Rapid Virus Titration by QPCRPublished on March 31, 2016
Knowing the infectious titre of a recombinant virus stock is really important prior to testing for protein production in insect cells. It enables you to add the optimal quantity of virus to initiate virus infection. Too much virus and you are wasting your stock. Too little virus and you won’t establish a synchronous infection. For some proteins this may not matter, as the virus produced by the initial round of replication will infect all other cells and result in protein production. However, if your protein target is particularly unstable, you may lose some of the product via its degradation in the population of cells initially infected. We will return to the question of how much virus to add to cells to optimize protein production in a later blog. In the current offering we will address how you can rapidly determine the infectious titre of a virus stock.
In our last blog we addressed the use of a traditional plaque assay-based titration system. While this is probably the gold standard for measuring virus infectivity, it is time consuming. From start to finish it requires a minimum of 3 days to get your results. In those far off days of leisurely academic research this may not have been too much of a problem. You could do your titration, wander off for a few games of tennis and a drink in the student bar, then come back to stain and read your plaque assay. It is a different story today as everyone wants results yesterday – even the academics. So while the plaque titration method still has its place, what is really needed is a technique that can provide results in near real time.
One of the most convenient ways to determine baculovirus infectivity is to use a test based on quantitative (q) PCR. Most laboratories have access to qPCR thermocyclers so it is a method accessible to all. We market baculoQUANT™ for this purpose. Our kit baculoQUANT kits provide all reagents required to perform a virus titration in under three hours with less than one hour hands on time required. A small quantity (80µl) of virus is pelleted using a microfuge, treated with a lysis buffer for a short time using a normal thermocycler and then introduced directly to the qPCR assay, where it is compared to a standard virus sample we provide. Accuracy is comparable to the traditional plaque assay method. Using baculoQUANT™ you can harvest your virus, titrate your virus and then inoculate cells to begin tests for protein expression all in the same day. The baculoQUANT™ kit converts qPCR values to pfu/ml, the accepted units of virus infectivity.
It is very important to note that measuring the infectious titre of a recombinant virus stock via qPCR is essentially assessing the quantity of virus DNA in a sample. In newly prepared virus stocks this will be directly related to the infectivity of the virus particle. However, as most recombinant virus stocks are stored at 4°C, there can be a gradual decline in their infectivity over time. Therefore, we recommend that virus stocks more than 3 months old are not titrated with the qPCR method. We have a suspicion that the nature of the recombinant protein being expressed by the virus can affect the stability of virus stocks. For example, plasma membrane-bound proteins can be incorporated into the virus that buds from cells. This may have an effect on virus stability.
Virus storage is an issue that we will return to in a future blog. Meanwhile, if you have any issues about virus storage namely, concerns about safe keeping of precious recombinants, we offer a secure virus cryo-storage service at very reasonable annual cost. Our next blog will concern factors you should consider when testing recombinant protein production by your newly amplified virus stock, which we alluded to in the opening paragraph of this post.