Original Paper
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Acta Biochim Biophys
Sin 2006, 38: 669-676
doi:10.1111/j.1745-7270.2006.00216.x
Quantitative determination of cucumber mosaic virus
genome RNAs in virions by Real-Time Reverse
Transcription-Polymerase Chain Reaction
Jun-Li Feng1, Shao-Ning CHen1, Xiang-Shan Tang1, Xian-Feng Ding1, Zhi-You Du2, and Ji-Shuang Chen1,2*
1
Institute of Bioengineering, Zhejiang Sci-Tech University, Hangzhou 310018,
China;
2
College of Life Sciences, Zhejiang University, Hangzhou 310029, China
Received: May 7,
2006
Accepted: June 30,
2006
*Corresponding
author: Tel/Fax, 86-571-86843196; E-mail, [email protected]
Abstract A real-time RT-PCR procedure using the
green fluorescent dye SYBR Green I was developed for determining the absolute
and relative copies of cucumber mosaic virus (CMV) genomic RNAs contained
in purified virions. Primers specific to each CMV ORF were designed and
selected. Sequences were then amplified with length varying from 61 to 153 bp.
Using dilution series of CMV genome RNAs prepared by in vitro transcription
as the standard samples, a good linear correlation was observed between their
threshold cycle (Ct) values and the logarithms of the
initial template amounts. The copies of genomic RNA 1, RNA 2, RNA 3 and the
subgenomic RNA 4 in CMV virions were quantified by this method, and the ratios
were about 1.00:1.17:3.58:5.81. These results were confirmed by Lab-on-a-chip and northern blot hybridization assays. Our work is the first
report concerning the relative amounts of different RNA fragments in CMV
virions as a virus with tripartite genome.
Key words real-time reverse transcription-polymerase chain reaction; cucumber
mosaic virus; genomic RNA; quantitative determination
Cucumber mosaic virus (CMV) is a species
of the Cucumovirus genus within the family Bromoviridae. It has
been long known that the virus has a genome structure consisting of three
single-stranded messenger sense RNAs (RNA 1, 2 and 3) and two subgenomic RNAs
(RNA 4A and RNA 4). RNA 1 and RNA 2 encode components of viral RNA-dependent
RNA polymerase (RdRp), the 1a and 2a proteins, with putative helicase and
polymerase activities, respectively. RNA 3 encodes the 3a movement protein and
the coat protein (CP), and the latter is expressed from RNA 4 as subgenomic
RNA. In addition, the 2b protein is expressed from 3‘-proximal
sequences of RNA 2, via subgenomic RNA 4A. This protein is a pathogenicity-determinant
and plays a role in the long-distance movement of CMV [1,2].CMV has a very large host range, which is estimated to be over 1000 species
in 85 families and has a worldwide distribution [3]. While the crop losses
caused by CMV have increased greatly in past decades, its replication and
pathogenicity mechanisms remain mostly undiscovered. Several publications have
mentioned that CMV genomic RNAs do not accumulate in equimolar amounts in
hosts, and that the relative ratios between them have a strong effect on the
level of virulence and the level of virus accumulation [4–6]. Therefore, a
rapid and sensitive method for reliably quantifying the amount of each CMV
genomic RNA would aid the understanding of the mechanisms of viral replication
and pathogenicity, as well as the interaction with hosts.The amounts of CMV genomic RNAs were previously quantified by
electrophoresis, northern blot
hybridization and RT-PCR [7]. However, these methods vary in their usefulness
for accurate quantification, being either time consuming, laborious,
insensitive, or having large results variations. Real-time RT-PCR is a new
method developed in recent years. By adding the fluorescent signal to the
reaction mixture, the whole PCR process is monitored through the increase of
fluorescence and the absolute amount of target is calculated from a
calibration curve. Due to its high sensitivity and reproducibility, real-time
RT-PCR has been considered the most reliable method currently available and is
used widely in gene (e.g. transgene or pathogen) detection and quantification,
and gene expression studies [8,9]. Its applications in virus researches are increasing, and many
economically important plant viruses have been detected by this method [10–13]. But most of
these studies are related simply to the qualitative detection of viruses,
taking advantage of its broad quantification range (?7 magnitudes) and low titration limitation. To date, there is no
report on the quantification of different RNA fragments in multipartite
viruses.This paper describes the development of a real-time RT-PCR assay
with SYBR Green I, which allows an accurate quantification of CMV genomic RNAs.
