Journal of Virological Methods 83 (1999) 27 – 33

www.elsevier.com/locate/jviromet

A solvent-free, rapid and simple virus RNA-release method

for potato leafroll virus detection in aphids and plants by
reverse transcription polymerase chain reaction
Rudra P. Singh *
Agriculture and Agri-Food Canada, Potato Research Centre, P.O. Box 20280, E3B 4Z7 Fredericton, NB, Canada

Received 19 April 1999; received in revised form 12 July 1999; accepted 12 July 1999

Abstract

A one-step, rapid and economical method for potato leafroll virus (PLRV) RNA release that is applicable to the
use on a microcentrifuge scale is described. Discs (3– 6 mm diameter) from leaves, petioles, stems, and tubers of
potato plants were incubated in microcentrifuge tubes with detergent solution. The supernatants were used directly for
reverse transcription (RT) and polymerase chain reaction (PCR). Of the seven nonionic detergents of the TritonX
series evaluated, Triton X405R was the most effective, although X-405 and X-100R were also effective in releasing
PLRV RNA. Application of the detergent method for detecting PLRV in greenhouse-grown potato organs (leaves,
petioles, stems, tubers) and in field-grown tubers was demonstrated and compared to the multi-step phenol method.
When individual aphids, Myzus persicae, were ground in 20 ml of detergent solution and supernatants were used for
RT-PCR, virus was detected in single aphids in undiluted solutions and up to a dilution of 1:4. The concentration of
PLRV RNA released by the detergent method was substantially lower than that released by the phenol method.
However, the detergent method was sensitive enough to detect PLRV from potato leaves, petioles, and stems 2 weeks
after graft inoculation. The detergent method was rapid and economical, and has potential for large-scale application.
The extracts survived over 37 days at room temperature, thus making it possible to mail extracts from remote areas
lacking specialised RT-PCR facilities to a central laboratory for PLRV testing. © 1999 Elsevier Science B.V. All
rights reserved.

Keywords: Luteovirus detection; Nonionic detergents; Nucleic acid extraction; Vectors; Virus detection

1. Introduction and with hexagonal outlines; it is transmitted by

Potato leafroll virus (PLRV) is a luteovirus
with isometric particles of 26 mm in diameter
* Tel.: +1-506-4523260; fax: + 1-506-4523316.
E-mail address: singhr@em.agr.ca (R.P. Singh)
aphids in a persistent, non-propagative manner
(Peters and Jones, 1981). Leafroll is an important
disease economically in potatoes throughout the
world. Since vegetative parts (the tubers) are used
as planting material in most countries, a tuber-
borne PLRV infection can propagate indefinitely.

