There are many methods available for the titration of recombinant baculoviruses. One of the most convenient is the use of the quantitative polymerase chain reaction (QPCR), which assesses how much virus DNA is present in a freshly-amplified stock. This is compared with a standard curve derived from a control virus stock to give an infectious titre expressed as Q plaque forming units (pfu) per millilitre. OET Ltd market the baculoQUANT™ all-in-one virus extraction and titration kit for this purpose. It supplants the use of time consuming and technically difficult virus infectivity titrations based on plaque assays or end point estimations (TCID50’s). While the plaque assay remains the gold standard in many ways for determining infectivity titres it does cause problems for many scientists who only perform it on an occasional basis. Conversely, most laboratories will have experience of performing PCR/QPCR in the course of their research projects and are ideally suited for adopting baculoQUANT titration of recombinant baculoviruses.
The key features of the baculoQUANT™ all-in-one kit are:
• Titre accuracy comparable to plaque assay method • Less than one hour hands-on time • Compatible with any AcMNPV-based baculovirus system containing gp64 virus gene • Single step virus DNA extraction: just add lysis solution (no spin columns) • QPCR Primers/Probe provided as a single reagent mix: just add water • Agilent high performance, ultra sensitive master mix reagent included • Plaque-assay titrated virus internal standards included for standard curve generation • 100 reaction kit allows for up to 24 titrations in triplicate with 3 standard curves.
What are the limitations of the QPCR method? It does have to be performed on virus stocks that were produced recently. We recommend virus stocks should not be older than 3 months. This is because the method does not distinguish between infectious and non-infectious virus particles – it just detects virus DNA. And that really is the only disadvantage of the method. Obviously, your laboratory has to have a QPCR instrument, but these are now very common and available for relatively low sums of money.
Why bother with titrating virus infectivity at all? If you don’t you run the risk of trying to obtain expression of your target gene using a non-optimized system. Many users of the baculovirus expression system just rely on infectious virus titres always being 10E8 pfu/ml or greater – because that’s what the manuals say you should achieve! In practice, infectious virus titres vary according to the quality of your host cells, the amount of virus used as an inoculum and to some extent the nature of the protein that you are producing. Although recombinant baculoviruses produce most of the target protein in the very late phase of virus gene expression, after the major burst of infectious budded virus (BV), some protein is made during this burst and can affect BV yields. This means that if you assume an infectious titre of 10E8 pfu/ml but in reality your stock is only 2 x 10E7 pfu/ml your calculations on the amount of virus to use in test for protein expression will be in error.
To conclude, we always recommend to customers that they determine the infectivity titre of their recombinant baculovirus stock. If you use our baculoQUANT™ all-in-one kit the process is very quick, simple and easy to perform. You can harvest your virus stock and determine its infectivity all in the same day.
Contact us at OET Ltd if you would like to receive further advice and information about any aspect of recombinant baculovirus expression vectors.
コメント