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Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans
Published on: March 19, 2015
Rapid direct identification of Cryptococcus neoformans from pigeon droppings by nested PCR using CNLAC1 gene
1Seoul Metropolitan Government Research Institute of Public Health & Environment, Gwacheon, Gyeonggi 137-131, Republic of Korea.
Abstract:
Isolation and identification of Cryptococcus neoformans and pathogenic yeast-like fungi from pigeon droppings has been taken for a long time and requires various nutrients for its growth. In this study, we attempted to establish a rapid direct identification method of Cr. neoformans from pigeon dropping samples by nested-PCR using internal transcribed spacer (ITS) CAP64 and CNLAC1 genes, polysaccharide capsule gene and laccase-associated gene to produce melanin pigment, respectively, which are common genes of yeasts. The ITS and CAP64 genes were amplified in all pathogenic yeasts, but CNLAC1 was amplified only in Cr. neoformans. The ITS gene was useful for yeast genotyping depending on nucleotide sequence. Homology of CAP64 genes among the yeasts were very high. The specificity of PCR using CNLAC1 was demonstrated in Cr. neoformans environmental strains but not in other yeast-like fungi. The CNLAC1 gene was detected in 5 serotypes of Cr. neoformans. The nested-PCR amplified up to 10(-11) μg of the genomic DNA and showed high sensitivity. All pigeon droppings among 31 Cr. neoformans-positive samples were positive and all pigeon droppings among 348 Cr. neoformans-negative samples were negative by the direct nested-PCR. In addition, after primary enrichment of pigeon droppings in Sabouraud dextrose broth, all Cr. neoformans-negative samples were negative by the nested-PCR, which showed high specificity. The nested-PCR showed high sensitivity without culture of pigeon droppings. Nested-PCR using CNLAC1 provides a rapid and reliable molecular diagnostic method to overcome weak points such as long culture time of many conventional methods.
Insights
A new nested-PCR method rapidly identifies Cryptococcus neoformans in pigeon droppings using the CNLAC1 gene. This molecular diagnostic offers high sensitivity and specificity, overcoming the limitations of traditional culture methods.
Area of Science:
- Mycology
- Molecular Biology
- Diagnostic Microbiology
Background:
- Cryptococcus neoformans is a pathogenic yeast found in pigeon droppings.
- Traditional identification methods are time-consuming and require extensive culturing.
- A rapid and specific diagnostic tool is needed for environmental surveillance.
Purpose of the Study:
- To develop a rapid, direct identification method for Cryptococcus neoformans in pigeon droppings.
- To utilize nested-PCR targeting specific genes for enhanced sensitivity and specificity.
- To evaluate the diagnostic performance compared to conventional methods.
Main Methods:
- Nested-PCR assay designed using internal transcribed spacer (ITS), CAP64, and CNLAC1 genes.
- CNLAC1 gene selected for its specificity to Cryptococcus neoformans.
- Testing on environmental pigeon dropping samples and comparison with culture-based methods.
Main Results:
- The CNLAC1 gene was exclusively amplified in Cryptococcus neoformans, demonstrating high specificity.
- Nested-PCR achieved high sensitivity, detecting down to 10(-11) μg of genomic DNA.
- The method accurately identified all Cryptococcus neoformans-positive and negative samples without prior culturing.
Conclusions:
- Nested-PCR using the CNLAC1 gene is a rapid, sensitive, and specific molecular diagnostic method for Cryptococcus neoformans.
- This approach significantly reduces diagnostic time compared to conventional culture techniques.
- The method is suitable for direct detection in environmental samples like pigeon droppings.