Establishment of TaqMan probe real-time PCR for detection of Cryptococcus neoformans / 国际检验医学杂志

ABSTRACT

Objective To establish TaqMan probe real‐time Fluorescent Quantitative PCR in detecting Cryptococcus neoformans genomic DNA ,and to provide important method for detection of cryptococcal meningitis .Methods According to the Cryptococcus neoformans ITS‐rDNA sequences obtained from NCBI ,specific primers and probe were designed based on the conserved sequences , a specific 114 bp fragment was amplified by primers and probe ,then recombinant plasmid was constructed .Eight different concen‐trations from 1 .42 × 10 copy/μL to 1 .42 × 10 copy/μL were used as templates by 2 μL .In the optimum reaction condition ,the sen‐sitivity ,specificity and repeatability were evaluated and standard curve was established .15 clinical cryptococcal meningitis strains i‐solated from clinical diagnosis patients were detected .Results The real‐time PCR showed high sensitivity and specificity and was a‐ble to detect 2 .84 × 102 copies of plasmid DNA .The detection sensitivity was 2 .84 × 102 copies plasmid DNA by real‐time PCR ,no amplification curve was detected with human genomic DNA ,other fungus ,bacterias and viruses .The CV of inter‐assay were 2 .86% ,1 .48% and 1 .36% respectively with excellent reproducibility .Fifteen clinical isolated strains could be detected accurately . Conclusion A method of detection of Cryptococcus neoformans DNA by real‐time PCR is established successfully with high sensi‐tivity and specificity ,and the results are stable ,could diagnose cryptococcal meningitis rapidly and early .