Computational protocol: Detection of a Yersinia pestis gene homologue in rodent samples

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Protocol publication

[…] This work was part of a EU project (FP7 WildTech) to develop and use a microarray to detect zoonotic pathogens in rodent tissues. A sequence of the pPCP1 plasmid of the Y. pestis genome (CP000310.1) was obtained from the NCBI database for microarray probe design. Probes were designed using two publicly available software packages: OligoWiz (http://www.cbs.dtu.dk/services/OligoWiz/) and Unique Probe Selector (http://array.iis.sinica.edu.tw/ups/). All probes were checked for suitability using an in silico BLAST analysis. The results of the in silico analysis at the time indicated that the probe sequences were specific to Y. pestis and no cross-hybridisation should occur with eukaryotic or prokaryotic species. Primers were designed using the software Primer3 (http://bioinfo.ut.ee/primer3-0.4.0/). The sequence of each oligonucleotide probe specific to Y. pestis is given in . During the confirmatory testing, both real-time PCR and end-point PCR were used. The primers used in standard end-point PCR and real-time PCR are shown in . These probes were evaluated thoroughly for specificity using reference samples of genomic DNA from Y. pestis NCTC5923 Type strain and non-related pathogens before screening took place. The microarray platform used was the ArrayStrip from Alere Technologies GmbH (Jena, Germany).Four different rodent species (R. rattus, R. norvegicus, Mus musculus and Apodemus sylvaticus) were screened for a number of zoonotic pathogens. Tissue samples were obtained from Vancouver (Canada), Liverpool (UK), and Lyon (France) as part of other studies. Automated nucleic acid extraction was performed on the samples using the QIAcube (Qiagen, Hilden, Germany) and the kit (Cador Pathogen Mini Kit; Qiagen, Hilden, Germany). Liver, kidney and lung tissues were available from each rodent sampled from Vancouver and Lyon, and extracted nucleic acid from each tissue was pooled to make a single sample per individual animal which was tested on the array. Only liver and kidney samples were available from the rodents sampled from Liverpool, and again, extracted nucleic acid was pooled to make a single sample. depicts the sequence enrichment and microarray hybridisation used in this study. […]

Pipeline specifications

Software tools OligoWiz, Primer3
Application qPCR
Organisms Rattus norvegicus, Rattus rattus, Mus musculus
Diseases Yersinia Infections