One-step reverse transcription loop-mediated isothermal amplification assay for detection of Apple chlorotic leaf spot virus

Dandan Peng; Jipeng Xie; Wei Qiang; Kai-Shu Ling; Liyun Guo; Zaifeng Fan; Tao Zhou

doi:10.1016/j.jviromet.2017.07.002

One-step reverse transcription loop-mediated isothermal amplification assay for detection of Apple chlorotic leaf spot virus

J Virol Methods. 2017 Oct:248:154-158. doi: 10.1016/j.jviromet.2017.07.002. Epub 2017 Jul 15.

Authors

Dandan Peng¹, Jipeng Xie¹, Wei Qiang¹, Kai-Shu Ling², Liyun Guo¹, Zaifeng Fan¹, Tao Zhou³

Affiliations

¹ State Key Laboratory of Agrobiotechnology and Department of Plant Pathology, China Agricultural University, No. 2 West Yuanmingyuan Road, Beijing 100193, China.
² U.S. Department of Agriculture - Agricultural Research Service, U.S. Vegetable Laboratory, 2700 Savannah Highway, Charleston, SC 29414, USA.
³ State Key Laboratory of Agrobiotechnology and Department of Plant Pathology, China Agricultural University, No. 2 West Yuanmingyuan Road, Beijing 100193, China. Electronic address: taozhoucau@cau.edu.cn.

PMID: 28720542
DOI: 10.1016/j.jviromet.2017.07.002

Abstract

A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of Apple chlorotic leaf spot virus (ACLSV). In this method, a set of four primers was designed based on the conserved regions in the coat protein gene of ACLSV, and the primers were synthesized for the RT-LAMP assay using total RNA extracted from ACLSV-infected leaf tissues. The optimal reaction temperature and assay time were determined to be 64°C and 75min, respectively. The sensitivity of RT-LAMP reactions was reliable up to a maximum dilution of 1:3125, which was more sensitive than the RT-PCR assay. The successful application of RT-LAMP to field-collected apple samples demonstrated its potential for broader applications in effectively diagnosing diseases and, consequently, its potential to control ACLSV from spreading further, particularly in many developing countries around the world. To our knowledge, this is the first application of RT-LAMP for the detection of ACLSV.

Keywords: Apple chlorotic leaf spot virus; Detection; Latent infection; RT-LAMP; Trichovirus.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

DNA Primers
Flexiviridae / genetics
Flexiviridae / isolation & purification*
Malus / virology
Nucleic Acid Amplification Techniques / methods*
Plant Diseases / virology*
RNA, Viral / genetics
Reverse Transcription
Sensitivity and Specificity
Temperature

Substances

DNA Primers
RNA, Viral