using a modified cetyltrimethylammonium bromide
(CTAB) protocol as described by Gawel and Jarret [10]
and James et al. [11]. Dry leaf tissue (0.05 g) was ground
in liquid nitrogen using pestle and mortar while adding
300 µl of the CTAB extraction buffer. The slurry was
then incubated at 65˚C for 15 minutes, spinned at 13,000
rpm and 750 µl transferred to a fresh tube. This was then
mixed with an equal volume of chloroform/isoamylal-
cohol (24:1) and centrifuged for 5 minutes at 13,000 rpm.
Nucleic acids in the aqueous phase were then pelleted
using isopropanol and centrifuged for another 5 minutes
at 13,000 rpm. The DNA pellet was then washed with
500 µl of 70% ethanol, and resuspended in 100 µl of
nuclease-free water. The DNA was left at 4˚C overnight
to fully dissolve. The total nucleic acids extracted were
treated with 10 mg/ml of RNAse A by incubating with 2
µl of RNase A with 100 µl of total nucleic acids at 37˚C
for 2 hr. The RNase A reaction was terminated by incu-
bating the mixture at 65˚C for 10 minutes. The nucleic
acids were stored at 20˚C.
The quality of the nucleic acid extracts was tested by
both electrophoresis on 1% agarose gels and PCR for
house-keeping genes using a number of randomly se-
lected samples. DNA integrity was ascertained by carry-
ing out actin gene PCR. A PCR master mix of 12.55 µl
comprised of 1.25 µl of PCR buffer, 0.5 µl dNTPs, 0.75
µl of 50 mM MgCl2, 0.25 µl of banana actin forward and
reverse primers, 0.05 µl Taq, 8.75 µl SDW and 1 µl of
the total nucleic acid extract. The PCR cycling conditions
for the actin gene PCR were an initial denaturation at
94˚C for 2 minutes, 35 cycles (94˚C for 20 seconds, 57˚C
for 20 seconds and 72˚C for 30 seconds) and a final ex-
tension at 72˚C for 3 minutes. Electrophoresis for actin
gene PCR products was carried out on 1.5% ethidium
bromide-stained agarose gels with Tris-Acetate EDTA
(TAE) as running buffer.
The thirty two DNA samples were then separately
subjected to three BSV indexing procedures (RCA, stan-
dard PCR and IC-PCR). IC-PCR was separately carried
out using both degenerate and specific primers. Each
treatment was replicated three times. These procedures
are outlined in the following sections.
2.4. Rolling Circle Amplification
The full Banana streak virus genome was amplified and
isolated using the TempliPhi Kit (GE Healthcare) ac-
cording to James et al. [11]. Two mixes were prepared.
In master mix 1 (MM1), 5 µl of TempliPhi Sample
Buffer was mixed with 1 µl of the isolated sample and 1
µl of primer mixture (details shown in Table 1) in an
appropriate reaction vessel. This was then heated at 95˚C
for 3 minutes to denature the DNA followed by cooling
at 4˚C. Denaturation is necessary because BSV is double
stranded. Master Mix 2 (MM2) was prepared by mixing
5 µl of TempliPhi Reaction Buffer and 0.2 µl of Tem-
pliPhi Enzyme Mix. Five microlitres of this TempliPhi
premix (mix 2) was transferred to the cooled, denatured
sample (MM1) then incubated at 30˚C for 18 hours. Af-
ter this incubation period, the enzyme (Phi29 DNA po
lymerase) was heat-inactivated at 65˚C for 10 minutes.
The samples were then be cooled and stored at 4˚C.
Ten microlitres of the TempliPhi product from each of
the samples were then incubated separately with Stu I
(Gibco BRL, Eggenstein) for 2 hours. Stu I was preferred
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Rolling Circle Amplification Is More Sensitive than PCR and Serology-Based
Methods in Detection of Banana streak virus in Musa Germplasm
1583
Table 1. RCA degenerate primers.
