Development of a DNA-based microarray for the detection of zoonotic pathogens in rodent species

Abstract

The demand for diagnostic tools that allow simultaneous screening of samples for multiple pathogens is increasing because they overcome the limitations of other methods, which can only screen for a single or a few pathogens at a time. Microarrays offer the advantages of being capable to test a large number of samples simultaneously, screening for multiple pathogen types per sample and having comparable sensitivity to existing methods such as PCR. Array design is often considered the most important process in any microarray experiment and can be the deciding factor in the success of a study. There are currently no microarrays for simultaneous detection of rodent-borne pathogens. The aim of this report is to explicate the design, development and evaluation of a microarray platform for use as a screening tool that combines ease of use and rapid identification of a number of rodent-borne pathogens of zoonotic importance. Nucleic acid was amplified by multiplex biotinylation PCR prior to hybridisation onto microarrays. The array sensitivity was comparable to standard PCR, though less sensitive than real-time PCR. The array presented here is a prototype microarray identification system for zoonotic pathogens that can infect rodent species.

Keywords: ArrayStrip; ArrayTube; Development; Microarray; Rodent; Zoonoses.

Fig. 1

Images produced after hybridisation of various Salmonella species on WT_Rodents_2_1.0 array. The spots indicated by arrows are the biotin markers. The solid square and rectangular areas are the orientation markers. A. S. Gallinarum hybridisation following random amplification. B. S. Dublin hybridisation following random amplification. C. S. Pullorum hybridisation following random amplification. D. S. Enteritidis hybridisation following random amplification. E. S. Hadar hybridisation following random amplification. F. S. typhimurium amplification following random amplification. G. S. typhimurium amplification with primers Salm/flag/1366055/F and Salm/flag/1366482/R (S. typhimurium-specific). H. S. typhimurium amplification with primers Salm/CDP/2167279/F and Salm/CDP/2005357/R (Generic Salmonella species).

Fig. 2

A. Agarose gel electrophoresis image produced after amplification of nucleic acid of individual pathogens using the multiplex primer mix. B. Profile produced after hybridisation of a mixture of all the pathogens following amplification with the multiplex primer mix for which reference samples were available.

Fig. 3

Real-time PCR sensitivity testing was performed with serial dilutions of Y. pestis. Sample A was a negative control sample (water). The copy number in samples B to F was 3.47 × 10⁹, 1.76 × 10⁷, 8.57 × 10⁴, 4.39 × 10², and 1. Array images after hybridisation of the standard PCR products from the same Y. pestis amplification on WT_Yersinia_01 are also shown. Biotin markers are indicated by arrows, and Y. pestis probes that showed hybridisation are circled.