We perform validated measurements of both DNA and RNA viruses. We use the digital droplet technique (ddPCR) which relies on tiny droplets dispersed in oil. To detect an RNA virus, the target RNA is first copied into DNA and then amplified (RT-ddPCR).
We offer ddPCR or RT-ddPCR for several of our model viruses: the retroviruses HIV-1 and X-MuLV, the herpes virus PRV and the parvovirus MVM. Please inquire if you need a ddPCR measurement with any other of our model viruses.
PCR is a method where a specific DNA sequence, the target, is amplified by a primer pair. The amplification can be detected by a sequence specific probe coupled to a fluorophore and its quencher. Each time the target sequence is copied a fluorophore is released. Provided there are no PCR-inhibitors or other obstacles, the change in bulk fluorescent signal from the sample is proportional to the increase in target sequence.
In digital PCR the reaction is divided into a large number of tiny volumes before amplification, typically 10 000 to 20 000 per reaction. These are known as partitions or droplets, depending on the technique used to create them. The amplification is run by temperature cycling as in conventional PCR and the signal is recorded separately for each droplet. Based on signal strength each droplet is classified as positive or negative, and assuming Poisson distribution, the target concentration in the sample can be calculated in absolute terms. The result is expressed as genome equivalents (geq) per volume.
The fact that dPCR only needs to establish whether a droplet is positive or not makes it very resilient to inhibitors. Furthermore, there is no need to establish and maintain a standard curve, as the frequency of positive droplets is the only variable needed to calculate the target concentration in the reaction mixture. This makes dPCR an ideal technique to measure virus removal from complex process materials.
Digital PCR is also a valuable complement to conventional infectivity assays. An illustration of this is virus validation of antibody capture by protein A. In this method the antibody is captured and washed on a membrane or column and then released by low pH. The capture will separate the antibody from any virus that will not bind, while the low pH will inactivate many enveloped viruses. Applying a combination of dPCR and an independent measurement of low pH induced virus inactivation will allow both virus removal and inactivation to be used in the overall estimation of the virus reducing capacity of the process. In this way, two virus reducing steps may be gained from a single process step.
The Vironova Biosafety services team have a broad research background and experience of a wide range of biologics. We can help you to plan a specific study or solve a tricky problem. Whatever your need, you can contact Vironova Biosafety by sending an e-mail to firstname.lastname@example.org or by using the form on our contact us page.