A multiplex polymerase chain reaction microarray assay to detect bioterror pathogens in blood

Abstract

Heightened concern about the dangers of bioterrorism requires that measures be developed to ensure the safety of the blood supply. Multiplex detection of such agents using a blood-screening DNA microarray is a sensitive and specific method to screen simultaneously for a number of suspected agents. We have developed and optimized a multiplex polymerase chain reaction microarray assay to screen blood for three potential bioterror bacterial pathogens and a human ribosomal RNA gene internal control. The analytical sensitivity of the assay was demonstrated to be 50 colony-forming units/ml for Bacillus anthracis, Francisella tularensis, and Yersinia pseudotuberculosis (surrogate for Yersinia pestis). The absence of any false-positives demonstrated high analytical specificity. Screening B. anthracis-infected mouse blood samples and uninfected controls demonstrated effectiveness and specificity in a preclinical application. This study represents proof of the concept of microarray technology to screen simultaneously for multiple bioterror pathogens in blood samples.

Figure 1

Agarose gels of multiplex PCR products stained with ethidium bromide. A: Gel-analyzing PCR products from primary multiplex reactions containing primer pairs for the human rRNA gene as well as all three pathogen primer pairs for detection of B. anthracis, F. tularensis, and Y. pseudotuberculosis (surrogate for Y. pestis). Lane M is the 50-bp GeneRuler (Fermentas) with selected band sizes in bp at left. In lane 1, the template for the reaction was no DNA as a negative control, the remaining lanes contain products from reactions with template DNA from a blood sample spiked with the following amounts of Y. pseudotuberculosis bacteria: lane 2, 5 CFU/ml; lane 3, 50 CFU/ml; lane 4, 500 CFU/ml; lane 5, 5000 CFU/ml; lane 6, blood alone. The arrow indicates the position of the Y. pseudotuberculosis amplicon DNA and the arrowhead indicates the position of the human rRNA amplicon DNA. B: Gel-analyzing PCR products from secondary multiplex reactions each using PCR products from a primary reaction as template, the primer pair for the human rRNA gene and one pathogen primer pair. The lanes are sets of three using the same template in which the first lane has the B. anthracis primer pair, the second lane has the F. tularensis primer pair, and the third has the Y. pestis primer pair. Lanes 1 to 3, template is negative control reaction shown in A, lane 1. Lanes 4 to 6, template is primary PCR reaction with blood alone. Lanes 7 to 9, template is primary PCR reaction with Y. pseudotuberculosis, 5 CFU/ml. Lanes 10 to 12, template is primary PCR reaction with Y. pseudotuberculosis, 50 CFU/ml. Lanes 13 to 15, template is primary PCR reaction with Y. pseudotuberculosis, 500 CFU/ml. Arrows indicate the position of DNA fragments that correspond to the Y. pseudotuberculosis amplicon and the human rRNA amplicon as in A.

Figure 2

Layout of the pathogen detection microarray probes and hybridization results. A: Image of one array hybridized with the Cy5-QC oligo. B: Image of one of the two replicate arrays in a microarray assay of blood spiked with B. anthracis (50 CFU/ml) labeled with Cy3. C: F. tularensis (50 CFU/ml) labeled with Cy5. D: Y. pseudotuberculosis (50 CFU/ml) labeled with Cy5.