The Establishment Of Real-time Fluorescent Quantitative PCR Assays For The Detection Of Candida And Cryptococcus Neoformans

objective: The experiment was carried out to establish real-timefluorescent quantitative polymerase chain reaction（FQ-PCR） assay forthe detection and strain identification of Candida species andCryptococcus neoformans rapidly，accurately and specificity. At the sametime quantitative analysis of each reaction took shape. These fungi areclinical common species including Candida albicans, Candida glabrata,Candida tropicalis, Candida parapsilosis and Cryptococcus neoformans.The results of those tests will give doctors conductions in the practice.Methods: The standard fungal strains were obtained from CGMCC andACCC, and were cultured on the substrate. Cell suspensions wereprepared with0.9%saline and adjusted to about107CFU/ml, and thenThe fungal suspensions will be10-fold serial diluted from107to100CFU/ml which will be used in the establish of the FQ-PCR assay of thefungal tests. GenBank（NCBI） was searched for sequences of the genes oflots of fungi species, bacteria, virus and human. At the end verified thespecific regions each of those five species. Specific primers and probes for those fungi were designed by the Primer Premier5.0and BeaconDesigner7.7. Seven different DNA Isolation methods were compared tochoose the best fungal DNA isolation method. Those methods containone-step method, lyticase digestion method, glass beads to break the wallmethod, Biospin membrane bound method, paramagnetic particle method,concentration method and heating after concentration. The PCR reactionswere carried out under the different concentrations of Mg²⁺, dNTPs,primers and probes to find out the best condition of each PCR reaction.Specific experiments, repeatability experiments and sensitivity analysiswere taken out in each of those five PCR reactions. At the last,86clinicalstrains of all the five fungal species were tested with those assaysestablished in the experiment.Results：Five funguses’ primers and probes were designed successfully.The best DNA isolation method of candida and Cryptococcus neoformanswas heating after concentration. The best condition of each PCR reactionwas sought out. Under the best PCR condition, each of the five PCRreactions would go on well with specific amplification but withoutcross-reaction among the other fungi, bacteria, viruses and human gens.In the experimental range, Ct value has linear relationship with theconcentration, R²>0.995. The sensitivity of Candida albicans, Candidaglabrata, Candida parapsilosis and Cryptococcus neoformans FQ-PCRreaction will achieve5CFU/ml with the good repeatability and stability. That was50CFU/ml to Candida tropicalis. All of the real-time protocolstested were highly reproducible, specific and sensitive. The coefficient ofvariation of Ct value in each Repetitive experimental was less than5%.The positive rate of the clinical fungi test was100%. Neither falsepositives nor cross-reactions were detected in any protocol.Conclusions: Heating after concentration method was the best methodof Candida and Cryptococcus neoformans’ DNA isolation which wassuitable for the FQ-PCR reaction. The establishment of those FQ-PCRassays was based on the success of primer and probe designing. Eachassay was a useful tool for the rapid, accurate identification of Candidaspecies and Cryptococcus neoformans. All of the FQ-PCR reactions wereadvanced in linear range, repeatability and stability. All the clinicalstrains were tested correctly without crossing reaction and that ensuredthe reliability of those assays. This technology will be variously used inthe diagnosis of invasive fungal infection.