Identification of pathogenic Helicobacter species by chaperonin-60 differentiation on plastic DNA arrays

Abstract

A microarray method for bacterial species identification based on cpn60 and 16S rDNA hybridization was developed. Specific cpn60 or 16S rDNA oligonucleotides from various Helicobacter or Campylobacter species were printed and immobilized onto a proprietary plastic solid support. Using universal primers, fragments derived from either cpn60 or 16S rDNA genes from single isolates or from a complex human waste sludge DNA sample spiked with Helicobacter pylori were biotinylated and hybridized to the plastic slide. Subsequent querying with a streptavidin-horseradish peroxidase conjugate followed by color development using tetramethylbenzidine resulted in accurate Helicobacter species identification with no cross-hybridization to either the 16S rDNA or the cpn60 sequence of a closely related strain of Campylobacter jejuni. The combination of a nonfluorescence visual detection system with a polymer-based DNA microarray slide has resulted in a molecular tool that should prove useful in numerous applications requiring rapid, low-cost bacterial species identification.

Plastic Polymers for Efficient DNA Microarray Hybridization: Application to Microbiological Diagnostics

“…However, these investigators reported only hybridizations with labeled oligonucleotides, and the specific hybridization signal-to-background ratios were relatively low. Others have avoided this issue by using nonfluorescent detection technologies such as radiolabeling (13,22) or enzymatic reactions (17), but none of these detection technologies fulfill the diagnostic requirement in terms of both rapidity and safety. In this work, we report ultrasensitive microarray hybridization of fluorescently labeled PCR-amplified target DNA using two high-quality plastic substrates for molecular diagnostic purposes.…”

“…The only other reported study of room temperature hybridization on plastic supports was performed using a 16-h protocol, and the hybridization signal-to-background ratios obtained were low (approximately 10) (4). Others have performed hybridization experiments on plastic supports at 30 to 65°C, requiring special heating devices to carry out the hybridizations (5,6,15,17,23,25). In addition, stability tests demonstrated that when stored in a vacuum desiccator, the chemically activated plastic slides were stable for at least 10 weeks.…”

“…Therefore, their usefulness for molecular diagnostic purposes, in which the target nucleic acids are mainly PCR-amplified genetic materials, has not been demonstrated. PCR-amplified bacterial and human genomic DNAs have both been used in plastic microarray hybridizations (11,15,17,22). However, these hybridizations were carried out using complex detection methods based on the use of radioactive isotopes, enzymatic assays, or biotin-labeled targets, which are incompatible with rapid and simple molecular diagnostics.…”

Development of a DNA Microarray for Enterococcal Species, Virulence, and Antibiotic Resistance Gene Determinations among Isolates from Poultry

“…Therefore, this approach could not be applied to the current virulence array when trying to match the length of the virulence factors and antibiotic resistance genes designed previously. Another more genetically diverse candidate gene, cpn60 (heat shock protein), also frequently used in bacterial taxonomy (10,34) was also eliminated for the same reasons as the 16S rRNA sequence since 28-mers were the longest species-specific probes that could be designed for enterococci.…”

“…Confirmation of selected enterococcal isolates was performed by sequencing a segment of the cpn60 gene amplified by the universal cpn60 primers H729 (5Ј-CGC CAG GGT TTT CCC AGT CAC GAC GAI III GCI GGI GAY GGI ACI ACI AC-3Ј) and H730 (5Ј-AGC GGA TAA CAA TTT CAC ACA GGA YKI YKI TCI CCR AAI CCI GGI GCY TT-3Ј), including the M13F and M13R sequences, respectively (underlined) (34).…”