Development of a Real-time Fluorescence Loop-mediated Isothermal Amplification Assay for Rapid and Quantitative Detection of Ustilago Maydis

Overview

Journal Sci Rep

Specialty Science

Date 2017 Oct 19

PMID 29042629

Citations 11

Authors

Yanyong Cao

Lifeng Wang

Liping Duan

Jingjing Li

Juan Ma

Shuna Xie

Lei Shi

Huiyong Li

Affiliations

Soon will be listed here.

Abstract

The common smut of corn, caused by Ustilago maydis is a troublesome disease of maize. Early and accurate detection of U. maydis is essential for the disease management. In this study, primer set Pep-2 was selected for LAMP (loop-mediated isothermal amplification) from 12 sets of primers targeting three U. maydis effector genes See1, Pit2 and Pep1 according to primer screening. The optimal concentrations of Bst DNA polymerase and Mg as well as inner/outer primer ratio of the LAMP reaction system were screened by combining a single factor experiment and an orthogonal design arrangement. The specificity of this real-time LAMP (RealAmp) assay was confirmed by negative testing for other pathogens. The detection sensitivity of the RealAmp assay was 200 times higher than that of detection through conventional PCR. Results of the RealAmp assay for quantifying the genomic DNA of U. maydis were confirmed by testing with both artificially and naturally infected samples. In addition, the RealAmp reaction could be conducted via an improved tube scanner to implement a "electricity free" assay from template preparation to quantitative detection. The resulting assay could be more convenient for use in the field as a simple, rapid, and effective technique for monitoring U. maydis.

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