Large scale multiplex PCR improves pathogen detection by DNA microarrays.
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.


CONCLUSIONS
Our data provide a proof of principle for the improvement of detection of pathogen DNA by microarray hybridization by using LSplex PCR.
Palka-Santini
Maria
M
Institute for Medical Microbiology, Immunology and Hygiene, Medical Faculty, University of Cologne, Germany. mpalka-santini@mmm.com
Cleven
Berit E
BE
Eichinger
Ludwig
L
Krönke
Martin
M
Krut
Oleg
O
D001419:Bacteria / Q000235:genetics / Q000302:isolation & purification*; D002176:Candida albicans / Q000235:genetics / Q000302:isolation & purification*; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis* / Q000235:genetics; D013293:Enterococcus faecalis / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics / Q000302:isolation & purification; D005798:Genes, Bacterial; D006801:Humans; D007711:Klebsiella pneumoniae / Q000235:genetics / Q000302:isolation & purification; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011513:Proteus mirabilis / Q000235:genetics / Q000302:isolation & purification; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D013210:Staphylococcus / Q000235:genetics / Q000302:isolation & purification
D017931:DNA Primers; D004269:DNA, Bacterial