Real Time Polymerase Chain Reaction (RT-PCR) Technique for the Detection of Vertically-Transmitted Pathogens
May 02 2013
Specific Pathogen Free (SPF) eggs are commonly used as the substrate for production of many vaccines. The Center for Veterinary Biologics (CVB) in the US and the European Pharmacopoeia in Europe both have regulations on the qualifications for SPF eggs. VS Memo 800.65 and PhEur 5.2.2 describe a critical subset of avian pathogens that, if present in a flock, disqualifies the flock as SPF and unsuitable for use in vaccine manufacture. Testing for these avian pathogens relies almost exclusively on serologic methods to detect antibodies generated in response to exposure to these pathogens. The PhEur 5.2.2 notes that at the end of the production period serologic testing of the flock must again be performed four weeks after the last egg collection that was used for vaccine manufacture. However, there is a provision that allows for testing suitable sample materials within four weeks of the final egg collection, for the presence of vertically-transmissible agents using validated nucleic acid amplification techniques as described in PhEur 2.6.21.
The Molecular Diagnostics Laboratory at Charles River recently developed and optimized the RT-PCR for the pathogens that are considered capable of vertical transmission as defined by the PhEur 5.2.2: Avian Adenovirus Group I, Avian Adenovirus Group III, Avian Encephalomyelitis Virus, Avian Leukosis Viruses (subgroups A, B, J), Avian Reovirus, Avian Reticuloendotheliosis Virus, Chicken Anemia Virus, Mycoplasma gallisepticum, Mycoplasma synoviae and Salmonella pullorum. In this application the RT-PCR is used as a limit test (positive or negative results) for each pathogen. The limit of detection, specificity and robustness were determined using cloacal swabs from SPF flocks that were spiked with each pathogen at different dilutions.
The results show that the RT-PCR was able to detect less than 47 infectious units of any of the organisms tested. Also, the primers were non-reactive to 200 negative cloacal swabs making the assay specific for each pathogen. In order to demonstrate robustness, a simple, intentional variable was used by not vortexing the samples after the addition of lysis buffer. Spiked samples remained positive even with the intentional change. Therefore, based on our validation, the sensitivity and specificity of these assays are adequate for the detection of infections in SPF flocks.
To learn more, contact us at askcharlesriver@crl.com or click here to view our poster titled “The Use of RT-PCR for the Detection of Vertically Transmitted Pathogens in SPF Flocks.”
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