Biomedical subjects

Nikolay Sergeev

Publications and source records attributed to Nikolay Sergeev.

4 recordsLinked to original sources

PubMed2006

New mosaic subgenotype of varicella-zoster virus in the USA: VZV detection and genotyping by oligonucleotide-microarray.

A rapid and sensitive microarray-based method was used to distinguish the three major circulating genotypes of varicella-zoster virus (VZV). The method analyzes five variable positions located in a 447-nucleotide variable region 1 of open reading frame 22 (ORF 22r1); these single nucleotide polymorphisms (SNP) display in stably occurring patterns specific to each of the VZV genotypes established in previously published studies. Pairs of short oligonucleotide probes (oligoprobes) with sequences corresponding to all of the observed SNP were used to detect specific sequences. Fluorescently labeled ssRNA samples for hybridization with a chip were prepared by in vitro T7 polymerase driven transcription of the amplicons of ORF 22r1, followed by chemical labeling with Cy5 into RNA sample. Ratios between fluorescent hybridization signals from each pair of oligoprobes were used to assess the sequence at each SNP. We evaluated six reference VZV strains and 130 VZV clinical specimens to validate the method. The microarray method accurately identified strains isolated in the US in 2001-2002, representing all major genotypes as determined using more extensive sequence analysis, correctly assigning strains to genotypes E (81.5%), J (3%) and M (15.5%). In addition, a new M variant (M3) was identified.

Carbocyanines↗

PubMed2005

Microarray analysis of Bacillus cereus group virulence factors.

Bacillus cereus, B. thuringiensis and B. anthracis are closely related medically and economically important bacterial species that belong to the B. cereus group. Members of the B. cereus group carry genes encoding several important virulence factors, including enterotoxins, phospholipases and exotoxins. Since it is difficult to differentiate among B. cereus group members, and because Bacillus virulence factors are very important for pathogenesis, we explored the use of microarray-based detection of virulence factor genes as a tool for strain identification and for determining virulence. Our method requires an initial multiplex PCR amplification step, followed by identification of the PCR amplicons by hybridization to an oligonucleotide microarray containing genes for all three types of Bacillus virulence factors including B. anthracis virulence factors. The DNA chip described here contains 21 identical arrays used for analysis of seven samples in triplicates. Using the arrays, we found that virulence factors are present in several combinations in the strains analyzed. This work also demonstrates the potential of oligonucleotide microarrays for medical, food safety and biodefense analysis of microbial pathogens.

Bacillus cereus↗

PubMed2004

Multipathogen oligonucleotide microarray for environmental and biodefense applications.

Food-borne pathogens are a major health problem. The large and diverse number of microbial pathogens and their virulence factors has fueled interest in technologies capable of detecting multiple pathogens and multiple virulence factors simultaneously. Some of these pathogens and their toxins have potential use as bioweapons. DNA microarray technology allows the simultaneous analysis of thousands of sequences of DNA in a relatively short time, making it appropriate for biodefense and for public health uses. This paper describes methods for using DNA microarrays to detect and analyze microbial pathogens. The FDA-1 microarray was developed for the simultaneous detection of several food-borne pathogens and their virulence factors including Listeria spp., Campylobacter spp., Staphylococcus aureus enterotoxin genes and Clostridium perfringens toxin genes. Three elements were incorporated to increase confidence in the microarray detection system: redundancy of genes, redundancy of oligonucleotide probes (oligoprobes) for a specific gene, and quality control oligoprobes to monitor array spotting and target DNA hybridization. These elements enhance the reliability of detection and reduce the chance of erroneous results due to the genetic variability of microbes or technical problems with the microarray. The results presented demonstrate the potential of oligonucleotide microarrays for detection of environmental and biodefense relevant microbial pathogens.

Bacteria↗

PubMed2004

Simultaneous analysis of multiple staphylococcal enterotoxin genes by an oligonucleotide microarray assay.

Staphylococcal enterotoxins (SEs) are a family of 17 major serological types of heat-stable enterotoxins that are one of the leading causes of gastroenteritis resulting from consumption of contaminated food. SEs are considered potential bioweapons. Many Staphylococcus aureus isolates contain multiple SEs. Because of the large number of SEs, serological typing and PCR typing are laborious and time-consuming. Furthermore, serological typing may not always be practical because of antigenic similarities among enterotoxins. We report on a microarray-based one-tube assay for the simultaneous detection and identification (genetic typing) of multiple enterotoxin (ent) genes. The proposed typing method is based on PCR amplification of the target region of the ent genes with degenerate primers, followed by characterization of the PCR products by microchip hybridization with oligonucleotide probes specific for each ent gene. We verified the performance of this method by using several other techniques, including PCR amplification with gene-specific primers, followed by gel electrophoresis or microarray hybridization, and sequencing of the enterotoxin genes. The assay was evaluated by analysis of previously characterized staphylococcal isolates containing 16 ent genes. The microarray assay revealed that some of these isolates contained additional previously undetected ent genes. The use of degenerate primers allows the simultaneous amplification and identification of as many as nine different ent genes in one S. aureus strain. The results of this study demonstrate the usefulness of the oligonucleotide microarray assay for the analysis of multitoxigenic strains, which are common among S. aureus strains, and for the analysis of microbial pathogens in general.

Base Sequence↗