DNA probes for the rapid identification of medically important Candida species using a multianalyte profiling system.

Recently, a new flow cytometric technology to detect multiple DNA target sequences in a single microtiter well plate was developed [multianalyte profiling (MAP) System, Luminex Corp., Austin, TX]. DNA probes, directed to the internal transcribed spacer 2 region of ribosomal DNA, were therefore designed to detect and differentiate PCR amplicons from six medically important Candida species using this system.

Comparison of real-time PCR protocols for differential laboratory diagnosis of amebiasis.

Specific identification of Entamoeba spp. in clinical specimens is an important confirmatory diagnostic step in the management of patients who may be infected with Entamoeba histolytica, the species that causes clinical amebiasis. Distinct real-time PCR protocols have recently been published for identification of E.

Real-time reverse transcription-polymerase chain reaction assay for SARS-associated coronavirus.

A real-time reverse transcription-polymerase chain reaction (RT-PCR) assay was developed to rapidly detect the severe acute respiratory syndrome-associated coronavirus (SARS-CoV). The assay, based on multiple primer and probe sets located in different regions of the SARS-CoV genome, could discriminate SARS-CoV from other human and animal coronaviruses with a potential detection limit of <10 genomic copies per reaction. The real-time RT-PCR assay was more sensitive than a conventional RT-PCR assay or culture isolation and proved suitable to detect SARS-CoV in clinical specimens.

Evaluation of dual-labeled fluorescent DNA probe purity versus performance in real-time PCR.

Real-time PCR technology using dual-labeled fluorescent oligonucleotide probes allows for sensitive, specific, and quantitative determination of mRNA or DNA targets. Historically, dual-labeled probes have been the most expensive reagent in real-time PCR because of the postsynthesis high-performance liquid chromatography (HPLC) and/or gel purification steps required due to limitations in traditional synthesis chemistry. The recent availability of quencher reagents that allow the 3' quencher incorporation as part of the on-machine synthesis has presented the possibility that probes, when carefully synthesized, may be used without extensive postsynthesis purification.

Temperature-mediated heteroduplex analysis performed by using denaturing high-performance liquid chromatography to identify sequence polymorphisms in Mycobacterium tuberculosis complex organisms.

PCR products containing sequence polymorphisms were prepared from six mycobacterial genes, denatured, mixed with reference PCR products, and reannealed; the mixtures were then examined with a denaturing high-performance liquid chromatography system (WAVE) equipped with a temperature-controlled alkalated polystyrene divinyl benzene column. Mismatching of bases in heteroduplexes of the PCR products causes elution patterns of the DNA from the column to be altered. The six mycobacterial genes studied were oxyR, in which a specific polymorphism (G(1031)A) is found only in certain species of the Mycobacterium tuberculosis complex, and five genes in which mutations associated with antituberculosis drug resistance have been found.

Development and evaluation of real-time PCR-based fluorescence assays for detection of Chlamydia pneumoniae.

Chlamydia pneumoniae is an important respiratory pathogen recently associated with atherosclerosis and several other chronic diseases. Detection of C. pneumoniae is inconsistent, and standardized PCR assays are needed.

Towards understanding the evolution of the human commensal yeast Candida albicans.

Allelic frequencies and relationships for one dimorphic locus and three unlinked polymorphic loci have been determined for 114 unrelated isolates of Candida albicans, including 14 laboratory reference strains and 50 strains from each of two geographic regions. Although there was no indication of geographical partitioning, there were significant correlations for specific allelic pairs among loci and little evidence that any alleles were in Hardy-Weinberg equilibrium. This gives additional support for the concept that the primary mode of genetic inheritance in this species is clonal, with other intracellular genetic events playing a lesser role in the creation of genomic diversity.