Their copy number ratios in virions were calculated and the results were comfirmedconfirmed
by lab-on-a-chip and northern blot hybridization assays.
Materials and methods
Plants and viruses
Nicotiana tobacum Huangmiaoyu tobacco plants were maintained
under greenhouse conditions with a day length of 16 h at day/night temperature
of 25 ?C/20 ?C. The young tobacco plants (four- to six-leaf stage) were used
for virus inoculation by Fny-CMV (kindly donated by Peter Palukaitis, Scottish Crop Research
Institute, Dundee, UK). Virions were purified from infected tobacco 17 d after
inoculation [14]. Fny-CMV genomic RNAs were extracted from virions and from
infected or healthy tobacco plants with the method as previously described [15].
The RNA samples were treated with DNase I and stored at –80 ?C.
Primer design and optimization
Two to four sets of primers were designed for each Fny-CMV ORF based on
their sequences. Primers were first designed using Primer 5.0 software (PE Applied Biosystems, Foster City, USA) and then selected manually based on
melting temperature (Tm), position and GC content in
the last five bases. Virtual RT-PCR was run to screen for amplification
efficiency and primer dimmer formation. The best primer set for each ORF was
selected for subsequent experiments (Table 1).
Preparation of standard
samples
To quantify Fny-CMV genomic RNAs, three sets of standard samples
were transcribed from biologically active cDNA clones of Fny-CMV RNA 1
(pFny109), Fny-CMV RNA 2 (pFny209), and Fny-CMV RNA 3 (pFny309) in vitro
with T7 polymerase (Promega, Madison, USA), respectively. Standard samples
of RNA 1 were used with 1a primer pair 1a-F/R, RNA 2 with 2a and 2b primer
pairs 2a-F/R and 2b-F/R, RNA 3 with 3a and CP primer pairs 3a-F/R and CP-F/R in
quantitative real-time RT-PCR. Transcription products were treated with DNase
I firstly, and then RT negative PCR products were analyzed in triplicates to
confirm there was no fluorescence resulting from either DNA template residue or
RT step. Finally they were quantified to 400 mg/ml and stored at –80 ?C. The copy
numbers could be determined by Equation 1.
To quantify Fny-CMV genomic RNAs, three sets of standard samples
were transcribed from biologically active cDNA clones of Fny-CMV RNA 1
(pFny109), Fny-CMV RNA 2 (pFny209), and Fny-CMV RNA 3 (pFny309) in vitro
with T7 polymerase (Promega, Madison, USA), respectively. Standard samples
of RNA 1 were used with 1a primer pair 1a-F/R, RNA 2 with 2a and 2b primer
pairs 2a-F/R and 2b-F/R, RNA 3 with 3a and CP primer pairs 3a-F/R and CP-F/R in
quantitative real-time RT-PCR. Transcription products were treated with DNase
I firstly, and then RT negative PCR products were analyzed in triplicates to
confirm there was no fluorescence resulting from either DNA template residue or
RT step. Finally they were quantified to 400 mg/ml and stored at –80 ?C. The copy
numbers could be determined by Equation 1.
Eq. 1
where N was the copy number per ml, C was the
concentration of sample (mg/ml), K was the length of target gene (nucleotide), 1.6601?10–18 was the transfer constant
between Dalton and mg.