Primer name Primer sequence (5’-3’)
1A CTNTAYGARTGGYTNGTNATGCCNTTYGG
4’ TCCAYTTRCANAYNSCNCCCCANCC
BadnaFP ATGCCITTYGGIITIAARAAYGCICC
BadnaRP CCAYTTRCAIACISCICCCCAICC
BSV2292 ATGARYTAHATWAGRTGYTMSCC
BSV2826 TYYWGAAARCATGGTGG GRGARGA
BSV3298 YTCCCAYCTTTCRAAKACYTT
BSV3517 KRATMTTYTWTYTDGAARATCC
BSV3700 KTGGBAGTTTKGTRAAGARYTC
BSV4030 TGCARRTGYTWYGCYTGYGGAGA
BSV6652 GAAAARRTMTGYGCNTAYGCVAG
over other restriction enzymes due to its capacity to gen-
erate distinct polymorphic profiles for the BSV genome.
A 20 µl aliquot of the digested TempliPhi product was
mixed with 2 µl of the loading dye (30 mg 0.42% bro-
mophenol blue; 84 mg 0.42% xylene xyanol; 12 ml 60%
glycerol) and electrophoresed for about 20 minutes at
100 V on a 1% SYBR Safe-stained agarose gel using 1 ×
Tris Boric EDTA or Tris Acetate EDTA as the running
buffers. The gel was photographed under ultra violet (UV)
illumination with Gel Doc (BIO-RAD) Software (USA).
Internal standards for BSV isolates generated using Stu I
(New England BioLabs) were used to identify the iso-
late(s) present in each sample.
2.5. Immuno-Capture Polymerase Chain
Reaction
Immuno-capture Polymerase Chain Reaction (IC-PCR)
involved extraction of the leaf sap from all the asympto-
matic samples and concentration carried out as described
by Harper et al. [12]. One gram of silica gel-dried leaf
sample was ground in 5ml of the BSV extraction buffer.
Before carrying out the IC-PCR on all the samples, opti-
mization was done for the antibody and antigen in con-
centrations of 5:1000, 10:1000 and 15:1000 in the car-
bonate coating buffer. The optimization was carried out
on infected symptomatic tissues. Thin-walled propylene
microfuge tubes were coated with polyclonal antibodies.
The tubes were then washed three times with 100 µl of
PBS-Tween-20. Sap extract (about 100 µl) of the sam-
ples was added to each tube then incubated at 37˚C for 3
hours. The tubes were again washed twice with PBS-T,
once with sterile distilled water (SDW) and then dried
briefly before carrying out PCR directly in the tubes [12].
The PCR step was carried out using both degenerate
and isolate-specific (Gold finger) primers in separate
reactions. Two pairs of different types of BSV degener-
ate primers (badna and Harper’s 1A/4’) were first evalu-
ated. The badna pair appeared to give more consistent
results and was therefore used for this experiment. A 20
µl PCR mix contained 10 µl of Go Taq Green master mix
(Promega), 0.5 µl of each primer at a concentration of 10
pmol/µl, 8 µl of SDW and 1 µl of DNA. The PCR cycle
conditions were an initial denaturation at 94˚C for 5 min-
utes, 5 cycles (94˚C for 30 seconds, 37˚C for 30 seconds
and 72˚C for 1 minute), followed by 30 cycles (94˚C for
30 seconds, 50˚C for 30 seconds and 72˚C for 1 minute)
and a final extension at 72˚C for 10 minutes. The PCR
products were electrophoresed on a 1.5% SYBR Safe-
stained agarose gel as described earlier.
2.6. Direct PCR
Direct PCR was carried out on the 32 samples using the
Badna degenerate primers only. The PCR mix comprised
of 5 µl of Top Taq master mix (Qiagen), 0.25 µl of each
primer at a concentration of 10 pmol/µl, 3.5 µl of SDW
and 1 µl of DNA. The PCR cycling regimes were an ini-
tial denaturation at 94˚C for 3 minutes, 35 cycles (94˚C
for 30 seconds, 50˚C for 30 seconds and 72˚C for 1 min-
ute) and a final extension at 72˚C for 3 minutes. The
PCR products were electrophoresed on a 1.5% ethidium
bromide-stained agarose gel as described earlier.
