Development of Real-Time Isothermal Amplification Assays for On-Site Detection of Phytophthora infestans in Potato Leaves

Abstract

Real-time loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) assays were developed targeting the internal transcribed spacer 2 region of the ribosomal DNA of Phytophthora infestans, the potato late blight causal agent. A rapid crude plant extract (CPE) preparation method from infected potato leaves was developed for on-site testing. The assay’s specificity was tested using several species of Phytophthora and other potato fungal and oomycete pathogens. Both LAMP and RPA assays showed specificity to P. infestans but also to the closely related species P. andina, P. mirabilis, P. phaseoli, and P. ipomoeae, although the latter are not reported as potato pathogen species. No cross-reaction occurred with P. capsici or with the potato pathogens tested, including P. nicotianae and P. erythroseptica. The sensitivity was determined using P. infestans pure genomic DNA added into healthy CPE samples. Both LAMP and RPA assays detected DNA at 50 fg/μl and were insensitive to CPE inhibition. The isothermal assays were tested with artificially inoculated and naturally infected potato plants using a Smart-DART platform. The LAMP assay effectively detected P. infestans in symptomless potato leaves as soon as 24 h postinoculation. A rapid and accurate on-site detection of P. infestans in plant material using the LAMP assay will contribute to improved late blight diagnosis and early detection of infections and facilitate prompt management decisions.

Loop Mediated Isothermal Amplification: Principles and Applications in Plant Virology

“…Thus, several protocols in plant pathology were developed, where crude plant saps were used as template. This implementation accelerated the time for the diagnosis, avoiding any previous step for samples preparation and DNA or RNA purification [26,[34][35][36][37][38][39]. In that way, diagnosis time was speeded up for 2-3 h, which was necessary to complete the extraction of DNA/RNA of target organism.…”

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

“…Similarly, NaOH (Karakkat et al, 2018;Qian et al, 2018), standard ELISA grinding buffer (Miles et al, 2015), and TE buffer (Ahmed et al 2018) have also been used for maceration. 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).…”

Rapid and Visual Detection of the Emerging Novel Duck Reovirus by Using a Specific and Sensitive Reverse Transcription Recombinase Polymerase Amplification Method

“…Recombinase polymerase ampli cation (RPA) is an emerging next-generation molecular diagnostic technique, which considered as a simple, rapid and cost-effective isothermal ampli cation method [17]. RPA is a considerably simpler technique and does not require a thermal cycler and could be completed at a low temperature (37 ℃ to 42 ℃) in less than 20 min in time [18,19]. RPA has been widely applied in the clinical diagnosis of animal and plant viral including porcine circovirus-2 [20], porcine parvovirus [21], foot-and-mouth disease virus [22,23], avian in uenza [24], potato virus Y [25], maize chlorotic mottle virus [26] and apple stem grooving virus [27].…”