Development and validation of a diagnostic microbial microarray for methanotrophs

Abstract

The potential of DNA microarray technology in high-throughput detection of bacteria and quantitative assessment of their community structures is widely acknowledged but has not been fully realised yet. A generally applicable set of techniques, based on readily available technologies and materials, was developed for the design, production and application of diagnostic microbial microarrays. A microarray targeting the particulate methane monooxygenase (pmoA) gene was developed for the detection and quantification of methanotrophs and functionally related bacteria. A microarray consisting of a set of 59 probes that covers the whole known diversity of these bacteria was validated with a representative set of extant strains and environmental clones. The potential of the pmoA microarray was tested with environmental samples. The results were in good agreement with those of clone library sequence analyses. The approach can currently detect less dominant bacteria down to 5% of the total community targeted. Initial tests assessing the quantification potential of this system with artificial PCR mixtures showed very good correlation with the expected results with standard deviations in the range of 0.4-17.2%. Quantification of environmental samples with this method requires the design of a reference mixture consisting of very close relatives of the strains within the sample and is currently limited by biases inherent in environmental DNA extraction and universal PCR amplification.

Introduction to Microarray‐Based Detection Methods

“…glass and silicon), spacer molecules with a length of more than 50 A can be used to physically separate the probes from the microchip surface [130,156]. These are typically C 6 -C 12 alkane spacers and/or 5-15 thymidine or adenine residues added to the tethered end of the oligo probe [7,24,61,201].…”

“…buffers, salt concentrations, and temperature). Strategies to overcome problems arising thereof include the acquisition of melting curves for every individual probe [104]; the careful design of probes with similar predicted hybridization properties (usually combined with the application of two to three probes per targeted group) [24,147,201]; the addition of tetramethylammonium chloride that equalizes the melting temperature of different probes by stabilizing the AT base pairs; composition of the hybridization solution [120]; or the use of highly redundant probe sets with multiple probes to target each specific group of microorganisms [189].…”

“…Several methods have been suggested to alleviate this problem, such as the use of helper oligonucleotides [130] and a two-probe proximal chaperon detection system [158] to mitigate the effects of target secondary structure hindrances, an appropriate labeling method [63], and a protocol to achieve optimized target lengths [126,164,197]. Since long targets can form secondary and tertiary structures that hinder efficient probe-target duplex formation, the sizes of the target molecule and its amplicon are often reduced via chemical, enzymatic or thermal fragmentation methods [24,87,104,126,138,158]. Liu et al have recently elegantly reviewed these issues and experimentally demonstrated that microarray hybridizations with short rRNA fragments were more dependent on target sequence than on the competition between probe-target interaction and RNA self-folding [103].…”

Application of COMPOCHIP Microarray to Investigate the Bacterial Communities of Different Composts

“…Nucleic acid microarrays offer the possibility to analyze a specifically selected array of microorganisms, concerning their presence or absence in a particular environmental sample, in a single experiment [9]. Microarray technology has been applied successfully to study microbial commu-nities in several habitats such as municipal wastewater [29], landfill methanotroph communities [43], and trinitrotoluenecontaminated soils [17].…”

Influence of Dangling Ends and Surface-Proximal Tails of Targets on Probe-Target Duplex Formation in 16S rRNA Gene-Based Diagnostic Arrays

“…Some of these factors include the characteristics of the fluorescent molecules used to label the target, the labeling efficiency, the length of the probe, the target secondary structure, and hybridization conditions (3,4,6,9,15,22,28,(33)(34)(35)37). However, factors such as the interaction of dangling ends and surface-proximal tails of targets with background DNA have not yet been systematically characterized.…”