Dataset Information

Large scale multiplex PCR improves pathogen detection by DNA microarrays.

ABSTRACT:

Background

Medium density DNA microchips that carry a collection of probes for a broad spectrum of pathogens, have the potential to be powerful tools for simultaneous species identification, detection of virulence factors and antimicrobial resistance determinants. However, their widespread use in microbiological diagnostics is limited by the problem of low pathogen numbers in clinical specimens revealing relatively low amounts of pathogen DNA.

Results

To increase the detection power of a fluorescence-based prototype-microarray designed to identify pathogenic microorganisms involved in sepsis, we propose a large scale multiplex PCR (LSplex PCR) for amplification of several dozens of gene-segments of 9 pathogenic species. This protocol employs a large set of primer pairs, potentially able to amplify 800 different gene segments that correspond to the capture probes spotted on the microarray. The LSplex protocol is shown to selectively amplify only the gene segments corresponding to the specific pathogen present in the analyte. Application of LSplex increases the microarray detection of target templates by a factor of 100 to 1000.

Conclusion

Our data provide a proof of principle for the improvement of detection of pathogen DNA by microarray hybridization by using LSplex PCR.

SUBMITTER: Palka-Santini M

PROVIDER: S-EPMC2631447 | biostudies-literature | 2009 Jan

REPOSITORIES: biostudies-literature

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Publications

Large scale multiplex PCR improves pathogen detection by DNA microarrays.

Palka-Santini Maria M Cleven Berit E BE Eichinger Ludwig L Krönke Martin M Krut Oleg O

BMC microbiology 20090103

<h4>Background</h4>Medium density DNA microchips that carry a collection of probes for a broad spectrum of pathogens, have the potential to be powerful tools for simultaneous species identification, detection of virulence factors and antimicrobial resistance determinants. However, their widespread use in microbiological diagnostics is limited by the problem of low pathogen numbers in clinical specimens revealing relatively low amounts of pathogen DNA.<h4>Results</h4>To increase the detection pow ...[more]

PMID: 19121223

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Project description:BACKGROUND: DNA microarrays are rapidly becoming a fundamental tool in discovery-based genomic and biomedical research. However, the reliability of the microarray results is being challenged due to the existence of different technologies and non-standard methods of data analysis and interpretation. In the absence of a "gold standard"/"reference method" for the gene expression measurements, studies evaluating and comparing the performance of various microarray platforms have often yielded subjective and conflicting conclusions. To address this issue we have conducted a large scale TaqMan Gene Expression Assay based real-time PCR experiment and used this data set as the reference to evaluate the performance of two representative commercial microarray platforms. RESULTS: In this study, we analyzed the gene expression profiles of three human tissues: brain, lung, liver and one universal human reference sample (UHR) using two representative commercial long-oligonucleotide microarray platforms: (1) Applied Biosystems Human Genome Survey Microarrays (based on single-color detection); (2) Agilent Whole Human Genome Oligo Microarrays (based on two-color detection). 1,375 genes represented by both microarray platforms and spanning a wide dynamic range in gene expression levels, were selected for TaqMan Gene Expression Assay based real-time PCR validation. For each platform, four technical replicates were performed on the same total RNA samples according to each manufacturer's standard protocols. For Agilent arrays, comparative hybridization was performed using incorporation of Cy5 for brain/lung/liver RNA and Cy3 for UHR RNA (common reference). Using the TaqMan Gene Expression Assay based real-time PCR data set as the reference set, the performance of the two microarray platforms was evaluated focusing on the following criteria: (1) Sensitivity and accuracy in detection of expression; (2) Fold change correlation with real-time PCR data in pair-wise tissues as well as in gene expression profiles determined across all tissues; (3) Sensitivity and accuracy in detection of differential expression. CONCLUSION: Our study provides one of the largest "reference" data set of gene expression measurements using TaqMan Gene Expression Assay based real-time PCR technology. This data set allowed us to use an alternative gene expression technology to evaluate the performance of different microarray platforms. We conclude that microarrays are indeed invaluable discovery tools with acceptable reliability for genome-wide gene expression screening, though validation of putative changes in gene expression remains advisable. Our study also characterizes the limitations of microarrays; understanding these limitations will enable researchers to more effectively evaluate microarray results in a more cautious and appropriate manner.

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Dataset Information

Large scale multiplex PCR improves pathogen detection by DNA microarrays.

Background

Results

Conclusion

Publications

Large scale multiplex PCR improves pathogen detection by DNA microarrays.

Similar Datasets

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