3. Results
Actin gene PCR for DNA samples selected randomly
gave expected 664 bp amplicons as shown in Figure 1,
confirming that the DNA was intact and of PCR-quality.
An evaluation of two degenerate primer pairs to be used
for IC-PCR and direct PCR showed that the badna prim-
ers were better. Results by the badna primers were re-
producible and the bands on agarose gel were more dis-
tinct and clearer (Figure 2) than those for 1A/4’ [12].
Results of the thirty two asymptomatic (but possibly
BSV-infected) samples indexed for BSV using IC-PCR
(using both degenerate and Gold finger primers), direct
PCR and TempliPhi (RCA) are shown in Figures 3(a)-(c)
and Table 2. Direct PCR gave 93.8% detection with the
badna degenerate primers. TempliPhi (RCA) detected the
virus in 37.5% of the samples compared to the 31.3%
with IC-PCR (degenerate primers) and the 14.1% detec-
tions with IC-PCR (Gold finger primers). Analysis of
variance (ANOVA) for comparison of the three tech-
niques showed that at P < 0.05, there was significant dif-
ference among all the means. The ANOVA also showed
that at P < 0.05, some of the 32 asymptomatic samples
differed significantly from one another.
Copyright © 2012 SciRes. AJPS
Rolling Circle Amplification Is More Sensitive than PCR and Serology-Based
Methods in Detection of Banana streak virus in Musa Germplasm
1584
Figure 1. Actin gene PCR for DNA samples selected ran-
domly. Mr is 1 kb Plus molecular marker.
Figure 2. Gel picture showing performance of two BSV
degenerate primer pairs. Five BSV-symptomatic samples
were used for this evaluation. Lanes 1-5 represent 1A/4’
primers while lanes 6-10 represent badna primers. Mr is a 1
kb ladder.
This latter observation was expected since direct PCR
is known to give false positives for all Musa tissues.
Only samples 21 and 27 tested negative with direct/
standard PCR. IC-PCR with goldfinger primers showed
476 bp amplicons on a 1.5% ethidium bromide-stained
agarose gel. These positives were obtained only in sam-
ples number 10, 12, 19, 20, 27 and 28.
However, the detections observed in samples 27 and
28 were confirmed as erroneous by a repeat of the same
experiment, IC-PCR with degenerate primers and by the
rolling circle amplification (RCA) technique. IC-PCR
with degenerate primers indicated that the two samples
(27 and 28) were negative for BSV. This was also con-
firmed by RCA.
The analysis presented in Table 3 points out clearly
that direct PCR had the highest detection limit (mean =
0.94 ± 0.00). TempliPhi exhibited a higher mean detec-
tion limit than IC-PCR. IC-PCR with Gold finger spe-
cific primers gave the lowest mean detection level at 0.14
± 0.001.
4. Discussion
The superiority of badna primers over Harper’s 1A/4’
can be explained on the basis of the high genomic vari-
ability inherent within the BSV species. Both badna and
1A/4’ primers are designed to target the replicase (re-
verse transcriptase/RNase H) region of the BSV ORF III.
It is possible that natural molecular rearrangements have
(a)
(b)
(c)
Figure 3. (a) RCA for samples 1-13. Mr is a 10 kb ladder; (b)
IC-PCR with Gold Finger primers for samples 1-12; (c)
IC-PCR with Badna dege nerate pr imers for samples 1-10. +
is a positive control (banana leaf sample infected with
BSMysV isolate). Mr for B and C is a 1 kb ladder .
occurred over time within this region of the genome,
lowering the capacity of the 1A/4’ primers. This may
however be remedied by use of touchdown PCR to de-
termine a suitable annealing temperature. However, in
this study, the classical annealing temperature of 50˚C
was used for all BSV PCRs.
