A one-step real time RT-PCR assay for quantifying rice stripe virus in rice and in the small brown planthopper (Laodelphax striatellus Fallen)

Journal of Virological Methods 151 (2008) 181–187

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Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet

A one-step real time RT-PCR assay for quantifying rice stripe virus in rice and in the small brown planthopper (Laodelphax striatellus Fallen) Xun Zhang, Xifeng Wang ∗ , Guanghe Zhou State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, No. 2, West Yuan Ming Yuan Road, Beijing 100094, China

a r t i c l e

i n f o

Article history: Received 10 December 2007 Received in revised form 11 May 2008 Accepted 14 May 2008 Available online 30 June 2008 Keywords: Rice stripe virus (RSV) Small brown planthopper (Laodelphax striatellus) Rice One-step real time RT-PCR Quantitative

a b s t r a c t Rice stripe virus (RSV) is an important pathogen affecting rice production in subtropical and temperate regions. One-step real time RT-PCR methods using the TaqMan probe are described for quantitative detection of RSV in rice tissues and in Laodelphax striatellus Fallen, the small brown planthopper (SBPH). Primers and probe for specific detection of RSV were designed within the conserved region identified within the coat protein (CP) gene sequence. A DNA fragment was amplified for mimicking the complementary RNA by PCR-based gene assembly, and was used for generation of standard RNA templates. A sensitivity assay showed that the detection limit of the assay was 20 copies, and the standard curve had a linear range from 20 to 2 × 105 copies, with good reproducibility. An internal control assay designed to target the rice ubiquitin 5 gene (UBQ5) was included in detecting RSV in different infected rice tissues. Simultaneously, a real time RT-PCR assay was used to detect the RSV CP gene in viruliferous SBPH. The results showed that the numbers of RSV CP genes in different tissues were variable. Accumulation of the RSV CP gene was greater in rice leaf and SBPH thoraco-abdominal tissue than in rice stem and SBPH head, respectively. As determined by an end-point dilution comparison, one-step real time RT-PCR was close to 104 -fold more sensitive than the indirect enzyme-linked immunosorbent assay (ELISA) for RSV detection. This quantitative detection assay provides a valuable tool for diagnosis and molecular studies of RSV biology. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Rice stripe virus (RSV), a typical member of the genus Tenuivirus, causes rice stripe disease, which is one of the most serious rice diseases in subtropical and temperate regions (Toriyama, 1983, 1986; Lin et al., 1990). In China, losses of 20–30% have been observed commonly in regions where Oryza sativa var. japonica is grown. There were severe crop losses in 2001, 2003, 2004, and 2006 in Jiangsu, Shandong, Anhui and Henan provinces due to climatic changes, changes in cultivation practice, and loss of variety resistance (Zhou et al., 2004; Zhang et al., 2007). RSV virons are not enveloped and contain four segments of linear singlestranded RNA, RNA1, RNA2, RNA3, and RNA4. RNA1 encodes an RNA dependent RNA polymerase (Toriyama et al., 1994). RNA2, RNA3 and RNA4 are ambisense in their coding strategy (Zhu et al., 1991, 1992; Takahashi et al., 1993). The coated protein is encoded by the viral complementary RNA3 (Takahashi et al., 1993). RNA4 encodes the major nonstructural protein (NCP) (on viral RNA), also called stripe disease-specific protein (SP), which may accumulate

∗ Corresponding author. Tel.: +86 10 62815928; fax: +86 10 62896114. E-mail address: [email protected] (X. Wang). 0166-0934/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2008.05.024

