• biosensor
• diagnostic assay
• gold nanoparticles/DNA probe
• Macrophomina phaseolina
• rapid colorimetric detection
• Recombinase Polymerase Amplification (RPA)

Macrophomina phaseolina (Tassi) Goid. is a soil and seed-borne polyphagous fungal pathogen infecting more than 500 cultivated and wild plant species worldwide, including economical important crops such as legumes and vegetables. M. phaseolina shows the ability to adapt to different agro-ecological conditions, suggesting a great genetic diversity within this species. Although considerable research related to the biology and ecology of M. phaseolina has been conducted, it continues to cause severe economic losses in many crops. The fungus is likely to become more important under climate change scenarios of increased heat and drought stress and may find favourable conditions in Italy, Spain, France and central Europe. In this context, the early diagnosis and accurate detection of the pathogen is an essential step in plant disease management. The Recombinase Polymerase Amplification (RPA) technique is an isothermal DNA amplification method and is becoming a popular molecular tool for the rapid, sensitive, portable and cost-effective detection of pathogens. RPA, operating at 37-42°C with minimal sample preparation, allows the amplification of the target sequence in 20-30 minutes even in the presence of some known PCR inhibitors. Furthermore, RPA has been successfully integrated with different end point detection strategies without the need for agarose gel electrophoresis.
This study describes the development of a RPA assay for the specific detection of M. phaseolina and the use of DNA-functionalized gold nanoparticles as probes for the end point detection of RPA amplicons.
The RPA reaction was performed using a TwistAmp™ lyophilized kit. Two species-specific primer pairs for M. phaseolina, based on the rDNA intergenic spacer (IGS) sequence following the manufacturer instructions (30 bp), were designed. Conditions of the assay selected for optimization were: temperature, mixing, incubation time and use of betaine. The optimized RPA protocol successfully amplified the expected 248 bp product in all the M. phaseolina isolates tested (20). In order to confirm the targeted sequence the amplicons were purified and sequenced in both directions. Primer specificity was also checked and confirmed using seven different non-target organisms isolated from melon tissues.
The RPA amplicons were first analysed by agarose gel electrophoresis in order to verify the presence of the expected product and then were used to develop a colorimetric assay with a DNA-gold nanoparticle (AuNPs) probe. Freshly synthesized AuNPs (13 nm) were functionalized with a thiolated detection probe designed on the RPA amplicon sequence. Visual detection of the amplification products was performed via hybridization at 65°C for 5 min with the DNA-labelled nanogold probe, followed by salt-induced AuNP aggregation that would normally yield a visible red (positive) to blue (negative) colour change. The hybridization of M. phaseolina RPA amplicons with the AuNPs probe followed by salt addition (MgSO4 final concentration 50 mM) maintained the original red colour while the negative controls turned blue. Colour changes were compared by naked eye and by UV-visible spectrum analysis using a µDrop plate.
The promising results obtained suggest that the developed assay may represent a simple and rapid tool for the molecular detection of M. phaseolina.