METHODS: A hydrolysis probe for a real-time PCR assay was designed to recognize a specific DNA sequence within the P. knowlesi small subunit ribosomal RNA gene. The sensitivity, linearity and specificity of the assay were determined using plasmids containing P. knowlesi DNA and genomic DNA of P. falciparum, P. knowlesi, P. malariae, P. ovale and P. vivax isolated from clinical samples. DNA samples of the simian malaria parasites Plasmodium cynomolgi and Plasmodium inui that can infect humans under experimental conditions were also examined together with human DNA samples.
RESULTS: Analytical sensitivity of the P. knowlesi-specific assay was 10 copies/μL and quantitation was linear over a range of 10-106 copies. The sensitivity of the assay is equivalent to nested PCR and P. knowlesi DNA was detected from all 40 clinical P. knowlesi specimens, including one from a patient with a parasitaemia of three parasites/μL of blood. No cross-reactivity was observed with 67 Plasmodium DNA samples (31 P. falciparum, 23 P. vivax, six P. ovale, three P. malariae, one P. malariae/P. ovale, one P. falciparum/P. malariae, one P. inui and one P. cynomolgi) and four samples of human DNA.
CONCLUSIONS: This test demonstrated excellent sensitivity and specificity, and adds P. knowlesi to the repertoire of Plasmodium targets for the clinical diagnosis of malaria by real-time PCR assays. Furthermore, quantitation of DNA copy number provides a useful advantage over other molecular assays to investigate the correlation between levels of infection and the spectrum of disease.
OBJECTIVE: The aim of this study is to screen potential yeast, which has the ability to inhibit the growth of Aspergillus brasielensis (isolate A1) and Aspergillus flavus section flavi (isolate A17) isolated from apple fruits.
METHODS: Antagonism test using YMA dual culture medium using in vitro assays and ITS rDNA identification were performed.
RESULTS: The result showed that 3 out of 19 yeast isolated from Cerbera manghas L, T1, T3 and T4, demonstrated the potential ability as a biocontrol agent. ITS rDNA identification demonstrated that T1 has a similarity to Rhodotorula mucilaginosa while T3 and T4 were identified as Aureobasidium sp. nov. The 3 isolates exhibited the ability to reduce the growth of A. brasiliensis sensu lato better than dithane 0.3% with a Disease Incidence (DI) of 100% and a Disease Severity (DS) value of 45%. Only isolate T1 and T3 were able to reduce decay symptoms in apples inoculated with A. flavus sensu lato (with DO and DS were 100% and 25%, respectively) compared to dithane pesticides 0.3%.
CONCLUSION: This study indicated that competition between nutrients occurs between pathogenic molds and under-yeast in vitro and in vivo conditions. However, further studies in the future might be able to elucidate the 'killer' activity and interaction with the pathogen cells and the bio-product production using Rhodotorula mucilaginosa and Aureoubasidium namibiae strains to control postharvest diseases.