A reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for detecting the pathogen of maize rough dwarf disease in China
Maize rough dwarf disease (MRDD) is one of the most serious viral diseases of maize in China. The object of this study is to develop a reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid and sensitive detection of MRDD pathogen. Four RT-LAMP primers were designed on the basis of S10. Template RNA from infected leaves were used for RT-LAMP which were carried out under isothermal conditions at 61 °C for 90 min. RT-LAMP products were analyzed by electrophoresis in agarose gels or visual inspection of color change after staining with calcein. Sensitivity of the RT-LAMP assay was 100-fold higher than RT-PCR. This method can distinguish RBSDV from SRBSDV and the vectors carrying RBSDV from Rice stripe virus (RSV). The RT-LAMP assay shows excellent potential for application in the rapid and sensitive detection of MRDD pathogens in field samples or vector.
KeywordsMRDD Rapid detection RT-LAMP Calcein Diagnosis
Maize rough dwarf disease (MRDD) is a severe and widely spread disease caused by three closely related members of the genus Fijivirus, family Reoviridae: Rice black streaked dwarf virus (RBSDV), Mal de Rio Cuarto virus (MRCV) and Maize rough dwarf virus (MRDV) (Lenardon et al. 1998; Bai et al. 2002; Dovas et al. 2004). In China, the MRDD pathogen is RBSDV (Wang et al. 2003), which is also transmitted efficiently to rice (Wang et al. 2006a), wheat (Zhang et al. 2001), barley (Zhou et al. 2010) and other cereals (Wang et al. 2006a) by small brown planthoppers (Laodelphax striatellus Fallén, SBPH) (Ruan et al. 1984). Recently, Southern rice black-streaked dwarf virus (SRBSDV) is a new species in the genus Fijivirus Group 2 within the family Reoviridae (Zhang et al. 2008; Zhou et al. 2008), which is transmitted efficiently to rice and maize by the white backed planthopper (WBPH, Sogatella furcifera). Maize infected by SRBSDV showed the same symptoms as infected by RBSDV (Zhou et al. 2008). In China, the occurrence of MRDD showed a significant upward trend with the changes in climate, environment and adjustment of planting structures in recent years (Tao et al. 2013). The accurate and efficient detection of pathogens is essential for forecasting and controlling the spread of MRDD.
Several methods for detecting RBSDV were developed, such as biological detection (Zhang et al. 2001), electron microscopy (Zhou et al. 1998), enzyme-linked immunosorbent assay (ELISA) (Wang et al. 2006b) and RT-PCR (Wu et al. 2000). RT-PCR is more accurate but may not be suitable for a rapid testing within a short period of time. Thus, a simple and convenient method should be developed for detecting the MRDD pathogen. The reverse-transcription loop-mediated isothermal amplification (RT-LAMP) reaction occurs under isothermal conditions (Notomi et al. 2000). The results of the reaction can be observed by adding a fluorescent indicator calcein or through the direct visualization of precipitation in the tubes (Tomita et al. 2008; Mori et al. 2001). RT-LAMP was widely applied for the detection of pathogens (Keizerweerd et al. 2015; Hu et al. 2016; Yoshikawa et al. 2014; Gao et al. 2015; Nair et al. 2016). Here, the RT-LAMP assay was developed for rapid and sensitive detection of the MRDD pathogen of maize and the small brown planthopper.
Materials and methods
Maize samples and vector
Eight maize samples showing stunting and dark-green leaves symptoms were collected from the fields of Jiangsu Academy of Agricultural Sciences in the summer of 2015, and four maize samples showing stunting and dark-green leaves symptoms were collected from the fields of Hainan province of China in 2014.
SBPH with RSV were preserved previously. SBPH without RSV were fed on rice seedlings (Wuyujing 3) (Zhou et al. 2012a). When SBPH grew to 3rd instar, they were placed on fresh rice seedlings infected with RBSDV for 3 days to achieve viral infection. The surviving SBPH were fed on rice seedlings (Wuyujing 3) for 12-15d to cycle through the circulative period (Zhou et al. 2011).
All samples were tested by RT-PCR (Ji et al. 2011), and positive samples were stored at −80 °C.
