Abstract text (incl. figure legends and references)
Introduction: In past, EM was involved with the detection and identification of a broad range of viruses some of which have impact on human, veterinary and wildlife health. However, since pathogen identification was performed mainly by molecular biology the use of diagnostic EM declined radically. With whole genome sequencing and metagenomic approaches the perspectives for molecular pathogen diagnostics changed from a primer-based testing system to a kit-independent diagnostic tool [Metzker, M.L. (2010): Genetic 11:31-45]. But molecular biology can still detect only genetic components of a pathogen whereas EM gives you a morphological picture of the virus. In the present study the method of EM virus screening followed by PCR described by Johnson at al. 2006 [J. Virol. Methods. 134:92-98] was further developed.
Material & methods: Viruses of different genera were investigated by negative staining in a TEM (Zeiss EM 906) at 80kv. Negative staining with 0,5% UA, 0,5% PTA or 4% NdAC was performed with pure or inactivated samples. Samples were provided from feces, cell supensions and biopsy material. Some samples were inactivated with 4% PFA; final PFA-concentration was 2%. For some samples the enrichment method by ultracentrifugation at 84000g (Beckmann airfuge) was used. The stained grids were exposed to the EM beam for 30-180min. Some grids were also stored 1 week before further processing. After EM analysis, virus RNA or DNA was obtained from the virus particles adhering to the 400 mesh copper grid(s) by placing the grid(s) into a vial and adding 180µl lysis buffer. The grid(s) were incubated in lysis buffer for 10min. Then 200µl of PBS was added and the solution with the grid(s) was vortexed for 1 min. The grid(s) was/were then removed before the sample was processed for nucleic acid amplification. The further steps of (RT-)PCR were routinely performed.
Results: Virus detection with EM combined with PCR was successful – in nearly all samples viruses could be detected by both methods. Since it is not possible to quantify the correct amount of viruses on a grid we will only present here a qualitative result (yes/no decision). Exposure to the electron beam does not significantly affect the diagnosis. Storage of grids for a short time (1 week) before processing to the lysis buffer also had no influence except for a slight decrease in viral copies. Enrichment by airfuge or eludation of more than one grid in 180µl lysis buffer naturally resulted in an increase of viral copies. Virus nucleic acids could be detected by PCR in pure or PFA inactivated samples. Viruses were also detected by PCR after using different stains. All used staining solutionsdid not inhibit essentially the amplification process. This is important in so far as NdAC (Neodymium (III)acetate), not toxic and easy to use, is an excellent stain for negative staining [Kuipers J. & Giepmans B, 2020. Histochemistry and Cell Biology 153:271-277]. It replaces the toxic and radioactive uranyl acetate.
Conclusion: Molecular biologists and electron microscopists should be aware that their methods bear a lot of uncertainties in their technical procedure. The results show that a combined EM/Molecular Biology method based on the same sample material eluted from a grid can be performed in an easy and fast procedure. Thus - with the simultanous use of both methods - diagnosis can be confirmed and false negative or positive diagnostic results can be prevented.