Reverse transcription loop-mediated isothermal amplification (RT-LAMP) for detection of coconut cadang-cadang viroid (CCCVd) variants in oil palm
2020
Madihah Ahmad Zairun | Maizatul Suriza Mohamed | Idris Abu Seman
The coconut cadang-cadang viroid (CCCVd) is the causal agent of orange spotting (OS) disease in commercial oil palm (Elaeis guineensis) plantations in Malaysia. At present, the methods used to detect OS are time-consuming and can be inaccurate. Here, we describe the development of a rapid, sensitive and specific method of detecting CCCVd variants in oil palm leaves using a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method. RT-LAMP detected CCCVd variants at a concentration of 2 ng μl-1 in less than 1 hr at 63°C using total ribonucleic acid (RNA) of leaf extracts; whereas a minimum of 20 ng μl-1 was required to detect CCCVd using conventional polymerase chain reaction (PCR). The utility of RT-LAMP as OS detection in plantations was evaluated using symptomatic and asymptomatic oil palm leaf samples from nurseries and field sites. RT-LAMP successfully detected CCCVd in OS-infected samples. Leaves with OS-like symptoms caused by nutrient deficiencies or leaf spot disease other than OS produced a negative result. The RT-LAMP results were confirmed by conventional PCR, indicating that RT-LAMP is a valuable tool, sensitive and rapid method of diagnosing OS in oil palm plantations that could be performed by plantation personnel for OS disease management in oil palm plantations.
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