Validation of a Pan-fungal polymerase chain reaction (PCR) assay for the detection and identification of medically important fungi in a diagnostic laboratory

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dc.contributor.advisorMoodley, Clintonen_ZA
dc.contributor.advisorBamford, Colleenen_ZA
dc.contributor.authorNtuli, Sindile Venessaen_ZA
dc.date.accessioned2018-04-24T13:49:16Z
dc.date.available2018-04-24T13:49:16Z
dc.date.issued2018en_ZA
dc.description.abstractThe laboratory diagnosis of fungal infection is complicated, based on the microscopic detection, culture isolation, and detection of serological response. In recent years, there has been an increase in the utilization of molecular diagnostic techniques for the detection and accurate identification of fungal pathogens. This was a laboratory accuracy study evaluating the performance of a selected pan-fungal PCR using 70 previously identified reference fungal isolates. The DNA yield and purity of three different DNA extraction methods was assessed, using 6 representative fungal isolates. The ZR Fungal/Bacterial DNA MicroPrep™ produced a median concentration of 17.28 ng/μl,which was significantly higher (p value = 0.0079) than the MagNA pure LC DNA Isolation Kit III and QIAamp DNA Mini Kit, which produced median yields of 11.08ng/μl and 3.54 ng/μl, respectively. The selected pan-fungal PCR was optimized PCR and successfully performed on 62 of the 70 reference isolates. A selection of 56 amplicons were submitted for DNA sequence determination. Of all the sequences queried on the NCBI and Ribosomal Development Project (RDP) databases, 95/111(86%) were concordant with the results obtained from the reference laboratory. Study findings have shown that the selected pan-fungal PCR, coupled with DNA sequence analysis is an excellent diagnostic tool for the identification of medically relevant fungi. This assay, in combination with conventional culture, is useful for the rapid and accurate identification of fungal isolates. Future work will involve evaluating the utility of this assay for the detection and identification of medically relevant fungi in deep tissue biopsies.en_ZA
dc.identifier.apacitationNtuli, S. V. (2018). <i>Validation of a Pan-fungal polymerase chain reaction (PCR) assay for the detection and identification of medically important fungi in a diagnostic laboratory</i>. (Thesis). University of Cape Town ,Faculty of Health Sciences ,Division of Medical Microbiology. Retrieved from http://hdl.handle.net/11427/27816en_ZA
dc.identifier.chicagocitationNtuli, Sindile Venessa. <i>"Validation of a Pan-fungal polymerase chain reaction (PCR) assay for the detection and identification of medically important fungi in a diagnostic laboratory."</i> Thesis., University of Cape Town ,Faculty of Health Sciences ,Division of Medical Microbiology, 2018. http://hdl.handle.net/11427/27816en_ZA
dc.identifier.citationNtuli, S. 2018. Validation of a Pan-fungal polymerase chain reaction (PCR) assay for the detection and identification of medically important fungi in a diagnostic laboratory. University of Cape Town.en_ZA
dc.identifier.ris TY - Thesis / Dissertation AU - Ntuli, Sindile Venessa AB - The laboratory diagnosis of fungal infection is complicated, based on the microscopic detection, culture isolation, and detection of serological response. In recent years, there has been an increase in the utilization of molecular diagnostic techniques for the detection and accurate identification of fungal pathogens. This was a laboratory accuracy study evaluating the performance of a selected pan-fungal PCR using 70 previously identified reference fungal isolates. The DNA yield and purity of three different DNA extraction methods was assessed, using 6 representative fungal isolates. The ZR Fungal/Bacterial DNA MicroPrep™ produced a median concentration of 17.28 ng/μl,which was significantly higher (p value = 0.0079) than the MagNA pure LC DNA Isolation Kit III and QIAamp DNA Mini Kit, which produced median yields of 11.08ng/μl and 3.54 ng/μl, respectively. The selected pan-fungal PCR was optimized PCR and successfully performed on 62 of the 70 reference isolates. A selection of 56 amplicons were submitted for DNA sequence determination. Of all the sequences queried on the NCBI and Ribosomal Development Project (RDP) databases, 95/111(86%) were concordant with the results obtained from the reference laboratory. Study findings have shown that the selected pan-fungal PCR, coupled with DNA sequence analysis is an excellent diagnostic tool for the identification of medically relevant fungi. This assay, in combination with conventional culture, is useful for the rapid and accurate identification of fungal isolates. Future work will involve evaluating the utility of this assay for the detection and identification of medically relevant fungi in deep tissue biopsies. DA - 2018 DB - OpenUCT DP - University of Cape Town LK - https://open.uct.ac.za PB - University of Cape Town PY - 2018 T1 - Validation of a Pan-fungal polymerase chain reaction (PCR) assay for the detection and identification of medically important fungi in a diagnostic laboratory TI - Validation of a Pan-fungal polymerase chain reaction (PCR) assay for the detection and identification of medically important fungi in a diagnostic laboratory UR - http://hdl.handle.net/11427/27816 ER - en_ZA
dc.identifier.urihttp://hdl.handle.net/11427/27816
dc.identifier.vancouvercitationNtuli SV. Validation of a Pan-fungal polymerase chain reaction (PCR) assay for the detection and identification of medically important fungi in a diagnostic laboratory. [Thesis]. University of Cape Town ,Faculty of Health Sciences ,Division of Medical Microbiology, 2018 [cited yyyy month dd]. Available from: http://hdl.handle.net/11427/27816en_ZA
dc.language.isoengen_ZA
dc.publisher.departmentDivision of Medical Microbiologyen_ZA
dc.publisher.facultyFaculty of Health Sciencesen_ZA
dc.publisher.institutionUniversity of Cape Town
dc.subject.otherMedical Microbiologyen_ZA
dc.titleValidation of a Pan-fungal polymerase chain reaction (PCR) assay for the detection and identification of medically important fungi in a diagnostic laboratoryen_ZA
dc.typeMaster Thesis
dc.type.qualificationlevelMasters
dc.type.qualificationnameMMeden_ZA
uct.type.filetypeText
uct.type.filetypeImage
uct.type.publicationResearchen_ZA
uct.type.resourceThesisen_ZA
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