Highly sensitive multiplex PCR for convenient quantification and differentiation of canine <i>Oomycota</i> pathogens: <i>Pythium insidiosum</i> , <i>Lagenidium giganteum</i> f. <i>caninum,</i> and <i>Paralagenidium karlingii</i>

<i>Oomycota</i> mammalian pathogens, including <i>Pythium</i> (<i>P</i>.) <i>insidiosum</i>, <i>Lagenidium</i> (<i>L</i>.) <i>giganteum</i> f. <i>caninum</i>, and <i>Paralagenidium</i> (<i>Para</i>.) <i>karlingii</i>, are increasingly recognized as causes of life-threatening infections in dogs, with diagnosis complicated by their clinical and phenotypic resemblance to fungal pathogens. Traditional diagnostic methods, such as serology, histopathology, and culture, often lack sensitivity and specificity, delaying appropriate management. Here, we developed a multiplex PCR assay with remarkable sensitivity, detecting as few as one DNA copy per reaction for <i>P. insidiosum</i> and <i>Para. karlingii</i> and 10 copies per reaction for <i>L. giganteum</i>. Validation on pure isolates from ATCC and USDA-ARS and other clinical isolates confirmed the assay's specificity, correctly identifying all three pathogens and showing no cross-reactivity in non-target isolates. Interestingly, the developed multiplex <i>Oomycota</i> PCR revealed that 45.5% (40/88) of serum samples positive for anti-<i>Pythium</i> antibody contained <i>Pythium</i> DNA. Additionally, among 24 suspected anti-<i>Pythium</i> antibody-positive samples, 54.2% (13/24) tested positive for <i>Pythium</i> DNA, while 8.3% (2/24) contained <i>Lagenidium</i> DNA. By allowing simultaneous detection and differentiation of these pathogens in a single reaction, the multiplex PCR significantly reduces diagnostic time and resource requirements. This highly sensitive diagnostic tool holds promise for enhancing the speed and accuracy of <i>Oomycota</i> infection diagnosis in dogs, as well as other mammalian species, including humans. This could enable the prompt implementation of species-specific diagnosis followed by early intervention, ultimately resulting in improved clinical outcomes.IMPORTANCEThis study addresses a critical gap in the diagnosis of life-threatening infections caused by <i>Oomycota</i> pathogens<i>-Pythium insidiosum</i>, <i>Lagenidium giganteum</i> f. <i>caninum</i>, and <i>Paralagenidium karlingii</i>-in dogs. These pathogens, often misdiagnosed as fungal infections due to overlapping clinical and phenotypic features, require accurate differentiation for appropriate treatment. Current diagnostic methods, including serology, histopathology, and culture, are time-consuming, lack specificity, and are prone to inconclusive results. The newly developed multiplex PCR assay offers a transformative solution by enabling simultaneous detection and differentiation of these pathogens with remarkable sensitivity and specificity. This tool not only reduces diagnostic time but also enhances the accuracy of pathogen identification, paving the way for earlier intervention and improved clinical outcomes. Moreover, its potential application in other mammalian species, including humans, underscores its broader significance in managing <i>Oomycota</i> infections.