Development of Reverse Transcription-Loop Mediated Isothermal Amplification (RT-LAMP) Technique for the Detection of Zucchini Yellow Mosaic Virus (ZYMV)

Abstract

Zucchini yellow mosaic virus (ZYMV) is one of the major viruses that cause disease in cucurbits.
The objective of this study was to develop a reverse transcription-loop mediated isothermal amplification (RT-LAMP) technique for the detection of ZYMV in infected cucurbits that uses primers that are specific to the nucleotide sequences of the ZYMV coat protein gene (CP) gene. The RT-LAMP assay took only an hour to detect ZYMV.
The RT-LAMP product of the tested samples was able to be visualized by staining directly in the tube with SYBR Green I, and no reaction was detected from the tissues of healthy plants and the tissues of the plants that were infected with Papaya ringspot virus (PRSV). The RT-LAMP products were analyzed by agarose gel electrophoresis, and ladder-like DNA fragments were observed in various sizes. The specificity of the RT-LAMP assay was assessed by sequencing of the RT-LAMP products. The result showed that the nucleotide sequences of the RT-LAMP products were those of the CP gene of ZYMV. The sensitivity of the RT-LAMP assay was higher than that of the reverse transcription-polymerase chain reaction technique (RT-PCR) by 10 fold serial dilution. The results showed that the RT-LAMP technique is a simple and accurate diagnostic method for the detection of ZYMV in cucurbits.