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Castelnuovo Rangone, Italy

Gentilini F.,University of Bologna | Turba M.E.,Genefast Srl
Veterinary Journal | Year: 2013

Two single tube real-time PCR methods were designed to genotype the mutation responsible for von Willebrand disease type I (von Willebrand factor c.7437G.> A) in Doberman Pinscher dogs: (1) the Divergent PCR assay, which is a modification of the bi-directional PCR amplification of a specific allele (BI-PASA) technique, and (2) a minor groove binder (MGB) real-time PCR assay using fluorescently labelled probes. There was complete agreement between the genotypes determined using the two real-time PCR methods and the results of sequencing of PCR products generated by conventional PCR from genomic DNA purified from the blood of 27 Doberman Pinscher dogs. The Divergent PCR assay yielded reliable results with ≥6.4. ng genomic DNA per reaction and the MGB real-time PCR assay yielded reliable results with ≥150. pg genomic DNA per reaction. Both real-time PCR methods are suitable for routine genetic testing for the von Willebrand disease type I mutation using blood samples. © 2013 Elsevier Ltd. Source

Di Cicco E.,University of Camerino | Paradis E.,Vancouver Aquarium Marine Science Center | Stephen C.,Center for Coastal Health | Stephen C.,University of Calgary | And 2 more authors.
Journal of Zoo and Wildlife Medicine | Year: 2013

A severe outbreak of scuticociliatosis occurred in Australian pot-bellied seahorse, Hippocampus abdominalis (Lesson, 1872), kept at the Vancouver Aquarium Marine Science Centre (Vancouver, British Columbia, Canada). Clinical signs included anorexia, lethargy, irregular respiration, and death. Cytology and histopathology revealed a high number of histophagous ciliated protozoa within the tissues. The parasite, identified as Philasterides dicentrarchi, was observed in several internal organs that appeared edematous and hemorrhagic upon postmortem examination. Severe histopathologic lesions were reported in particular in the ovary, the kidney, and the intestine. This infection was successfully treated with metronidazole via bath therapy. No further evidence of this parasite was found in the treated fish. © 2013 American Association of Zoo Veterinarians. Source

Turb M.E.,University of Bologna | Turb M.E.,Genefast Srl | Zambon E.,University of Bologna | Zannoni A.,University of Bologna | And 2 more authors.
Journal of Clinical Microbiology | Year: 2012

A fundamental role for the endosymbiotic bacteria Wolbachia pipientis in the pathogenesis of Dirofilaria immitis infections has emerged in recent years. Diagnostic opportunities arising from this breakthrough have not yet been fully exploited. This study was aimed at developing conventional and real-time PCR assays to carry out a molecular survey in a convenience sample of cats living in an area where D. immitis is endemic and to evaluate the detection of bacterial DNA in blood as a surrogate assay for diagnosing filaria-associated syndromes in cats. COI and FtsZ loci were used as targets for D. immitis and Wolbachia PCR assays, respectively, and real-time TaqMan PCR assays were used only for Wolbachia. A convenience sample of 307 disease-affected or healthy cats examined at a University facility were PCR tested, and their medical records were investigated. Conventional nested PCR for Wolbachia amplified the endosymbionts of both D. immitis and D. repens, while real-time PCR was highly specific only for the former. Observed prevalences of 0.3 and 10.4% were found using conventional nested PCR assays for D. immitis and real-time PCR for Wolbachia, respectively. Similar prevalences were established using the Wolbachia nested PCR (98% concordance with real-time PCR). The group of Wolbachia-positive samples had a significantly higher proportion of subjects with respiratory signs (29.0% versus 9.7%; P = 0.002). The findings of this study indicate that a highly sensitive PCR assay can be used to detect the Wolbachia organism in the peripheral blood of cats with respiratory signs. Copyright © 2012, American Society for Microbiology. All Rights Reserved. Source

Gentilini F.,University of Bologna | Turba M.E.,Genefast Srl
Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis | Year: 2014

A novel technique, called Divergent, for single-tube real-time PCR genotyping of point mutations without the use of fluorescently labeled probes has recently been reported. This novel PCR technique utilizes a set of four primers and a particular denaturation temperature for simultaneously amplifying two different amplicons which extend in opposite directions from the point mutation. The two amplicons can readily be detected using the melt curve analysis downstream to a closed-tube real-time PCR.In the present study, some critical aspects of the original method were specifically addressed to further implement the technique for genotyping the DNM1 c.G767T mutation responsible for exercise-induced collapse in Labrador retriever dogs. The improved Divergent assay was easily set up using a standard two-step real-time PCR protocol. The melting temperature difference between the mutated and the wild-type amplicons was approximately 5. °C which could be promptly detected by all the thermal cyclers. The upgraded assay yielded accurate results with 157. pg of genomic DNA per reaction. This optimized technique represents a flexible and inexpensive alternative to the minor grove binder fluorescently labeled method and to high resolution melt analysis for high-throughput, robust and cheap genotyping of single nucleotide variations. © 2014 Elsevier B.V. Source

The present invention relates to a method for the detection of nucleic acid synthesis and/or amplification, characterised in that the method includes adding at least one colorimetric metal indicator and at least one bland magnesium chelator to a reaction mixture for nucleic acid amplification. The present invention further relates to a kit for carrying out such a method and to the use thereof in the health, food and agricultural or veterinary fields.

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