in infected plants (Toriyama, 1986; Kakutani et al., 1990; Zhu et al., 1992). RSV has been transmitted in most rice-growing areas by Laodelphax striatellus Fallen, the small brown planthopper (SBPH), and three other delphacid species, Unkanodes sapporona, Unkanodes albifascia, and Terthron albovittatum, in circulative and persistent mode (Toriyama, 1986). The virus can replicate in the SBPH ovary and is transmitted transovarially (vertically) to offspring by eggs (Toriyama, 1986). SBPH, especially in high density, can cause damage to rice plants when sucking the sap. Even at a much lower density, the vector of RSV SBPH can lead to more significant yield losses by virus infection (Hibino, 1996). Therefore, information of the numbers of viruliferous SBPH in a given local population is very important for forecasting and spray warning schemes to advise farmers on the potential threat to their crops (Kisimoto, 1993). It is too late to take measures when the disease symptoms appear and extensive infection occurs. A reliable diagnosis is essential for the development of efficient strategies of viral disease control. Many identification procedures are available for RSV detection in rice plants and SBPH (Koenig and Lesemann, 2001; Wong, 2001), including: direct observation (Ma, 1999); Western blotting (Toriyama, 1986; Qu et al., 1999); SPAELISA (square protein A enzyme-linked immunosorbent assay) (Xu

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et al., 1996); DIBA (dot-immunobinding assay) (Qian et al., 1994); and RT-PCR (reverse transcription polymerase chain reaction) (Cai et al., 2003). However, direct observation is not suitable for largescale and routine diagnosis because it is time-consuming (14–18 days for infected rice to show disease symptoms) (Ma, 1999), laborintensive, and has low levels of sensitivity and specificity (Toriyama, 1986; Qu et al., 1999). Western blotting, SPA-ELISA and DIBA techniques are more economical and better suited for large numbers of samples and have been used to forecast epidemics of rice stripe disease (Qian et al., 1994; Xu et al., 1996), but these techniques are limited by the supply and quality of antiserum (or monoclonal antibody) or specific probe, as well as by the type of sampling (Koenig and Lesemann, 2001). RT-PCR detects only RSV segment RNA1 in SBPH, but not RNA2 or RNA4 because of lower genomic RNA content (Cai et al., 2003). So, a sensitive and reliable method is required to detect RSV in rice and vector. Real time PCR technology has already proved to be efficient for the detection of plant RNA and DNA viruses, including: tomato spotted wilt virus (genus Tospovirus) (Roberts et al., 2000; Boonham et al., 2002); maize streak virus (genus Mastrevirus) (Lett et al., 2002), potato yellow vein virus (family Closteroviridae) (Lopez et al., 2006), grapevine leafroll-associated virus (family Closteroviridae) (Monique et al., 2007) Citrus tristeza virus (Susans et al., 2007) and tomato yellow leaf curl Sardinia virus (family Geminiviridae) (Giovanna et al., 2008). Here, we report for the first time a sensitive and reliable one-step real time RT-PCR procedure for RSV detection in infected rice tissues and viruliferous SBPH. The sensitivity, reproducibility and quantitation of this new detection method for RSV are assessed and compared with indirect ELISA techniques. 2. Materials and methods 2.1. Virus sources, insect vector and antiserum The isolate of RSV used here was obtained in 2004 in Yuanyang County, Henan Province, China from a rice plant showing typical stripe symptoms. The isolate was identified as RSV by indirect ELISA using antiserum, RT-PCR using primers located in RSV RNA3 (Wu, 2001), and sequence assay (GenBank accession number DQ299151), and then inoculated into the susceptible rice cultivar Wuyujing No. 3 by aviruliferous SBPH to increase virus concentration. The rice plants were later tested for RSV by indirect ELISA. Leaves were collected from RSV-positive plants displaying typical symptoms of RSV infection, and stored frozen at −70 ◦ C. Laboratory virus-free and viruliferous SBPH were reared on rice seedlings (2–3 cm tall) in glass vessels at 26 ◦ C with a 14 h light/10 h dark cycle. SBPH collected from an RSV-infected rice field in Zhengzhou, Henan Province of China were stored frozen at −70 ◦ C. The monoclonal antibody against RSV was provided by Dr. Y. Zhou (Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences) (Zhou et al., 2004). The goat anti-mouse IgG (H + L) alkaline phosphatase was purchased from Promega (Madison, WI, USA). 2.2. Primers and probe design In order to develop a real-time PCR detection system, the design of primers and probes for the TaqMan one-step real time RT-PCR assay was done according to specific criteria, such as maintaining a specific distance between the primers and probe, a high G + C content in the probe sequence, and a shorter amplification production. Two different systems of primers and probes were designed, one for detecting RSV (RSV system) and one for the rice housekeeping gene (internal control system). For the RSV system, pairs