0166-0934/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 6 - 0 9 3 4 ( 9 9 ) 0 0 1 0 2 - 0
28 R.P. Singh / Journal of Virological Methods 83 (1999) 27–33
One of the primary methods of managing PLRV
infection in potato crops is the use of certified
virus-free tuber as ‘seed’ for planting. Since
PLRV-carrying aphids from another region could
introduce virus during the growing season to an
otherwise virus-free potato crop, monitoring of
aphid flights and identification of the aphid popu-
lations carrying PLRV is necessary for the timely
application of pesticides. Reverse transcription
polymerase chain reaction (RT-PCR) has been
adapted for both aspects (plants and aphids) of
PLRV monitoring (Spiegel and Martin, 1993;
Singh et al., 1995, 1996, 1997; Singh, 1998).
Successful applications of an RT-PCR for
PLRV detection depends on the effective recovery
of viral RNA from plant parts and aphids and the
absence of PCR inhibitors in the nucleic acid
extracts. To achieve this, most nucleic acid extrac-
tion procedures involve homogenisation of tis-
sues, which also releases PCR inhibitors in the cell
homogenates. In addition, these procedures are
multi-step and require several hours to several
days to prepare nucleic acids. Therefore, a proce-
dure was sought for nucleic acid extraction for
PLRV-infected tissues that would minimise or
eliminate tissue homogenisation and at the same
time release enough PLRV RNA in solution to be
detected by RT-PCR. This approach was
prompted by the observation that high concentra-
tions of nonionic detergents have been incorpo-
rated in rapid sample-preparation protocols for
PCR without inhibitory effect (Weyant et al.,
1990). Some of the ionic and nonionic detergents
have been used successfully for the extraction of
PLRV RNA (Spiegel and Martin, 1993; Singh et
al., 1995, 1996; Singh, 1998). In addition a non-
ionic detergent, TritonX-100, has been shown to
release filter-bound viral RNA after a short incu-
bation at room temperature (Olmos et al., 1996).
Therefore, it was prudent to evaluate Triton series
of detergents for PLRV RNA release and detec-
tion of PLRV by RT-PCR. This study reports a
simple, rapid and economical method of PLRV
RNA preparation from potato tissues without
homogenisation and from aphids with homogeni-
sation using nonionic detergents, particularly the
TritonX-405R.
2. Materials and methods
2.1. Virus source, aphid transmission, and
PLRV-infected tubers
PLRV-infected field plants were collected lo-
cally. Virus was transferred by aphid to virus-free
potato (Solanum tuberosum) plantlets. Aphids
(Myzus persicae Sulzer), the most efficient vector
of PLRV, were reared at 20–24°C under a 16-h
photoperiod of 3 to 5 K lux on potato cv
Katahdin. Fourth-instar nymphs and adult
apterae were used for virus acquisition on PLRV-
infected sources for 3–4 days. Five aphids from
the PLRV-exposed group were transferred to
virus-free plantlets of potato cvs Red Pontiac,
Russet Burbank, and Shepody for a 2–3 day
inoculation feeding period. Aphid-inoculated
plants were assayed by RT-PCR at weekly inter-
vals. In addition, potato infesting aphids (Aphis
nasturtii, M. persicae, and Macrosiphum euphor-
biae) and other unidentified aphids collected from
yellow pan traps in potato fields in Prince Edward
Island (PEI) were assayed for PLRV with RNA
extracted by the detergent method (see below).
PLRV-infected field-grown tubers were obtained
from Cavendish Farms (PEI) and potato fields in
New Brunswick. Potato plants infected with
PLRV within 2 weeks of graft or aphid inocula-
tion to over 4 months after infection were used to
evaluate the sensitivity of the detergent method.
2.2. Detergent solutions and RNA release
The detergents, nonionic Tritons (poly-
oxyethylene ethers) used as surface-active com-
pounds, were purchased from Sigma–Aldrich
Canada Ltd., Oakville, Ont. These were: TritonX-
100 (t-Octylphenoxypolyethanol), X-100R (Tri-
tonX-100 hydrogenated to reduce UV
