Development of field-applicable tests for rapid and sensitive detection of Candidatus Phytoplasma oryzae. (October 2017)

• Record Type: Journal Article
• Authors: Wambua, Lillian; Schneider, Bernd; Okwaro, Allan; Wanga, Joseph Odhiambo; Imali, Olive; Wambua, Peninah Nduku; Agutu, Lavender; Olds, Cassandra; Jones, Chris Stephen; Masiga, Daniel; Midega, Charles; Khan, Zeyaur; Jores, Joerg; Fischer, Anne
• Is Part Of: Molecular and cellular probes. Volume 35(2017)
• Page Start: 44
• Page End: 56
• Publication Date: 2017-10
• Subjects: Candidatus Phytoplasma oryzae -- Napier grass -- Diagnostics -- Recombinase polymerase amplification -- Lateral flow assay; Molecular probes -- Diagnostic use -- Periodicals; Pathology, Cellular -- Technique -- Periodicals; Cell Biology -- Periodicals; Molecular Biology -- Periodicals; Sondes moléculaires -- Utilisation diagnostique -- Périodiques; Cytopathologie -- Technique -- Périodiques; 572
• DOI: 10.1016/j.mcp.2017.06.004
• Languages: English
• ISSNs: 0890-8508

Abstract

Abstract: Napier grass Stunt Disease (NSD) is a severe disease of Napier grass ( Pennisetum purpureum ) in Eastern Africa, caused by the leafhopper-transmitted bacterium Candidatus Phytoplasma oryzae. The pathogen severely impairs the growth of Napier grass, the major fodder for dairy cattle in Eastern Africa. NSD is associated with biomass losses of up to 70% of infected plants. Diagnosis of NSD is done by nested PCR targeting the phytoplasma DNA, which is difficult to perform in developing countries with little infrastructure. We report the development of an easy to use, rapid, sensitive and specific molecular assay for field diagnosis of NSD. The procedure is based on recombinase polymerase amplification and targets the imp gene encoding a pathogen-specific immunodominant membrane protein. Therefore we followed a two-step process. First we developed an isothermal DNA amplification method for real time fluorescence application and then transferred this assay to a lateral flow format. The limit of detection for both procedures was estimated to be 10 organisms. We simplified the template preparation procedure by using freshly squeezed phloem sap from Napier grass. Additionally, we developed a laboratory serological assay with the potential to be converted to a lateral flow assay. Two murine monoclonal antibodies with high affinity and specificity to the immunodominant membrane protein IMP of Candidatus Phytoplasma oryzae were generated. Both antibodies specifically reacted with the denatured or native 17 kDa IMP protein. In dot blot experiments of extracts from infected plant, phytoplasmas were detected in as little as 12, 5 μg of fresh plant material. Highlights: Development of molecular and serological assays for detection of Candidatus Phytoplasma oryzae. Both assays show high specificity and sensitivity. The recombinase polymerase assay is applicable in the field directly from sap extracted from plants and is combined with a lateral flow device. This is the first description of an RPA based assay applied to phytoplasma detection.