SYBR Green I reverse
transcription-polymerase chain reaction
For RT-PCR, cDNA was synthesized in 10 ml reaction buffer
containing 2 ml 5?M-MLV buffer, 0.5 ml specific
primer (2 mM), 0.25 ml M-MLV RTase (200 U/ml; Takara, Takara, Japan), 0.25 ml RNase
inhibitor (40 U/ml; Takara) and 1 ml template RNA. The thermal
profile for RT was 42 ?C for 15 min and 95 ?C for 2 min.The PCR was carried out in a 96-well plate in a reaction volume of
25 ml
containing 12.5 ml 2?Premix EX Taq buffer (Takara), 0.5 or 1 ml 50?SYBR Green I nucleic acid fluorescent dye (Takara), 2 ml template cDNA,
and forward and reverse primers at final concentrations varying from 0.1 mM to 1 mM. The thermal
profile for PCR was 95 ?C for 10 s, followed by 40 cycles of 95 ?C for 10 s and
60 ?C for 30 s. Immediately after the final PCR cycle, a melting curve analysis
was carried out to determine the specificity of the reaction by incubating the
reaction mixture at 95 ?C for 15 s, annealing at 60 ?C for 20 s, and then
slowly increasing the temperature to 95 ?C over 20 min [16]. The Ct used in the real-time PCR quantification
was defined as the PCR cycle number that crossed an arbitrarily chosen signal
threshold in the log phase of the amplification curve. Standard curve for each ORF was generated using dilution series of
standard samples as the RT-PCR template. The RNA extracted from virions was
diluted 100 times and amplified along with standard samples under optimal
concentrations, and the copy number of each ORF was calculated from standard
curves according to its Ct value. Each sample
had three replicates and all reactions were replicated three times
independently to ensure the reproducibility of the results. The RNA extracted
from healthy tobacco plants was used as a negative control. After the PCR, data
were viewed and analyzed using the ABI 7000 Sequence Detection Software (PE
Applied Biosystems). For each sample, the amplification plot and the
corresponding dissociation curve were examined. Due to the variation in length
and nucleotide composition, each amplicon had a unique Tm value [17].
Lab-on-a-chip assay
Lab-on-a-chip assay
was carried out in Aglient 2100 Bioanalyzer System (Agilent technologies, Palo Alto, USA),
following the manufacturer’s instructions of RNA 6000 Nano Reagents and
Supplies (Agilent technologies).
The concentrations of Fny-CMV genomic RNAs were analyzed using 2100 Expert
Software (Agilent technologies)
and the relative copy number ratios of them were calculated based on the
molecular weight.
Northern blot hybridization
The standard samples and viral RNAs were denatured with formamide/formaldehyde
prior to electrophoresis on a 1.6% agarose gel. Denaturation, electrophoresis,
blotting to nitrocellulose membranes, hybridization, washing of the blot and
autoradiography were carried out by standard procedures [18]. Probes were synthesized
by Random primer label kit (TaKaRa). The RNA extracted from infected and
healthy tobacco plants were used as positive and negative controls,
respectively. The hybridization was replicated three times.
Results
Optimization of real-time RT-PCR
The real-time RT-PCR was first optimized by varying the
concentrations of primers and SYBR Green I dye. It was found that 0.5 ml of SYBR Green
dye was optimal for the reaction. Increasing the SYBR Green I dye concentration
in the reaction mixture resulted in an error reading of the fluorescent signal
from the amplified DNA. Also, 0.2 mM each of forward and reverse primers was the
optimum concentration, as this gave the highest reporter fluorescence and the
lowest Ct value.
Specificity of real-time
RT-PCR
Real-time RT-PCR amplification of Fny-CMV ORFs with each primer set
produced the expected amplicon. The predicted RT-PCR product was confirmed by
agarose gel electrophoresis (data not shown). Dissociation curve analysis also
demonstrated that each of the primer pairs tested amplified a single PCR
product with a distinct Tm value.