IC-PCR technique (both with specific and degenerate
primers) amplified BSV sequences from partially puri-
fied virus extraction for some of the samples, though
with minimal reproducibility. IC-PCR with the gold fin-
ger virus specific primers gave a 476 bp fragment while a
580 bp amplicon was observed with the badna degener-
ate primers. The direct implication
Is that some of the samples had been infected by BSV
isolates other than the Gold finger isolate. This con-
firmed earlier reports of high genomic heterogeneity
among BSV isolates [8]. The lower percentage detection
Copyright © 2012 SciRes. AJPS
Rolling Circle Amplification Is More Sensitive than PCR and Serology-Based
Methods in Detection of Banana streak virus in Musa Germplasm
Copyright © 2012 SciRes. AJPS
1585
Table 2. Details of 32 asymptomatic banana samples assayed for BSV using 3 virus indexing methods with each replicated
thrice per sample.
Sample number Cultivar Genotype Direct PCR
(degenerate primers)IC-PCR
(degenerate primers)IC-PCR
(Gold finger primers) RCA
1 Mysore AAB +++ - - -
2 Mysore AAB +++ - - -
3 Gold finger AAAB +++ - - -
4 Mysore AAB +++ - - -
5 Nshule EAH-AAA +++ -+- - +++
6 Nshule EAH-AAA +++ +++ - +++
7 Musera EAH-AAA +++ +++ - +++
8 Mysore AAB +++ - - -
9 Gold finger AAAB +++ - - -
10 Gold finger AAAB +++ +++ ++- +++
11 Gold finger AAAB +++ - - +++
12 Nshule EAH-AAA +++ +++ -++ +++
13 FHIA 18 AAAB +++ - - -
14 FHIA 18 AAAB +++ - - -
15 Solio EAH-AAA +++ +++ - +++
16 Solio EAH-AAA +++ +++ - +++
17 Sukari ndizi AAB +++ - - -
18 Musera EAH-AAA +++ - - +++
19 Solio EAH-AAA +++ +++ +++ +++
20 Nusu Ng’ombeEAH-AAA +++ +++ +++ -
21 FHIA
18 AAAB --+ +++ - +++
22 Solio EAH-AAA +++ +++ - ++-
23 FHIA 18 AAAB +++ - - -
24 Solio EAH-AAA +++ - - -
25 Sukari ndizi AAB +++ - - -
26 FHIA 18 AAAB +++ - - -
27 Cavendish EAH-AAA --- - -+- -
28 FHIA 18 AAAB +++ - -++ -
29 FHIA 18 AAAB +++ - - -
30 FHIA 18 AAAB +++ - - -
31 FHIA 18 AAAB +++ - - -
32 Solio EAH-AAA +++ --+ - -
+
Positive detection; - Negative detection.
Rolling Circle Amplification Is More Sensitive than PCR and Serology-Based
Methods in Detection of Banana streak virus in Musa Germplasm
1586
Table 3. Comparison based on least significant difference
(LSD) test for the four detection tec hniques.
Technique N** Means*
Direct PCR 96 0.94 ± 0.00
TempliPhi 96 0.38 ± 0.019
IC-PCR (degenerate) 96 0.33 ± 0.042
IC-PCR (Gold finger) 96 0.14 ± 0.001
LSD0.05 = 0.0354. *Any two means are significantly different if their differ-
ence is the greater than LSD value; **N is the total number of replicates.
with Gold finger primers points out to the fact that this
primer pair could not amplify thee target sequences of all
the BSV isolates. The badna primers are designed to
degenerately target the replicase (RNaseH/RT) region of
all members of the Badnavirus genus. Other than Banana
streak virus, many other members of the Badnavirus ge-
nus are known to exist [13]. Since these viruses are not
hosted by banana, it is unlikely that they were amplified
by the badna degenerate primers used in this study. To
reinforce this further, the antisera used during the im-
muno-capture step of IC-PCR were specific to BSV.
Although all the 32 samples were asymptomatic, it
was possible to detect BSV in some samples. This is be-
cause the 32 samples were collected from a field which
four months before sampling, was highly endemic with
banana streak disease. The implication is that most of the
32 samples were actually infected with BSV but had
masked the symptoms probably due to changes in envi-
ronmental conditions.
