Abstract
Huanglongbing disease caused by ‘Candidatus Liberibacter asiaticus’ is a serious threat to citrus production at global level. Its management largely relies on accurate detection and taking preventive measures to check its spread through sensitive and reliable diagnosis. The available standard nucleic acid-based detection assays rely on use of purified DNA as template and are cumbersome. This study reports a simplified extraction protocol of citrus phloem tissues for rapid recombinase polymerase amplification (RPA) assay-based detection of ‘Candidatus Liberibacter asiaticus’ (CLas)-associated with huanglongbing (HLB). Simplified crude plant extract prepared in phosphate buffer and NaOH:EDTA (using mortar pestle or extraction bag without the use of liquid nitrogen) was successfully employed in the RPA assay. Developed assay targeted specific outer membrane protein (OMP) and ribosomal protein (RP) genomic regions of CLas and could detect its infection up to 10−9 to 10−11 dilution of plant extract and up to 1−10 ag µl−1 of DNA of CLas positive citrus tissues and plasmid DNA having respective gene insert, thus exhibiting sufficient sensitivity. Assay was highly specific and showed no cross-reactivity with the common pathogens infecting citrus exhibiting high reliability. Developed RPA assay was validated using 447 citrus samples collected across the Indian citrus belts, where 79.19 % were detected CLas positive and was more sensitive as compared to PCR wherein 0.45 % to 3.80 % samples were detected false negative. The developed RPA assay rapidly detected CLas infection in simply prepared crude plant extract in cost-effective manner and can be performed at low isothermal temperature in resource-poor laboratories for prevalence studies, bud wood certification and production of CLas-free planting materials of citrus.
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