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Validation of polymerase chain reaction assays as replacement of the Mouse Antibody Production (MAP) Test
Frank Bootz and Felix R. Wolf1
Institut für Labortierkunde, Universität Zürich, 8057 Zürich Switzerland
1present address: Research Anmal Resources Center, Medical College of Cornell University, New York 10021, USA
Keywords: mice; viruses; molecular biology: pcr; reduction; replacement; antibody production test; diagnostic approaches: viruses
Duration: 1 year Project Completion: 2002
Background and Aim
Rodent viruses may be transmitted through contaminated biological materials such as transplantable tumors, cell lines, etc. Such inadvertent transmission may cause endemic infections in the recipient colony, resulting in clinical disease and/or compromised research results. To avoid this, biological material must be screened for pathogens before it is introduced into rodents. This is currently done using the mouse antibody production (MAP) or the rat antibody production (RAP) test. In a previous project (3R Project 45-96, Homberger et al.), we established alternative assays using PCR technology to detect these viruses directly in the biological material. We were able to show that the complete spectrum of agents detectable by the traditional MAP/RAP test could be covered by these new assays. Since then, over 50 cell lines and tumour samples have been tested. Having demonstrated their potential, the aim of this study is to validate these PCR assays and compare them directly to the MAP/RAP tests (Kraft 1996, Rehbinder et al. 1996).
Method and Results
The validation was carried out using 1) routine samples submitted to our diagnostic lab for MAP-testing as well as 2) experimentally spiked samples. All virus strains (described in project 45-96) were cultured under their specific optimal growth conditions. The virus material was titrated to a concentration of between 103 and 10-3 infectious particles (IP/ml) and injected into mice (MAP-test) and collected for DNA/RNA-extraction (PCR) at all dilutions. The limits of detection (sensitivity) of the two techniques were compared.
For 6/16 viruses, the conventional PCR technique seems to be more sensitive and more specific in detecting murine viruses. In 12/14 viruses, the. RT-PCR (Real-Time PCR) is more sensitive than the MAP-test. In 2/14 cases all three detection methods have the same sensitivity. Furthermore, the PCR technology had a much shorter turnover time (two days) than the MAP test (one month to obtain seroconversion plus a few days for the serology results).
Conclusions and Relevance for 3R
In the past, the MAP-test was shown to be more sensitive than the direct isolation of virus particles grown in cell culture (de Souza & Smith 1989). Our study affirms that with PCR technology, the sensitivity of in vitro techniques can be sufficiently improved as to provide a viable alternative to the in vivo MAP-tests. The PCR-based technology was able to detect all viruses found in the context of the MAP-test, although the specific sensitivity pattern was somewhat different.
The advantages of the PCR technique are its speed (2 days vs. 30 days for the MAP-test) and its contribution to the principles of 3R as a replacement technique. Currently, between 10 and 50 animals are used per MAP-test. The final results of this project will show whether the in vivo test can be completely replaced by the PCR technique. A final contribution to the principles of 3R: healthy, standardised, pathogen-free laboratory animals are a prerequisite in achieving reproducible animal experiments. With an effective in vitro method to monitor the pathogen free status of laboratory rodents, the number of animals used in in vivo experiments can also be reduced (Rehbinder et al. 1996).
(see also 3R-INFO-BULLETIN Nr. 20)
Published updated Version 20/2007 (pdf)
1. Tischhauser, ME, Popovic, D and Homberger FR (1999) Replacement of mouse and rat antibody production (MAP/RAP) test by polymerase chain reaction asays. Inaugural Dissertation, University of Zurich.
2. Kraft V (1996) What is the real impact of rodent viruses? Scandinavian Journal of Laboratory Animal Science 23, 159-63-
3. Rehbinder C, Baneux P, Forbes D, et al. (1996) FELASA recommendations for the health monitoring of mouse, rat, hamster, gerbil, guinea pig and rabbit experimental units. Laboratory Animals 30, 193-208.
4. Bootz, F. und Sieber, I. (2002). “Ersatzmethode für den Maus/Ratte Antikörper Produktions Test. Sensitivitätsvergleich zwischen in vitro und in vivo Methode.” ALTEX 19, Suppl. 1, 76 - 86.
5. Popovic, D. (1998). “Die Polymerase-Kettenreaktion (PCR) als Ersatz für den Mäuse-und Ratten-Antikörper-Produktionstest.” (Inaugural Dissertation, University of Zürich).
6. Tischhauser, M. E. (1999). “Replacement of mouse and rat antibody production (MAP/RAP) test by polymerase chain reaction.” (Inaugural Dissertation, University of Zürich).
7. Sieber, I.U. (2001) “Ersatzmethode für den Maus-Antikörper-Produktionstest. Ein Sensitivitärsvergleich verschiedener PCR-Assays mit dem Tierversuch.” (Inaugural Dissertation, University of Zürich).
8. Bootz F., I. Sieber, D. Popovic, M. Tischhauser, F.R. Homberger. 2003. Comparison of the sensitivity of in vivo antibody production tests with in vitro PCR-based methods to detect infectious contamination of biological materials, Lab. Anim. 37: 341-351.