of primers for preparation of viral RNA standards were designed by the PRIMER PREMIER software (Applied Biosystems) as follow. The first pair of primers: RCP-F: 5 -CTA GTC ATC TGC ACC TTC TGC C-3 (upstream) and RCP-R: 5 -ATG GGT ACC AAC AAG CCA GCC-3 (downstream) correspond to nucleotides of the RSV coat protein (CP) gene (GenBank accession number DQ299151), and were expected to amplify a full-length CP gene fragment of 969 bp. For fluorescence detection, one primer–probe combination was selected from combinations proposed by the PRIMER EXPRESS software (Applied Biosystems) according to the manufacturer’s instructions. The first primer–probe combination was designed: 266F: 5 -TGA AAG TGG CGG CTG GAA-3 (upstream), 285T: 5 -CAC AGA GGC TTC AAC CTT GGT GTC GA-3 (TaqMan probe), and 334R: 5 -CCA CCG AGG ACA CTA TCC CAT A-3 (downstream) targeting the conserved region within the RSV CP gene (GenBank accession number DQ299151). For the rice housekeeping gene, the sequence of the rice ubiquitin 5 gene (GenBank accession number AK061988) was selected because its expression was the most stable across all the tissue samples examined (Mukesh et al., 2006). UBQ-F: 5 -CTC GCC GAC TAC AAC ATC C-3 (upstream) and UBQ-R: 5 -AGG GCA TCA CAA TCT TCA CA-3 (downstream) correspond to nucleotides of the rice ubiquitin 5 gene, and were expected to amplify a fragment of 460 bp. The primer–probe combination: 533F: 5 -AGT GCG GCC TCA CCT ACG T-3 (upstream), 557T: 5 -ACC AGC AGG CTT AGG CGT AGG CT-3 (TaqMan probe) and 581R: 5 -CCG CCC CCA AAG AAC AG-3 (downstream) was designed to target the rice ubiquitin 5 gene as an internal control. All TaqMan probes were labeled with 6-carboxyfluorescein (FAM, excitation wavelength 494 nm, emission wavelength 521 nm) at the 5 end and Black Hole Dark Quencher 1 (BHQ-1) at the 3 end. 2.3. RNA extraction Total RNA from rice tissue (stored at −70 ◦ C) and individual SBPH were extracted using the RNAiso Reagent (TaKaRa Biotech., Dalian, China) according to the manufacturer’s instruction. The concentration of each RNA sample was measured with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, USA). Only the RNA samples with an A260 /A280 ratio (an indication of protein contamination) of 1.9–2.1 and an A260 /A230 ratio (an indication of reagent contamination) greater than 2.0 were used for the analysis. The integrity of RNA samples was assessed by agarose gel electrophoresis. 2.4. Preparation of viral RNA standards for one-step real time RT-PCR assay The sensitivity of the assay was examined. In order to determine the absolute number of RSV CP gene copies in infected rice tissue and viruliferous SBPH, RNA transcripts were synthesized in vitro, and serial dilutions were used in real time RT-PCR assays to generate external standard curves. A 975 nt cDNA fragment (including a primer recognition site) from the RSV CP gene was cloned into pGEM T-easy (Promega, WI, USA) according to the manufacturer’s instructions, and transformed into competent cells of Escherichia coli strain JM110. The presence of inserted PCR products was monitored by gel electrophoresis of restriction enzyme cleavage, PCR screening and sequence assay. Purified plasmid DNA was measured by NanoDrop ND-1000 (NanoDrop Technologies, USA), then linearized by restriction enzyme cleavage for the in vitro transcription. Positive strand RNA was transcribed using the RiboMAX Large Scale RNA Production Systems-T7 Kit (Promega, WI, USA) according to the manufacturer’s protocol, using 1 ␮g of linearized plasmid DNA as template. RNA was treated with

0.010 20 0.011 2.17 × 102 0.011 2.21 × 103

2.5. One-step real time RT-PCR assay and optimization

0.064 2.11 × 105 0.365 2.10 × 106 1.469 2.09 × 107 1.614 2.22 × 108

c

a

b

Healthy rice leaf. RSV-infected rice leaf. Mean calculated using all repetitions.

