Short communicationOne-step reverse transcription loop mediated isothermal amplification assay for sensitive and rapid detection of Cucurbit chlorotic yellows virus
Introduction
Cucurbit chlorotic yellows virus (CCYV) is a newly discovered cucurbit-infecting crinivirus within the Closterviridae (Abrahamian et al., 2012, Gu et al., 2011, Huang et al., 2010, Okuda et al., 2010). The virus is transmitted by the Sweet potato whitefly, Bemisia tabaci of both the B and Q biotypes in a semi-persistent manner (Gyoutoku et al., 2009). The infected plants showed yellowing, mottling, and chlorosis on their lower leaves. Since its initial report in Japan, the disease has been reported in mainland China (Gu et al., 2011), Taiwan (Huang et al., 2010), Sudan (Hamed et al., 2011), and Lebanon (Abrahamian et al., 2012).
Accurate and efficient pathogen detection is essential for forecasting and controlling the spread of CCYV. Since CCYV is a newly identified virus, only one immunodiagnostic method is currently available (Kubota et al., 2011). The virus, which is solely transmitted by B. tabaci (Gyoutoku et al., 2009), is present at very low titers in the phloem of the host plant, thus necessitating the development of a highly sensitive detection method.
Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a sensitive diagnostic method for high specificity DNA amplification. This method, which has been adapted to diagnose various pathogens including many human, animal and plant viruses, is attractive due to the relatively short reaction time under isothermal conditions (Arita et al., 2009, Lee et al., 2011, Notomi et al., 2000, Varga and James, 2006, Zhou et al., 2012), especially for detecting viruses in a host (Zhao et al., 2010). The CCYV genome consists of two single-stranded RNA components designated as RNA1 and RNA2 (Okuda et al., 2010), which are 8607 bp and 8041 bp in length, respectively. Here, an RT-LAMP assay was developed and optimized for detection of CCYV using total RNA isolated from melon leaves.
Section snippets
Plant material and virus sources
Whiteflies, which act as CCYV vectors, were collected in 2012 from CCYV-infected melon fields in Henan provinces and released on healthy melon and cucumber seedlings in a growth chamber at 25 °C. Melon leaves showing symptoms were collected and used in this study.
RT-LAMP primer design
Based on the conserved sequences in the CCYV genome, primers for use in RT-LAMP were designed using PrimerExplorer V4 software (available at http://primerexplorer.jp/elamp4.0.0/index.html) using default settings (Table 1). Primers were
Optimization of the reaction conditions for one-step RT-LAMP assay for CCYV detection
The optimal temperature and reaction time of RT-LAMP assay were explored for CCYV detection. The reaction was carried out at 60, 61, 62, 63, 64, and 65 °C, using the 10−2 dilution of the RNA samples extracted from CCYV-infected melon leaves (Fig. 1(A) and (B)). When the reaction was performed at 60 and 65 °C for 60 min, it produced a low concentration of amplification products. However, when the reaction was performed at 62 and 64 °C the amplification products exhibited a clear ladder-like pattern
Discussion
CCYV is a novel cucurbit-infecting crinivirus, which was initially reported in Japan in 2010 (Okuda et al., 2010) and has spread rapidly worldwide. CCYV-infection results in significant economic losses for cucurbit plant farmers. Therefore, it is necessary to establish a reliable and efficient diagnostic method for rapid detection of this virus.
In this study, the one-step RT-LAMP method was established for the detection of CCYV in melon leaves. The optimal conditions for CCYV detection by
Acknowledgments
Financial support for this study has been provided by Joint Funding from the National Nature Foundation of China (U12041968) and the Postdoctoral Science Foundation of China (2013M530338).
References (13)
- et al.
One-step reverse transcription loop-mediated isothermal amplification assay for rapid detection of Cymbidium mosaic virus
J. Virol. Methods
(2011) - et al.
Use of reverse transcription loop-mediated isothermal amplification for the detection of Plum pox virus
J. Virol. Methods
(2006) - et al.
Reverse transcription loop-mediated isothermal amplification of DNA for detection of Barley yellow dwarf viruses in China
J. Virol. Methods
(2010) - et al.
Reverse transcription loop-mediated isothermal amplification of RNA for sensitive and rapid detection of southern rice black-streaked dwarf virus
J. Virol. Methods
(2012) - et al.
First report of Cucurbit chlorotic yellows virus on cucumber in Lebanon
Plant Dis.
(2012) - et al.
Development of a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) system for a highly sensitive detection of enterovirus in the stool samples of acute flaccid paralysis cases
BMC Infect. Dis.
(2009)