Real-time reverse transcription recombinase polymerase amplification (RT-RPA) assay for detection of cassava brown streak viruses

Abstract

Cassava brown streak disease (CBSD) caused by Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) is the most economically important viral disease of cassava. As cassava is a vegetatively propagated crop, the development of rapid and sensitive diagnostics would aid in the identification of virus-free planting material and development of effective management strategies. In this study, a rapid, specific and sensitive real-time reverse transcription recombinase polymerase amplification (RT-RPA) assay was developed for real-time detection of CBSV and UCBSV. The RT-RPA was able to detect as little as 2 pg/µl of purified RNA obtained from infected cassava leaves, a sensitivity equivalent to that obtained by quantitative real-time reverse transcription PCR (qRT-PCR), within 20 min at 37 °C. Further, the RT-RPA detected each target virus directly from crude leaf and stem extracts, avoiding the tedious and costly isolation of high-quality RNA. The developed RT-RPA assay provides a valuable diagnostic tool that can be adopted by cassava seed certification and virus resistance breeding programs to ensure distribution of virus-free cassava planting materials to farmers. This is the first report on the development and validation of crude sap-based RT-RPA assay for the detection of cassava brown streak viruses (UCBSV and CBSV) infection in cassava plants.

Keywords: Cassava brown streak viruses; Early virus detection; Isothermal amplification; Rapid diagnosis; Reverse transcriptase recombinase polymerase amplification (RT-RPA).

Figure 1

Amplifications for designed RPA primers for detection of cassava brown streak virus (CBSV) and Uganda cassava brown streak virus (UCBSV). G1 and G2 represent amplifications using RPA primers targeting the coat protein (CP) and cylindrical inclusion (CI) genes, respectively, for CBSV. G3 and G4 represent amplifications using primers targeting the CP and CI genes, respectively, for UCBSV. Lanes A, B and C: CBSV infected plant samples; lane D: Dual (CBSV and UCBSV) infected plant sample; Lanes E and F: UCBSV infected plant samples; lane G: Health control plant; lane NT: Non-template control; lane + ve: Dual (CBSV and UCBSV) infected positive plant sample; and L: 100 bp ladder (New England Biolabs).

Figure 2

Amplification curves for real time RT-RPA assay for detection of cassava brown streak viruses. (a, b) represents amplification curves for detection of CBSV and UCBSV, respectively, as observed in Eppendorf’s Mastercycler ep realplex instrument.

Figure 3

Specificity of RT-RPA assays for specific detection of cassava brown streak viruses by agarose gel electrophoresis and amplification curves. (a, b): Detection specificity of UCBSV- and CBSV-specific RT-RPA assay, respectively. Lane M, 100 bp ladder (New England Biolabs); Lanes 1 and 2, Sweet potato mild mottle virus (SPMMV), Ipomovirus; Lanes 3 and 4, Cowpea-aphid-born mosaic virus (CABMV), Potyvirus; Lanes 5 and 6, Healthy control; and Lanes 7 and 8, UC + (Dual [UCBSV and CBSV] infected positive control). (c) and (d): Real-time RT-RPA amplification curves for specificity using RPA primers for UCBSV and CBSV, respectively. Curves (i) and (ii) shows specific amplification for positive UCBSV samples; (v) and (vi) shows specific amplification for positive CBSV samples; (iii) and (vii), shows negative amplification for a sample infected with Sweet potato mild mottle virus (SPMMV), Ipomovirus; (iv) and (viii) shows negative amplification for a sample infected with Cowpea-aphid-born mosaic virus (CABMV), Potyvirus.

Figure 4

Cassava leaf and stem samples collected from the quarantine greenhouse showing characteristic CBSD symptoms. (a, b) feathery chlorosis and yellowing along the leaf veins; and (c) a young cassava stem with brown lesion symptoms.