absorption), X-114 (TritonX-114), X-405 (Tri-
tonX-405 aqueous solution), X-405R (TritonX-
405, hydrogenated to reduce UV absorption), XL
80N (TritonX-low-foaming liquid), and WR 1339
(a nonionic liquid polymer of alkyl aryl polyether
alcohol). In preliminary tests Triton X-100 was
tested at concentrations of 0.3, 0.5 and 0.7%
solution in water to determine the optimum con-
 R.P. Singh / Journal of Virological Methods 83 (1999) 27–33
centration for RNA release. The unadjusted pH
values of seven detergent solutions ranged from
4.32 for X-100R to 6.25 for X-114. All solutions
were also tested at pH 7.5.
Discs from potato leaves, petioles, stems, and
tubers were prepared using a cork borer (3 – 6 mm
internal diameter). The cut surfaces of tuber discs
were maximised by cutting a longitudinal strip
first, then preparing discs from the strip, or using
two 3-mm discs instead of one. Leaf discs were
punched out from mid-vein or from other promi-
nent veinal area. The weights of 6-mm discs from
different portions of the plant were: leaves (6 – 10
mg), petioles (35–69 mg), stems (60 – 90 mg), and
tubers (50–100 mg). The discs were incubated in
microcentrifuge tubes with 100 – 200 ml of deter-
gent solution. Evaluations included incubation at
25°C (room temperature), 37°C, or heating 1 – 2
min in a microwave. Samples were either shaken
or left undisturbed. The tube contents were cen-
trifuged at 12,000 rpm for 15 min in an Eppen-
dorf centrifuge and supernatant was removed for
RT (the detergent method). In addition, three
types of comparative virus release tests were
made. (1) Samples of leaf, petiole, stem, and tuber
discs were incubated in water alone; (2) the viral
RNA from the identical plant samples was ex-
tracted by the widely used, proteinase K-phenol/
chloroform method (the phenol method) (Singh et
al., 1996); and (3) plant discs were ground prior to
incubation in detergent solution. Individual
aphids were homogenised in 20 ml of detergent
solution using a mechanical pestle (Mandel Scien-
tific Company Ltd., Guelph, Ontario, Canada),
and centrifuged as above. Healthy plant material
and aphids not exposed to infected plants were
always included in tests. Presence of virus was
detected by RT-PCR.
2.3. Re6erse transcription and polymerase chain
reaction
The sense primer 5%-CGCGCTAACA-
GAGTTCAGCC-3% and the antisense primer 5%-
GCAATGGGGGTCCAACTCAT-3% were those
used in an earlier study (Singh et al., 1995). For
RT, 2.5 ml of detergent extract was added to 7.5 ml
of reaction mixture. The final assay mixture con-
29
tained 50 mM Tris–HCl (pH 8.3), 75 mM KCl,
10 mM DTT, 3 mM MgCl2, 0.5 mM each of
dNTP, 5 U of RNasin (Promega, Madison, WI),
0.1 mg of antisense primer, and 200 U of MMLV
reverse transcriptase (M-MLV RT, Gibco BRL
Life Technologies, Gaithersburg, MD). Samples
were incubated at room temperature for 10 min to
allow primer annealing and transferred to a 42°C
incubator for 1 h. The reaction was stopped by
heating the samples to 95°C for 2 min. Aliquots (5
ml) of the RT reaction were transferred to tubes
containing 20 ml of the PCR mixture. The final
conditions for the PCR assay were as follows: 10
mM Tris–HCl (pH 8.3), 50 mM KCl, 1.5 mM
MgCl2, 200 mM each dNTPs, 0.1 mg each of
antisense and sense primers, and 1.25 U of Ampli-
taq DNA polymerase (Perkin–Elmer Cetus, Nor-
walk, CT). Samples were overlaid with one drop
of mineral oil and amplified at 35 cycles using a
PTC-200 thermocycler (M J Research, Water-
town, MA). Each cycle consisted of denaturation
at 94°C, primer annealing at 55°C, primer exten-
sion at 72°C (each step of 1 min duration), and
final extension at 72°C (10 min). Aliquots (10 ml)
of the PCR reaction solutions were analysed on a
2% agarose gel containing 0.5 mg/ml of ethidium
bromide. The bands were visualised under UV
light and photographed by an imaging system
(Alpha Innotech IS 1000, San Leandro, CA).
3. Results
3.1. Virus RNA release protocol
A series of experiments were carried out with
