. M. Idris, I. Torres-Jerez, G. K. Banks, and S. D. Wyatt, 2001. The core
region of the coat protein gene is highly useful for establishing the provisional
identification and classification of Begomoviruses. Arch Virol (2001) 146: 1581–1598
Rampersad, S. N., and Umaharan, P. 2003. Detection of begomoviruses in clarified plant
extracts: A comparison of standard, direct-binding, and immunocapture polymerase chain
reaction techniques. Phytopathology 93:1153-1157.
1
International nomenclature for degenerate primers
R = G or A
K= G or T
Y = T or C
D = G or A or T
N = G or A or T or C
S = G or C
V = G or A or C
W = A or T
B = G or T or C
M = A or C
H = A or T or C
Wyatt, S. D., and Brown, J. K. 1996. Detection of subgroup III geminivirus isolates in
leaf extracts by degenerate primers and polymerase chain reaction. Phytopathology
86:1288-1293.
Date of modification:Wednesday, February 17, 2016