Loop-mediated Isothermal Amplification (LAMP) for Identification of Pythium insidiosum

•High morbidity and mortality of the Pythium insidiosum infection are due to delayed diagnosis.•Our primary concern regarding molecular diagnosis is the lack of costly equipment in remote areas, where pythiosis is prevalent.•We successfully developed a loop-mediated isothermal amplification (LAMP) assay for the rapid detection of P. insidiosum.•LAMP is a suitable and efficient assay for detecting the P. insidiosum infection in resource-limited laboratories. Pythium insidiosum causes a life-threatening condition called pythiosis. High morbidity and mortality of pythiosis are consequences of delayed diagnosis. We aimed to develop a loop-mediated isothermal amplification (LAMP) assay for the rapid detection of P. insidiosum for use in remote areas, where pythiosis is prevalent. We designed four LAMP primers to amplify the rDNA sequence. A side-by-side comparison evaluated performances of LAMP and the previously-established multiplex PCR (M-PCR), using gDNA samples extracted from colonies of P. insidiosum (n = 28) and other fungi (n = 54), and tissues of animals with (n = 16) or without (n = 13) pythiosis. LAMP demonstrated a 50% shorter assay duration (1.5 h) and a 10-fold lower limit of detection (10-4 ng) than did M-PCR. Based on colony-extracted gDNAs, LAMP and M-PCR correctly reported P. insidiosum in all 28 samples, providing 100% sensitivity. While M-PCR did not amplify all fungal controls (100% specificity), LAMP falsely detected one organism (98% specificity). Based on the clinical samples, LAMP and M-PCR provided an equivalently-high specificity (100%). However, LAMP showed a markedly-higher sensitivity than that of M-PCR (88% vs. 56%). LAMP is a simple, useful, efficient assay for the detection of P. insidiosum in clinical specimens and pure cultures in resource-limited laboratories.