Viruses And Fungi

Nearly all of the techniques discussed so far have been largely applied to the typing of bacterial pathogen strains. However, typing systems have been employed to examine viral pathogen strains and fungal pathogen strains. So it is worth discussing these pathogens as well. By and large the systems to genetically type viral pathogens and fungal pathogens are not as widespread in use as those implemented for bacterial species.

Viruses pose a unique set of problems for the development of typing regimes. However, these problems are not insurmountable. For example, many viruses use RNA instead of DNA as their genetic material, which renders direct DNA fingerprinting of viruses such as foot-and-mouth disease virus, influenza, or measles virus impossible. In order to employ RFLP-type methods on these viruses, the viral genetic material must first be converted to DNA and then amplified using a PCR step. This process, called reverse transcription (RT)-PCR, utilizes the enzyme reverse transcriptase to convert the viral RNA genome to DNA, which can then be amplified by PCR.43 However, for viruses with smaller genomes the number of bands produced may be few. Viruses possessing small genomes (viral genomes can range in size from a few thousand bases to several hundred thousand) may best be typed by direct sequence analysis, both for cost and efficiency. Also, by using sequence analysis from many similar viral isolates, SNP loci can be identified for use as typing markers. In the near future as more and more viral isolates are sequenced, the development of rapid typing procedures should proceed at an accelerated pace.

Although fungi possess DNA as their genetic material, their genomes are more complex and larger than those of viruses or bacteria. With regard to typing pathogenic fungi, two techniques predominate: RFLP analysis, which has already been discussed, and a PCR-based technique called random amplified polymorphic DNA (RAPD).44,45 RAPD has also been used with some success in typing bacterial strains.1 RAPD uses a multitude of oligonucleotide primers, 10 nucleotides in length, consisting of random sequence composition to amplify fragments throughout the genome by PCR. Numerous primer sets must be tested to achieve the desired levels of variability in the datasets to produce meaningful discriminatory typing results.44 The effectiveness of RAPD typing varies widely with primer sets and fungal species and is not optimal for all fungal species.44 Finally, it can be expected that many of the identification assays used for eukaryotes, such as humans, are likely to be used on fungi.

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