Elsevier

Journal of Virological Methods

Volume 195, January 2014, Pages 63-66
Journal of Virological Methods

Short communication
One-step reverse transcription loop mediated isothermal amplification assay for sensitive and rapid detection of Cucurbit chlorotic yellows virus

https://doi.org/10.1016/j.jviromet.2013.08.037Get rights and content

Abstract

A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of Cucurbit chlorotic yellows virus (CCYV). In this procedure, a set of four primers matching a total of six sequences in the coat protein gene region of CCYV was synthesized for the RT-LAMP assay using total RNA extracted from CCYV-infected melon leaf tissues, and the optimum reaction temperature and assay time were determined. The sensitivity assay showed that the virus was detectable in RT-LAMP reactions at dilutions of 1 × 10−11, which was 105 times more sensitive than the RT-PCR assay. The RT-LAMP assay for CCYV and Sweet potato chlorotic stunt virus (SPCSV) exhibited high specificity for CCYV. This simple and sensitive method has potential for detection of CCYV in samples collected in the field.

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).

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