Background
The small brown planthopper (SBPH),
Laodelphax striatellus Fallén (Delphacidae: Hemiptera), is a serious sap-sucking pest of agricultural crops. More importantly, it acts as an insect vector to transmit multiple plant viruses and causes severe yield losses. For example, it transmits reoviruses (RBSDV and maize rough dwarf virus) [
1,
2], tenuiviruses (rice stripe virus, RSV) [
3], rhabdoviruses (barley yellow striate mosaic virus and northern cereal mosaic virus) [
4,
5], and cripaviruses (Himetobi P virus, HiPV) [
6]. Both RSV and RBSDV are transmitted by SBPH in a persistent, circulative and propagative manner. RSV can be transmitted by SBPH from mother to offspring [
7], while RBSDV cannot. The rice stripe disease caused by RSV damaged over 957,000 hectares of paddy fields in 2003 and 1,571,000 hectares in 2004, accounting for 80% of the rice fields and a 30–40% yield loss in China [
8]. RBSDV not only infects rice plants to cause rice black-streaked dwarf disease, but also causes maize rough dwarf disease in maize [
1]. These viral diseases have been economically destructive in the rice- and maize- growing areas in China for decades [
9].
Rapid diagnosis of virus in vector is important for viral disease forecast and control. Various approaches have been developed for detection of RNA virus in its insect vector, including biological inoculation [
10,
11], direct observation using electron microscopes [
3,
12], antibody-based serological method [
13], and other molecular detection methods. Biological inoculation method is time consuming and labor-intensive; for example, it takes approximately one month for the plants to show disease symptoms after inoculation with RBSDV via viruliferous insects [
11]. Electron microscopes are very expensive and require specialty-trained personnel to operate them. In addition, the results of electron microscopy usually need to be confirmed by other methods [
12]. Serological methods, such as enzyme-linked immunosorbent assay, are economical for detection of high throughput samples [
14], but they are limited by the specificity and availability of antibodies against the virus. Deep sequencing and qRT-PCR and are of high sensitivity and specificity [
15‐
17], but expensive. RT-PCR is a rapid, specific and reliable assay to detect RNA viruses [
18,
19], especially for viruses that do not have antibodies available.
Traditional RT-PCR assays usually require purified RNA for reverse transcription. Isolation of RNA with commercial kits is expensive and time consuming. Besides, as SBPH is a small insect, measuring approximately 2–4 mm long, it is challenging to purify RNA from an individual SBPH. In this study, we developed a simplified RT-PCR assay for RNA virus detection in a single SBPH without RNA isolation. The sensitivity and reliability of this detection method are assessed and compared with those of traditional RT-PCR.
Methods
Preparation of SBPH used for virus detection
SBPHs free of RSV and RBSDV has been continuously maintained in our lab over ten years. A RSV-viruliferous SBPH population, with a RSV infection rate higher than 80%, was screened and reared in the lab on rice seedlings grown in 1 L beakers at 25 °C with a photoperiod of 16 h /8 h (light/dark).
The RBSDV-infected SBPH vectors were prepared as described previously [
20]. Non-viruliferous SBPHs were fed with rice black streaked dwarf diseased plants for three days, and then transferred to healthy rice seedlings for two weeks to pass the latent period. The SBPHs were subsequently collected for RBSDV detection. RSV-free and RSV-viruliferous SBPHs in 2nd to 4th instar were mixed and used for HiPV detection and duplex RT-PCR assay.
The SBPHs reared on rice seedlings were collected and frozen in −20 °C for 5 min. A single SBPH was placed in a 0.2 mL centrifuge tube, washed with 100 μL sterile H2O, and ground with sterile wet toothpicks in 30 μL sterile H2O. After centrifugation at 12,000 g for 1 min, the supernatant from individual SBPH was immediately transferred to new 200 μL centrifuge tube and used for reverse transcription.
RT-PCR
The crude RNA extract was used as template for simplified RT-PCR. The cDNA was synthesized using M-MuLV 1st strand cDNA synthesis kit (Sangon Biotech, P.R. China) according to the manufacturer’s protocol. The procedure is as followed: 11 μL crude sample and 1 μL random primer (Random 6, 0.2 μg/μL) were mixed and incubated at 65 °C for 5 min, the mixture then was transferred onto ice for 30 s immediately. After a short centrifuge, 4 μL 5 × M-MuLV reverse transcriptase buffer, 2 μL dNTP mix (10 mM), 1 μL RNase inhibitor (20U/μL) and 1 μL M-MuLV RT (200 U/μL) were added. The tubes were incubated in 25 °C for 10 min, 42 °C for 1 h, and 70 °C for 10 min. The resulting cDNA could be stored in −20 °C or applied to virus PCR detection directly.
