Species-Level Identification of Orthopoxviruses with an Oligonucleotide Microchip

Abstract

A method for species-specific detection of orthopoxviruses pathogenic for humans and animals is described. The method is based on hybridization of a fluorescently labeled amplified DNA specimen with the oligonucleotide DNA probes immobilized on a microchip (MAGIChip). The probes identify species-specific sites within the crmB gene encoding the viral analogue of tumor necrosis factor receptor, one of the most important determinants of pathogenicity in this genus of viruses. The diagnostic procedure takes 6 h and does not require any sophisticated equipment (a portable fluorescence reader can be used).

Development of a DNA Microarray for Detection and Identification of Fungal Pathogens Involved in Invasive Mycoses

“…In view of the increasing number of fungal species isolated from clinical samples as potential pathogens, a broad-spectrum detection system as it is represented by the microarray technology would be desirable. Until today, diagnostic DNA microarrays were applied for the identification of viruses (5,(27)(28)(29)56), bacteria (14,24,45,46,49), and mechanisms of resistance to certain antibiotics (15,18,55). To our knowledge, this study describes for the first time the application of the DNA microarray technology in combination with a quantification and data-processing method for the rapid and reliable detection of fungal pathogens.…”

Diversity of Human Immunodeficiency Virus Type 1 Subtype A and CRF03_AB Protease in Eastern Europe: Selection of the V77I Variant and Its Rapid Spread in Injecting Drug User Populations

“…Oligonucleotide microarrays have already been proven to be a powerful diagnostic tool for identifying genetic polymorphisms (24,29,41). Moreover, several approaches based on hybridization technology have already been described for genotypic analysis of HIV-1 drug resistance (42,43).…”

“…To obtain such information, we analyzed HIV-1 genetic variants, along with conventional sequencing, by using oligonucleotide microarrays that have already been proven to be used as a diagnostic tool for identifying nucleic acid polymorphisms (24,29,41).…”

Real-Time PCR System for Detection of Orthopoxviruses and Simultaneous Identification of Smallpox Virus

“…The concentrations of the VAR DNAs used in the test panel ranged from 100 fg per l to 1 ng per l and included total viral and cellular DNAs from cell lysates and crust material as well as purified viral DNAs. Nucleic acids of species other than VAR were extracted either from infected tissue culture cells or from purified preparations by using conventional chemistry (QiaAmp DNA Mini kit; Qiagen, Hilden Germany) or as described earlier (12,18). Parapoxvirus strain D 1701, avipoxvirus strain HP-1, and tanapoxvirus TP-1, as well as one strain each of herpes simplex virus types 1 and 2, Epstein-Barr virus, varicella-zoster virus, and cytomegalovirus, were also included.…”

“…DNA maps and several other molecular biologic features, including full genome sequencing, have been applied and have provided definitive information for virus classification (4,24,25,26). Various PCR methods have been used to identify and subtype the available OPVs by using consensus primers combined with restriction cleavage and/or sequencing of amplicons (6,10,13,18,23) or oligonucleotidebased microchip technology (11,12,20). Today, real-time PCR is even more efficient because it combines amplification and detection of target DNA in one vessel, thereby eliminating any time-consuming post-PCR procedures and potentially limiting possible contamination events (5,8,9).…”