PCR Application in Dermatophyte Identification
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PCR Application in Dermatophyte Identification

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Questions and Answers

What is dermatophytosis commonly known as?

  • Tinea (correct)
  • Acne
  • Eczema
  • Psoriasis

    • Which genus is represented by a single species?

  • Trichophyton
  • Microsporum
  • Epidermophyton (correct)
  • Candida

    • What has contributed to the increased understanding of dermatophytes?

  • Historical records
  • Traditional treatments
  • Molecular diagnostic methods (correct)
  • Clinical interviews

    • Which dermatophyte species has been most commonly isolated in the USA in the 1990s?

    <p>T. rubrum</p> Signup and view all the answers

    What has historically made dermatophytes seem less significant as human pathogens?

    <p>Infections are generally mild.</p> Signup and view all the answers

    What change has occurred regarding the geographical distribution of dermatophyte species?

    <p>There are more global variations now.</p> Signup and view all the answers

    Which technique is mentioned for the rapid identification of dermatophyte isolates?

    <p>Polymerase Chain Reaction</p> Signup and view all the answers

    How many recognized species are there in the genus Trichophyton?

    <p>15</p> Signup and view all the answers

    What condition is caused by infection of keratinised tissues by keratinophilic fungi?

    <p>Dermatophytosis</p> Signup and view all the answers

    Which three genera are classified as dermatophytes?

    <p>Epidermophyton, Microsporum, Trichophyton</p> Signup and view all the answers

    What disadvantage is commonly associated with traditional laboratory methods for identifying dermatophytes?

    <p>They are too slow or non-specific.</p> Signup and view all the answers

    What is one of the advantages of using arbitrarily primed PCR for dermatophyte identification?

    <p>It provides rapid and precise identification.</p> Signup and view all the answers

    How many dermatophyte species or subspecies can be distinguished using one of the random primers in AP-PCR?

    <p>Up to 20</p> Signup and view all the answers

    Which two random primers were used in separate reaction tubes to identify a larger number of dermatophyte species?

    <p>OPD18 and OPAA17</p> Signup and view all the answers

    What is the main component of the tissues affected by dermatophytes?

    <p>Keratin</p> Signup and view all the answers

    What method traditionally involved in dermatophyte identification has limitations?

    <p>Microscopy and in-vitro culture</p> Signup and view all the answers

    What is a key challenge associated with conventional identification methods for dermatophytes?

    <p>They can be influenced by external factors.</p> Signup and view all the answers

    What type of agar is NOT mentioned as useful for culturing dermatophytes?

    <p>Blood agar</p> Signup and view all the answers

    What is the purpose of reducing the stringency of the primer annealing temperature in AP-PCR?

    <p>To allow for random primer matching with the genome.</p> Signup and view all the answers

    Why might subsequent manipulation after amplification be unnecessary in AP-PCR?

    <p>The detailed sequences of genes do not need to be known.</p> Signup and view all the answers

    What outcome occurs when annealing and priming events take place within a certain distance in AP-PCR?

    <p>The sequence between the matching sites is amplified.</p> Signup and view all the answers

    What implication does a perfect match of two-to-three nucleotides from the primer to the template have?

    <p>It can result in effective annealing and priming.</p> Signup and view all the answers

    Which fungi were included in the distinct DNA band patterns observed?

    <p>Scytalidium and Fusarium</p> Signup and view all the answers

    What is a consequence of using costly and time-consuming culture media for dermatophyte identification?

    <p>Extended wait times for results may occur.</p> Signup and view all the answers

    How many dermatophyte species or subspecies were tested in the study?

    <p>25</p> Signup and view all the answers

    Which method allows for differentiation of micro-organisms based on DNA patterns?

    <p>Agarose gel electrophoresis</p> Signup and view all the answers

    Which genetic testing method was used to differentiate the fungi?

    <p>AP-PCR</p> Signup and view all the answers

    Which of the following species could not be differentiated from another depending on the mentioned varieties?

    <p>T. mentagrophytes var. interdigitale and var. mentagrophytes</p> Signup and view all the answers

    What was the highest DNA product size obtained for T. equinum var. autotraphicum using OPAA11?

    <p>2.5 kb</p> Signup and view all the answers

    What does AP-PCR stand for?

    <p>Arbitrary Primer - Polymerase Chain Reaction</p> Signup and view all the answers

    Which of the following species displayed a DNA product size of 1.7 kb?

    <p>T. ajelloi</p> Signup and view all the answers

    Which DNA primer generated the most consistent results across multiple species?

    <p>OPD18</p> Signup and view all the answers

    Which dermatophyte species is identified by OPAA17?

    <p>T. ajelloi</p> Signup and view all the answers

    Which dermatophyte species has all identification options marked as 'Yes'?

    <p>M. cookei</p> Signup and view all the answers

    Which of the following species of dermatophytes can be identified by OPD18?

    <p>T. tonsurans</p> Signup and view all the answers

    Which dermatophyte species shows a 'No' for identification through OPAA11?

    <p>T. gourdvillii</p> Signup and view all the answers

    What is the identification status of T. mentagrophytes var. mentagrophytes for OPAA17?

    <p>Yes</p> Signup and view all the answers

    Which of the following species cannot be identified by OPD18?

