The RT-PCR identification and sequence analysis of Apple chlorotic leaf spot virus from apple cultivars in Jiaodong Peninsula, China

Abstract

A set of specific primer pairs was utilized to detect Apple chlorotic leaf spot virus (ACLSV) from seven different apple cultivars in Jiaodong Peninsula via reverse transcription polymerase chain reaction (RT-PCR), and the sequence of ACLSV genome was analysed. The results indicate that: (1) High-purity total RNA could be successfully isolated using plant RNA rapid extraction kit. The ratios of A260/A280 varied between 1.8 and 2.1. The fragmentation in agarose gel was good and the 28S and 16S bands were clear, which suggested that the extracted RNA had better quality and could be used for RT-PCR. (2) The amplified products by RT-PCR were approximately 220 bp, which showed the tested samples were infected by ACLSV in this study. (3) Sequencing analysis showed that the lengths of the target fragments were 217 bp, and the sequence identity rate ranged from 85.7% to 99.1% at the nucleotide level aligned with the corresponding sequences of other ACLSV strains in National Center for Biotechnology Information.