Quantification of Fny-CMV
genomic RNAs by real-time RT-PCR
Each ORF was amplified clearly and reproducibly by real-time RT-PCR
(Fig. 1). The assay was proved to be highly reproducible, as
demonstrated by low Ct standard deviation values
between triplicates and a high correlation coefficient of the standard curves (R2>0.99) (Fig.
2). Based on respective Ct values, the copy number of each Fny-CMV ORF was calculated with
intra-group coefficient variations (CVs) of 0.76%–6.24% and inter-group CVs
of 2.51%–11.37% [19]. Copy numbers of Fny-CMV RNAs 1 and 3 in virions were
represented by the amounts of 1a ORF and 3a ORF, and RNA 4 was obtained by
subtracting the amounts of 3a ORF from those of CP ORF (Table 2). A
difference between the copy number of 2a ORF and that 2b ORF was observed
[(4.87±0.33)?107 vs.
(5.57±0.25)?107, CV=9.57%],
but it was in the range of inter-group CVs, indicating absence of RNA 4A in
Fny-CMV virions. Therefore, the copy number of RNA 2 was represented by the
average amounts of 2a ORF and 2b ORF, and the copy number ratios between
Fny-CMV RNA 1, 2, 3 and 4 in virions were determined to be
1.00:(1.17±0.11):(3.58±0.20):(5.81±0.31).
Comparison of the
quantification results between real-time RT-PCR, Lab-on-a-chip and Northern blot hybridization
assays
To confirm the quantification results of real-time RT-PCR, RNAs
extracted from virions were also analyzed in parallel by lab-on-a-chip and northern
blot hybridization assays. The results of lab-on-a-chip were shown in Fig. 3. Based
on the electropherogram, the relative concentrations of Fny-CMV RNA 1, 2, 3 and
4 were obtained, and the copy number ratios between them were deduced to be
1.00:(1.23±0.08):(3.68±0.15):(5.79±0.65).
The results of northern
blot hybridization are shown in Fig. 4. Due to different hybridization
efficiencies, the RNA 1, RNA 2 and RNA 3 standard samples with equal
concentration (Fig. 4, lane 4) yielded different hybridization
intensity. Thus when the relative concentration of each virus RNA was
calculated, its hybridization intensity was divided by that of corresponding
standard sample to eliminate this difference, and the copy number ratios
between Fny-CMV RNA 1, 2, 3 and 4 were determined to be
1.00:(1.20±0.10):(4.03±1.07):(6.19±2.51). The copy number ratios of Fny-CMV genomic RNAs in virions determined
by real-time RT-PCR, lab-on-a-chip and northern blot hybridization are compared in Table 3,
revealing the largest result variations of northern
blot hybridization.
Discussion
There are two general approaches for real-time PCR: the specific and
non-specific fluorescent reporting chemistries. Both display similar levels of
sensitivity [20]. The use of a specific probe-based assay such as TaqMan-PCR
requires high complementarity for probe binding, which might result in a
failure to detect a high sequence variability in the probe-binding region,
while non-specific assays using intercalating dyes such as SYBR Green I were
found to be more reliable, flexible, simple, and of lower cost for detecting
nucleic acid targets characterized by sequence variability, especially for RNA
viruses. SYBR Green I is a minor groove DNA binding dye with a high affinity
for double-strand DNA (dsDNA) and exhibits fluorescence enhancement upon
binding to dsDNA. The accumulation of amplified DNA is measured by determining
the increase in fluorescence over time, and this is followed by confirmation of
results by melting curve analysis [21]. In this study, real-time RT-PCR with
SYBR Green I was used for quantificating ORFs of Fny-CMV genomic RNAs, and the
results showed that it is reliable in determining the copy number ratios
between them.The disadvantages of real-time RT-PCR (SYBR Green I) quantification
was its indiscriminate binding to any dsDNA, which could result in fluorescence
readings in the primer dimers and non-specific amplification, so the
dependability of the assays relied greatly on the specificity of the
amplification [22]. Therefore in this study, primers for each ORF were
selected, separate RT and PCR steps were adopted, and Taq Hot Start DNA
polymerase (Takara) was used to minimize dimer
formation and non-specific amplification. The different RT and PCR efficiency
between primer sets was eliminated by carrying out RT-PCR of standard and test
samples at the same time, and variation among reactions was avoided by ROX
Reference Dye (Takara) in PCR mixture. Due to these
efforts, the established quantification system had a high sensitivity and
specificity.Standard curves indicated that the PCR efficiencies for 1a ORF, 2a
ORF, 2b ORF, 3a ORF and CP ORF (E=10–1/slope–1) were 125%, 97%, 105%,
98% and 80%, respectively. These values reflected less than optimal PCR
conditions for 1a ORF and CP ORF. But this is not entirely unexpected, as the
amplification of template by PCR is a process involving multiple components,
including structure of target gene, amount of templates, primers, ions,
nucleotides, enzyme activity, and reaction temperature. All of them are likely
to be dynamically changed as the reaction progresses and to subsequently affect
amplification efficiency. The Ct value for the
first dilution gradient of 1a ORF was below 10, which is unreliable in data
analysis and also will influence the calculation of amplification efficiency.
