Optimization and clinical validation of a pathogen detection microarray

Abstract

Pathogen detection by microarray <p>New design and optimization of pathogen detection microarrays is shown to allow robust and accurate detection of a range of pathogens. The customized microarray platform includes a method for reducing PCR bias during DNA amplification.</p> Abstract DNA microarrays used as 'genomic sensors' have great potential in clinical diagnostics. Biases inherent in random PCR-amplification, cross-hybridization effects, and inadequate microarray analysis, however, limit detection sensitivity and specificity. Here, we have studied the relationships between viral amplification efficiency, hybridization signal, and target-probe annealing specificity using a customized microarray platform. Novel features of this platform include the development of a robust algorithm that accurately predicts PCR bias during DNA amplification and can be used to improve PCR primer design, as well as a powerful statistical concept for inferring pathogen identity from probe recognition signatures. Compared to real-time PCR, the microarray platform identified pathogens with 94% accuracy (76% sensitivity and 100% specificity) in a panel of 36 patient specimens. Our findings show that microarrays can be used for the robust and accurate diagnosis of pathogens, and further substantiate the use of microarray technology in clinical diagnostics.

Report for Development of a Census Array and Evaluation of the Array to Detect Biothreat Agents and Environmental Samples for DHS

“…In prior work, we have found that the length of longest perfect match (PM) is a strong predictor of hybridization intensity, and that for probes at least 50 nt long, PM≤20 bp have signal less than 20% of that with a perfect match over the entire length of the probe (2). This is similar to results from a systematic study of viral probe hybridization characteristics by (3). Therefore, for each target family we eliminated regions with perfect matches to sequences outside the target family.…”

Viral Nucleic Acids in Live-Attenuated Vaccines: Detection of Minority Variants and an Adventitious Virus

“…Nucleic acid samples were reverse transcribed to cDNA using tagged random primers as described above (36), except that primer A2 (5Ј-GATGAGGGAAGATGGGGNNNNNNNNN-3Ј) was used (39). The cDNA was synthesized into double-stranded cDNA and then amplified by random PCR using primer B2 (5Ј-GATGAGGGAAGATGGGG-3Ј).…”

Epidemiology and Diagnosis of Human Respiratory Syncytial Virus Infections

“…Microarray analysis has the capability to offer multiplex detection, Multiple microarray platforms exist, including printed double-stranded DNA and oligonucleotide arrays, in situsynthesized arrays, high-density bead arrays, electronic microarrays, and suspension bead arrays (Wong et al, 2007). In general terms, probes are synthesized and immobilized as discrete features, or spots.…”