A system to simultaneously detect tick-borne pathogens based on the variability of the 16S ribosomal genes

Particle and Fibre Toxicology

Imrich Barák²  Marketa Derdáková¹  Jana Melničáková² 

[1] Institute of Parasitology, Slovak Academy of Sciences, Hlinkova 3, 040 01, Košice, Slovak Republic;Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravska cesta 21, 845 51, Bratislava, Slovak Republic

关键词: Quantitative PCR;    DNA microarray;    Tick-borne bacteria;   

Others  :  824579
DOI  :  10.1186/1756-3305-6-269

 received in 2013-08-01, accepted in 2013-09-12,  发布年份 2013

【 摘 要 】

Background

DNA microarrays can be used to quickly and sensitively identify several different pathogens in one step. Our previously developed DNA microarray, based on the detection of variable regions in the 16S rDNA gene (rrs), which are specific for each selected bacterial genus, allowed the concurrent detection of Borrelia spp., Anaplasma spp., Francisella spp., Rickettsia spp. and Coxiella spp.

Methods

In this study, we developed a comprehensive detection system consisting of a second generation DNA microarray and quantitative PCRs. New oligonucleotide capture probes specific for Borrelia burgdorferi s.l. genospecies and Candidatus Neoehrlichia mikurensis were included. This new DNA microarray system required substantial changes in solution composition, hybridization conditions and post-hybridization washes.

Results

This second generation chip displayed high specificity and sensitivity. The specificity of the capture probes was tested by hybridizing the DNA microarrays with Cy5-labeled, PCR-generated amplicons encoding the rrs genes of both target and non-target bacteria. The detection limit was determined to be 10³ genome copies, which corresponds to 1–2 pg of DNA. A given sample was evaluated as positive if its mean fluorescence was at least 10% of the mean fluorescence of a positive control. Those samples with fluorescence close to the threshold were further analyzed using quantitative PCRs, developed to identify Francisella spp., Rickettsia spp. and Coxiella spp. Like the DNA microarray, the qPCRs were based on the genus specific variable regions of the rrs gene. No unspecific cross-reactions were detected. The detection limit for Francisella spp. was determined to be only 1 genome copy, for Coxiella spp. 10 copies, and for Rickettsia spp., 100 copies.

Conclusions

Our detection system offers a rapid method for the comprehensive identification of tick-borne bacteria, which is applicable to clinical samples. It can also be used to identify both pathogenic and endosymbiontic bacteria in ticks for eco-epidemiological studies, tick laboratory colony testing, and many other applications.

【 授权许可】

   
2013 Melničáková et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

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