How to evaluate the propagation of DNA virus? what's methods can be used? Quantitive-PCR to detect DNA of the cell culture at different time post infection and RT-PCR to detect the RNAs about replication. which one is more suggested ?
Your question is too general. What is “your” virus? Do you have information about its replication strategy and life cycle? What is the aim of your study - are you trying to evaluate the influence of some factors (antiviral agents, etc) on viral replication and life cycle? Do you want to obtain more information about replication of “your” virus or you ask just about “virus quantification”?
In order to study “viral propagation” we must have serious knowledge in the field of virology and as more as possible information about our virus (or similar viruses).
We need to prepare adequate experimental design – this is not only the question of equipment available and cocktail of methods that we can perform. First of all, this is “philosophy”, strategy... Such investigation is a multistep process and we will need information, respectively we must take decisions at each step. For example:
A) We need a permissive cell culture (as a Model system) to allow the implementation of the full virus cycle and obtaining of mature and infectious viral particles. There is a difference between permissive cell cultures on one hand and susceptible cell cultures on the other (susceptible cultures allow absorption and entry of the virus but do not allow the implementation of the full cycle) – each permissive cell line is sensitive, but the opposite is not true.
B) We need viral isolate/stock – it is better to know its infectious titer in order to use the adequate multiplicity of infection (MOI, this is the ratio of viral particles to cells) then you infect the cells. If you use higher MOI for example, you can obtain a high percentage of defective virus particles.
C) We can cultivate our cell-virus system in plates or petri dishes or …, but we have to prepare many (enough) repetitions (you will decide how many you need, but it is always better to prepare at least 1-2 more - just in case). For each cell-virus repetition you will need a control (non-infected cells).
D) At different periods of time post infection (that is why we have to know the replication strategy of the virus, we have to follow the full cycle period) we can take one or more repetitions (ant their controls) and we can analyze them immediately or store (after freezing) for later analyses. How to analyze these samples – we have to do it at different levels (DNA – RNA-Protein) and by different methods. For example, protein detection is very important (in many DNA viruses there are “early” and “late” genes and proteins and we have to follow their expression) – we can do this by Western blotting or some other immunological assay but it is always informative and very helpful to perform some (immunofluorescence) microscopy - it will allow us to localize the protein in the cell. Of, course we will need specific antibodies for such kind of experiments. For some viruses it could be a problem, I know.
E) Electron microscopy will be also very helpful. In this case we can (and we have to) “make photos” of our virus at different steps of its replication and morphogenesis – absorption, entry... as well as assembly and exit.
F) Finally, we have to do “virus quantification”. And, you can use for this purpose different modern methods including quantitave PCR. But according to my opinion it will be of benefit to determine also the infectious viral titer using some classical “old” methods – we can use different methods (depending on the virus) for this purpose – Plaque assay, Endpoint dilution assay (Method of Reed and Muench for example), etc. Yes, these methods are “old” and “classical” but their advantage is that they show “in reality” the full biological capacity of the virus (measuring only number of mature infectious viral particles). It is always better to combine and compare results obtained in different ways. Good luck!
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