Development of a recombinase polymerase based isothermal amplification combined with lateral flow assay (HLB-RPA-LFA) for rapid detection of "Candidatus Liberibacter asiaticus".

Dilip Kumar Ghosh,Sunil B Kokane,Manali R Motghare,Amol D Kokane,Sumit Bhose,Ashish J Warghane,Ashwani Kumar Sharma,M Krishna Reddy,Leandro Peña

doi:10.1371/journal.pone.0208530

Abstract

Huanglongbing (HLB) or citrus greening is highly destructive disease that is affecting the citrus industry worldwide and it has killed millions of citrus plants globally. HLB is caused by the phloem limited, Gram negative, non-culturable, alpha-proteobacterium, ‘Candidatus Liberibacter asiaticus’. Currently, polymerase chain reaction (PCR) and real time PCR have been the gold standard techniques used for detection of ‘Ca. L. asiaticus’. These diagnostic methods are expensive, require well equipped laboratories, not user-friendly and not suitable for on-site detection of the pathogen. In this study, a sensitive, reliable, quick and low cost recombinase polymerase based isothermal amplification combined with lateral flow assay (HLB-RPA-LFA) technique has been developed as a diagnostic tool for detection of ‘Ca. L. asiaticus’. The assay was standardized by designing the specific primer pair and probe based on the conserved 16S rRNA gene of ‘Ca. L. asiaticus’. The assay was optimized for temperature and reaction time by using purified DNA and crude plant extracts and the best HLB-RPA-LFA was achieved at the isothermal temperature of 38°C for 20 to 30 min. The efficacy and sensitivity of the assay was carried out by using field grown, HLB-infected, HLB-doubtful and healthy citrus cultivars including mandarin, sweet orange cv. mosambi, and acid lime. The HLB-RPA-LFA did not show cross-reactivity with other citrus pathogens and is simple, cost-effective, rapid, user-friendly and sensitive. Thus, the HLB-RPA-LFA method has great potential to provide an improved diagnostic tool for detection of ‘Ca. L. asiaticus’ for the farmers, nurserymen, disease surveyors, mobile plant pathology laboratories, bud-wood certification and quarantine programs.

Highlights

The present study has focused on the development of a rapid and user friendly recombinase polymerase amplification (RPA) combined with lateral flow assay-based diagnostic technique for detection of ‘Ca. L. asiaticus’ in citrus plants and its working principle is illustrated in figure (Fig 1)
In silico analysis was performed for specificity and cross reactivity of RPA-lateral flow assays (LFA) primers using the genome of different Candidatus species and other citrus pathogens with primer-BLAST software
In the present study, an HLB-RPA-LFA assay has been established for speedy visual detection of ‘Ca. L. asiaticus’ which requires minimum instrumentation and has the potential to be used as a point of care diagnostic tool

Summary

Introduction

‘Candidatus Liberibacter spp.’ causes the economically devastating disease of citrus known as Huanglongbing (HLB) or citrus greening disease [1, 2]. Numerous diagnostics techniques have been implemented for the detection of ‘Ca. L. asiaticus’ including biological indexing [11], enzyme-linked immunosorbent assay (ELISA) [12; [13], polymerase chain reaction (PCR) [14], real time-polymerase chain reaction (RT-PCR) [15, 16, 17, 18, 19], DNA-labeled hybridization probes [20] and loop mediated isothermal amplification (LAMP) [21, 22]. A number of RPA assays have been developed for rapid detection of plant pathogens such as Yam mosaic virus (YMV), Yam mild mosaic virus (YMMV) [27], Phytophthora spp. The present study has focused on the development of a rapid and user friendly RPA combined with lateral flow assay-based diagnostic technique for detection of ‘Ca. L. asiaticus’ in citrus plants and its working principle is illustrated in figure (Fig 1)

Materials and methods

Results

Discussion