more than 340 stem-disc samples from PLRV-in-
fected potato plants, where discs were incubated
in detergent solution and the supernatants were
tested by RT-PCR for virus detection. Of the
three detergent concentrations tested, the 0.5%
Triton X-100 was the most reproducible. At this
concentration, PLRV samples were detected with
98–100% accuracy (83–85 of the 85 discs tested),
while at 0.3 and 0.7% concentrations the percent-
ages of PLRV detection were 68 and 71%, respec-
tively. To determine which detergents were
suitable for virus release, three tests were con-
30 R.P. Singh / Journal of Virological Methods 83 (1999) 27–33
ducted using stem discs of PLRV-infected and
healthy plants. A total of 60 discs were used for
each detergent and 0.5% concentration of each
detergent was used. Triton X-405R released
PLRV in all discs (60/60), Triton X-405 and Tri-
ton X-100R released virus in 54 of the 60 discs,
and Triton X-100 released virus in 48 of the 60
discs. The remaining detergents released PLRV
from less than 50% of the discs.
Incubation of stem discs at room temperature
or at 37°C did not affect the percentage of PLRV
positive samples or the RNA yield. Heating sam-
ples in a microwave for 1 – 2 min reduced the
number of positive samples (data not shown).
Similarly, duration of incubation between 20 and
30 min or adjustment of pH to 7.5 did not make
any difference in the detection. Since room tem-
perature can vary from place to place, incubation
at 37°C for 30 min was used throughout the study
for uniformity.
For aphids, grinding of a single aphid in deter-
gent solution was more reliable than incubating
the aphids in solution. Individual aphids were
ground in microcentrifuge tubes containing 20 ml
of Triton X-405R solution, incubated for 20 min,
the aphid homogenates were centrifuged and the
supernatants were used for RT without any fur-
ther purification. In one test, where 20 aphids
from PLRV-infected and 20 from healthy plants
were tested, 11 aphids from PLRV infected plants
were found positive for PLRV. None of the
aphids collected from healthy plants contained
PLRV (data not shown).
3.2. Suitability of plant organs
Discs from potato leaves, petioles, stems, and
tubers were incubated in a Triton X-405R solu-
tion and tested by RT-PCR for PLRV. Results of
a typical experiment is shown in Fig. 1. PLRV
was detected from leaves, petioles, stems and tu-
bers (Fig. 1, upper panel, three samples for each
organ). In comparison to the detergent method,
the bands were of higher intensity from the phe-
nol-extracted RNA preparations (Fig. 1, lower
panel).
In a separate test, 300 samples from potato
plants infected with PLRV for 4 months were
extracted by both detergent and phenol methods.
In the detergent method, PLRV was detected in
92–95% of the stem, leaf, and petiole samples and
only in 70% of the tuber samples. When com-
pared with the phenol method of nucleic acid
extract, stems were 98% positive for PLRV while
leaves and tubers were 96% positive (150 samples
tested, data not shown). To improve the detection
sensitivity of tubers, two 3-mm discs were incu-
bated in a smaller amount of detergent solution
(100 ml instead of 200 ml) and compared as above.
It was observed that stems, petiole and leaves
were more suitable sources for PLRV RNA re-
lease than tubers for PLRV detection by the
detergent method. Homogenisation of tissue and
incubation in detergent solution improved the
band intensity. PLRV specific bands from stem
discs were obtained from water incubated materi-
als. However, this happened in 1–2% of cases.
3.3. Concentration of 6iral RNA in extracts of
the detergent method
Since the viral RNA released in the detergent
method is not concentrated by ethanol precipita-
Fig. 1. RT-PCR amplification of PLRV from various plant
organs by two methods of extract preparation. Top panel: M,
molecular size markers; three lanes with three different sam-
ples, extracted from tuber, stem, leaf and petiole by the