The four oligonucleotide primers used in the RT-LAMP assay for the detection of MRDD
FIP(F1c + F2)
BIP(B1c + B2)
Total RNA was extracted from the maize leaves or SBPH using TRIzol® Reagent (Invitrogen, CA, USA) according to the manufacturer’s protocol with modifications for the volume of the reagents added. Briefly, approximately 100 mg of leaf tissue or a single SBPH that had cycled through the circulative period were mixed with 1.0 mL or 350 μL TRIzol reagent, respectively, for 5 min. At the final step, the RNA was resuspended in 35 μL or 20 μL DEPC-treated water, respectively. The RNA concentration was determined by spectrophotometric analysis (Thermo NanoDrop 2000C).
RT-LAMP was carried out using maize total RNA. In a total volume of 25 μL, the reaction contained 1 μL of maize RNA or 2 μL of SBPH RNA, 0.2 μM of F3 and B3, 1.6 μM of FIP and BIP, 8 mM of MgSO4, 0.8 M of betaine (Sigma), 1.4 mM of dNTPs, 0.5 mM of MnCl2, 50 μM of calcein (Sangon Biotech), 1 μL of Bst DNA polymerase (8 U/μL, NEB, USA), 0.5 μL of M-MuLV reverse transcriptase (200 U/μL, Fermentas), 0.5 μL of RNase Inhibitor (40 U/μL, Fermentas), 20 mM of Tris–HCl (pH 8.8, 25 °C), 10 mM of KCl, 10 mM of (NH4)2SO4, 0.1% TritonX-100, and supplemental DEPC-treated water. The mixture was incubated at 61 °C for 90 min, followed by 5 min at 80 °C.
The RT-LAMP products were evaluated by direct visual inspection of color change in the reaction tube as well as by examining the presence or absence of the characteristic white pyrophosphate precipitate in each reaction tube following centrifugation. In addition, 2 μL of products were checked for purity and size by ethidium bromide staining after agarose gel electrophoresis (1% agarose, TAE).
Sensitivity and specificity of RT-LAMP
To determine the specificity of the RT-LAMP, total RNA from maize leaf infected with SRBSDV or RBSDV was applied independently to the reaction mix. The optimized reaction system was used under the appropriate conditions for carrying out the RT-LAMP assay. Water served as the blank controls. An observation was made for whether the color changed and whether the precipitate emerged after the reaction finished.
Sensitivity of the RT-LAMP amplification compared with RT-PCR
Specificity of RT-LAMP
The disease caused by RBSDV has recently became one of the most damaging maize crop disease in China and led to significant economic loss (Zhang et al. 2008; Zhou et al. 2008, 2012a, b). MRDD need to be monitored and diagnosed at the early stages for forecasting and controlling the spread of virus diseases. RT-LAMP is a sensitive diagnostic technique based on amplification of reverse transcribed cDNA in a relatively short time and at a single reaction temperature, and there is no time loss for temperature change as in PCR or real-time PCR (Notomi et al. 2000). Electrophoresis equipment and gel imaging systems were also not necessary for the RT-LAMP. This method has been widely used in detection of various pathogens, especially for detection of viruses which cause similar symptoms in a host and show a high nucleotide homology (Zhao et al. 2010). In this study, four RT-LAMP primers were designed on the basis of S10, and the concentrations of the primers, the best reaction temperature and time for detecting the virus were observed, respectively. The specificity and sensitivity of RT-LAMP were tested. MRDD pathogeny could be detected in total RNA extracts diluted up to 1.1 × 10−6 μg, which is one hundred times more sensitive than the RT-PCR method.
In this study, calcein was used as the fluorescence indicator, the detection results could be directly judged by observing the color changes. Calcein was added into the reaction system before the reaction began (Dong et al. 2015; Luo et al. 2016; Jiang et al. 2017), which avoided the false positives resulted from the reaction product contamination easily occurred from opening the lids. In the RT-LAMP assay for detection of SRBSDV (Zhou et al. 2012a) and RBSDV (Zhou et al. 2012b), SYBR Green I as the fluorescence indicator was added after the reaction was finished by opening the lids, that increased the rate of false positives. A rapid and specific RT-LAMP method was established for detection of MRDD pathogen in maize and a single small brown planthopper. The RT-LAMP may be useful for preventing MRDD occurrence and spread.
This work was supported by National Natural Science Foundation of China (NSFC)-International Rice Research Institute (IRRI) project (31761143012); The National Key R&D Program of China (2017YFD0100400); Six talent peaks project of Jiangsu Province (NY-056); 333 high level talent training project of Jiangsu Province (BRA2018081).
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