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Original Paper
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Acta Biochim Biophys
Sin 2006, 38: 669-676 |
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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 inter�action 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 hybridiza�tion 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.
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 fluo�rescence 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.
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