Development of a recombinase polymerase amplification combined with lateral flow dipstick assay for rapid and sensitive detection of bean common mosaic virus

Xin Chen,Zhe Yin,Danyu Shen,Xiaoyan Cui,Jiachao Qin,Huatao Chen,Xuehao Chen

doi:10.1186/s42483-021-00080-3

Abstract

Bean common mosaic virus (BCMV) is one of the most widespread and damaging viruses of cultivated legumes in the world. In addition to serious yield reduction and germplasm decline, BCMV infection also makes legumes more vulnerable to other pathogens. Early diagnosis of the virus is particularly important in limiting its spread. Recombinase polymerase amplification (RPA) is a novel isothermic amplification technology. The whole reaction can be done outside the laboratory environment after the nucleic acid sample is obtained. In this study, we established a rapid and sensitive RPA combined with the lateral flow dipstick (LFD) assay for detection of BCMV, based on the conserved BCMV coat protein (CP) gene sequence. Specific primers and a probe were designed, which amplify ~ 150 bp CP fragments from BCMV-infected samples under a constant temperature of 37 °C for 20 min. The end-labeled amplification products were detected by high-affinity LFD within 5 min. Sensitivity of this RPA-LFD assay was 1000 times greater than that of the conventional polymerase chain reaction (PCR) assay. Furthermore, when the primers/probe were used against related potyviruses including soybean mosaic virus (SMV), bean yellow mosaic virus (BYMV), and turnip mosaic virus (TuMV), the three potyviruses were not detected, indicating that the assay was BCMV species-specific. The RPA-LFD assay was also successfully applied for the detection of seed-borne BCMV in beans. The RPA-LFD assay has great potential application in the rapid diagnosis of BCMV in the field.

Highlights

Bean common mosaic virus (BCMV, genus Potyvirus) is a pathogen of common beans (Phaseolus vulgaris), as well as other cultivated and wild legumes (Morales 2006)
Establishment of Recombinase polymerase amplification (RPA)-lateral flow dipstick (LFD) assay for the detection of BCMV To evaluate the simplicity and rapidity of RPA-LFD assay for the detection of BCMV in legume plants, BCMV-infected soybean samples exhibiting severe mosaic symptoms were collected from different experimental farms in Jiangsu Province, China
polymerase chain reaction (PCR) results demonstrated that a 150-bp product corresponding to the partial BCMV coat protein (CP) gene was amplified from diseased soybean plants, but not from healthy soybean plants (Fig. 1a)

Summary

Introduction

Bean common mosaic virus (BCMV, genus Potyvirus) is a pathogen of common beans (Phaseolus vulgaris), as well as other cultivated and wild legumes (Morales 2006). Isolates of BCMV have been identified and characterized from different countries, including USA, India, China, Iran, South Korea, Turkey, and Australia, based on analysis of complete or partial genome sequences (Flores-Estévez et al 2000; Saqib et al 2005; Damayanti et al 2008; Cui et al 2014; Seo et al 2015; Jang et al 2018). An RT-PCRbased method combined with restriction enzyme analysis was first used to distinguish between BCMV and its (2021) 3:3 closest relative, bean common mosaic necrosis virus (BCMNV) (Saiz et al 1994). Flores-Estévez et al (2003) used RT-PCR with primers based on the viral coat protein (CP) gene sequence to differentiate BCMV from BCMNV. A disadvantage of conventional RT-PCR-based assays for BCMV is their unsuitability for rapid detection in the field

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