Rapid and specific detection of apple chlorotic leaf spot virus in pear by reverse-transcription recombinase polymerase amplification.

Abstract

Apple chlorotic leaf spot virus (ACLSV) is an important virus infecting fruit trees. It causes serious economic losses in the global production of fruit trees belonging to the genera Prunus and Malus and can be vegetatively transmitted during propagation. In this study, an isothermal reverse transcription-recombinase polymerase amplification (RT-RPA) assay method was developed for detecting ACLSV in pear leaves. A set of RT-RPA primers showed high rapidity, sensitivity, and specificity in ACLSV detection. The RT-RPA assay was performed at a single, constant temperature of 42°C, could be completed in approximately 10min, and did not exhibit cross-reactivity with other common pear viruses. This RT-RPA assay was 100-fold more sensitive than regular RT-PCR. The optimized RT-RPA assay was further used to detect ACLSV in field-collected pear samples. These advantages make RT-RPA a promising diagnostic tool for determining ACLSV infection in pear certification programs. Keywords: apple chlorotic leaf spot virus; detection; pear; reverse transcription-recombinase polymerase amplification.