0.036 – Mean of A405 c Number of RSV genomic RNA copies

183

2 U of DNase I (Promega, WI, USA) for 15 min at 37 ◦ C, purified using a Transcript RNA Clean Up Kit (TaKaRa, Dalian, China). The purified RNA was quantified using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, USA). A dilution series from 2 × 105 to 20 copies per 2 ␮l was made for each transcript and stored frozen at −70 ◦ C. The parallel program was carried out for preparation of viral RNA standards of the rice UBQ5 gene, and a dilution series was made of 106 to 102 copies per 2 ␮l.

0.023 2.10 × 104

1/106 th 1/102 th 1/10th Positive controlb Negative controla

Table 1 Detection of RSV in 10-fold serial dilutions of the crude sap from infected rice by indirect ELISA and by one-step real time RT-PCR

1/103 th

1/104 th

1/105 th

1/107 th

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One-step real time RT-PCR reactions were performed in a final volume of 20 ␮l using the One-step PrimeScript RT-PCR Kit (TaKaRa Biotech., Dalian, China) according to the manufacturer’s instructions. The reactions, carried out with 2 ␮l of total RNA from insect and 0.2 ␮l of total RNA from plant, were performed on the Bio-Rad iCycler IQ Real-Time PCR Detection System. During the amplification process, the fluorescence intensity of the reporter dye (FAM), and of the quencher dye (BHQ-1) was recorded. These data allowed calculation of the normalized reporter signal, which is linked to the amount of product amplified. The threshold cycle (Ct-values, number of cycles for the fluorescence to reach the threshold) refers to the number of amplification cycles required for a significant increase in the reporter’s fluorescence. The data were analyzed with iCycler IQ Real-Time PCR Detection System Software Version 3.1. Protocol optimization was recommended for developing a good real time PCR detection system. According to the manufacturer’s recommendations, the primer was introduced initially at 100 nM in real time RT-PCR reactions. The upstream and downstream primers were subjected to an optimization of concentration using a 5 × 5 matrix of 50 nM, 100 nM, 200 nM, 300 nM, and 400 nM for each concentration of primer. This procedure was carried out using RNA resulting from in vitro transcription. The optimum concentration was found to be 200 nM for both upstream and downstream primers. The concentration of the TaqMan probe was then optimized in order to reduce the quantity used in reactions. Detection of the RSV and UBQ5 target by real time RT-PCR was efficient and reproducible with 200 nM TaqMan probe. The parameters of the reaction program were examined to determine the most suitable program. Different reaction temperatures and times were studied. The optimum denaturation time was 10 s at 95 ◦ C (the manufacturer’s recommendation was 5 s). Annealing–extension time and temperature were 60 s at 60 ◦ C, the same as the manufacturer’s recommendations. The most suitable program and parameter was reverse transcription of the viral RNA at 42 ◦ C for 5 min. PCR performed with the hot-start Taq polymerase included an enzyme activation step (95 ◦ C for 5 s) followed by 45 cycles of denaturation/annealing–extension (10 s at 95 ◦ C; 60 s at 60 ◦ C). Viral RNA transcripts, prepared as described above, were used in 10-fold serial dilutions to generate standard curves and to determine the assay efficiency and the quantification of viral target in the unknown samples. 3. Results 3.1. Standard curve The linear range of quantification of the one-step real time RTPCR assay for RSV genomic RNA was determined by using 10-fold serial dilutions of the standard ssRNA ranging from 20 to 2 × 105 copies to determine the end-point limit of detection and the linearity of the assay (Fig. 1A and B). Ct-values were measured in

