Methicillin resistance in Staphylococcus aureus is a frequent cause of nosocomial and community-acquired infections. Accurate, rapid epidemiologic typing is crucial to the identification of the source and spread of infectious disease and could provide detailed information on the generation of methicillin-resistant S. aureus (MRSA) strains. The high degree of genetic relatedness of MRSA strains has precluded the use of more conventional methods of genetic fingerprinting. A rapid DNA fingerprinting method that exploits PCR amplification from a DNA repeat sequence in MRSA is described. The random chromosomal distribution of this repeat sequence provides an ideal target for detecting DNA fragment patterns specific to individual MRSA strains. Two PCR fingerprinting methods which use an oligonucleotide primer based on a repetitive sequence found in Mycoplasma pneumoniae are presented. The repetitive element sequence-based PCR (rep-PCR) and fluorophore-enhanced rep-PCR (FERP) can identify epidemic strains among background MRSA. The combination of oligonucleotide primers labeled with different fluorescent dyes allowed simultaneous FERP fingerprinting and mecA gene detection. Eight different fingerprint patterns were observed in MRSA strains collected from different sources. These techniques provide a rapid discriminatory means of molecular epidemiologic typing of MRSA involved in nosocomial infections.

The genome of Bacillus anthracis is extremely monomorphic, and thus individual strains have often proven to be recalcitrant to differentiation at the molecular level. Long-range repetitive element polymorphism-PCR (LR REP-PCR) was used to differentiate various B. anthracis strains. A single PCR primer derived from a repetitive DNA element was able to amplify variable segments of a bacterial genome as large as 10 kb. We were able to characterize five genetically distinct groups by examining 105 B. anthracis strains of diverse geographical origins. All B. anthracis strains produced fingerprints comprising seven to eight bands, referred to as "skeleton" bands, while one to three "diagnostic" bands differentiated between B. anthracis strains. LR REP-PCR fingerprints of B. anthracis strains showed very little in common with those of other closely related species such as B. cereus, B. thuringiensis, and B. mycoides, suggesting relative heterogeneity among the non-B. anthracis strains. Fingerprints from transitional non-B. anthracis strains, which possessed the B. anthracis chromosomal marker Ba813, scarcely resembled those observed for any of the five distinct B. anthracis groups that we have identified. The LR REP-PCR method described in this report provides a simple means of differentiating B. anthracis strains. Anthrax is often a fatal bacterial infection that results afterBacillus anthracis spores enter a suitable host either via inhalation or ingestion or through the contamination of a wound or abrasion (8). Since death occurs within a few days after the onset of symptoms, B. anthracis presents a very serious threat if deployed as a biological weapon (13). Naturally occurring human B. anthracis infections are rare and generally result from contact with contaminated animals or their products (5).Due to the highly monomorphic nature of B. anthracis, differentiation of strains from diverse origins has proven to be very difficult. DNA fingerprinting of B. anthracis strains using ribotyping has shown some strain-to-strain variations, presumably due to changes in the organization and number of rrn loci (18). However, due to insufficient numbers of variant strains, ribotyping was found to have limited discriminatory potential.Neither arbitrarily-primed PCR fingerprints (1) nor amplified fragment length polymorphisms fingerprints (15) have been useful in discriminating between B. anthracis strains, although some genetic diversity has been observed. In this case, the diversity was due to the presence of a variable-number tandem repeat (14) sequence in the open reading frame (ORF) vrrA. Recently, seven additional loci containing these sequences were discovered in B. anthracis, leading to the identification of six genetically distinct groups of B. anthracis (16).The presence of multiple copies of highly conserved repetitive extragenic palindromic (REP) sequences in DNA (12) has been identified in a number of microorganisms (28). The technique of REP-PCR takes advantage of the fact that multiple copies of these REP sequences...

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A repetitive sequence-based PCR (rep-PCR) technique was developed to characterize the genotypic relatedness among Candida rugosa isolates. Two repetitive sequences, viz., Care-2 and Com29 from Candida albicans, were used to design primers Ca-21, Ca-22, and Com-21, respectively. When used alone or in combination, these primers generated discriminatory fingerprints by amplifying the adjacent variable regions of the genome. Twenty-three isolates from burn patients, eight from other human sources, and four C. rugosa isolates pathogenic in animals were placed into nine fingerprinting groups. Different primers placed these isolates into identical groups, indicating that rep-PCR is a specific and reproducible technique for molecular characterization of C. rugosa. Moreover, these primers unequivocally discriminated among other important Candida species such as C. albicans, C. glabrata, C. tropicalis, C. krusei, C. parapsilosis, C. kefyr, and C. lusitaniae. These data confirm the conservation of repetitive sequences in Candida species. Because of its ease and sensitivity, rep-PCR offers a relatively rapid and discriminatory method for molecular typing of C. rugosa in outbreaks.

Partially purified extracts of soluble Hardy-Passey mouse meldnoma tyrosinase yielded three precipitin lines upon immunoelectrophoresis. Two immunochemically homologous but electrophoretically heterologotis precipitin lines possessed tyrosinase activity whereas a more anodic line was observed which did not display tyrosinase activity.Tyrosinase from serum of patients with metastatic malignant melanomas cross-reacted with antiserum to mouse melanoma tyrosinase, and one precipitin line was seen. Imtmtnoelectrophoresis of mixed suspensions of human serum and mouse melanoma tyrosinase yielded two tyrosinase-anlibody complexes which intersected in a manner suggesting that the human and mouse melanoma tyrosinases possessed certain related antigenic groups.