detergent method. Bottom panel: M, molecular size markers,
three lanes with the same three samples as in upper panel,
extracted from tuber, stem and leaf by the phenol method.
 R.P. Singh / Journal of Virological Methods 83 (1999) 27–33
Fig. 2. RT-PCR analysis of various dilutions of the extract
prepared by two methods and from plant and aphid sources.
Left side, top and bottom are extracts from stem discs. Two
samples were used for twofold dilutions in upper and fourfold
dilutions in the bottom samples. Right side, top and bottom,
extracts are from individual aphids. Samples were diluted
twofold. M, molecular size markers.
tion, the extracts obtained by the detergent and
phenol methods were compared by diluting the
preparations and carrying out RT-PCR. The ex-
tracts obtained from five stem discs and five tuber
discs were diluted twofold to 1:2, 1:4, 1:8, 1:16,
and 1:32 for the RNA from the detergent method
and diluted fourfold to 1:4, 1:16, 1:64, 1:256,
1:512, and 1:1024 for the RNA from the phenol
method prior to RT-PCR. As shown (Fig. 2) the
band intensity obtained from extract of detergent
method was low and PLRV was detected only up
to a dilution of 1:32. On the other hand detection
of PLRV from nucleic acid preparations by the
phenol method remained unaffected by dilutions
up to 1:1024 and band intensity was higher than
the detergent method (Fig. 2).
3.4. Detection of PLRV in aphids by the
detergent method
For the detection of luteoviruses in aphids, the
RNA extraction methods are multi-step, involve
the use of organic solvents and ethanol precipita-
tion, and are time consuming (Singh et al., 1996;
Canning et al., 1996; Naidu et al., 1998). Since,
preliminary tests showed that PLRV in aphids can
be detected by the shorter detergent method,
aphids exposed to PLRV-infected Russet Burbank
plants for 3–4 days were tested individually. Each
aphid was ground in 20 ml of Triton X-405R
31
solution, and the supernatants were tested by
RT-PCR as undiluted as well as diluted to 1:2,
1:4, 1:8, and 1:16 with water. PLRV was detected
from all 60 aphids (M. persicae) exposed to
PLRV-infected plants and tested individually
(data not shown). When diluted samples were
tested, PLRV bands were observed up to the 1:4
to 1:8 dilutions. Fig. 2 shows a representative test
result. In comparison, PLRV was also detected in
all 60 aphid extracts prepared by the phenol
method, however, at higher dilution of up to 1:32
(Fig. 2). About 700 aphids from fields were also
tested by the detergent method. PLRV was de-
tected in potato-infesting aphid species of A. nas-
turtii (8/100); M. euphorbiae (13/250); M. persicae
(25/100). None of the 250 aphids grouped as
‘others’ contained PLRV.
3.5. Detection of PLRV in newly infected plants
As shown in Figs. 1 and 2, the intensity of
PLRV bands in detergent-extracted plant and
aphid material is less than that obtained by the
phenol method. The question arises whether
newly infected plants can be identified by this
method. Potato plants were inoculated by aphids
and grafts and the virus was monitored on a
weekly basis. PLRV was detected in five of the
five plants within 2 weeks of graft-inoculation.
Infection was detected in leaves and petiole discs
of three plants in the first new axillary growth
below the graft union, and in two of the petiole
discs. At 4 weeks after inoculation, virus was
detected in all tissues. However, in new leaves
there was a difference in concentration, two were
faint (leaf) and three were intense (petiole) (Fig.
3). In aphid-inoculated potato plants, virus was
not detected until 6 weeks by the detergent
method in leaf and petiole discs.
3.6. Sur6i6al of PLRV RNA in detergent extract
at room temperature
The detergent-released extracts from leaves,
stems, petioles, and tubers were stored at room
temperature up to 50 days. Fractions were re-
moved at 7, 17, 24, 37 and 50 days after storage
and assayed by RT-PCR for PLRV. A total of 80
32 R.P. Singh / Journal of Virological Methods 83 (1999) 27–33