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Fig. 1. Standard curve for TaqMan real time RT-PCR amplification of standard RSV ssRNA (viral transcripts). (A) Amplification plots showing the testing in duplicate of a 10-fold dilution series containing standard ssRNA at: 2 × 105 (a); 2 × 104 (b); 2 × 103 (c); 2 × 102 (d); 2 × 101 (e) template copies/reaction. The threshold (T) of normalized reporter fluorescence used for Ct calculation is represented with a black horizontal line. (B) and (D) Standard curve showing a linear relationship between standard ssRNA concentrations and Ct. Plots are Ct-values vs. log standard ssRNA concentrations (copies/reaction) generated from mean data of experiments performed in triplicate. (C) Amplification plots showing the testing in duplicate of a 10-fold dilution series containing standard ssRNA at: 106 (a); 105 (b); 104 (c); 103 (d); 102 (e) template copies/reaction.

duplicate and plotted against the known copy numbers of the standard sample. The standard curve covered a linear range of five orders of magnitude. The slope (−3.352) and the correlation coefficient (R2 = 0.999) of the standard curve showed that this assay could be used to quantify target RNA in infected rice tissue and individual viruliferous SBPH. Dilution curves were obtained with total RNA from RSV-infected rice and their amplification efficiency was sim-

ilar to that of the standard curves (data not shown). This real time RT-PCR assay enabled detection of as few as 20 RSV CP gene copies in rice total RNA extracts. A standard curve of quantification of UBQ5 gene in rice was developed as described above (Fig. 1C and D). The linear range was 106 to 102 copies, the slope was −3.145, and the correlation coefficient was 0.995.

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185

Table 3 Detection and absolute quantification of RSV genomic RNA copies in different tissues from viruliferous individual SBPH by quantitative one-step real time RT-PCR

Fig. 2. Comparison between end-point dilutions of indirect ELISA and one-step real time RT-PCR detection of RSV in infected rice leaf extracts. Indirect ELISA was performed on crude sap and PCR diagnosis was performed on total nucleic acids extracts. () Quantification (number of copies in sample) data resulting from real time RT-PCR assay. () Absorbance (405 nm) obtained using indirect ELISA assay. ET and PT are indirect ELISA and real time RT-PCR thresholds for RSV detection, respectively. (A) Corresponds to the difference of sensitivity between indirect ELISA and real time RT-PCR.

3.2. Comparison of sensitivity between indirect ELISA and real time RT-PCR In order to assess whether the real time RT-PCR assay had greater sensitivity than indirect ELISA in RSV detection, the endpoint dilution of the two techniques was carried out with plant extracts (Frederic et al., 2003). Tenfold serially diluted fractions of crude sap (using the grinding buffer) or of total RNA extracts (using DEPC-treated water) from RSV-infected rice leaf were produced and tested using the two detection techniques. In indirect ELISA, a positive reaction (twice that of the negative control) was recorded only when the crude sap of 100 mg of virus-infected leaf tissue was diluted 10-fold and 102 -fold. The values of A405 were 1.469 and 0.365, respectively, and 0.036 for the negative control (Table 1). Simultaneously, total RNA from 100 mg of infected leaf tissue was quantified after 10-fold serial dilution. For total RNA diluted 107 -fold, the measurable number of copies was about 20, reaching the minimum limit of the quantitative assay. These results suggested that the dilution limit for indirect ELISA was 103 -fold, but RSV genome RNA at a corresponding dilution was detected easily by real time RT-PCR assay. Comparing the results, real time RT-PCR was close 104 -fold more sensitive than indirect ELISA (Fig. 2). 3.3. Detection and quantitation of RSV in rice tissues One-step real time RT-PCR was carried out for detection and absolute quantification of RSV genomic RNA copies in different rice tissues. An equal fresh weight (100 mg) of leaf and stem tissue was collected from the same plant infected by RSV for RNA extraction. Table 2 Detection and absolute quantification of RSV CP gene copies in different tissues from viruliferous rice by quantitative one-step real time RT-PCR Sample