Specific virus primers were used for PCR amplification to detect viruses in a single SBPH (Table
1). PCRs were performed with a final reaction volume of 20 μL, containing 5 μL cDNA, 10 μL 2 × Taq Master Mix (Vazyme Biotech, P.R. China), 0.5 μL each of the primers. The initial denaturation (95 °C, 5 min) was followed by 40 cycles of 95 °C for 30 s, 58 °C for 30 s, 72 °C for 1 min, and a final extension step at 72 °C for 10 min. A plasmid containing the RSV CP gene was used as PCR positive control for detection of RSV and a plasmid with the RBSDV P10 gene for RBSDV. Crude extracts from non-viruliferous SBPH were used as negative controls. The PCR products were evaluated by agarose gel electrophoresis.
Table 1
Primers used for RT-PCR or qRT-PCR amplification of RSV, RBSDV and HiPV
RSV CP-F1 | ATGGGTACCAACAAGCCAGC | EF198700 | 936 |
RSV CP-R1 | CTAGTCATCTGCACCTTCTG | | |
RBSDV P10-F | ATGGCTGACATAAGACTCGA | NC_003733 | 1677 |
RBSDV P10-R | TCATCTTGTCACTTTGTTTA | | |
HiPV-F | CTGGACAACATGATATTAGA | AB183472 | 678 |
HiPV-R | CTATTTCCCAGTTCCAAG | | |
RSV CP-F2 | GCCACTCTAGCTGATTTGCA | EF198700 | 167 |
RSV CP-R2 | GTGTCACCACCTTTGTCCTT | | |
Dot immunobinding assay (DIBA)
Crude extracts (1 μL) were dotted individually onto a nitrocellulose membrane (0.2 μm pore size, Pall) and allowed to dry at room temperature. Nonspecific sites were blocked with blocking buffer containing 2% skim milk in PBST (137 mM NaCl, 2 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH7.5, 0.05% Tween-20) at 37 °C for 30 min. The membrane was subsequently incubated in blocking buffer containing RSV- or RBSDV-specific monoclonal antibody at 1:5,000 dilution at 37 °C for 1.5 h. After wash for three times (each 5 min) with PBST, membranes were immersed in secondary antibody conjugated with HRP (Sigma-Aldrich, USA) at 1:2,000 dilution with PBST containing 2% skim milk. After another round of washes (3 times), the membranes were developed in a freshly prepared substrate solution containing 6 mg 4-chloro-1-naphtol, 2 mL ethanol, and 7 μL of 30% H2O2 in 10 mL PBS. The crude extracts of the individual SBPHs that developed well-defined dots and those with no dots on the membranes were kept in −20 °C, and used as positive and negative controls for the DIBA assay.
Comparison of sensitivity of virus detection with and without RNA isolation
To compare the detection sensitivities of the simplified RT-PCR using crude extract and the traditional RT-PCR using purified RNA, RSV RNA in individual SBPHs was prepared by these two methods and detected using RSV CP primers. Individual SBPHs were ground as described above. An aliquot of 11 μL supernatant was immediately transferred to a new tube for centrifugation and then the crude extract was used for reverse transcription. Another aliquot of 11 μL supernatant was used for Trizol RNA isolation. The reverse transcribed products were evaluated by PCR and qRT-PCR.
As SBPHs cannot be completely ground by toothpick, the tissue in the same volume of the supernatant (11 μL) may not be equal. In order to ensure that the two methods processed the same amount of tissue sample, the RSV-viruliferous SBPHs were treated in another way. Every 10 SBPHs at the third instar stage were collected in a 1.5 mL centrifuge tube and ground in liquid nitrogen using a pestle. Then 100 μL RNase-free ddH2O were added. After thorough vortexing, 11 μL suspensions was transferred to a new tube as crude extract and another 11 μL suspension was used for Trizol RNA isolation.
Trizol RNA isolation
Isolation of total RNAs with Trizol (Invitrogen, USA) was operated according to the manufacturer’s protocol with some modifications. An aliquot of 11 μL of supernatant was transferred to a new 1.5 mL tube. Subsequently 120 μL Trizol and 26 μL chloroform were added and mixed well. After centrifugation of the tube at 12,000 g for 15 min at 4 °C, the upper aqueous phase was immediately transferred into a new 1.5 mL Eppendorf tube. An equal volume of isopropanol was added to the aqueous layer, vortexed and placed in room temperature for 10 min. After centrifugation at 12,000 g for 10 min at 4 °C, the supernatant was removed and the RNA pellet was rinsed with 200 μL 75% ethanol twice. The RNA was dried and resuspended in 11 μL RNase-free ddH2O. The total RNA was used for reverse transcription or stored at −70 °C.
qRT-PCR
qRT-PCR was performed with an IQ5 Real-Time PCR System (Bio-Rad, USA) in a final reaction volume of 20 μL, containing 8 μL ddH
2O, 0.5 μL of each primer, 1 μL of cDNA template and 10 μL of 2 × SYBR@premix Ex Taq II (Tli Rnase H plus) (TaKaRa, including SYBR Green I, TaKaRa Ex Taq HS, dNTP mixture, Mg
2+ and Tli RnaseH). The PCR was run as described previously [
20].
