PCR amplification of a putative gene for exo-1,3-β-glucanase to identify the pathogenic oomycete Pythium insidiosum

Abstract

Abstract Background: Pythium insidiosum is the etiologic agent of pythiosis, a life-threatening infectious disease. Diagnosis of pythiosis is difficult and often delayed. Early diagnosis can lead to prompt treatment, and therefore a better prognosis for patients with pythiosis. Molecular diagnostic techniques are useful if microbiological and immunological assays are not available, or in cases of suspected pythiosis that test negative by other methods. So far, PCR identification of P. insidiosum has been largely relied on amplification of the rDNA region. Objective: To evaluate the diagnostic performance of Dx3 and Dx4 primers specific for a putative gene for exo- 1,3-β-glucanase (PinsEXO1), which encodes a specific immunogen of P. insidiosum, for rapid single-round PCR identification of P. insidiosum, in comparison with the previously-reported rDNA-specific primers, ITSpy1 and ITSpy2. Materials and Methods: Genomic DNA (gDNA) from 35 P. insidiosum isolates and 48 control organisms were prepared to evaluate the diagnostic performance of the PinsEXO1- and rDNA-specific primers. Results: When amplifying the control gDNA by using the Dx3/4 and ITSpy1/2 primer sets, no PCR product was observed, indicating that both primer sets had 100% detection specificity. When amplifying the P. insidiosum gDNA, the Dx3/4 primers provided an expected 550-bp amplicon for all 35 isolates, while the ITSpy1/2 primers provided an expected 230-bp amplicon for only 32 isolates. Thus, detection sensitivity of the Dx3/4 and ITSpy1/2 primer sets were 100% and 91%, respectively. Conclusion: By using the Dx3/4 primers, PinsEXO1 was an alternative, efficient, and novel PCR target for rapid single-round PCR identification of P. insidiosum.