However, it is to be noted that quantification using the standard curve method
may be used without detailed optimization [23,24].To evaluate the efficiency of real-time RT-PCR, the amounts of
Fny-CMV genomic RNAs in virions were also determined by lab-on-a-chip
and northern blot hybridization.
RNA 4A was not detected in virions by all three methods, which was consistent
with the reports that RNA 4A could not be encapsidated by strains of CMV
subgroup I, or only at very low levels. The copy number ratios determined by three
methods were compared, indicating that these methods correlated with each
other, except the larger variations of northern
blot hybridization. The results of the Lab-on-a-chip assay were similar to those of the real-time RT-PCR, but
the major constraint of this method was the high purity requirements on test
samples. While the levels of viral RNAs in the host were relatively low, it was
impossible to study the amounts of viral RNAs in plant tissues by this method
directly. Northern blot hybridization also got the ratios closed to those of
other methods, but it has larger variations in quantification results resulting
from its own defects. For real-time RT-PCR, the amounts of target genes are
accurately recorded as Ct values and analyzed in a standard
format. This makes the assay considerably less subjective than other methods
and more suitable for quantification assay.
In conclusion, the real-time RT-PCR assay presented here offers a
sensitive and rapid method for high throughput detection and quantification of
Fny-CMV genomic RNAs. To our knowledge, this is the first report for
quantification of different RNA fragments in tripartite virus. The accurate
quantification property of this assay could be useful to monitor viral
replication kinetics, such as the changes of copy number ratios between genomic
RNAs in the progress of an infection, the effects of satellite RNA on helper virus,
the response to antiviral therapy, and the evaluation of viral tolerance levels
in new breeding programs. Northern blot hybridization also got the ratios
closed to those of other methods, but it has larger variations in
quantification results resulting from its own defects. For real-time RT-PCR,
the amounts of target genes are accurately recorded as Ct
values and analyzed in a standard format. This makes the assay considerably
less subjective than other methods and more suitable for quantification assay.