Results of this study show that the TempliPhi tech-
nique has a greater capacity to reliably detect BSV than
the other assayed techniques. This is expected because
the TempliPhi technique allows for selective amplifica-
tion of all circular DNAs within the tissue being indexed
[14]. The high sensitivity, fidelity and processivity of
Phi29 DNA polymerase, the enzyme involved in Tem-
pliPhi amplification [15] ensures accurate and reliable
diagnosis by means of this method. A number of factors
can be used to explain the lower percentage detection by
IC-PCR; the low reproducibility always inherent with
IC-PCR, the high serological and genomic heterogeneity
known to exist among BSV isolates [8] and probably the
inactivity and/or non-specificity of the antisera. More-
over, there appears to be a link between genetic and se-
rological diversity; the former leads to the latter [16].
Since genetic diversity of Banana streak virus has been
shown to exist in Kenya [17], it is likely that serological
variability among BSV isolates exists with almost the
same measure as the genetic diversity.
Apart from the increased sensitivity and reliability of
TempliPhi compared to IC-PCR and standard PCR,
TempliPhi has the advantage of allowing the identity of
the isolate infecting the sample to be known. This can be
an avenue for carrying out more accurate distribution
studies for other viruses with circular DNA genomes.
Results for direct PCR and IC-PCR using degenerate
primers were not in agreement. Direct PCR gave a
93.75% detection limit compared to the 31.25% with IC-
PCR for the 32 asymptomatic samples. This finding con-
firms earlier reports of possible integration of badnavirus
sequences in their host genomes [4]. Much as most of the
detections by direct PCR can therefore be safely classi-
fied as false positives, recent studies have tentatively
shown that direct PCR for detection of BSV can be reli-
able for tissues without balbisiana (B) components in
their genomes [11]. Banana cultivars of AAA and EAH-
AAA genomes can be reliably indexed for BSV by
means of direct PCR. Direct PCR for sample 27 (AAA
cavendish) was indeed a confirmation of this finding.
There was no detection of BSV for this sample using all
the 3 techniques (direct PCR, IC-PCR and TempliPhi).
More so, the positive detections observed with direct
PCR for tissues with AAA genomes were confirmed by
the other techniques. Therefore direct PCR in this case
can be relied upon. However, the negative result ob-
served for sample 21 was certainly erroneous since it was
not in agreement with the results of the other techniques.
The likely explanation is that the DNA for this sample
was not of PCR quality.
As yet, it is not clear why the integrated sequences are
never amplified by direct PCR in tissues exclusively
containing acuminata (A) genome but earlier studies
have established that the numerous infectious BSV en-
dogenous pararetroviruses (integrated sequences) of dif-
ferent BSV species are restricted to the B genome [18].
According to these studies, the integrated sequences in A
genomes are usually non-infectious; they cannot be acti-
vated by the tissue culture stress. This suggests that the
non-detectability of these endogenous pararetroviruses in
A genomes is linked to the position of these sequences in
the genome.
It is an undisputed fact that rapid and reliable detection
of Banana streak virus has been an enormous challenge
over the last two decades. Although the RCA technique
offers a remedy to this problem, the solution seems to be
only partial. The long incubation period, need for incuba-
tion and electrophoresis apparatus required for the RCA
method have meant that this technique cannot be used in
the field for rapid indexing of banana samples, a need
which has remained unaddressed for a long time. It
would be worthwhile for future research to focus on es-
tablishing a method based on the rolling circle amplifica-
tion principle that will circumvent the long incubation
periods and the electrophoretic procedures.
Copyright © 2012 SciRes. AJPS
Rolling Circle Amplification Is More Sensitive than PCR and Serology-Based
Methods in Detection of Banana streak virus in Musa Germplasm
1587
5. Acknowledgements
We would like to thank the Queensland University of
Technology (QUT), Australia, through the Banana 21
project for funding this work. Special thanks also go to
the Kenya Agricultural Research Institute, Njoro for
availing laboratory facilities.
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