A rapid field-based assay using recombinase polymerase amplification for identification of Thrips palmi, a vector of tospoviruses

Abstract

Thrips palmi (Thysanoptera: Thripidae) is an important pest of vegetables, ornamentals, and legumes worldwide. Besides damage caused by feeding, it transmits several tospoviruses. Identification of T. palmi at an early stage is crucial in implementing appropriate pest management strategies. Morpho-taxonomic identification of T. palmi based on the adult stage is time-consuming and needs taxonomic expertise. Here, we report a rapid, on-site, field-based assay for identification of T. palmi based on recombinase polymerase amplification (RPA), its first application in insects. RPA primers designed based on 3′ polymorphisms of the Internal Transcribed Spacer 2 region efficiently discriminated T. palmi without any cross-reactivity to other predominant thrips species. RPA was performed with crude DNA, extracted from single T. palmi simply by crushing in sterile distilled water and could be completed within 20 min by holding the reaction tubes in the hand. The assay was further simplified by using fluorescent as well as colorimetric dyes thus eliminating the gel-electrophoresis steps. The presence of T. palmi was visualized by a change in color from dark blue to sky blue. The assay was validated with known thrips specimens and found to be effective in diagnosing the presence of T. palmi in natural vegetation. This on-site, rapid assay for diagnosis of T. palmi can be used by non-expert personnel in the field of quarantine and pest management. Keywords RPA • Melon thrips • Insect vector • Tospovirus transmission • Thrips diagnostics Key message • Thrips palmi an important pest of vegetables and ornamentals transmitting several tospoviruses • Morpho-taxonomic identification stage-specific, timeconsuming, and needs taxonomic expertise • Rapid, on-site, field-based assay for identification of T. palmi using recombinase polymerase amplification • Assay uses crude extract of T. palmi, completed within 20 min by holding the reaction tubes in hand without use of sophisticated laboratory instruments • The presence of T. palmi visualized by a change in reaction color • Useful for non-expert personnel in field-based identification, quarantine, and adopting suitable pest management strategies

Species‐specific primers for rapid detection of Monochamus saltuarius, an effective vector of Bursaphelenchus xylophilus in China

“…(Notomi et al., 2000), the high sensitivity and amplification rate may increase the rate of false positive results (Bhadra et al., 2018; Li et al., 2012; Liu et al., 2012; Peyrefitte et al., 2008; Tsai et al., 2018; Wang et al., 2020; Zhang et al., 2015). In addition, the complex primer design makes the development of LAMP primer time‐consuming (Priti et al., 2021; Zou et al., 2020). In light of the urgently requirement for the identification of M. saltuarius , SS–PCR detection is highly desirable, which would provide a simpler primer design process to accelerate this progress.…”

Development of a Polymerase Spiral Reaction-Based Isothermal Assay for Rapid Identification of Thrips palmi

“…A LAMP assay developed for T. palmi works well for on-site detection ( Przybylska et al, 2015 ). Recently, we reported an RPA-based assay for on-site identification of T. palmi that uses recombinase, single-stranded DNA binding protein, and strand displacing polymerase to amplify the target DNA at isothermal conditions ( Priti et al, 2021 ).…”

“…The assays also suffer resource-limited settings and are not portable. Isothermal amplification-based assays like loop-mediated isothermal amplification (LAMP) ( Przybylska et al, 2015 ) and recombinase polymerase amplification (RPA) ( Priti et al, 2021 ) for rapid identification of T. palmi are advantageous in this context. LAMP and RPA do not require any sophisticated laboratory equipment and can be performed at the field level within a short time.…”

Frontiers Approaches to the Diagnosis of Thrips (Thysanoptera): How Effective Are the Molecular and Electronic Detection Platforms?

“…RPA has been successfully applied for the detection of several animal and plant pathogens [ 194 , 195 , 196 , 197 , 198 ]. We have developed an RPA protocol for field-based identification of T. palmi [ 199 ]. This RPA is performed with crude extract from a single T. palmi in sterile distilled water.…”

“…The presence of T. palmi can be visualized by the appearance of fluorescence or by a change in color from dark blue to sky blue. The RPA assay is highly sensitive; it can detect as little as 0.2 ag of target DNA which is 10 9 -fold more sensitive than PCR using the same primers [ 199 ]. This on-site, rapid in-field assay for diagnosis of T. palmi is easy to use by non-expert personnel in plant biosecurity and pest management.…”