Development of field-applicable tests for rapid and sensitive detection of Candidatus Phytoplasma oryzae

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.

Review: a comprehensive summary of a decade development of the recombinase polymerase amplification

“…The former is to mix all the RPA reagents to initiate the reaction, the latter is to prevent from local depletion of the reaction reagents, thereby increasing the reaction rate. Wambua et al 79 reported that threshold fluorescence values were reached in 5-8 minutes when agitation was performed after 4 minutes, whereas the time to reach detectable levels ranged between 8 and 14 minutes without this agitation. In addition, constant shaking throughout the RPA reaction has been shown to further accelerate the RPA reaction rate, achieve more stable positive results and improve sensitivity, especially when the template concentration is close to the limit of detection.…”

“…200 words, to perform RPA amplification directly from the crude sample (in the same tube). RPA has been able to amplify nucleic acids directly from crude samples, such as plant tissue extract, 111,203 crude sap, 79,112 soil and water samples, 71 food samples (eggs and chicken meat) 119 and vaginal swab lysate. 137 Nevertheless, Choi and co-workers were the first to demonstrate a fully integrated RPA assay on foil-based microfluidics that was facilitated by the one-step assay approach.…”

RPA-PCR couple: an approach to expedite plant diagnostics and overcome PCR inhibitors

“…Besides maceration, extraction buffers have been employed to release nucleic acid from leaf disks (Si Ammour et al, 2017;Silva et al, 2018). Moreover, squeezed sap has been directly utilized for RPA (Wambua et al, 2017).…”

Immunological detection of the Weligama coconut leaf wilt disease associated phytoplasma: Development and validation of a polyclonal antibody based indirect ELISA

“…In contrast, serologic detection of phytoplasmas using specific antiserum was used as an economical and convenient method that allows analysis of many samples within a short time period [13]. Phytoplasma specific antibodies were first produced in 1974, with the use of a partially purified phytoplasma preparation as the immunogen [13,14].…”

“…In contrast, serologic detection of phytoplasmas using specific antiserum was used as an economical and convenient method that allows analysis of many samples within a short time period [13]. Phytoplasma specific antibodies were first produced in 1974, with the use of a partially purified phytoplasma preparation as the immunogen [13,14]. Since then, enriched or partially purified phytoplasma preparations from infected plants have been used to raise polyclonal and monoclonal antibodies against several phytoplasma strains [13,15,16].…”