Development of a closed tube loop-mediated isothermal amplification (LAMP) assay to detect Hirschmanniella oryzae (Tylenchida: Pratylenchidae) in exported aquatic plants

Abstract

Rice root nematode, Hirschmanniella oryzae is an important plant-parasitic nematode inflicting substantial destruction on economically important agricultural crops worldwide and invading an extensive range of plant hosts, including aquatic plants. However, morphological identification of the nematode has encountered limitations. This study developed a loop-mediated isothermal amplification (LAMP) assay targeting the genetic sequence of H. oryzae 28S ribosomal RNA gene as an alternative method. The LAMP assay demonstrated remarkable speed that the outcome could be achieved within 40 min at an isothermal temperature, 65 °C. The assay could interpret the fluorescence (SYBR safe) and color (SYBR green I) in a pre-reaction mixture, ensuring reliability and minimizing contamination risk during post-amplification. Our assay has high specificity and enables accurate differentiation from other nematode species in diverse aquatic plant samples. The sensitivity was higher than PCR and real-time PCR as low as 4.61 fg/μL. The development of fluorescent and colorimetric closed tube LAMP assay highlights it as a valuable tool that is a simplified, sensitive and rapid diagnostic method for detecting aquatic pest nematode, H. oryzae, at point-of-service level. The implementation of this method holds promise for future applications in plant quarantine departments and offers a significant advancement in the diagnosis of infected plants.