References
1 Roossinck MJ. Cucumber mosaic virus, a model for
RNA virus evolution. Mol Plant Pathol 2001, 2: 59–63
2 Gal-On A, Canto T, Palukaitis P.
Characterization of genetically modified cucumber mosaic virus expressing
histidine-tagged 1a and 2a proteins. Arch Virol 2000, 145: 37–50
3 Palukaitis P, Gar?ca-Arenal F. Cucumoviruses.
Adv Virus Res 2003, 62: 241–323
4 Duggal R, Rao AL, Hall TC. Unique nucleotide
differences in the conserved 3‘ termini of brome mosaic virus
RNAs are maintained through their optimization of genome replication. Virology
1992, 187: 261–270
5 Kwon CS, Chung WI. Differential roles of the
5‘ untranslated regions of cucumber mosaic virus RNAs 1, 2, 3 and
4 in translational competition. Virus Res 2000, 66: 175–185
6 Sivakumaran K, Bao Y, Roossinck MJ, Kao CC.
Recognition of the core RNA promoter for minus-strand RNA synthesis by the
replicases of Brome mosaic virus and Cucumber mosaic virus. J
Virol 2000, 74: 10323–10331
7 Gal-On A, Kaplan I, Palukaitis P.
Differential effects of satellite RNA on the accumulation of cucumber mosaic
virus RNAs and their encoded proteins in tobacco vs zucchini squash with
two strains of CMV helper virus. Virology 1995, 208: 58–66
8 Mackay IM, Arden KE, Nitsche A. Real-time PCR
in virology. Nucleic Acids Res 2002, 30: 1292–1305
9 Valasek MA, Repa JJ. The power of
real-time PCR. Adv Physiol Educ 2005, 29: 151–159
10 Boonham N, Smith P, Walsh K, Tame J, Morris J,
Spence N, Bennison J et al. The detection of Tomato spotted wilt
virus (TSWV) in individual thrips using real time fluorescent RT-PCR
(TaqMan). J Virol Methods 2002, 101: 37–48
11 Balaji B, Bucholtz DB, Anderson JM. Barley
yellow dwarf virus and cereal yellow dwarf virus quantification by
real-time PCR in resistant and susceptible plants. Phytopathology 2003, 93:
1386–1392
12 Delanoy M, Salmon M, Kummert J. Development of
real-time PCR for the rapid detection of episomal Banana streak virus
(BSV). Plant Dis 2003, 87: 33–38
13 Zhu JY, Zhu, SF, Liao XN, Gao, BD. Detection
of tomato ringspot virus by real-time
fluorescent RT-PCR one step assay. Acta Phytopathol Sinica 2003, 33: 338–341
14 Chen JS, Chai LH, Wu P. Change of relative
loading of cucumber mosaic virus genomic RNA and satellite RNA in
systemical-infection. Prog Biochem Biophys 2003, 30: 285–289
15 Jin B, Chen JS. Coexistence and competition of
two satellite RNAs of cucumber mosaic virus in systemic hosts. Wei Sheng
Wu Xue Bao 2005, 45: 209–212
16 Wilhelm J, Hahn M, Pingoud A. Influence of DNA
target melting behavior on real-time PCR quantification. Clin Chem 2000, 46:
1738–1743
17 Varga A, James D. Real-time RT-PCR and SYBR
Green I melting curve analysis for the identification of Plum pox virus
strains C, EA, and W: effect of
amplicon size, melt rate, and dye translocation. J Virol Methods 2006, 132: 146–153
18 Sambrook J, Russell DW. Molecular Cloning: A
Laboratory Manual. 3rd ed. New York: Cold Spring Harbor Laboratory Press 2001
19 Panicker G, Myers ML, Bej AK. Rapid detection
of Vibrio vulnificus in shellfish and Gulf of Mexico water by real-time PCR.
Appl Environ Microbiol 2004, 70: 498–507
20 Bustin SA, Nolan T. Pitfalls of quantitative
real-time reverse-transcription polymerase chain reaction. J Biomol Tech 2004,
15: 155–166
21 Heid CA, Stevens J, Livak KJ Williams PM. Real
time quantitative PCR. Genome Res 1996, 6: 986–994
22 Gachon C, Mingam A, Charrier B. Real-time PCR:
what relevance to plant studies?
J Exp Bot 2004, 55: 1445–1454
23 Ginzinger DG. Gene quantification using real-time
quantitative PCR: An emerging technology hits the mainstream. Experimental
Hematology 2002, 30: 503–512
24 Liu W, Saint DA. A new quantitative method of real time reverse
transcription polymerase chain reaction assay based on simulation of polymerase
chain reaction kinetics. Analytical Biochemistry 2002, 302: 52–59