Hybridization probes for conventional DNA fingerprinting used as single primers in the polymerase chain reaction to distinguish strains of Cryptococcus neoformans

W Meyer, T G Mitchell, E Z Freedman, R Vilgalys
1993 Journal of Clinical Microbiology  
In conventional DNA fingerprinting, hypervariable and repetitive sequences (minisatellite or microsatellite DNA) are detected with hybridization probes. As demonstrated here, these probes can be used as single primers in the polymerase chain reaction (PCR) to generate individual fingerprints. Several conventional DNA fingerprinting probes were used to prime the PCR, yielding distinctive, hypervariable multifragment profiles for different strains of Cryptococcus neoformans. PCR fingerprinting
more » ... h the oligonucleotide primers (GTG)5, (GACA)4, and the phage M13 core sequence (GAGGGTGGXGGXTCT), but not with (CA)8 or (CT)8, generated DNA polymorphisms with all 42 strains of C. neoformans investigated. PCR fingerprints produced by priming with (GTG)5, (GACA)4, or the M13 core sequence differentiated the two varieties of C. neoformans, C. neoformans var. neoformans (serotypes A and D) and C. neoformans var. gattii (serotypes B and C). Furthermore, strains of serotypes A, D, and B or C could be distinguished from each other by specific PCR fingerprint patterns. These primers, which also successfully amplified hypervariable DNA segments from other species, provide a convenient method of identification at the species or individual level. Amplification of polymorphic DNA patterns by PCR with these primers offers several advantages over classical DNA fingerprinting techniques, appears to be more reliable than other PCR-based methods for detecting polymorphic DNA, such as analysis of random-amplified polymorphic DNA, and should be applicable to many other organisms. The identification of medically important fungi is based on morphological and physiological characteristics and is often difficult and time-consuming. Because the frequency of and mortality due to opportunistic mycoses are increasing among patients with AIDS, hematologic malignancies, and transplants, there is an urgent need for improved methods to identify fungal pathogens (15, 22) . Novel molecular approaches for the genetic identification of fungal strains and species appear to offer advantages of simplicity, speed, and accuracy. * Corresponding author. isolates are serotype A, strains of serotype A may vary in several pathobiological respects (3, 29) , and the ability to identify individual strains would be advantageous. Several studies have documented variation at the DNA
doi:10.1128/jcm.31.9.2274-2280.1993 fatcat:6eeu7rl4qnhaba4vbxuetrmvni