Discussion
Plants are infected by a wide range of viruses that cause economic losses and pose threats to certain agricultural industries. Hemipteran insects act as the main vectors for the plant viruses, transmitting as much as 55% of the described plant viruses. Planthoppers in Hemiptera mainly transmit RNA viruses: among the 18 viruses they transmit, 14 are RNA viruses [
22]. Analysis of virus infection in the insect vector is important for preventing the potential disease threat. In this study, we developed a simplified RT-PCR method without RNA isolation for RNA virus detection in a single SBPH. The virus RNA preparation can be completed in approximately 2 min and requires no pre-treatments for RNA purification. This simplified method may be expanded for detection of RNA viruses in other insects in Hemiptera with similar insect size, such as aphids, leafhoppers, brown planthopper and whitebacked planthopper.
The traditional RT-PCR for RNA virus detection contains a laborious RNA isolation process [
18]. There are several methods to isolate RNA from insects, including using commercial SV total RNA isolation system (Promega) [
23], Trizol reagent (Invitrogen) [
24], and the RNAiso Reagent (TaKaRa) [
15]. The RNA samples isolated by these methods are of high quality with limited contamination and can produce well-defined amplification products in RT-PCR [
25]. However, it is expensive to use these commercial kits. Besides, it is time-consuming and inconvenient to extract RNA from a single SBPH because planthoppers are of small size. Our method offers an effective way to detect viruses in a large number of planthopper samples by eliminating the expensive and laborious RNA isolation process.
The quality of RNA is considered as one of the important factors that affect the accuracy of RT-PCR. RNA samples with their A260/A280 ratios falling between 1.8-2.0 are considered to be of good quality and selected for RT-PCR and subsequent analysis [
26]. The A260/A280 ratios of crude extracts of SBPH in our assay are below 1.8 (data not show) and the crude extracts are presumed to contain contaminating components, such as DNA, protein and lipid, which might inhibit RT-PCR reactions. However, the simplified RT-PCR successfully detected the RSV, RBSDV and HiPV in SBPH (Figs.
1,
2 and
3). Moreover, the cDNA prepared by the described protocol can be used for duplex RT-PCR and qRT-PCR for virus detection (Figs.
4, Fig.
6c and d). These results suggest that the contaminants in crude extracts do not affect the detection results, which is in agreement with previous studies showing that viruses could be detected specifically in plant samples by RT-PCR using unpurified RNA [
27]. One possible explanation is that the contaminants in planthopper extracts that can inhibit RT-PCR reactions are present at very low concentrations, not high enough to cause problems in RT-PCR.
On the other hand, we found that the reverse transcription method affects the result of the simplified RT-PCR. When we first incubated the crude extract at 65 °C with random primer (Random 6) and then proceeded to reverse transcription as described in the Methods, the RNA virus was successfully detected (Figs.
1,
2 and
3). However, when the cDNA was obtained by using the primeScript™ RT Master Mix (Takara, contains every components except the RNA template for reverse transcription) without heat treatment, the RNA virus could not be detected by the simplified RT-PCR. We hypothesize that virus particles in SBPH may require heat disruption at 65 °C to release RNA for cDNA synthesis. Alternatively, contaminating inhibitors may be inactivated by treatment at 65 °C.
The sensitivity of the detection method is another important factor for virus detection. Our results show that the simplified RT-PCR method bears the sensitivity sufficient to reliably detect RSV, RBSDV and HiPV in a single SBPH (Figs.
1,
2 and
3). Both RSV and HiPV were detected when cDNA samples were diluted up to 10
3 fold (Fig.
5). These results suggest that the simplified method can be practically applied to virus detection using a single SBPH. The relative abundance of RSV analyzed by qRT-PCR using Trizol-isolated RNA was 7 fold higher than that using RNA in crude extract (Fig.
6d). One explanation is that the Trizol reagent disrupts insect tissues more thoroughly to release RNA from tissues whereas some amounts of RNA may be lost together with tissues during centrifugation in the simplified method.
Conclusions
A simplified RT-PCR method was developed for detection of RNA virus in a single SBPH by preparing virus RNA in an easy and fast procedure. Our results demonstrated that crude extracts of SBPHs could be used as the template for RT-PCR. The viral RNA prepared by this method was also suitable for duplex RT-PCR and qRT-PCR detection. This protocol reduces the use of costly reagents, shortens the sample processing time, and improves the efficiency of virus detection. As the method is highly simplified and of sufficient sensitivity, it provides a useful tool to the investigation of epidemics of viral diseases in the early stage by enabling easy detection of viruses within a single insect vector.
Acknowledgements
We thank Dr. Mawsheng Chern of the University of California, Davis, for the advice and help in English editing of the manuscript.
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