and RNA (Thomson and Dietzgen, 1995). How-

Fig. 3. Detection of PLRV from grafted plants using extracts
prepared by detergent method. Left side, first two lanes,
contained extracts from stem discs; the next three contained
from petiole discs. M, molecular size marker; right side, first
two samples (faint bands) from leaf discs and the next three
were from petiole discs.
samples (leaf, 15; petiole, 25; stem, 25; and tuber
15) was tested every time. Samples from leaves,
petioles, and stems remained positive for PLRV in
80 – 96% of cases after 7 days, while tuber samples
lost PLRV rapidly (Table 1). There was a gradual
loss of RT-PCR positive samples with increase in
storage. None of the tubers extracts contained
PLRV after 37 days; and only 24% of petiole;
30% leaves and 44% of the stem samples con-
tained PLRV after 50 days (Table 1).
4. Discussion
A single-step virus RNA-release protocol with a
buffer (100 mM Tris – HCl, 1.0 M KCl, 10 mM
EDTA) and incubation of tissue at 60 or 90°C has
been described for plant viruses containing DNA
ever, this protocol has not been evaluated for any
luteoviruses in plants and aphids. The one-step
procedure described in this study, consisting of
incubation of plant-tissue discs in a detergent
solution at 37°C for 30 min and using the eluate
without further treatment for RT and PCR, is the
simplest procedure described for an RNA virus. It
is particularly so, in view of its application to
PLRV, a luteovirus, which occurs in low concen-
tration in its hosts (Martin, 1995). In addition, the
procedure is applicable to small discs of leaf,
stem, and tuber, and to single aphids. The proce-
dure can be further improved by proper selection
of samples. For example, in a comparison of discs
with or without prominent veins, leaves with finer
veins did not contain detectable PLRV while discs
prepared from major veins increased the percent-
age of detection. Similarly, the increase in the cut
surfaces of tubers by including two 3-mm discs
facilitated more PLRV detection compared to a
single disc of 6 mm.
The nature of the virus material released in the
liquid medium is not clear. However, amplifica-
tion of PLRV-specific bands in water-incubated
infected-plant discs indicates that the released ma-
terial could be viral RNA. Assuming that the
released material is viral RNA, the detergents
could facilitate either further release of viral
RNAs over and above the ones released in water
alone or protect the released RNAs from degrada-
tion by ribonucleases. Only nonionic detergents
were evaluated in this study, both ionic and non-
ionic detergents could probably release the viral

Table 1
Survival of PLRV RNA in detergent extract at room temperature

Survival in days Leaves Petiole Stems Tubers

a
No % No a % No a % No a %

7 14/15 93 20/25 80 24/25 96 5/15 33

17 11/15 73 14/25 56 17/25 68 4/15 27
24 10/15 67 10/25 40 17/25 68 4/15 27
37 8/15 53 7/25 28 13/25 52 1/15 7
50 5/15 33 6/25 24 11/25 44 0 0

a
Number of samples positive by RT-PCR/number tested.
 R.P. Singh / Journal of Virological Methods 83 (1999) 27–33
RNAs. However, the ionic detergents such as
sodium deoxycholate, sodium dodecyl sulphate,
and Sarkosyl have been shown to inhibit PCR at
concentrations of less than or equal to 0.06%
(Weyant et al., 1990; Singh, unpublished data).
Comparison of the phenol and detergent meth-
ods showed that the concentration of viral RNAs
released by the detergent method is considerably
lower than that of the phenol method (Fig. 2).
Similar observations have been made with non-
tissue homogenisation methods when compared
to those of tissue-homogenisation methods
(Thomson and Dietzgen, 1995). However, the low
yield of released RNA may not be a handicap for
the detection of PLRV in most cases. Detection of
PLRV from newly inoculated plants was
achieved. Successful detection (Fig. 3) of virus
within 2 weeks of graft-inoculation indicates that
the sensitivity of the detergent method is high
enough to detect new infections and sensitivity
was also higher than the enzyme-linked immuno-
sorbent assay, which detected PLRV in the same
plants 5 weeks after graft-inoculation (data not
shown). In addition, the detergent method is sen-
sitive enough to detect PLRV in single aphids
collected from fields, and those exposed to PLRV-
infected plants under controlled conditions (Fig.
2) but may not be as useful as other methods
(Singh et al., 1995) for batch-aphid tests.
The material cost of the detergent method for
nucleic acid extraction is 1% compared to the
phenol method. It requires 2 h to prepare 90
samples for RT-PCR as opposed to 12 – 16 h by
the phenol method. The procedure can be
adopted to conduct initial extractions in less so-
phisticated laboratories, and the extracted RNA
can be mailed to a central laboratory for comple-
tion of the specialised RT-PCR. The extracts ob-
tained by the detergent method survive in 80 – 96%
of petiole, leaves and stem extracts maintained at
room temperature for 7 days (Table 1).
33
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