qPCR methods excel at detecting small amounts of nucleic acids, but by its nature the technique is vulnerable to contamination and amplification bias. Particularly when using complex samples such as clinical blood samples, there is a risk that contamination will introduce an amplification bias or that the wrong template entirely will be amplified.
Reginald Beer, Ph.D., a principal investigator at Lawrence Livermore National Laboratory (LLNL), presented his work on monodisperse picoliter PCR. LLNL studies viral and bacterial pathogens that threaten the population and food supply. “Viruses mutate quickly, constantly probing the immune defenses,” said Dr. Beer. “It’s hard to find causes of agents in new pandemics. When people first present sick, it’s hard for people in public health organizations to know what they’re sick with.” The goal is to reduce the time to treatment in a disease outbreak by optimizing diagnostic methods using qPCR. To this end, LLNL has harnessed microfluidic technologies to create chip qPCR.
Running qPCR in sample volumes of 10 picoliters not only reduces sample volumes and reagent volumes, it isolates the nucleic acids in the droplets and reduces PCR amplification bias. Standard or bulk PCR works quite well for oligonucleotides at high concentrations, but not so well when the concentration of target DNA or RNA is low and the background is complex.
A 10 picoliter droplet is 1,000,000 times smaller than a standard PCR sample volume. Poisson statistics predicts that this droplet will only contain one copy of viral DNA (or none) at typical concentrations, Dr. Beer said. That means that there is only one possible template in each reaction. Not every droplet will amplify, because not every droplet will contain DNA.
In their first publication, using qPCR with vaccinia, Dr. Beer and his team were able to come within 6% of the actual titer of the sample on the bench qPCR system. “Generally, when you talk to people in the field they are happy to get 40%,” he explained.