Ct ± S.D.a

Number of copies (X ± S.E.)b

RSV GUc /UBQ5 GU

Leaf UBQ5 RSV

20.31 ± 0.18 20.99 ± 0.12

2.51 × 108 ± 1.93 × 107 1.31 × 105 ± 1.29 × 104

5.22E−4

Stem UBQ5 RSV

21.39 ± 0.17 24.31 ± 0.27

1.13 × 108 ± 9.78 × 106 1.59 × 104 ± 1.97 × 103

1.41E−4

a Average threshold cycle (Ct) and standard deviation (S.D.) obtained from 30 samples. b Average number of RSV CP gene copies (X) per 100 mg of sample and standard error (S.E.). c Number of genomic units.

Sample

Ct ± S.D.a

CV%b

Number of copies (X ± S.E.)c

SBPH (female) Thoraco-abdominal Head

22.31 ± 0.52 36.01 ± 0.49

2.33 1.36

1.97 × 107 ± 2.13 × 106 N/Ad

SBPH (male) Thoraco-abdominal Head

27.21 ± 0.55 36.63 ± 0.32

2.02 0.87

9.22 × 105 ± 8.97 × 103 N/Ad

a Average threshold cycle (Ct) and standard deviation (S.D.) obtained from 30 different samples. b Coefficient of variation (CV%) between assays. c Average number of RSV CP gene copies (X) per sample and standard error (S.E.). d Ct-values are below the standard curve minimum limit and do not allow quantification.

RSV and the UBQ5 gene were quantified (Table 2). In the UBQ5 gene assay, the Ct-value obtained was 20.31 ± 0.18 in rice leaf tissue and 21.39 ± 0.17 in stem tissue; gene copies were 2.51 × 108 ± 1.93 × 107 in leaf, and 1.13 × 108 ± 9.78 × 106 in stem. In the RSV gene assay, the Ct-value obtained was 20.99 ± 0.12 in leaf and 24.31 ± 0.27 in stem; gene copies were 1.31 × 105 ± 1.29 × 104 in leaf, and 1.59 × 104 ± 1.97 × 103 in stem. The value of RSV GU/UBQ5 GU in the leaf tissue was 5.22E−4 and 1.41E−4 in the stem tissue. 3.4. Detection and quantitation of RSV in individual insects RSV genomic RNA in a single SBPH was detected using indirect ELISA and one-step real time RT-PCR. Using the indirect ELISA, none of 40 insects collected from the RSV-infected rice field was considered as viruliferous. However, using the one-step real time RT-PCR assay, the numbers of RSV genomic RNA copies ranged from 3.23 × 105 to 20 in individual insects, with only three exceptions. To assess RSV genomic RNA accumulation in different viruliferous SBPH tissues, total RNA of the head and of the thoraco-abdominal tissue from the same insect were extracted and quantification of RSV genomic RNA was carried out (Table 3). For female insects, the Ct-value of the thoraco-abdominal tissue was 22.31 ± 0.52 and the number of copies was 1.97 × 107 ± 2.13 × 106 . For male insects, the Ct-value of the thoraco-abdominal tissue was 27.21 ± 0.55, and the number of copies was 9.22 × 105 ± 8.97 × 103 . However, the number of copies in head tissue was not quantified because the Ct-values were below the detection threshold of real time RT-PCR. 4. Discussion In order to control rice stripe disease, estimates of the proportion of infected plants and viruliferous SBPH can be achieved through reliable methods for RSV detection and quantitation. A quantitative one-step real time RT-PCR protocol was developed that gives a rapid and reliable estimate of the number of RSV RNA3 segment copies in total RNA extracts from infected plants and SBPH. The decrease of threshold for virus detection leads to an improvement of control schemes for plant virus diseases, especially for perennial cultures where control is often based on the early eradication of infected plants and viruliferous vector insects (Thresh, 1988). The success of such methods depends mostly on the possibility of early diagnosis of the infection. Molecular detection methods (PCR, RT-PCR and hybridization assays) have allowed the detection of pathogens in infected hosts earlier than can be achieved by serological or biological detection protocols (Korschineck et al., 1991; Muller et al., 2001). Rapid and quantita-

