What Is The Most Effective Method For Distinguishing Between Stachybotrys Chartarum And Aspergillus Versicolor When Analyzing Environmental Samples Using QPCR, Given The Potential For Co-colonization And Overlapping DNA Sequences In Water-damaged Buildings?

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To distinguish between Stachybotrys chartarum and Aspergillus versicolor using qPCR in water-damaged buildings, follow this structured approach:

  1. Target Selection:

    • Utilize genetic markers with sufficient variation, such as the ITS region, β-tubulin, or calmodulin genes, to ensure species-specific detection.

  2. Primer Design:

    • Design primers specific to each species. Use bioinformatics tools to check for cross-reactivity and test primers with pure cultures to confirm specificity.

  3. Use of Probes:

    • Incorporate probes (e.g., TaqMan) to enhance specificity by targeting unique sequences, reducing false positives.

  4. Quantification:

    • Employ standard curves with known DNA concentrations for accurate quantification. Consider multiplex qPCR for simultaneous detection, ensuring no primer interference.

  5. Post-PCR Analysis:

    • Perform melting curve analysis to differentiate amplicons based on melting temperatures, aiding in species identification.

  6. Validation:

    • Validate the method using environmental samples with confirmed presence of each fungus to ensure reliability.

This comprehensive strategy ensures accurate and reliable distinction between the two fungi, even in co-colonizing environments.