    <p>T. mentagrophytes var.interdigitale</p> Signup and view all the answers

    Which variety of T. mentagrophytes shows partial identification capabilities?

    <p>var. interdigitale</p> Signup and view all the answers

    Which dermatophyte is identified by OPAA11 but not by OPD18?

    <p>T. soudanense</p> Signup and view all the answers

    What does AP-PCR measure in dermatophytes?

    <p>Genotypic characteristics</p> Signup and view all the answers

    What was observed in the odd colony morphology of certain T. violaceum isolates?

    <p>They displayed identical band patterns to typical isolates</p> Signup and view all the answers

    Why is nucleotide sequence analysis important in AP-PCR?

    <p>It elucidates precise structures and functions of gene regions</p> Signup and view all the answers

    What can major DNA bands obtained through AP-PCR help design?

    <p>Specific primers for dermatophyte species</p> Signup and view all the answers

    What characteristic of AP-PCR enhances its utility in dermatophyte identification?

    <p>Its robustness and reproducibility</p> Signup and view all the answers

    Which of the following fungal species was specifically mentioned in the content?

    <p>T. rubrum</p> Signup and view all the answers

    What aspect of dermatophyte isolates did the research specifically highlight?

    <p>Their evolutionary variability in gene regions</p> Signup and view all the answers

    What is a future perspective mentioned regarding dermatophyte identification?

    <p>Application of specific primers in detection systems</p> Signup and view all the answers

    Study Notes

    Application of PCR to Dermatophyte Fungi Identification

    • Dermatophytes infect keratinized tissues (skin, hair, nails) in humans and animals, causing dermatophytosis (tinea or ringworm).
    • Current methods for identifying dermatophytes are slow and lack specificity.
    • Nucleic acid amplification technology, like PCR, allows for rapid and precise identification.
    • Arbitrarily Primed PCR (AP-PCR) using random primers (OPAA11, OPD18, OPAA17, OPU15) distinguishes dermatophyte species based on band patterns in agarose gels.
    • Combining two random primers (OPD18 and OPAA17) in separate reactions increased the number of identified species to 23 out of 25.

    Introduction

    • Dermatophytes are keratinophilic fungi from three closely related genera: Epidermophyton, Microsporum, and Trichophyton.
    • These fungi colonize keratinized tissues, aided by proteolytic enzymes, leading to inflammation and dermatophytosis.
    • Epidermophyton has a single species (E. floccosum), while Microsporum and Trichophyton are complex with many species and variants.
    • Clinical symptoms from different species are not sufficient for easy differentiation, hence a need for improved diagnostic methods.

    Conventional Diagnostic Methods

    • Current methods rely on microscopic identification of hyphae from lesion materials and subsequent culture.
    • Microscopy is not species specific, and is relatively insensitive (up to 15% false negatives).
    • Culture for identification takes 10-15 days for most, and longer for unusual/slow-growing isolates, and depends on specific culture media (cost and time consuming).
    • External factors like temperature variations and chemotherapy can affect phenotypic characteristics, making interpretation and identification less reliable.

    Nucleic Acid-Based Techniques

    • Genotypic-based methods are more specific and less influenced by external factors.
    • PCR based diagnostics, including AP-PCR, improve diagnoses.
    • AP-PCR, exploiting random primers, amplifies unique DNA fragments which allows differentiation of microorganisms by characteristic band patterns from gel electrophoresis, eliminating the need for further manipulation after amplification.
    • AP-PCR is more sensitive and precise as it amplifies DNA sequences by billions of times within 30 cycles of denaturing, annealing, and extension.
    • AP-PCR doesn't need a viable organism. Useful for 20-year-old samples.
    • Use of short random primers (usually 10 nucleotides) at relatively low temperature can identify organisms through AP-PCR.
    • AP-PCR has a clear advantage over conventional techniques.

    AP-PCR

    • Using random primers, 230 isolates from 25 dermatophyte species and subspecies (16 Trichophyton, 8 Microsporum, and 1 Epidermophyton) were examined.
    • 16 species could be distinguished with OPAA11; 19 Species with OPD18; 20 species with OPAA17; 19 species with OPU15.
    • A combination of two primers further enhances identification. OPAA11 and OPD18 identified 21 species, and OPD18 and OPAA17 identified 23 species.
    • AP-PCR time is considerably less than conventional methods (takes ~ 1 day).
    • Non-dermatophyte fungi showed different band patterns, enabling differentiation.

    Future Perspectives

    • AP-PCR provides a rapid solution for identification for dermatophytes with varied morphological characteristics.
    • Species-specific primers are needed for better identification.
    • Further development of DNA extraction procedures for clinical materials can be used for improved diagnosis.
    • Sequence analysis of amplified DNA fragments will help determine the exact structures and functions of the involved genes, and establish evolutionary relationships among dermatophytes.

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    Related Documents

    Application of PCR to Dermatophyte Fungi Identification (2000) PDF

    Description

    Explore the use of PCR technology for the rapid identification of dermatophyte fungi that cause skin infections. This quiz delves into the mechanisms of Arbitrarily Primed PCR and its effectiveness in distinguishing species of fungi like Epidermophyton, Microsporum, and Trichophyton. Test your knowledge on dermatophytosis and its impact on human and animal health.

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