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tive detection of virus in plant tissues or vector insects will help to reduce secondary spread of the pathogen through vector transmission and increase the efficiency of pesticide application, all of which require new, more sensitive methods with good precision and repeatability as diagnostic tools (Wang and Zhou, 2003). Quantitative real time PCR is a highly sensitive type of PCR, in which by detecting the fluorescence signal in each amplified cycle can have a higher level of sensitivity than that achieved by standard PCR. Also, it has provided a powerful guarantee for accurate quantification through use of the TaqMan probe. The choice of probes: sequence of probe primer, reporter dye and quencher dye are of central importance to the judgment of the reliability of the described method. In the present study, the most suitable was selected from 200 primers–probe combinations. Using this combination, quantitative real time PCR has excellent efficiency and repeatability. TAMRA (tetramethyl-6-carboxyrhodamine) was the universal quencher, but it limits the ultimate sensitivity and flexibility of FRET (fluorescence resonance energy transfer) assays because it contributes to an overall increase in background due to its own native fluorescence. We chose Black Hole Dark Quencher as a quencher dye because it has no native fluorescence, which ensures a higher signal-to-noise ratio. Previous studies showed that only RSV segment RNA1 and not RNA2 or RNA4 could be detected by RT-PCR in individual SBPH (Cai et al., 2003). The low concentration of RSV in individual SBPH was suggested as the possible reason. In this study, the RSV RNA3 segment was detected for the first time. Given the need for absolute quantification, the linear part of the standard curve was limited to 2 × 105 to 20 copies. The sensitivity of real time PCR was much higher than that of indirect ELISA because it could detect 20 copies of standard RNA. For the same weight of sample, end-point dilution factors were of the order of 103 for indirect ELISA and 107 for real time RT-PCR. Comparing the results, real time RT-PCR was close to 104 -fold more sensitive than indirect ELISA; so real time RT-PCR should be the better tool for detecting virus in a vector insect. Absolute quantification of RSV in individual SBPH showed the same difference between these methods. No SBPH collected in the field was identified as viruliferous by the indirect ELISA assay, but virus was detected and quantified in all parallel samples by one-step real time RT-PCR. For early forecasting, one-step real time RT-PCR is a valuable method for evaluating the rate and ability of the insect vector to acquire the virus. The present study established a real time RT-PCR assay that can be used for detection and absolute quantitation of RSV in rice tissues. The rice UBQ5 gene was selected as an internal control for the RSV quantification assay. As a housekeeping gene, UBQ5 was the most stable across all the tissues examined (Mukesh et al., 2006). RNA extract error can be eliminated from data analysis by quantitation of the UBQ5 gene. Comparison of RSV GU versus UBQ5 GU values showed that a greater RSV genomic RNA accumulation occurred in rice leaf tissue than in stem tissue. This result suggested that RSV had reproduced actively more in the leaf than in the stem. Absolute quantitation of RSV in different SBPH tissues was carried out. The results showed a higher number of virus copies in thoracoabdominal tissues than that in the head tissues of viruliferous SBPH. The number of copies was especially high in females, because the virus is able to replicate in the ovary. Further, the methods established in this study should prompt more studies of virus–vector interaction. In conclusion, compared with other routine detection methods, real time RT-PCR has considerable advantages in sensitivity, accuracy and large-scale identification. The protocol described above represents a useful tool for forecasting rice stripe disease accurately and for studying optimal control of vector–virus–plant interactions, and for identification of other virus strains carried by SBPH.

Acknowledgements We thank Dr. Yijun Zhou (Institute of Plant Protection, Jiangsu Academy of Agriculture Sciences) for kindly providing virus antibody and virus-free small brown planthoppers. Dr. Jiajian Xie (our Institute) provided technical support. Financial support was provided by the National Basic Research of China (973 contract 2006CB101903), 863 High Technology Program (2007AA10Z415) and the Natural Sci-Tech Supporting Project of China (2006BAD08A04).

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