Evaluation of Integrated Cell Culture-PCR C-PCR for ...

1. Evaluation of Integrated Cell Culture-PCR (C-PCR) for Virological Analysis of Environmental Samples. Article taken from: Journal of Applied Microbiology 2002, 93, 745-750 Authors: G.E. Greening, J. Hewitt, G.D. Lewis Presented by: Stephen Gurczynski

2. What is PCR? Polymerase Chain Reaction is a method used to amplify specific portions of genetic material. Uses a series of different temperatures to denature, anneal specific primers, and ultimately extend and replicate the target DNA. PCR is an often used technique because of its ability to amplify samples with low concentrations of nucleic acids to makes those samples easier to study.

4. What is Cell Culture PCR New technique where viral particles are inoculated first into a cell culture. The virus is then grown for a length of time (24-48h) Viral genomic material is then extracted straight from the cell culture and analyzed using PCR techniques.

5. Advantages of C-PCR During normal culturing and observation some infectious viruses do not exhibit cytopathic effects (CPE) C-PCR tests directly for viral nucleic acids in a cell culture whether or not the cells are exhibiting CPE. Thus C-PCR is both faster and often times more accurate than traditional techniques such as plaque assays.

6. The Experiment Sewage samples used Primary-treated effluent Tertiary-treated effluent Primary-settled sludge Marine Life samples used Naturally growing feral shellfish (mussels, clams, oysters) Viruses tested for Enterovirus Adenovirus

8. Methods Cont. Virus samples were extracted from either the sewage effluent, sludge, or shell fish gut tissue and then concentrated. Enterovirus samples inoculated into buffalo green monkey (BGM) cell cultures, Adenovirus samples inoculated into HEp-2 cell culture with pen/strep and fetal calf serum added. Samples were taken for both C-PCR and RT-PCR at 1d, 3d, and 5d intervals. Plaque assays (2-4d incubation time) were also carried out for both samples serving as a control.

9. Results 1 plaque forming unit (PFU) of virus PV2 was detected after 24h using C-PCR. 10 PFU were detected of other enteroviruses after 24h Comparatively it can take up to 5 days for CPE to be exhibited in cell culture/plaque assay technique. RT-PCR for enterovirus was still 10-100 more sensitive detecting .05-.1 PFU.

10. Results Cont. 1 PFU of adenovirus detected after only 3 days of incubation. 10 or more days are sometimes necessary to show any results with adenovirus in a normal plaque assay. Again normal PCR was 10 times more sensitive detecting .16 PFU.

12. C-PCR versus RT-PCR/PCR RT-PCR is proven to be the most sensitive method for detection so why not use it? Because RT-PCR looks at virus particles that are outside of the cell it provides no information about infectivity of virus particles. Even virus particles inactivated by hypochlorite can still give false positives about infectivity. Any type of inhibitory factors present in the sample will interfere with successful PCR. C-PCR however looks at developing nucleic acids inside of the infected cells thus it does give an accurate picture of infectivity.

13. Conclusion Pros and Cons Pros of C-PCR Results available after only 24-48 hours compared to multiple days with other methods. Provides an accurate picture as to the infectivity of viruses in a sample By reducing the amount of steps needed both time and money are saved. Because C-PCR looks at developing viral nucleic acids in a cell it can detect viruses that do not show CPE or viruses that are hard to culture.

14. Cons Inhibitory factors present in a sample can still interfere Not quite as sensitive as traditional (RT)-PCR techniques. Neither C-PCR or traditional PCR can give quantitative results as to the numbers of virus particles in a sample. Only plaque assays can do this. No PCR technique will let you look for an unknown virus.

15. Is C-PCR worth it? C-PCR seems to be a unique solution that is highly applicable to the screening of environmental samples. Has a high efficiency with enterovirus which is often present in contaminated environmental samples. The bottom line is that C-PCR is not suited for all situations and there are certain situations where other methods fair better. For screening certain viruses from environmental samples however C-PCR offers a cheap, accurate, and effective alternative.