Quantitative real-time PCR study on persistence of pDNA vaccine pVax-Hsp60 TM814 in beef muscles
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BioMed Central
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Quantitative real-time PCR study on persistence of pDNA vaccine pVax-Hsp60 TM814 in beef muscles Petr Orság1, Veronika Kvardová1, Milan Raška2, Andrew D Miller3, Miroslav Ledvina4 and Jaroslav Turánek*1 Address: 1Veterinary Research Institute, Department of Immunology, Brno, Czech Republic, 2Palacky University, Faculty of Medicine and Dentistry, Department of Immunology, Olomouc, Czech Republic, 3Imperial College Genetic Therapies Centre, Department of Chemistry, Imperial College London, London, SW7 2AZ, UK and 4The Institute of Organic Chemistry and Biochemistry, Prague, Czech Republic Email: Petr Orság - [email protected]; Veronika Kvardová - [email protected]; Milan Raška - [email protected]; Andrew D Miller - [email protected]; Miroslav Ledvina - [email protected]; Jaroslav Turánek* - [email protected] * Corresponding author
Published: 2 September 2008 Genetic Vaccines and Therapy 2008, 6:11
doi:10.1186/1479-0556-6-11
Received: 15 May 2008 Accepted: 2 September 2008
This article is available from: http://www.gvt-journal.com/content/6/1/11 © 2008 Orság et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract Background: Application of plasmid DNA for immunization of food-producing animals established new standards of food safety. The addition of foreign products e.g. pDNA into the food chain should be carefully examined to ensure that neither livestock animals nor consumers develop unpredicted or undesirable side-effects. Methods: A quantitative real-time PCR (QRTPCR) methodology was developed to study the biodistribution and persistence of plasmid DNA vaccine pDNAX (pVAX-Hsp60 TM814) in mice and beef cattle. The linear quantification range and the sensitivity of the method was found to be 10 – 109 copies per reaction (500 ng/gDNA) and 3 copies per reaction, respectively. Results: Persistence of pDNAX in mice muscle tissue was restricted to injection site and the amount of pDNAX showed delivery formulation dependent (naked pDNA, electroporation, cationic liposome complexes) and mouse age-dependent clearance form injection site but pDNAX was still detectable even after 365 days. The QRTPCR analysis of various muscle tissue samples of vaccinated beef bulls performed 242–292 days after the last revaccination proved that residual pDNAX was found only in the injection site. The highest plasmid levels (up to 290 copies per reaction) were detected in the pDNAX:CDAN/DOPE group similarly to mice model. No pDNA was detected in the samples from distant muscles and draining lymph nodes. Conclusion: Quantitative real-time PCR (QRTPCR) assay was developed to assess the residual pDNA vaccine pVAX-Hsp60 TM814 in mice and beef cattle. In beef cattle, ultra low residual level of pDNA vaccine was only found at the injec
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