RT-LAMP detection of Apple chlorotic leaf spot virus in apple and pear

A Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) assay was employed to develop a simple and efficient system for the detection of Apple chlorotic leaf spot virus (ACLSV) in apple and pear plants in India. A set of four primers targeting ACLSV coat protein (CP) gene were designed and used in RT-LAMP assay under isothermal condition at 57°C for 60 minutes. The RT-LAMP amplified products were visualized directly by adding SYBR gold nucleic acid fluorescent dye. The negative samples appeared dark orange in colour whereas in positive samples fluorescent yellowish green colour was observed under natural light. When compared to RT-PCR, the sensitivity of RT-LAMP was found to be similar. This technique successfully detected ACLSV in symptomatic and asymptomatic tissues of field grown apple and pear infected plants. This is the first attempt of ACLSV detection using RT-LAMP from India.