A reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for detecting the pathogen of maize rough dwarf disease in China

Yongjian Fan,Yijun Zhou,Wenjuan Shi,Ying Lan,Feng Sun,Xuejuan Li,Tong Zhou,Linlin Du

doi:10.1007/s13313-019-00649-9

Abstract

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.

Highlights

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 dwarfLinlin Du and Wenjuan Shi contributed to this work.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)
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)
Calcein was used as the fluorescence indicator, the detection results could be directly judged by observing the color changes

Summary

Introduction

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). A simple and convenient method should be developed for detecting the MRDD pathogen. 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). The RTLAMP assay was developed for rapid and sensitive detection of the MRDD pathogen of maize and the small brown planthopper

Methods

Results

Conclusion